CN1764722A - Cyclic AMP response element activator proteins and uses related thereto - Google Patents

Abstract

The invention discloses newly identified cyclic AMP response element activator proteins (CREAP proteins). It is contemplated herein that said proteins are suitable drug targets for the development of new therapeutics to prevent, treat or ameliorate pathological conditions related to abnormal activation of CRE-dependent gene expression or abnormal activation of chemokines. The invention relates to methods to prevent, treat or ameliorate said pathological conditions and pharmaceutical compositions therefor comprising modulators with inhibitory effect on CREAP protein activity and/or CREAP gene expression. The invention also relates to methods to identify compounds with therapeutic usefulness to treat said pathological conditions, comprising identifying compounds that can inhibit CREAP protein activity and/or CREAP gene expression.

Description

Cyclic AMP response element activator protein and related uses thereof

Background of the Invention

Cyclic-AMP response element binding protein (CREB), activating transcription factor 1 (ATF1), and cAMP response element regulator (CREM) are a subgroup of closely related proteins belonging to the basic domain leucine zipper (bZIP) transcription factor superfamily . They are central regulators of transcriptional control produced by a variety of extracellular stimuli, such as hormones, growth factors, neuropeptides and neurotransmitters, calcium, hypoxia and oxidative stress. It has been well documented, primarily through CREB studies, that phosphorylation of conserved serine residues within the kinase-inducible domain (KID) of these proteins leads to the transcriptional activation of a variety of target genes involved in the regulation of cell growth and differentiation, metabolism, reproduction and development, regulation of neuronal activity, and immune regulation. All of these target genes have a conserved cis-acting loop-AMP response element (CRE) with the TGACGTCA palindromic sequence or an asymmetric variant that includes a CRE half-site with the core sequence TGAC (see Mayr B, Montminy M ., Nat RevMol Cell Biol 2001 Aug;2(8):599-609).

Transcriptional regulation occurs when phosphorylated CREB/CREM/ATF1 homo- and/or heterodimers bind to CRE sites via the bZIP domain, while the KID domain binds effector molecules such as the 265kDCREB-binding protein CBP or p300 and related Pol II The basal transcription machinery is recruited near the transcription start site.

Extensive research has been devoted to identifying molecules linking cellular stimulation to CREB/CREM/ATF1 activation. The complexity of these activators is exemplified by the study of kinases for CREB phosphorylation. Originally, CREB was regarded as the sole transcriptional regulator of extracellular stimuli that increased cAMP, which in turn activated protein kinase A (PKA) for CREB phosphorylation. However, subsequent studies revealed that CREB proteins are also phosphorylated by pp90RSK in response to growth factors, MSK-1 in response to mitogens and stress, CAMK II/IV in response to Ca ⁺⁺ rise, and AKT in response to growth factors. Responses to hypoxia and survival signals. From these studies it is clear that the regulation of the activity of CREB/CRE/ATF1 family proteins in cells is highly complex to ensure specificity and sensitivity in generating appropriate cellular output from various extracellular stimuli.

Here we describe the genome-level cellular function screening of a large number of full-length human cDNA clones (representing 11,000 to 15,000 gene transcripts) for proteins that activate CRE-dependent gene expression. These data point to several hitherto unidentified CRE activators, including KIAA0616, a gene of previously unknown function and here renamed CREAP1. Applicants have also discovered that 2 more distinct human proteins are similar in structure and activity to CREAP1, these two human proteins are named here as CREAP2 and CREAP3, and the mouse and Drosophila homologues, all of which are so far Member of an unknown gene family that regulates CRE-dependent gene expression.

Applicants also report here the surprising discovery that CREAP1 is a member of other proteins, including enolpyruvate phosphate carboxykinase (PEPCK), amphiregulin, and chemokines such as IL-8 and Exodus-1/MIPα effective inducer. Likewise, it can be considered herein that the CREAP proteins of the present invention can be used as new drug targets for the treatment of pathological conditions associated with genes containing CRE sites within their promoter regions, as well as for the treatment of diseases associated with PEPCK, amphiregulin and chemokines, especially Diseases associated with abnormal activation of IL-8 and Exodus-1/MIPα. These diseases include but are not limited to osteoarthritis, psoriasis, asthma, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, cancer, pathological angiogenesis, diabetes, hypertension, chronic pain and other inflammatory and autoimmune and neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's diseases.

The present invention also provides methods of identifying modulators that inhibit or enhance CREAP activity and/or inhibit or enhance CREAP gene expression and the use of such modulators for the treatment of these diseases in human and veterinary patients. The present invention also provides a pharmaceutical composition comprising the modulator.

Brief description of the invention

The present application relates to the discovery of a new family of proteins referred to herein as CREAPs, which are activators of CRE-dependent transcription and inducers of chemokines. Likewise, members of this protein family are considered herein to be appropriate targets for the development of new therapeutic agents to prevent, treat or alleviate pathological conditions associated with aberrant activation of CRE-dependent gene expression or aberrant activation of chemokines, including but not Limited to osteoarthritis, psoriasis, asthma, COPD, rheumatoid arthritis, cancer, pathological angiogenesis, diabetes, hypertension, chronic pain, and other inflammatory and autoimmune diseases. Furthermore, since loss of CREB function has been shown to be associated with deficits in learning and neurodegeneration, agonists of the CREAP protein could be used to prevent, treat or alleviate neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's diseases. Therefore, in one aspect the present invention relates to a method for identifying a modulator for preventing, treating or alleviating said disease, comprising: a) determining in vitro, ex vivo or in vivo that a candidate modulator inhibits or enhances the activity of a CREAP protein and/or or inhibit or enhance the ability of CREAP protein expression, and may further comprise b) determination of identified CREAP modulator reversal in vivo, ex vivo or in vitro models of said pathological condition and/or on the expression of said pathological condition The ability to observe pathological effects in clinical studies of patients.

In another aspect, the present invention relates to a method for preventing, treating or alleviating pathological conditions associated with abnormal activation of CRE-dependent gene expression or abnormal activation of chemokines, which comprises administering an effective amount of a CREAP regulator to a patient when needed, Wherein the regulator, for example, can inhibit or enhance the activity of any one or more of the CREAP proteins or inhibit or enhance the expression of any one or more of the CREAP proteins, wherein the CREAP proteins are selected from CREAP1, CREAP2 and CREAP3.

In one embodiment, the modulator comprises any one or more substances selected from antisense oligonucleotides, triple-helix DNA, ribozymes, RNA aptamers, siRNA and double-stranded or single-stranded RNA, wherein the substances Designed to inhibit the expression of CREAP protein. In another embodiment, the modulator comprises an antibody to a CREAP protein or a fragment thereof, wherein said antibody or fragment thereof is capable of inhibiting the activity of said CREAP protein. In another embodiment of the invention, the modulator comprises a peptidomimetic of a CREAP protein, wherein said peptidomimetic is capable of inhibiting the activity of said CREAP protein. It is contemplated that one or more modulators described herein may be administered simultaneously.

In another aspect, the present invention relates to a method for treating, preventing or alleviating pathological conditions associated with abnormal activation of CRE-dependent gene expression or abnormal activation of chemokines, comprising administering to a patient in need thereof a medicament comprising an effective amount of a CREAP modulator combination. In one embodiment, the modulator inhibits or enhances the activity of a CREAP protein or inhibits or enhances the expression of a gene encoding said protein in a patient, wherein said CREAP protein is selected from CREAP1, CREAP2 or CREAP3. In one embodiment, the modulator comprises any one or more substances selected from antisense oligonucleotides, triple-helix DNA, ribozymes, RNA aptamers, siRNA and double-stranded or single-stranded RNA, wherein the substances Designed to inhibit the expression of CREAP protein. In another embodiment, a modulator comprises an antibody or peptidomimetic of a CREAP protein or fragment thereof, wherein said antibody or peptidomimetic is capable of, for example, inhibiting the enzymatic or other activity of said CREAP protein. It is contemplated herein that one or more modulators of one or more of said proteins may be administered simultaneously.

In another aspect, the present invention relates to a method comprising one or more CREAP modulators in an amount effective for treating, preventing or alleviating pathological conditions associated with aberrant activation of CRE-dependent gene expression or aberrant activation of chemokines in a patient in need thereof A pharmaceutical composition, wherein the modulator is capable of inhibiting or enhancing the activity of a CREAP protein and/or inhibiting or enhancing the expression of a protein encoding CREAP, wherein the CREAP protein is selected from CREAP1, CREAP2 or CREAP3. In another embodiment, the modulator includes any one or more substances selected from antisense oligonucleotides, triple-helix DNA, ribozymes, RNA aptamers, siRNA, and double-stranded or single-stranded RNA, wherein the The substance is designed to inhibit the expression of CREAP. In another embodiment, a modulator comprises an antibody or peptidomimetic of a CREAP protein or fragment thereof, wherein said antibody or peptidomimetic is capable of, for example, inhibiting the enzymatic or other activity of said CREAP protein.

In another aspect, the present invention relates to a pharmaceutical composition comprising a CREAP protein.

In another aspect, the present invention relates to a method of treating, preventing or alleviating pathological conditions associated with abnormal activation of CRE-dependent gene expression or abnormal activation of chemokines, the method comprising administering to a patient in need thereof a pharmaceutical combination comprising a CREAP protein things.

In another aspect, the present invention relates to a method of diagnosing a subject suffering from a pathological condition associated with abnormal activation of CRE-dependent gene expression or abnormal activation of chemokines, possibly using a CREAP modulator or exogenous CREAP protein for treatment, the diagnostic method comprising detecting the level of CREAP protein in a biological sample from said subject, wherein a subject with abnormal levels compared to a control would be a suitable candidate for treatment .

In another aspect, the present invention relates to a method of diagnosing a subject suffering from a pathological condition associated with abnormal activation of CRE-dependent gene expression or abnormal activation of chemokines, the subject may be treated with one or more A suitable candidate for treatment with a CREAP modulator or an exogenous CREAP protein, the diagnostic method comprising determining the level of CREAP protein mRNA in a biological sample from said subject, wherein the subject has an abnormal mRNA level compared to a control would be suitable candidates for treatment.

In another aspect, there is also provided a method for treating, preventing or alleviating pathological conditions related to abnormal activation of CRE-dependent gene expression or abnormal activation of chemokines, comprising: (a) measuring CREAP mRNA and/or or CREAP protein levels and (b) administering an amount of CREAP modulator or exogenous CREAP protein to a subject with abnormal levels of mRNA and/or CREAP protein compared to controls sufficient to treat, prevent or alleviate said pathological condition.

Another aspect of the present invention also provides assay methods and kits, which include the necessary components for detecting the expression of polynucleotides encoding CREAP proteins or the levels of CREAP proteins or fragments thereof in biological samples from patients, said kits Included are, for example, antibodies or peptidomimetics that bind CREAP protein or fragments thereof, or polynucleotide probes that hybridize to CREAP polynucleotides. In preferred embodiments, such kits also include instructions for use detailing the procedures by which the kit components are used.

The present invention also relates to the use of CREAP regulator or exogenous CREAP protein for the production of medicines for treating, preventing or alleviating pathological conditions associated with abnormal activation of CRE-dependent gene expression or abnormal activation of chemokines. Preferably, said pathological condition is an autoimmune disease or a neurodegenerative disease. In one embodiment, the regulator includes any one or more substances selected from antisense oligonucleotides, triple-helix DNA, ribozymes, RNA aptamers, siRNA, and double-stranded or single-stranded RNA, wherein The above substances are designed to inhibit the expression of the CREAP gene. In another embodiment, the modulator comprises one or more antibodies or fragments thereof to a CREAP protein, wherein said antibodies or fragments thereof are capable of, for example, inhibiting CREAP enzyme activity or other activity. In another embodiment, the modulator comprises one or more peptidomimetics of a CREAP protein, wherein the mimetic is capable of, for example, inhibiting CREAP enzyme activity or other activity.

The present invention also relates to exogenous CREAP proteins or modulators of CREAP proteins for use as medicaments. In one embodiment, the regulator includes any one or more substances selected from antisense oligonucleotides, triple-helix DNA, ribozymes, RNA aptamers, siRNA, and double-stranded or single-stranded RNA, wherein Said substances are designed to inhibit the expression of CREAP. In another embodiment, the modulator comprises one or more antibodies to CREAP or peptide mimetics or fragments thereof, wherein the antibodies, peptide mimetics or fragments thereof are capable of, for example, inhibiting the enzymatic activity of CREAP or other activities. In another embodiment, the modulator comprises one or more peptidomimetics of a CREAP protein, wherein the mimetic is capable of, for example, inhibiting the enzymatic or other activity of CREAP.

Since the correct polynucleotide sequences of CREAP2 and CREAP3 have not been disclosed so far, it is considered herein that the present invention also provides isolated polypeptides comprising the amino acid sequences shown in SEQ ID NO: 16 and SEQ ID NO: 25, respectively. In addition, the present invention provides isolated polypeptides comprising the amino acid sequences shown in SEQ ID NO: 16 and SEQ ID NO: 25 and fragments thereof. According to this aspect of the present invention, there are also provided novel polypeptides of human origin and their biologically, diagnostically or therapeutically useful fragments, variants, homologues and derivatives, variants and derivatives of fragments and combinations of the above-mentioned substances. analog.

The present invention can also obtain isolated nucleic acids comprising the nucleotide sequences encoding the CREAP proteins disclosed herein, specifically CREAP2 and CREAP3 and homologues and fragments and/or equivalents thereof, or substantially identical to those having the sequence of SEQ ID NO: A nucleic acid similar to the nucleic acid of the nucleotide sequence shown in ID NO: 15 and SEQ ID NO: 24. In a preferred embodiment, the isolated DNA is in the form of a vector molecule comprising at least a portion of the DNA of the invention, in particular comprising the DNA shown in SEQ ID NO: 1, SEQ ID NO. DNA composed of nucleotide sequences.

Another aspect of the invention provides methods of producing the above-mentioned polypeptides, polypeptide fragments, variants and derivatives, fragments of the variants and derivatives, and analogs of the above-mentioned substances. In a preferred embodiment of this aspect of the present invention, a method for producing the above-mentioned CREAP protein is provided, which includes culturing a host cell expressing a vector integrated therein under conditions sufficient to express the polypeptide in the host cell, the expression vector containing An exogenous source of nucleotide sequences for a polynucleotide, thereby causing expression of said polypeptide, and optionally recovering the expressed polypeptide.

In a preferred embodiment of this aspect of the invention, there is provided a method of producing a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 16 or SEQ ID NO: 25, the method comprising sufficient steps in Cultivating the host cell in which the expression vector comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 16 or SEQ ID NO: 25 is cultured under conditions in which the polypeptide is expressed in the host cell. An exogenously sourced nucleotide sequence of the polypeptide, thereby causing the production of the expressed polypeptide, and optionally recovering the expressed polypeptide. Preferably, in any such method, the exogenous source nucleotide sequence comprises or is the nucleotide sequence shown in SEQ ID NO: 1, the nucleotide sequence shown in SEQ ID NO: 15 or the nucleotide sequence shown in SEQ ID NO: Nucleotide sequence in ID NO:24. According to another aspect of the present invention, there are provided products, compositions or methods utilizing the polypeptides and polynucleotides described above for, inter alia, research, biological, clinical and therapeutic purposes.

In another aspect, the invention provides host cells, preferably vertebrate cells, especially mammalian cells, or bacterial cells capable of propagating in vitro, which cells, once grown in culture, are capable of producing NO: 2, SEQ ID NO: 16, the polypeptide of the amino acid sequence in SEQ ID NO: 25 or a fragment thereof, wherein the cell contains a transcriptional control DNA sequence, preferably a sequence other than the human CREAP transcriptional control sequence, wherein the transcriptional control sequence Controlling the transcription of DNA encoding a polypeptide having an amino acid sequence according to SEQ ID NO: 2, SEQ ID NO: 16, SEQ ID NO: 25 or a fragment thereof, including but not limited to an amino acid sequence comprising an active portion or fragment of a CREAP protein.

In another aspect, the invention relates to methods for introducing a nucleic acid of the invention into one or more tissues of a subject in need of treatment, with the result that cells within the tissue express or secrete one or more of the nucleic acids encoded by said nucleic acid. protein.

Description of drawings

Figure 1 illustrates that CREAP1 is a highly conserved protein and contains a potent transcriptional activation domain. Amino acid sequences of human CREAP1 and predicted mouse, fugu and Drosophila CREAP1-related genes are shown. Shading indicates identical and highly conserved amino acids. Conserved potential PKA phosphorylation sites are boxed. The first sequence represents human, the second mouse, the third fugu and the fourth Drosophila.

Figure 2 shows the amino acid sequences of the full-length cDNAs corresponding to human and Drosophila CREAP proteins. Amino acids were aligned using ClustalW and shading indicates conserved amino acids.

Detailed description of the invention

The invention described herein is not considered limited to particular methodology, protocols and reagents as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention in any way.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing the materials and methodologies reported in the publications which may be used in connection with the present invention.

In practicing the present invention, many conventional techniques of molecular biology are employed. These techniques are well known and described in, for example, Current Protocols in Molecular Biology, Volumes I, II and III, 1997 (eds. F.M. Ausubel); Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; DNA Cloning: A Practical Approach, Volumes I and II, 1985 (ed. D.N. Glover); Oligonucleotide Synthesis, 1984 (ed. M.L. Gait); Nucleic Acid Hybridization, 1985, (Hames and Higgins); Transcription and Translation , 1984 (edited by Hames and Higgins); Animal Cell Culture, 1986 (edited by R.I. Freshney); Immobilized Cells and Enzymes, 1986 (IRL Press); Perbal, 1984, A Practical Guide to Molecular res Cloning; Series, Methods in P Enzymology (Academic Inc.); Gene Transfer Vectors for Mammalian Cells, 1987 (eds. J.H. Miller and M.P. Calos, Cold Spring Harbor Laboratory); and Methods in Enzymology volumes 154 and 155 (Wu and Grossman, and Wu, eds., respectively).

Abbreviations:

ABIN-2 A20-binding inhibitor of NF-κB activation-2

ACT1 NFkB-activator protein 1

ANKRD3 Ankyrin repeat domain protein 3

AP-1 Activator protein 1

ARHGEF1 Rho guanine nucleotide exchange factor (GEF) 1

ATCC American Type Culture Collection

ATF activates transcription factor

BZIP Basic zone leucine zipper

C/EBP CCAAT/enhancer-binding protein

CAD constitutive activation domain

CAMK Ca ⁺⁺ /calmodulin-dependent protein kinase

cAMP Cyclic AMP

CBP CREB binding protein

CNS central nervous system

COPD chronic obstructive pulmonary disease

CR53 putative transcription factor CR53

CRE Cyclic AMP Effect Element

CREB cyclic AMP response element binding protein

CREB1 cAMP response element binding protein 1

CRE-BPa cAMP response element-binding protein

CREM cAMP response element regulator

ERK Extracellular signal-regulated kinase

EST Expressed Sequence Tag

Human homologue of HPH2 Drosophila protein Polyhomeotic (Ph)

HPH2 Human Polycomb homolog 2

HTS High throughput screening

IBMX 3-isobutyl-1-methylxanthine

ICER Inducible cAMP early repressor

IkBα Inhibitor of nuclear factor κ-B kinase α subunit

IKK IkBα kinase

IKKγ IkBα Kinaseγ

IL-1 Interleukin-1

IL-8 Interleukin-8

IL-8P-Luc IL-8 promoter-reporter drives luciferase expression

IL-24 Interleukin-24

KIAA0616 Hypothetical protein predicted from cDNA clone KIAA0616

KID Kinase Inducible Domain

MAP3K11 Mitogen-activated protein kinase kinase kinase 11

MAP3K12 Mitogen-activated protein kinase kinase kinase 12

MEK Mitogen-activated protein kinase/ERK kinase

MEKK Mitogen-activated protein kinase/ERK kinase kinase-1

MSK Mitogen and stress-activated protein kinase

NFAT activation of T cell nuclear factor

NF-IL-6 Nuclear factor-interleukin-6 transcription factor

NF-κB Nuclear Factor of κ Light Polypeptide Gene Enhancer in B-Cells

NPY Neuropeptide Y

NR2F2 Nuclear receptor subfamily 2, group F, member 2

Oct-1 octamer-binding transcription factor 1

Oct-1/C/EBP octamer-binding transcription factor 1/CCAAT/enhancer-binding protein

PCK1 Enolpyruvate phosphate carboxykinase I

PKA Cyclic AMP-dependent protein kinase

POL II RNA polymerase II

relA reticuloendothelial proliferation virus oncogene homologue A, aliased as NF-κB subunit 3,

p65

Rho-GEF-p114Rho-specific guanine nucleotide exchange factor p114

RIPK2 Receptor-interacting serine-threonine kinase 2

RLU relative luminescence unit

RSK ribosomal S6 kinase

TBP TATA-binding protein

TEF1 Thyroid-stimulating embryonic factor 1

TF transcription factor

TNFα Tumor necrosis factor-α

TRAF6 TNF receptor-associated factor 6

TSHα Thyroid-stimulating hormone alpha

VCAM1 Vascular cell adhesion molecule-1

XboxP X-box binding protein 1

As used herein and in the appended claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, an "antibody" is one or more antibodies and equivalents thereof known to those skilled in the art. Additionally, a CREAP protein or "CREAP", unless otherwise indicated, includes any one or more of the CREAP proteins disclosed herein, specifically any one or more of the human CREAP1-3 identified herein as belonging to the CREAP protein family peptide.

The ability of a substance to "modulate" a CREAP protein (eg, a "CREAP modulator") includes, but is not limited to, the ability of a substance to inhibit or enhance the activity of a CREAP protein and/or to inhibit or enhance expression of any one or more of such proteins. Such modulators include agonists and antagonists of CREAP activity. Such regulation may also be involved in enabling the ability of other proteins to interact with the CREAP protein, such as associated regulatory proteins or proteins modified by CREAP.

As used herein, the term "agonist" refers to a molecule (ie, modulator) that can directly or indirectly regulate a polypeptide (eg, CREAP polypeptide) and increase the biological activity of the polypeptide. Agonists can include proteins, nucleic acids, carbohydrates or other molecules. Modulators that enhance the gene transcription or biochemical function of a protein are substances that increase the transcription of said protein or stimulate its biochemical properties or activity, respectively.

The term "antagonist" or "inhibitor" as used herein refers to a molecule (ie, a modulator) that can directly or indirectly modulate a polypeptide (eg, a CREAP polypeptide) and block or inhibit the biological activity of the polypeptide. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or other molecules. Modulators that inhibit the expression or biochemical function of a protein are substances that reduce the gene expression or biological activity of said protein, respectively.

"Nucleic acid sequence" as used herein refers to oligonucleotides, nucleotides or polynucleotides and fragments or portions thereof, and also to DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded, and represent the sense or antisense strand.

The term "antisense" as used herein refers to a nucleotide sequence that is complementary to a specific DNA or RNA sequence. The term "antisense strand" is used herein to refer to the nucleic acid strand that is complementary to the "sense" strand. Antisense molecules can be produced by any method, including synthesis by linking the gene of interest in reverse orientation to a viral promoter that allows synthesis of the complementary strand. Once introduced into a cell, the transcribed strand combines with the native sequence produced by the cell to form a duplex. These duplexes then block further transcription or translation. The nomenclature "negative" is sometimes used to refer to the antisense strand and "positive" is sometimes used to refer to the sense strand.

As considered herein, antisense oligonucleotides, triple-helix DNA, RNA aptamers, siRNA, ribozymes, and double- or single-stranded RNA are designed to inhibit CREAP expression so that inhibitory molecules designed according to the selected nucleic acid sequence of the protein can elicit Endogenous CREAP produces specific inhibitory effect. For example, knowledge of the CREAP1 nucleotide sequence can be used without undue experimentation to design antisense molecules that hybridize most strongly to CREAP mRNA. Also, a ribozyme that recognizes a specific nucleotide sequence of a protein of interest and cleaves it can be synthesized (Cech. J. Amer. Med Assn. 260: 3030 (1988)). Techniques for designing such molecules for targeted inhibition of gene expression are well known to those skilled in the art.

The CREAP proteins disclosed herein include, but are not limited to, human CREAP1, CREAP2, and CREAP3 polypeptides, and any and all forms of these polypeptides include, but are not limited to, partial forms, homologues, isoforms, precursor forms from humans or any other species , a full-length polypeptide, a fusion protein comprising any of the above protein sequences or parts. Fragments of interest include, but are not limited to, those comprising amino acids that are particularly important for normal CREAP function, for example including amino acids 356-580. The sequence of CREAP1 and variants thereof can be found in Genbank accession numbers NM_025021 and AB014516. To the applicant's knowledge, the complete and correct sequences of CREAP2 and CREAP3 have not been published before; partial sequences can be found in Genbank (CREAP 2 accession numbers XM_117201 (DNA) and XP_117201 (protein) and CREAP3 accession numbers AK090443 (DNA) and BAC03424 ( protein)). CREAP homologues include those disclosed herein, and those homologues apparent to those skilled in the art, and are included within the scope of the present invention. CREAP proteins may also be considered to include those isolated from naturally occurring sources of any species such as genomic DNA libraries and genetically engineered host cells containing expression systems, or produced by chemical synthesis, using, for example, an automated peptide synthesizer, or combinations of such methods CREAP protein. Methods for isolating and preparing such polypeptides are well known in the art.

The term "sample" or "biological sample" as used herein is used in its broadest sense. Biological samples from a subject can include blood, urine, or other biological material, using which samples CREAP protein activity or gene expression can be determined.

As used herein, the term "antibody" refers to whole molecules and fragments thereof, such as Fa, F(ab') _x2 and Fv, which are capable of binding an epitopic determinant. Antibodies that bind the CREAP polypeptides disclosed herein can be prepared using the entire polypeptide or a fragment containing a small peptide of interest as an immunizing antigen. Polypeptides or peptides used to immunize animals can be derived from translation of RNA or chemical synthesis, and if desired, the altered peptides or peptides can be linked to a carrier protein. Commonly used carriers for chemically coupling peptides include bovine serum albumin and thyroglobulin. The coupled peptides are then used to immunize animals (eg mice, rats or rabbits).

The term "humanized antibody" as used herein refers to an antibody molecule in which amino acids in non-antigen-binding regions have been replaced to more closely resemble human antibodies, yet retain the original binding ability.

A peptidomimetic is a synthetically derived peptide or non-peptidic agent based on the knowledge of key residues of a subject polypeptide capable of mimicking normal polypeptide function. Peptidomimetics are capable of disrupting the binding of a polypeptide to its receptor or other proteins and interfering with the normal function of the polypeptide. For example, a CREAP mimetic will interfere with normal CREAP function.

A "therapeutically effective amount" is an amount of drug sufficient to treat, prevent or alleviate pathological conditions associated with abnormal activation of CRE-dependent gene expression or abnormal activation of chemokines.

As used herein, "associated regulatory proteins" and "associated regulatory polypeptides" refer to polypeptides involved in the regulation of CREAP proteins, which can be identified by those skilled in the art using conventional methods such as described herein.

"Pathological conditions associated with aberrant activation of CRE-dependent gene expression or aberrant activation of chemokines" includes, but is not limited to, diseases such as: osteoarthritis, COPD, psoriasis, asthma, rheumatoid arthritis, cancer, pathology angiogenesis, diabetes, hypertension, chronic pain and other inflammatory and autoimmune diseases, and neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's diseases. Aberrant activation can include overactivation, such as those in which mRNAs encoding CREAP proteins are upregulated or the protein products of these genes have enhanced activity in cells by increasing absolute amounts or specific activities, and in which CRE-dependent gene expression is downregulated or abnormally low activation of chemokines.

As recognized herein, the present invention encompasses methods of using the CREAP genes and gene products disclosed herein to discover agonists and antagonists, respectively, that induce or inhibit CRE-dependent genes. As used herein, "CRE-dependent" genes include those genes that depend on the loop amp response element acting through CRE-binding proteins such as CREB1, CREB2, CRE-BPa (for review, see Lonze, B., and Ginty, D. .(2002) Neuron 35, 605; Muller FU, Neumann J, Schmitz W., Mol Cell Biochem 2000 Sep; 212(1-2): 11-7 and Mayr B, Montminy M.Nat Rev Mol Cell Biol 2001 Aug; 2(8):599-609). These genes include, but are not limited to, genes critical for metabolic control such as PEPCK, uncoupling protein-1, neuroregulatory molecules such as galanin and tyrosine hydroxylase, and genes including insulin and amphiregulin growth factor. Chemokines activated by CREAP include IL-8 and Exodus1/MIP3α and by CRE include MIP-1β (Proffitt et al., 1995, Gene 152:173-179; and Zhang et al., 2002; J. Biol Chem , 277:19042-19048).

"Subject" refers to any human or non-human organism.

In its broadest sense, the term "substantially similar" or "equivalent", when used herein in reference to a nucleotide sequence, means a nucleotide sequence corresponding to a reference nucleotide sequence, wherein the corresponding sequence encodes a Polypeptides encoded by nucleotides have substantially the same structure and function as polypeptides, for example, only changes in amino acids that do not affect the function of the polypeptide. It is expected that substantially similar nucleotide sequences encode polypeptides encoded by the reference nucleotide sequence. The percent identity between a substantially similar nucleotide sequence and a reference nucleotide sequence is desirably at least 80%, more desirably at least 85%, preferably at least 90%, even more preferably at least 95%, even more preferably Ground is at least 99%.

A nucleotide sequence "substantially similar" to a reference nucleotide sequence hybridizes to the reference nucleotide sequence at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO ₄ , 1 mM EDTA, 50 Wash in 2×SSC, 0.1% SDS at 50° C., more desirably hybridize in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C., in 1×SSC at 50° C. , wash in 0.1% SDS, more desirably hybridize in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO ₄ , 1 mM EDTA at 50°C, wash in 0.5×SSC, 0.1% SDS at 50°C , preferably hybridized in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO ₄ , 1 mM EDTA at 50°C, washed in 0.1×SSC, 0.1% SDS at 50°C, more preferably at 50°C Hybridized in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO ₄ , 1 mM EDTA, washed at 65°C in 0.1×SSC, 0.1% SDS, the nucleotide sequence still encodes a functionally equivalent gene product. Generally, hybridization conditions can be highly stringent conditions or less highly stringent conditions. In the case where the nucleic acid molecule is a deoxyoligonucleotide ("oligo"), highly stringent conditions can refer to, for example, at 37°C (for 14-base oligos), 48°C (for 17-base oligos), 55°C Wash in 6xSSC/0.05% sodium pyrophosphate (for 20-base oligos) and 60°C (for 23-base oligos). Appropriate ranges of such stringent conditions for nucleic acids of varying composition are described by Krause and Aaronson (1991), Methods in Enzymology, 200:546-556, and also by Maniatis et al., cited above.

"Increased mRNA transcript level" refers to the expression of CREAP polypeptide of the present invention from the natural endogenous mRNA in appropriate tissues or cells of an individual suffering from abnormal activation of CRE-dependent gene expression or abnormal activation of chemokines The amount of mRNA transcribed from the human gene is greater than the control level, specifically at least about 2 times the amount of mRNA found in the corresponding tissue of a subject not suffering from the disease, preferably at least about 5 times, more preferably at least about 10 times, most preferably at least about 100 times. Such elevated mRNA levels can ultimately lead to increased levels of protein transcribed from such mRNA in individuals with the disease compared to healthy individuals.

As used herein, "host cell" refers to a prokaryotic or eukaryotic cell containing heterologous DNA that has been introduced by any method, such as electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, etc. cell.

"Heterologous" as used herein means a "different natural source" or represents a non-natural state. For example, if a host cell is transformed with DNA or a gene from another organism, particularly from another species, that gene is heterologous to the host cell and also heterologous to progeny cells of the host cell carrying the gene . Likewise, heterologous refers to a nucleotide sequence that is derived from and inserted into the same native cell type, but which is present in a non-native state, eg, at a different copy number, or under the control of different regulatory elements.

A "vector" molecule is a nucleic acid molecule into which a heterologous nucleic acid can be inserted which can then be introduced into a suitable host cell. A vector preferably has one or more origins of replication, and one or more sites into which recombinant DNA can be inserted. Vectors generally have convenient means by which cells containing the vector can be selected from those that do not, eg, these vectors encode drug resistance genes. Common vectors include plasmids, viral genomes and (mainly in yeast and bacteria) "artificial chromosomes".

"Plasmid" is used herein to generally begin with a lowercase p, followed by an uppercase letter and/or number, consistent with standard naming conventions familiar to those skilled in the art. The starting plasmids disclosed herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids by well-known routine methods, published procedures. Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known and readily available to those skilled in the art. Moreover, a skilled artisan can easily construct any number of other plasmids suitable for use in the present invention. The nature, construction and use of such plasmids and other vectors in the present invention will be apparent to those skilled in the disclosure.

The term "isolated" means that a material is removed from its original environment (eg, the natural environment if the material is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide separated from some or all co-occurring materials in the natural system is isolated even if they are subsequently introduced into the natural system middle. Such polynucleotides may be part of a vector and/or such polynucleotides or polypeptides may be part of a composition and still be isolated in that such vector or composition is not part of its natural environment.

As used herein, the term "transcriptional control sequence" refers to a DNA sequence, such as an initiation codon sequence, an enhancer sequence, and a promoter sequence, which induces, represses, or otherwise controls the protein-encoding nucleic acid to which they are operably linked. transcription of the sequence.

As used herein, a "human transcriptional control sequence" is any of those transcriptional control sequences that are found in the respective human chromosome and are generally found in association with any of the human genes encoding the various CREAP proteins of the present invention.

As used herein, a "non-human transcriptional control sequence" is any transcriptional control sequence not found in the human genome.

As used herein, a "chemical derivative" of a polypeptide of the invention is a polypeptide of the invention that contains additional chemical moieties that are not normally part of the molecule. This chemical moiety can improve the solubility, absorption, biological half-life, etc. of the molecule. The moiety may optionally reduce the toxicity of the molecule, remove or attenuate any undesired side effects of the molecule, and the like. Moieties capable of modulating such effects have been disclosed, for example, in Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa. (1980).

The present invention is based on the surprising discovery that a protein previously designated "KIAA0616" in the public sequence database and of as yet unknown function is a CRE-activating protein. Referred to herein as CREAP1, in addition to normally activating CRE-dependent transcription, this polypeptide is also capable of inducing a variety of disease-associated genes such as chemokines, enzymes such as PEPCK and growth factors such as amphiregulin.

In addition, a search of public databases revealed that 2 cDNAs and proteins (albeit with errors and/or only partial sequences) that were previously included without indicating function, XP 117201 and FLJ00364, encode proteins with CREAP1-like activity. Thus, the present invention includes the heretofore unpublished exact nucleotide sequences encoding polypeptides designated herein as CREAP2 and CREAP3 and which belong to the novel CREAP protein family, as outlined in detail herein.

Accordingly, the invention provides isolated polypeptides comprising the amino acid sequences shown in SEQ ID NO: 16 and SEQ ID NO: 25. Furthermore, the present invention provides isolated polypeptides consisting of the amino acid sequences shown in SEQ ID NO: 16 and SEQ ID NO: 25. For example, such a polypeptide may be a fusion protein comprising the amino acid sequence of CREAP2 or CREAP3. The text also relates to fusion proteins comprising CREAP1.

The invention also includes isolated nucleic acid or nucleotide molecules, preferably DNA molecules, in particular molecules encoding a CREAP protein, especially CREAP2 or CREAP3. Preferably, the isolated nucleic acid molecule of the invention, preferably a DNA molecule, encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 25. Also preferred is an isolated nucleic acid molecule, preferably a DNA molecule, encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 25.

The present invention also includes: (a) carrier, it comprises CREAP protein, especially the nucleotide sequence of human CREAP1, CREAP2 or CREAP3 or its part and/or its complementary sequence (i.e. antisense sequence); (b) carrier molecule, preferably It is a carrier molecule comprising a transcriptional control sequence, especially an expression vector, which comprises the coding sequence of any of the above-mentioned CREAP proteins, which is operatively linked with a regulatory element directing the expression of the coding sequence; and (c) a genetically engineered host cell, which comprises such as The carrier molecule described herein or at least a fragment of any of the above-mentioned nucleotide sequences is operably linked to regulatory elements that direct the expression of the coding sequence in a host cell. As used herein, regulatory elements include, but are not limited to, inducible and non-inducible promoters, enhancers, operators, and other elements that drive and regulate expression well known to those skilled in the art. Preferably, the host cell may be a vertebrate host cell, preferably a mammalian host cell, such as a human cell or a rodent cell, such as a CHO or BHK cell. Also preferably the host cell may be a bacterial host cell, especially an Escherichia coli (E.coli) cell

Particularly preferred host cells, specifically the above-mentioned types of cells, they are capable of propagating in vitro and producing CREAP polypeptides when grown in culture, specifically comprising or consisting of the amino acid sequence shown in SEQ ID NOs: 2, 16 or 25 A polypeptide consisting of, wherein said cell comprises at least one transcriptional control sequence that is not the transcriptional control sequence of a native endogenous human gene encoding said polypeptide, wherein said one or more transcriptional control sequences control the DNA encoding said polypeptide transcription.

The invention also includes fragments of any of the nucleic acid sequences disclosed herein. Nucleic acid sequence fragments encoding CREAP polypeptides can be used as hybridization probes for cDNA libraries to isolate full-length genes and to isolate other genes with high sequence similarity to CREAP genes that have similar biological activity as CREAP. Probes of this type preferably have at least about 30 bases and comprise, such as about 30 to about 50 bases, about 50 to about 100 bases, about 100 to about 200 bases, or more than 200 bases . Probes can also be used to identify cDNA clones corresponding to full-length transcripts and genomic single or multiple clones comprising the complete CREAP gene including regulatory and promoter regions, exons and introns . An example of a screen includes isolating the coding region of the CREAP gene by synthesizing oligonucleotide probes using known DNA sequences. Labeled oligonucleotides having a sequence complementary to the gene sequence of the invention are used to screen human cDNA, genomic DNA or mRNA libraries to determine which members of the library the probe hybridizes to.

In addition to the gene sequences described above, homologues of such sequences are disclosed, preferably the Drosophila, mouse and Fugu rubripres CREAP proteins are identified (see Examples below). Additional homologues can be identified and readily isolated by molecular biology techniques well known in the art without undue experimentation. In addition, there may be genes encoding proteins with extensive homology to one or more domains of such gene products at other genetic loci within the genome. These genes can also be identified by similar techniques.

For example, an isolated nucleotide sequence encoding a CREAP polypeptide of the invention can be labeled and used to screen a cDNA library constructed from mRNA obtained from an organism of interest. When the cDNA library is from a different type of organism than that from which the marker sequences are derived, the hybridization conditions will be less stringent. Alternatively, the labeled fragments can be used again to screen a genomic library from the organism of interest using appropriate stringency conditions. Such low stringency conditions will be well known to those skilled in the art and will vary predictably depending on the particular organism from which the library and marker sequences are derived. Guidance on such conditions is found, eg, in Sambrook et al., cited above.

In addition, previously unknown differentially expressed genotypic sequences can be isolated by PCR using 2 degenerate oligonucleotide primer sets designed based on the amino acid sequence within the gene of interest. The template for the reaction may be cDNA obtained by reverse transcription of mRNA prepared from human or non-human cell lines or tissues known or suspected of expressing differentially expressed gene alleles.

The PCR products can be subcloned and sequenced to ensure that the amplified sequences represent those of the differentially expressed gene-like nucleic acid sequences. The PCR fragments can then be used to isolate full-length cDNA clones by a variety of methods. For example, the amplified fragments can be labeled and used to screen phage cDNA libraries. Alternatively, labeled fragments can be used to screen genomic libraries.

PCR techniques can also be used to isolate full-length cDNA sequences. For example, RNA can be isolated from appropriate cell or tissue sources following standard procedures. The RNA is subjected to a reverse transcription reaction using oligonucleotide primers specific for the most 5' ends of the amplified fragments to initiate first strand synthesis. The resulting RNA/DNA hybrid is then "tailed" with guanine using a standard terminal transferase reaction, and the hybrid can be digested with RNase H and subsequently primed with poly-C primers to initiate second-strand synthesis. Therefore, the upstream cDNA sequence of the amplified fragment can be easily isolated. For a review of cloning strategies that can be used, see eg Sambrook et al., 1989, supra.

Where the identified gene is a normal or wild-type gene, the gene can be used to isolate mutant alleles of the gene. Such separation is preferred in processes and disorders known or suspected to have a genetic basis. Mutant alleles can be isolated from individuals known or suspected of having a genotype that promotes disease symptoms associated with an inflammatory or immune response. The mutant alleles and mutant allele products can then be used in the diagnostic assay systems described below.

For example, cDNA of mutant genes can be isolated by using PCR - a technique well known to those skilled in the art. In this case, by hybridizing the oligo-dT oligonucleotide to mRNA isolated from tissues known or suspected to be expressed in individuals putatively carrying the mutant allele, and by extending the new strand using reverse transcriptase The first cDNA strand can be synthesized. The second strand of cDNA is then synthesized using an oligonucleotide that specifically hybridizes to the 5' end of the normal gene. The product was then amplified by PCR using these two primers, cloned into an appropriate vector, and subjected to DNA sequence analysis by methods well known to those skilled in the art. By comparing the DNA sequence of the mutant gene to the normal gene, the mutation responsible for the loss or altered function of the mutant gene product can be identified.

Alternatively, genomic or cDNA libraries are constructed and screened using DNA or RNA, respectively, from tissues known or suspected to express the gene of interest in individuals suspected or known to carry the mutant allele. The normal gene, or any suitable fragment thereof, can be labeled and used as a probe to identify the corresponding mutant allele in the library. Clones containing this gene are then purified by methods routinely practiced in the art and subjected to sequence analysis as described above.

In addition, expression libraries are constructed using DNA isolated or cDNA synthesized from tissues known or suspected to express the gene of interest in individuals suspected or known to carry the mutant allele. In this manner, gene products produced by putative mutant tissues can be expressed and screened using standard antibody screening techniques as described below in conjunction with antibodies raised against the normal gene product. (For screening techniques see, e.g., Harlow, E., and Lane, 1988, "Antibodies: A Laboratory Manual," Cold Spring Harbor Press, Cold Spring Harbor.) In mutations that result in altered function (e.g., as a result of missense mutations), In cases where the gene product is expressed, a panel of polyclonal antibodies may cross-react with the mutated gene product. Library clones detected by their reaction with such labeled antibodies can be purified and sequenced as described above.

The present invention includes those proteins or fragments thereof encoded by the nucleotide sequences shown in any of the sequences of SEQ ID NO: 1, 15, 24, 26, 28, 31.

Furthermore, the present invention includes proteins representing functionally equivalent gene products. Such equivalent differentially expressed gene products may comprise deletions, additions or substitutions of amino acid residues within the amino acid sequences encoded by the differentially expressed gene sequences described above, but which result in silent changes resulting in functionally equivalent differentially expressed gene products. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathicity of the residues involved.

For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, Serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include Aspartic Acid and Glutamic Acid. As used herein, "functional equivalent" may refer to a protein or polypeptide capable of exhibiting substantially similar in vivo or in vitro activities to the endogenous differentially expressed gene product encoded by the above differentially expressed gene sequence. "Functionally equivalent" can also refer to proteins or polypeptides that are capable of interacting with other cells or extracellular molecules in a manner substantially similar to that an endogenous differentially expressed gene product would. For example, a "functionally equivalent" peptide will be able to reduce antibody binding in an immunoassay to the corresponding peptide of the endogenous protein (i.e., a peptide whose amino acid sequence has been modified to obtain a "functionally equivalent" peptide) or to the endogenous protein itself, Antibodies against the corresponding peptides of endogenous proteins are raised therein. An equimolar concentration of a functionally equivalent peptide will reduce the binding of the corresponding peptide above by at least about 5%, preferably between about 5% and 10%, more preferably between about 10% and 25%, even more preferably between Between about 25% and 50%, and most preferably between about 40% and 50%.

The data disclosed herein indicate that specific polypeptide fragments are critical for the activity of CREAP family proteins. For CREAP1-3, these regions are in particular the conserved amino-terminal 200 amino acids and carboxy-terminal 100 amino acids, each serving as several conserved domains. Particularly preferred polypeptides of the invention are those comprising an amino acid sequence corresponding to or contained within an evolutionarily conserved region, such as the terminal 75 amino acids of each protein; such as the region from amino acids 1-75, more specifically, an amino acid fragment of CREAP1 1-68, amino acid fragment 1-74 of CREAP2 and amino acid fragment 1-66 of CREAP3.

Therefore, these CREAP peptide fragments and nucleic acid fragments encoding the active part of the CREAP polypeptide disclosed herein, as well as vectors comprising the fragments also fall within the scope of the present invention. As used herein, a nucleic acid fragment encoding an active part of a CREAP polypeptide refers to a nucleotide sequence having fewer nucleotides than the nucleotide sequence encoding the entire amino acid sequence of a CREAP polypeptide, and the sequence encodes a protein having the activity of the CREAP protein described herein. A peptide (ie, a peptide having at least one biological activity of a CREAP protein). Generally, the nucleic acid encoding a peptide having CREAP protein activity will be selected from the bases encoding the mature protein. However, in some cases it may be desirable to select all or part of the peptides from the leader sequence portion of the nucleic acid of the CREAP protein. These nucleic acids may also include linker sequences, modified restriction enzyme sites, and other sequences useful for molecular cloning, expression, or purification, or for recombinant peptides having at least one biological activity of the CREAP protein. CREAP peptide fragments and nucleic acids encoding peptide fragments having CREAP protein activity can be obtained according to conventional methods.

In addition, antibodies against these peptide fragments can be prepared as described above. Modifications (such as amino acid substitutions) that may increase the peptide immunogenicity of these peptide fragments may also be used. Likewise, using methods familiar to those skilled in the art, the polypeptides of the CREAP proteins can be modified to contain signal or leader sequences or to be coupled to linkers or other sequences for ease of molecular manipulation.

Polypeptides of the invention can be produced by recombinant DNA technology using techniques well known in the art. Accordingly, there is provided a method of producing a polypeptide of the invention comprising culturing a host cell into which is incorporated an expression vector comprising a protein encoding the polypeptide comprising the polypeptide shown in SEQ ID NO under conditions sufficient to express the polypeptide in the host cell. : 2, 16, 25, 27, 29 and 30, preferably an exogenous source polynucleotide of the amino acid sequence of SEQ ID NO 2, 16 and 25, thereby causing the production of the expressed polypeptide. Optionally, the method further comprises recovering the polypeptide produced by the cells. In preferred embodiments of such methods, said polynucleotide of exogenous origin encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, 16, 25, 27, 29 and 30. Preferably, the exogenous source polynucleotide comprises any one of the nucleotide sequences shown in SEQ ID NO: 1, 15, 24, 26, 28 and 31.

Thus, described herein are methods of making polypeptides and peptides of the invention by expressing nucleic acids encoding the respective polypeptide sequences. Methods well known to those skilled in the art can be used to construct expression vectors containing protein coding sequences and appropriate transcriptional/translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, eg, the techniques described in Sambrook et al., 1989, supra and Ausubel et al., 1989, supra. Alternatively, RNAs capable of encoding protein sequences of differentially expressed genes can be chemically synthesized using, for example, a synthesizer. See, eg, the technique described in "Oligonucleotide Synthesis", 1984, by Gait, M.J., IRL Press, Oxford, which is hereby incorporated by reference in its entirety.

A variety of host-expression vector systems can be used to express differentially expressed genes encoding sequences of the invention. Such host-expression systems represent not only vehicles that can produce and subsequently purify the coding sequence of interest, but also cells that can present differentially expressed gene proteins of the invention in situ when transformed or transfected with the appropriate nucleotide coding sequence. These expression systems include but are not limited to microorganisms such as bacteria (such as Escherichia coli, Bacillus subtilis (B.subtilis)) transformed with recombinant phage DNA, plasmid DNA or cosmid DNA expression vectors that contain differentially expressed gene protein coding sequences; Yeast transformed with recombinant yeast expression vectors containing differentially expressed gene protein coding sequences (such as Saccharomyces, Pichia); recombinant virus expression vectors containing differentially expressed gene protein coding sequences (such as baculovirus ) infection or transfection of insect cell systems; infection with recombinant viral expression vectors containing protein coding sequences (e.g. cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with recombinant vectors, including plasmids containing protein coding sequences ( For example, a plant cell system transformed with a Ti plasmid); or a plant cell system containing a promoter from a mammalian cell genome (such as a metallothionein promoter) or a mammalian viral promoter (such as an adenovirus late promoter; a vaccinia virus 7.5K promoter or a CMV promoter) for recombinant expression constructs in mammalian cell systems (eg COS, CHO, BHK, 293, 3T3).

Expression of the CREAP protein of the invention can also be performed by the cell from the cell's native CREAP-encoding gene. Methods of such expression are described in detail in, for example, US Patent Nos. 5,641,670; 5,733,761; 5,968,502 and 5,994,127, all of which are expressly incorporated by reference herein in their entirety. Cells expressing CREAP have been induced by the methods of any one of US Pat. Nos. 5,641,670; 5,733,761; 5,968,502 and 5,994,127 to be implanted into desired tissue of a living subject to increase the local concentration of CREAP in that tissue. Such methods have therapeutic implications for eg neurodegenerative diseases in which loss of CREB function occurs, and likewise agonists and/or exogenous CREAP proteins can be used to prevent, treat or ameliorate said diseases.

In bacterial systems, a number of expression vectors can be advantageously selected depending on the use of the protein to be expressed. For example, when large quantities of such proteins are produced, for antibody production or for screening peptide libraries, vectors directing the expression of high-level fusion protein products that are readily purified may be desirable. In this regard, fusion proteins comprising a hexahistidine tag (Sisk et alk, 1994: J. Virol 68: 766-775) can be used as supplied by a number of vendors (eg Qiagen, Valencia, CA). Such vectors include, but are not limited to, the Escherichia coli expression vector pUR278 (Ruther et al., 1983, EMBO J.2: 1791), in which the protein coding sequence can be individually ligated into the vector and the lac Z coding region in reading frame, thereby generating a fusion protein; pIN vector (Inouye and Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke and Schuster, 1989, J. Biol. Chem. 264:5503-5509) and the like. The pGEX vector can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption to glutathione sepharose beads followed by elution in the presence of free glutathione. The pGEX vector is designed to contain a thrombin or Factor Xa protease cleavage site to enable release of the cloned target gene protein from the GST moiety.

A promoter region can be selected from any desired gene using a vector comprising a reporter gene transcription unit lacking a promoter region, such as chloramphenicol acetyltransferase ("CAT"), or luciferase transcription unit, located downstream of single or multiple restriction sites for the introduction of candidate promoter fragments, ie, fragments that may contain a promoter. For example, introduction of a fragment comprising a promoter into a vector at a restriction site upstream of the CAT gene results in CAT activity, which can be detected by standard CAT assays. Vectors suitable for this purpose are well known and readily available. Two such vectors are pKK232-8 and pCM7. Therefore, the promoters used for expression of the polynucleotide of the present invention include not only well-known and readily available promoters, but also promoters that can be easily obtained by the above-mentioned techniques using a reporter gene.

Known bacterial promoters suitable for expressing polynucleotides and polypeptides according to the invention include the E. coli lacI and lacZ promoters, T3 and T7 promoters, T5tac promoter, lambda PR, PL promoter and trp promoter. Eukaryotic promoters known to be suitable in this regard include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, retroviral LTR promoters, such as Rous sarcoma virus (Rous sarcoma virus) ( "RSV"), and metallothionein promoters, such as the mouse metallothionein-I promoter.

Among insect systems, Autographa californica nuclear polyhedrosis virus (AcNPV) is one of several insect systems that can be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The coding sequence can be cloned separately into non-essential regions of the virus (eg, the polyhedrin gene) and placed under the control of an AcNPV promoter (eg, the polyhedrin promoter). Successful insertion of the coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (ie, a virus lacking the protein coat encoded by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera cells in which the inserted gene is expressed (eg, see Smith et al., 1983, J. Virol. 46:584; Smith, US Patent No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems are available. Where adenovirus is used as the expression vector, the coding sequence of interest is ligated into the adenoviral transcriptional/translational control complex, such as the late promoter and tripartite leader sequence. This chimeric gene is then inserted into the genome of the adenovirus by in vitro or in vivo recombination. Insertion into non-essential regions of the viral genome (such as the El or E3 regions) will result in the production of recombinant viruses that are viable and capable of expressing the protein of interest in infected host cells (see, for example, Logan and Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655-3659). Specific initiation signals are also required for efficient translation of the inserted gene coding sequence. These signals include the ATG initiation codon and adjacent sequences. In cases where the complete gene, including its own initiation codon and adjacent sequences, is inserted into an appropriate expression vector, no additional translational control signals may be required. However, in cases where only part of the gene coding sequence is inserted, exogenous translational control signals must be provided, including the ATG initiation codon. Furthermore, the initiation codon must be in phase with the reading frame of the coding sequence of interest to ensure translation of the complete insert. These exogenous translational control signals and initiation codons can come from a variety of sources, both natural and synthetic. The efficiency of expression can be enhanced by inclusion of appropriate transcriptional enhancer elements, transcriptional terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:516-544). Other commonly used systems are based on SV40, retrovirus or adeno-associated virus. Selection of appropriate vectors and promoters for expression in host cells is a well-known procedure and the necessary techniques for vector construction, introduction of the vector into the host and expression in the host itself are routine in the art. Typically, a recombinant expression vector will include an origin of replication, a promoter derived from a highly expressed gene to direct transcription of downstream structural sequences, and a selectable marker to isolate cells containing the vector after exposure to the vector.

In addition, it is possible to select host cell strains which are capable of modulating the expression of the inserted sequences or of modifying and processing the gene products in a specific, purposeful manner. Such modification (eg, glycosylation) and processing (eg, cleavage) of protein products are important for protein function. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure correct modification and processing of the foreign protein expressed. For this purpose, eukaryotic host cells with the cellular machinery for proper processing of primary transcripts, glycosylation and phosphorylation of gene products can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and the like.

The present invention also includes recombinant CREAP peptides and peptide fragments having CREAP protein activity. The term "recombinant protein" refers to the protein of the present invention produced by recombinant technology, wherein usually the DNA encoding the active fragment of CREAP is inserted into a suitable expression vector, which is used to transform the host cell to produce the heterologous protein. Specifically, the recombinant peptide fragments having CREAP protein activity include CREAP protein fragments comprising the conserved amino-terminal 200 amino acids or carboxy-terminal 100 amino acids of CREAP1, 2 or 3. Said fragments include amino acid fragments 1-267 and 575-650 of CREAP1, amino acid fragments 1-280 and 615-693 of CREAP2 and amino acid fragments 1-279 and 545-619 of CREAP3 and the human CREAP1-3 as discussed above. A fragment of the region of amino acids 1-75.

The recombinant proteins of the present invention may also include chimeric or fusion proteins of CREAP and different polypeptides available according to techniques familiar to those skilled in the art (see, for example, Current Protocols in Molecular Biology; Ausubel et al., John Wiley &Sons;1992; PNAS 85: 4879 (1988)).

For long-term high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines can be engineered to stably express proteins of differentially expressed genes. Instead of using an expression vector containing a viral origin of replication, host cells are transformed with DNA and selectable markers controlled by appropriate expression control elements (eg, promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.). Following foreign DNA introduction, engineered cells are grown in enriched media for 1-2 days and then transferred to selective media. The selectable marker in the recombinant plasmid confers resistance against selection and allows the cell to stably integrate the plasmid into its chromosome and allow the cell to grow to form foci, which are cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines expressing proteins of differentially expressed genes. Such engineered cell lines are particularly useful for screening and evaluating compounds capable of affecting the endogenous activity of expressed proteins.

A number of selection systems can be used, including but not limited to herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, 1962, Proc. Natl. Acad. .Sci.USA 48:2026) and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk ^- , hgprt ^- or aprt ^- cells, respectively. Antimetabolite resistance can also be used as dhfr for conferring resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527), gpt (Mulligan and Berg, 1981, Proc.Natl.Acad.Sci.USA 78:2072) conferring resistance to mycophenolic acid, neo (Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1) and hygro (Santerre et al., 1984, Gene 30:147) selection basis for conferring resistance to hygromycin.

An alternative fusion protein expression system allows facile purification of native fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-8976). In this system, the gene of interest is subcloned into a vaccinia recombinant plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. Extracts from recombinant vaccinia virus-infected cells were applied to Ni ²⁺ nitriloacetic acid-agarose columns and proteins with histidine tags could be selectively eluted with imidazole-containing buffer.

When the protein of the present invention is used as a component in an analytical system such as those described below, the protein of the present invention may be directly or indirectly labeled to facilitate detection of the complex formed between the protein and the test substance. Any of a variety of suitable labeling systems that may be used include, but are not limited to, radioisotopes such as125I, enzymatic labeling systems that produce a detectable calorimetric signal or light when exposed to a substrate, and fluorescent labels.

When recombinant DNA techniques are used to generate proteins of the invention for use in such assay systems, fusion proteins may advantageously be engineered to facilitate labelling, immobilization, detection and/or isolation.

Indirect labeling involves the use of proteins capable of specifically binding the polypeptide of the invention, such as labeled antibodies. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments and fragments produced by a Fab expression library.

It is also contemplated that the CREAP proteins disclosed herein are useful drug targets for the development of therapeutic agents for the treatment of pathological conditions associated with aberrant activation of CRE-dependent gene expression or aberrant activation of chemokines. Such conditions include, but are not limited to, osteoarthritis, psoriasis, asthma, COPD, psoriasis, asthma, rheumatoid arthritis, cancer, pathological angiogenesis, diabetes, hypertension, chronic pain, and other inflammatory and autoimmune diseases and neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's diseases.

In addition to chemokines, data also indicate that CREAP proteins can induce other genes such as PEPCK and amphiregulin. Amphiregulin is an EGF-like growth factor associated with cancer. PEPCK is a limiting factor in the synthesis of glucose and is also required for gluconeogenesis, and blockade of PEPCK is generally considered a therapeutic approach for the treatment of diabetes. Likewise, pathological conditions that can be treated by the modulators of the invention are also contemplated herein to include conditions associated with aberrant activity or expression of these proteins.

In another aspect, the present invention relates to a method of identifying modulators useful for the treatment, prevention or amelioration of the above-discussed pathological conditions, said method comprising: a) determining the inhibition of a candidate modulator in vitro, ex vivo or in vivo Or the ability to enhance CREAP activity and/or inhibit or enhance CREAP expression and further comprising b) assaying the identified CREAP regulator reversal in an in vitro, ex vivo or in vivo model of said pathological condition and/or in the presence of The ability to observe pathological effects in clinical studies of patients with the pathological condition.

Modulators capable of inhibiting or enhancing CREAP protein activity and/or inhibiting or enhancing CREAP expression can be identified using routine screening assays (eg, in vitro, ex vivo, and in vivo). CREAP activity and CREAP levels in a subject can be determined by routine assay methods using a biological sample derived from the subject. Gene expression (eg, mRNA levels) of CREAP can also be determined using methods familiar to those skilled in the art, including, for example, conventional Northern analysis or commercially available microarrays. In addition, the effect of test compounds on CREAP levels and/or levels of related regulatory proteins can be detected using ELISA antibody-based assays or fluorescent labeling reaction assays. These techniques are readily used for high-throughput screening and are familiar to those skilled in the art.

The data gathered from these studies can be used to identify those modulators that are therapeutically useful for the treatment of the pathological conditions discussed above, e.g., according to conventional methods in conventional in vitro or in vivo models of said pathological conditions and/or in Inhibitory substances are further assayed in clinical trials in humans with the pathological condition in order to assess the ability of the compound to treat, prevent or ameliorate the pathological condition in vivo.

By applying key information about the active portion of a CREAP polypeptide, the present invention enables the development of modulators of CREAP function, such as small molecule agonists or antagonists, by employing rational drug design familiar to those skilled in the art.

In another aspect, the present invention relates to a method of preventing, treating or ameliorating the pathological conditions described herein, said method comprising administering to a subject in need thereof a pharmaceutical composition comprising an effective amount of a CREAP modulator. Such modulators include antibodies directed against CREAP polypeptides or fragments thereof. In some particularly preferred embodiments, the pharmaceutical composition comprises an antibody that is highly selective for a human CREAP polypeptide or a portion of a human CREAP polypeptide. Antibodies against a CREAP protein are capable of causing aggregation of the protein in a subject and thereby inhibiting or reducing the activity of the protein. Such antibodies may also inhibit or reduce CREAP activity, eg, by directly interacting with the active site or preventing access of substances to the active site. CREAP antibodies can also be used to inhibit CREAP activity by preventing protein-protein interactions that may be involved in CREAP protein regulation and are necessary for the protein's activity. Antibodies having inhibitory activity as described herein can be produced and identified according to standard assays familiar to those skilled in the art.

CREAP antibodies can also be used for diagnosis. For example, these antibodies can be used according to conventional methods in order to quantify CREAP protein levels in a subject; for example, increased levels indicate overactivation of CRE-dependent gene expression (eg, activation of genes with CRE in their promoter regions) and possibly Indicates the degree of hyperactivation and the corresponding severity of the associated pathological condition. Thus, different CREAP levels can be indicative of various clinical forms or severity of pathological conditions associated with aberrant CRE-dependent gene expression or aberrant activation of chemokines. Such information is useful for identifying subpopulations of patients with pathological conditions that may or may not be responsive to treatment with a CREAP modulator.

It is also contemplated herein that monitoring CREAP levels or activity and/or detecting CREAP expression (mRNA levels) can be used as part of a clinical trial process, eg, to determine the efficacy of a given treatment regimen. For example, patients who have been administered the drug are evaluated and the clinician can identify those patients with CREAP levels, activity and/or expression levels higher than expected (i.e. levels higher or lower than control patients not experiencing the relevant disease state or by clinical level of patients whose disease state has been sufficiently remitted from the intervention). Based on this information, the physician then adjusts the dosage, administration regimen, or type of drug prescribed.

Factors to be considered in optimizing patient treatment include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the site of delivery of the active compound, the particular type of active compound, the method of administration, the timing of administration, and the practitioner. other known factors. The therapeutically effective amount of active compound to be administered can be governed by such considerations and will be the minimum amount required to treat the particular pathological condition.

Since the CREAP gene family contains highly conserved key regions, it is expected that peptidomimetics of CREAP proteins could serve as CREAP regulators. Therefore, one embodiment of the present invention are peptides derived or designed from CREAP family proteins capable of blocking CREAP function. These peptidomimetics are expected to block the function of all highly related CREAP proteins. Suitable peptidomimetics can be produced according to conventional methods based on the understanding of regions of the polypeptide required for CREAP protein activity. Briefly, short amino acid sequences in proteins can be identified by routine structure-function studies such as deletion or mutation analysis of wild-type proteins. Once critical regions are identified, it can be expected that the regions are responsible for critical functions (eg protein-protein interactions) if they correspond to highly conserved parts of the protein. Small synthetic peptidomimetics designed to look like the critical region are expected to compete with the intact protein and thus interfere with its function. Synthetic amino acid sequences may consist of amino acids matching all or part of this region. Such amino acids can be replaced by other chemical structures that are similar to the original amino acids but confer better pharmacological properties such as higher inhibitory activity, stability, half-life or bioavailability.

Suitable antibodies against CREAP proteins or related regulatory proteins can be obtained from commercial sources or raised according to routine methods. For example, described herein are methods for generating antibodies capable of specifically recognizing epitopes of one or more differentially expressed genes. Such antibodies may include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') ₂ fragments, fragments produced by a Fab expression library, Anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above antibodies.

To raise antibodies against the CREAP polypeptides discussed herein, various host animals are immunized by injection of the polypeptides or portions thereof. Such host animals include, but are not limited to, rabbits, mice and rats. A variety of adjuvants can be used to enhance the immune response, depending on the host species, including but not limited to Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as Lysolecithin, polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol and potentially useful human adjuvants such as BCG (BCG) and Corynebacterium parvum.

Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the serum of animals immunized with an antigen, such as a target gene product or an antigenic functional derivative of the antigen. For the production of polyclonal antibodies, host animals as described above are immunized by injection of the polypeptide or a part thereof added with the above-mentioned adjuvant.

Monoclonal antibodies are homogeneous populations of antibodies directed against a specific antigen, which can be obtained by any of the techniques used to produce antibody molecules from cultured continuous cell lines. Such techniques include, but are not limited to, the hybridoma technique of Kohler and Milstein (1975, Nature 256:495-497; and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc.Natl.Acad.Sci.USA 80:2026-2030) and EBV-hybridoma technology (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R.Liss, Inc., pages 77-96 ). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. Hybridomas producing mAbs of the present invention can be cultured in vitro or in vivo. The in vivo production of high titer mAbs makes it the presently preferred method of production.

In addition, a technique developed for the production of "chimeric antibodies" by splicing genes of mouse antibody molecules with appropriate antigen specificity and human antibody molecules with appropriate biological activity (Morrison et al., 1984, Proc. Sci., 81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda et al., 1985, Nature, 314:452-454). A chimeric antibody is a molecule in which different portions are derived from different animal species, eg, a chimeric antibody having variable or hypervariable regions of a mouse-derived mAb and a human immunoglobulin constant region.

Alternatively, techniques for producing single-chain antibodies (US Patent No. 4,946,778; Bird, 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85: 5879-5883; and Ward et al., 1989, Nature 334:544-546) in order to generate differentially expressed genes-single-chain antibodies. Single-chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge resulting in a single-chain polypeptide.

Most preferably, techniques for producing "humanized antibodies" can be used to generate antibodies against the polypeptides, fragments, derivatives and functional equivalents disclosed herein. Such techniques are disclosed in U.S. Patent Nos. 5,932,448; 5,693,762; 5,693,761; 5,585,089; 5,530,101; 5,910,771; 5,569,825; 5,625,126;

Antibody fragments capable of recognizing specific epitopes can be produced by known techniques. Examples of such fragments include, but are not limited to, F(ab') ₂ fragments produced by pepsin digestion of antibody molecules and Fab fragments produced by reduction of disulfide bonds of F(ab') ₂ fragments. Alternatively, Fab expression libraries can be constructed (Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with specificity of interest.

Antibody detection as described herein can be achieved using standard ELISA, FACS analysis and standard imaging techniques in vitro or in vivo. Detection is facilitated by conjugating (ie, physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic complexes include streptavidin/biotin and avidin/ Biotin; examples of suitable fluorescent substances include umbelliferone, luciferin, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent substances include Luminol; examples of bioluminescent substances include luciferase, luciferin and aequorin, examples of suitable radioactive substances ^include125I , ^131I , ^{35S or3H} .

Particularly preferred for ease of detection are sandwich assays, of which there are many variations, all of which are included within the scope of the invention. For example, in a typical forward assay, unlabeled antibodies are immobilized on a solid substrate and the sample to be tested is contacted with the bound molecules. After an appropriate incubation period sufficient to allow formation of antibody-antigen binary complexes, a secondary antibody labeled with a reporter capable of inducing a detection signal is added and incubated for a time sufficient to form antibody-antigen-labeled antibody ternary complexes. Any unreacted material is then washed away and the presence of antigen can be determined by observing the signal or quantified by comparison to a control sample containing known amounts of antigen. Variations of forward assays include simultaneous assays, where sample and antibody are added simultaneously to the bound antibody, or reverse assays, where labeled antibody and sample to be tested are first mixed, incubated, and added to an unlabeled surface binding antibody. These techniques are well known to those skilled in the art and minor variations of the techniques will be apparent. As used herein, "sandwich assay" is intended to include all variations of the basic two-site technique. For the immunoassays of the present invention, the only limiting factor is that the labeled antibody is specific for the CREAP polypeptide or related regulatory protein or fragment thereof.

The most commonly used reporter molecules can be enzymes, molecules containing fluorophores or radionuclides. For enzyme immunoassays, the enzyme is usually attached to the secondary antibody via glutaraldehyde or periodate. However, as can be readily appreciated, there are many different connection techniques well known to those skilled in the art. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase. Substrates for use with specific enzymes are usually chosen to produce a detectable color change when hydrolyzed by the corresponding enzyme. For example, p-nitrophenyl phosphate is suitable for alkaline phosphatase conjugates; for peroxidase conjugates 1,2-phenylenediamine or toluidine are typically used. It is also possible to use fluorogenic substrates, in which a fluorescent product is obtained instead of the chromogenic substrates mentioned above. A solution containing the appropriate substrate is then added to the ternary complex. The substrate reacts with an enzyme linked to the secondary antibody, giving a quantitative visual signal that can be further quantified (usually by spectrophotometer) to assess the presence of the polypeptide or polypeptide fragment of interest in the serum sample amount.

Alternatively, fluorescent compounds such as fluorescein and rhodamine can be chemically coupled to antibodies without altering their binding capacity. When activated by irradiation with light of a specific wavelength, the fluorochrome-labeled antibody absorbs the light energy, induces an excitable state within the molecule, and subsequently emits characteristic longer wavelength light. The emitted light exhibits a characteristic color detectable visually using an optical microscope. Both immunofluorescence and EIA techniques are well established in the art and are particularly preferred for the methods of the invention. However, other reporter molecules such as radioisotopes, chemiluminescent molecules or bioluminescent molecules may also be used. It will be apparent to the skilled person how to alter the operation to suit the required use.

In another embodiment, a nucleic acid comprising a sequence encoding a CREAP protein or a functional derivative thereof may be administered for treatment by gene therapy. Gene therapy refers to treatment by administering a nucleic acid to a subject. In this embodiment of the invention, the nucleic acid yields its encoded protein that mediates the therapeutic effect by promoting normal CRE-dependent gene expression or normal activation of chemokines.

Any method for gene therapy available in the art can be used according to the present invention. Representative methods are described below.

In a preferred aspect, the treatment comprises a CREAP nucleic acid that is part of an expression vector that expresses the CREAP protein, or a fragment or chimeric protein thereof, in an appropriate host cell. In particular, such a nucleic acid has a promoter, possibly linked to the CREAP coding region, said promoter being inducible or constitutive and, optionally, tissue-specific. In another specific embodiment, nucleic acid molecules are used wherein the CREAP coding sequence and any other desired sequences are flanked by regions that promote homologous recombination at desired sites within the genome, thereby providing for intrachromosomal expression of the CREAP nucleic acid (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).

Delivery of nucleic acids into a patient can be direct, where the patient is directly exposed to the nucleic acid or nucleic acid delivery vehicle, or indirect, where cells are first transformed in vitro with the nucleic acid and then transplanted into the patient. These two approaches are known as in vivo or ex vivo gene therapy, respectively.

In certain embodiments, the nucleic acid is administered directly in vivo, wherein expression of the nucleic acid produces the encoded product. This can be achieved by any of numerous methods known in the art, for example by constructing it as part of an appropriate nucleic acid expression vector and administering it as an intracellular part, for example by using a defective or attenuated retro infection with a recording virus or other viral vector (see, e.g., U.S. Pat. Lipid or cell surface receptors or transfection reagents, encapsulated in liposomes, microparticles or microcapsules, or administered by linking them to peptides known to enter the nucleus, by linking them to receptor-mediated internalization Ligand-linked administration of endocytosis (see eg US Patents 5,166,320; 5,728,399; 5,874,297 and 6,030,954, all of which are incorporated by reference in their entirety) (this can be used to target cell types that specifically express the receptor), and the like. In another embodiment, a nucleic acid-ligand complex is formed wherein the ligand comprises a viral fusion peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In another embodiment, nucleic acids can be targeted in vivo for specific uptake and expression by cells by targeting specific receptors (see, e.g., PCT publications WO92/06180; WO92/22635; WO92/20316; WO93/ 14188 and WO93/20221). Alternatively, the nucleic acid can be introduced intracellularly by homologous recombination and integrated into the host cell DNA for expression (see, eg, US Pat.

In a specific embodiment, a viral vector comprising a CREAP nucleic acid is used. For example, retroviral vectors can be used (see, eg, US Patents 5,219,740; 5,604,090; and 5,834,182). These retroviruses have been modified to delete retroviral sequences not required for viral genome packaging and integration into host cell DNA. The CREAP nucleic acid to be used in gene therapy is cloned into a vector that facilitates gene delivery into patients.

Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are particularly attractive vehicles for delivering genes to the respiratory epithelium. Adenoviruses naturally infect the respiratory epithelium resulting in mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells.进行基于腺病毒的基因治疗的方法描述于例如美国专利5,824,544；5,868,040；5,871,722；5,880,102；5,882,877；5,885,808；5,932,210；5,981,225；5,994,106；5,994,132；5,994,134；6,001,557和6,033,8843，所有这些在文中全文引用作为参考.

The use of adeno-associated virus (AAV) in gene therapy has also been proposed. Methods of producing and utilizing AAV are described, for example, in US Patent Nos. 5,173,414; 5,252,479; 5,552,311; 5,658,785;

Another approach to gene therapy involves the transfer of genes into cells in tissue culture by methods such as electroporation, lipofection, calcium phosphate-mediated transfection, or viral infection. Typically, methods of transfer involve transferring a selectable marker into cells. The cells are then subjected to selective conditions to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to the patient. In this embodiment, the nucleic acid has been introduced into the resulting recombinant cell prior to its use in vivo. Such introduction can be performed by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with viral or phage vectors containing nucleic acid sequences, cell fusion, chromosome-mediated Gene transfer, minicell-mediated gene transfer, spheroplast fusion, and more. A variety of techniques are known in the art for introducing foreign genes into cells and can be used in accordance with the present invention, provided that essential developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid into the cell so that the nucleic acid can be expressed by the cell and preferably inherited and expressed by its progeny of cells.

The resulting recombinant cells can be delivered to a patient by methods known in the art. In a preferred embodiment epithelial cells are used for injection, eg subcutaneous injection. In another embodiment, recombinant skin cells are applied to a patient as a skin graft. Recombinant blood cells (eg, hematopoietic stem or progenitor cells) are preferably administered intravenously. The envisioned amount of cells used depends on the desired effect, patient condition, etc. and can be determined by one skilled in the art.

Cells into which nucleic acids can be introduced for the purpose of gene therapy include any desired cell type available, and include, but are not limited to, epithelial cells, endothelial cells, keratinocytes, fibroblasts, myocytes, hepatocytes; blood cells such as T lymphocytes; cells, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, especially hematopoietic stem or progenitor cells, e.g. from bone marrow, umbilical cord Those stem cells or progenitor cells obtained from blood, peripheral blood, fetal liver.

In preferred embodiments, the cells used in gene therapy are autologous to the patient.

In embodiments where recombinant cells are used for gene therapy, the CREAP nucleic acid is introduced into the cell such that the CREAP nucleic acid can be expressed by the cell or its progeny, and then the recombinant cell is administered in vivo for therapeutic effect. In a particular embodiment, stem or progenitor cells are used. According to this embodiment of the invention, any stem and/or progenitor cells capable of being isolated and maintained in vitro can potentially be used. Such stem cells include, but are not limited to, hematopoietic stem cells (HSCs), epithelial-lined stem cells of epithelial tissues such as the skin and digestive tract, embryonic cardiomyocytes, liver stem cells (see, e.g., WO94/08598) and neural stem cells (Stemple and Anderson, 1992, Cell 71:973-985).

Epithelial stem cells (ESCs) or keratinocytes can be obtained from epithelial-lined tissues such as skin and digestive tract by known methods (Rheinwald, 1980, Meth. Cell Bio. 21A: 229). In stratified epithelial tissues such as skin, renewal occurs by mitosis of stem cells in the germinal layer closest to the basement membrane. Stem cells within the epithelium lining the digestive tract provide a rapid turnover rate for this tissue. ESCs or keratinocytes obtained from the skin or lining epithelium of the gut of a patient or donor can be grown in tissue culture (Pittelkow and Scott, 1986, Mayo Clinic Proc. 61:771). If the ESC is provided by a donor, methods of suppressing host-versus-graft reactivity (eg, radiation, drug or antibody administration to promote appropriate immunosuppression) can also be used.

With regard to hematopoietic stem cells (HSCs), any technique that provides for the isolation, propagation and maintenance of HSCs in vitro may be used in this embodiment of the invention. Techniques that can accomplish this include (a) isolation and establishment of HSC cultures from bone marrow isolated from future hosts or donors, or (b) use of previously established long-term HSC cultures, which can be allogeneic or heterogeneous. Non-autologous HSCs are preferably used in combination with methods of suppressing immune responses to future host/patient transplants. In a specific embodiment of the invention, human bone marrow cells are obtained from the posterior iliac crest by needle aspiration (see, eg, Kodo et al., 1984, J. Clin. Invest. 73:1377-1384). In a preferred embodiment of the invention, highly enriched or substantially pure HSCs can be obtained. This enrichment can be accomplished before, during, or after long-term culture, and can be accomplished by any technique known in the art. By using, for example, the improved Dexter cell culture technique (Dexter et al., 1977, J.Cell Physiol.91:335) or the Witlock-Witte culture technique (Witlock and Witte, 1982, Proc.Natl.Acad.Sci.USA 79:3608- 3612) can establish and maintain long-term cultures of bone marrow cells.

In a specific embodiment, a nucleic acid to be introduced for the purpose of gene therapy comprises an inducible promoter operably linked to the coding region such that expression of the nucleic acid can be controlled by the presence or absence of an appropriate inducer controlling transcription.

The invention also relates to the use of the polynucleotides of the invention as diagnostic reagents. Specifically, the present invention relates to a method for diagnosing pathological conditions associated with abnormal activation of CRE-dependent gene expression or abnormal activation of chemokines, wherein said method comprises: detecting messenger RNA in a suitable human-derived tissue and abnormal transcription in the cell, such as increased transcription, the messenger RNA is transcribed from a natural endogenous human gene encoding an amino acid sequence comprising SEQ ID NO: 2, 16, 25, wherein the abnormal transcription can be diagnosed The person suffers from the above-mentioned diseases. Specifically, the natural endogenous human gene comprises the nucleotide sequences shown in SEQ ID NO: 1, 15, 24. In a preferred embodiment, such a method comprises comparing a sample of said suitable tissue or cell, or an isolated RNA or DNA molecule derived from said tissue or cell, with an isolated nucleotide sequence of at least about 20 nucleotides in length. Contact, wherein said nucleotide sequence can hybridize with the isolated nucleotide sequence encoding the polypeptide consisting of the amino acid sequences shown in SEQ ID NO: 2, 16, and 25 under high stringency conditions. Detection of elevated transcripts indicates that the subject is a suitable candidate for treatment with one or more CREAP modulators.

Detection of mutated forms of the CREAP protein associated with dysfunction provides a diagnostic tool that can augment or define the diagnosis of disease or susceptibility to disease arising from temporally or spatially underexpressed, overexpressed, or altered CREAP genes Express. Individuals carrying mutations in genes can be detected at the DNA level by a variety of techniques.

Nucleic acids for diagnosis, especially mRNA, can be obtained from cells of a subject, such as from blood, urine, saliva, biopsy or autopsy material. Genomic DNA can be used directly for detection or enzymatically amplified using PCR or other amplification techniques prior to analysis. RNA or cDNA can also be used in a similar manner. Deletions and insertions can be detected by changes in the size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to a labeled nucleotide sequence encoding a CREAP polypeptide of the invention. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperature. DNA sequence differences can also be detected by changes in the electrophoretic mobility of DNA fragments in gels with or without denaturing agents or by direct DNA sequencing (eg, Myers et al., Science (1985) 230:1242). Sequence changes at specific positions can also be revealed by nuclease protection assays such as RNase and S1 protection or chemical fragmentation methods (see Cotton et al., Proc Natl Acad Sci USA (1985) 85:4397-4401). In another embodiment, arrays of oligonucleotide probes comprising a polynucleotide sequence encoding a CREAP of the invention or fragments of such a nucleotide sequence can be constructed for efficient screening of, for example, genetic mutations. Array technology methods are well known and widely applicable and can be used to solve a variety of problems in molecular genetics including gene expression, genetic linkage and genetic variation (see e.g. M. Chee et al., Science, Vol. 274, No. 610 -613 pages (1996)).

Diagnostic assays provide methods for diagnosing or determining susceptibility to disease by detecting mutations in the CREAP gene using the methods described. Furthermore, such diseases can be diagnosed by a method comprising detecting abnormally reduced or elevated levels of a polypeptide or mRNA in a sample derived from a subject. Decreased or increased expression can be measured at the RNA level using any method well known in the art for quantifying polynucleotides, such as nucleic acid amplification such as PCR, RT-PCR, RNase protection, Northern blot and other hybridization methods. Assay techniques for determining levels of proteins, such as polypeptides of the invention, in a sample derived from a host are well known to those skilled in the art. Such assays include radioimmunoassays, competitive binding assays, Western blot analysis and ELISA assays.

Therefore in another aspect the present invention relates to a diagnostic kit comprising: (a) a polynucleotide of the present invention, preferably a nucleotide sequence of SEQ ID NO: 1, 15 or 24 or a fragment thereof;

(b) a nucleotide sequence complementary to the nucleotide sequence in (a);

(c) a polypeptide of the present invention, preferably a polypeptide of SEQ ID NO: 2, 16, 25 or a fragment thereof;

(d) against polypeptide of the present invention, preferably against the antibody of the polypeptide of SEQ ID NO:2,16,25; Or

(e) for CREAP protein, preferably the peptide mimetic of SEQ ID NO:2,16,25.

It will be understood that in any such kit, (a), (b), (c), (d) or (e) comprise the essential components. Such kits are useful in the diagnosis of disease or susceptibility to disease, especially pathological conditions associated with CRE-dependent gene expression, abnormal activation or abnormal activation of chemokines. It is also contemplated that the kit comprises components (a)-(e) designed to detect levels of CREAP-associated regulatory proteins or proteins modified by CREAP as discussed herein.

The nucleotide sequences of the invention are also valuable for chromosomal mapping. The sequence is specifically targeted to and hybridizes to a particular location on an individual's human chromosome. Chromosomal mapping of related sequences according to the present invention is an important first step in linking those sequences to gene-associated diseases. Once a sequence has been mapped to a precise chromosomal location, the sequence's physical location on the chromosome can be linked to genetic map data. Such data can be found, for example, in V. McKusick, Mendelian Inheritance in Man (available online through the Johns Hopkins University Welch Medical Library). Relationships between genes and diseases that have been mapped to the same chromosomal region are then identified by linkage analysis (co-inheritance of physically adjacent genes).

Differences in cDNA or genomic sequence between affected and non-affected individuals can also be determined. If a mutation is observed in some or all affected individuals but not in any normal individuals, the mutation may be the cause of the disease.

The pharmaceutical composition of the present invention may also contain a substance that inhibits the expression of CREAP protein at the nucleic acid level. Such molecules include ribozymes, antisense oligonucleotides, triple-helix DNA, RNA aptamers, siRNA, and double- or single-stranded RNA directed to the appropriate nucleotide sequence of the CREAP nucleic acid. These inhibitory molecules can be generated by those skilled in the art using routine techniques without undue burden and experimentation. Modification (eg, suppression) of gene expression can be achieved, for example, by designing antisense molecules, DNA or RNA directed against the control regions of the gene encoding the CREAP polypeptides discussed herein, ie, the promoter, enhancer and intron. For example oligonucleotides derived from the transcription start site, eg positions -10 to +10 of the start site, may be used. Nonetheless, all regions of the gene can be used to design antisense molecules in order to generate antisense molecules that hybridize most strongly to mRNA and such suitable antisense oligonucleotides can be generated and analyzed by standard assays familiar to those skilled in the art. Identification.

Similarly, expression inhibition of gene expression can be achieved using the "triple helix" base pairing method. Triple helix pairing is useful because it inhibits the ability of the double helix to open sufficiently to bind polymerases, transcription factors, or regulatory molecules. Recent advances in therapy using triple-stranded DNA are described in the literature (Gee, J.E. et al. (1994) Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.). These molecules can also be designed to block mRNA translation by preventing the transcript from binding to the ribosome.

Ribozymes, enzymatically active RNA molecules, can be used to inhibit gene expression by catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to a complementary target RNA and subsequent endonucleic cleavage. Examples that can be used include engineered "hammerhead" or "hairpin" motif ribozyme molecules that can be designed to specifically and efficiently catalyze gene sequences For example endonucleic cleavage of the CREAP1, CREAP2 or CREAP3 genes.

Specific ribozyme cleavage sites within any potential RNA target were initially identified by scanning the target molecule for ribozyme cleavage sites including the following sequences GUA, GUU and GUC. Once the ribozyme cleavage site has been identified, evaluate the short RNA sequence between 15-20 ribonucleotides corresponding to the region of the target gene containing the cleavage site for secondary factors that may render the oligo ineffective Structure. Candidate target suitability can also be assessed by testing accessibility to hybridization to complementary oligonucleotides using a ribonuclease protection assay.

Ribozyme methods involve exposing cells to ribozymes or inducing expression of such small RNA ribozyme molecules in cells (Grassi and Marini, 1996, Annals of Medicine 28:499-510; Gibson, 1996, Cancer and Metastasis Reviews 15: 287-299). Intracellular expression of hammerhead and hairpin ribozymes targeting mRNA corresponding to at least one of the genes discussed herein can be used to inhibit the protein encoded by said gene.

The ribozyme can be delivered directly into the cell in the form of an RNA oligonucleotide incorporating the ribozyme sequence or introduced into the cell as an expression vector encoding the ribozyme RNA of interest. Ribozymes are usually expressed in sufficient numbers in vivo to efficiently catalytically cleave mRNA and thereby alter intracellular mRNA abundance (Cotten et al., 1989, EMBO J. 8:3861-3866). Specifically, a ribozyme-encoding DNA sequence capable of encoding a DNA sequence designed according to conventional well-known rules and synthesized by, for example, standard phosphoramidite chemistry can be ligated into the anticodon stem of the gene encoding the tRNA and The restriction enzyme site of the loop, and then transformed into the target cell and expressed in the target cell by conventional methods in the art. Preferably, an inducible promoter (such as a glucocorticoid or tetracycline response element) is also incorporated into the construct so that expression of the ribozyme can be selectively controlled. For saturating use, highly active and constitutively active promoters can be used. tDNA genes (ie, genes encoding tRNAs) are useful in this application because of their small size, high transcription rate and ubiquitous expression in different types of tissues.

Thus, ribozymes are usually designed to cleave virtually any mRNA sequence and cells are usually transformed with DNA encoding such ribozyme sequences so that controlled and catalytically effective amounts of the ribozymes are expressed. Thus, the abundance of almost any RNA species within a cell can be altered or disturbed.

The ribozyme sequence may be altered in substantially the same manner as described for antisense nucleotides, eg, the ribozyme sequence may comprise modified base moieties.

RNA aptamers can also be introduced into cells or expressed within cells to alter the abundance or activity of RNA. RNA aptamers are specific RNA ligands for proteins that are capable of specifically inhibiting the translation of said proteins, such as Tat and Rev RNA (Good et al., 1997, Gene Therapy 4:45-54).

Gene-specific inhibition of gene expression can also be achieved using conventional double-stranded RNA technology. A description of this technique can be found in WO 99/32619, the disclosure of which is incorporated by reference in its entirety. Furthermore, siRNA technology has been demonstrated as a method to inhibit gene expression (Cullen, BR Nat. Immunol. 2002, Jul;3(7):597-9).

Antisense molecules, triple-helix DNA, RNA aptamers and ribozymes of the present invention can be prepared by any method known in the art for the synthesis of nucleic acid molecules. These techniques include those used in the chemical synthesis of oligonucleotides such as solid phase phosphite chemical synthesis. Alternatively, RNA molecules can be produced by in vitro and in vivo transcription of DNA sequences encoding the polypeptide genes discussed herein. Such DNA sequences can be integrated into a variety of vectors with appropriate RNA polymerase promoters such as T7 or SP6. Alternatively, cDNA constructs for constitutive or inducible synthesis of antisense RNA can be introduced into cell lines, cells or tissues.

In addition to the methods described above for inhibiting CREAP expression, it is contemplated that one can identify and employ small molecules or other natural products to inhibit in vivo transcription of the polypeptides discussed herein. For example, one skilled in the art can establish assays for CREAP1 , CREAP2 or CREAP3 using routine methods, which assays can be applied to samples obtained from the culture of cell lines. Using this assay, cell lines can be screened for those expressing the CREAP protein of interest. These cell lines can be cultured, for example, in 96-well plates. Comparison of the effects of known regulators of gene expression such as dexamethasone, phorbol esters, heat shock on primary tissue cultures and cell lines allows selection of the most suitable cell line for use. Screening will simply be culturing cells for a certain length of time in the presence of different compounds added to each well and then measuring CREAP activity/mRNA levels.

To facilitate the detection of CREAP in the assays described above, luciferase or other commercially available fluorescent proteins can be genetically fused to the promoters of CREAP1, CREAP2 or CREAP3 as appropriate marker proteins. Sequences such as those upstream of the ATG of the CREAP1 promoter can be identified from the genomic sequence data by BLAST analysis of the NCBI genome sequence (current GenBank accession number of the genomic contig sequence of CREAP1 is NT_011295) using the sequence of GenBank accession number NM_025021. The results give a sequence of at least 5 kb upstream of the ATG of CREAP1 that does not contain any unknown bases. Two pairs of nested PCR primers capable of amplifying fragments of 2 kb or longer from human genomic DNA can be easily designed and tested. The promoter fragment can be easily inserted into any promoter-less reporter gene vector designed for expression in human cells (e.g. Clontech Promoter-less Enhanced Fluorescent Protein Vectors pECFP-1, pEGFP-1 or pEYFP, Clontech, Palo Alto, CA). Screening would simply be incubating the cells for the appropriate length of time in the presence of the different compounds added to each well and then assaying for reporter gene activity. The effect of promising compounds on CREAP1 activity and/or mRNA levels in vivo is determined using previously described in vivo models of pathological conditions. Additional methodologies such as appropriate culture times, culture conditions, reporter assays and other methodologies that can be used to identify small molecules or other natural products that can be used to inhibit CREAP protein transcription in vivo will be familiar to those skilled in the art.

Furthermore, the cDNA encoding the CREAP protein and/or the CREAP protein itself can be used to identify other proteins, such as kinases, proteases or transcription factors, that are modified or indirectly activated by the CREAP protein in a cascade of reactions. The proteins thus identified can be used, for example, in drug screening to treat the pathological conditions described herein. In order to identify these genes downstream of the CREAP protein, it is envisioned, for example, that a skilled artisan could use routine methods to treat disease state model animals with specific CREAP inhibitors, sacrifice the animals, recover relevant tissues and isolate total RNA from these cells and identify them using standard microarray analysis techniques. Altered signal levels relative to control animals (animals not administered drug).

Additionally, conventional in vitro or in vivo assays can be used to identify possible genes responsible for overexpression of CREAP protein. These associated regulatory proteins encoded by the genes so identified can be used to screen for drugs that may be effective therapeutic agents for the treatment of the pathological conditions described herein. For example, conventional reporter gene assays can be used, wherein the CREAP protein promoter region is placed upstream of the reporter gene, the construct is transfected into suitable cells (eg, from ATCC, Manassas, VA) and assayed by detecting expression of the reporter gene using conventional techniques. Upstream genes that cause activation of the CREAP promoter.

It is also conceived herein that the skilled person can follow conventional methods by designing, for example, antibodies or peptidomimetics of CREAP proteins and/or designing inhibitory antisense oligonucleotides, triple-helix DNA, ribozymes, siRNA, Double-stranded or single-stranded RNA and RNA aptamers, inhibit the function and/or expression of genes of related regulatory proteins or proteins modified by CREAP proteins as a way to treat the pathological conditions described herein. Pharmaceutical compositions comprising such inhibitory substances for use in the treatment of said pathological conditions are also contemplated.

Additional embodiments of the present invention relate to the administration of pharmaceutical compositions in combination with a pharmaceutically acceptable carrier, excipient or diluent for the treatment of any of the pathological conditions described herein. Such pharmaceutical compositions may comprise antibodies, peptidomimetics, and/or CREAP modulators (eg, agonists, antagonists, or inhibitors of CREAP expression and/or function) of the CREAP protein or fragment thereof directed against a CREAP polypeptide or peptide fragment. These compositions can be administered alone or in combination with at least one other agent, such as a stabilizing compound, and the compositions can be administered in any sterile biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water . Compositions may be administered to a patient alone or in combination with other agents, drugs or hormones.

When those skilled in the art deem it to be medically advantageous, for example, where a CREAP functional agonist has a therapeutic effect on, for example, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease, it may be administered comprising a CREAP protein or Pharmaceutical composition of its fragments. Such pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.

The pharmaceutical compositions disclosed herein for preventing, treating or alleviating pathological conditions associated with abnormal expression of CRE-dependent genes or abnormal activation of chemokines can be administered to patients in a therapeutically effective amount. A therapeutically effective amount refers to that amount of the compound sufficient to prevent, treat or ameliorate the condition.

The compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (through the mouth or nose) or topically, orally, buccally, parenterally or rectally.

For oral administration, pharmaceutical compositions may take the form of, for example, tablets or capsules, which are conventionally prepared with pharmaceutically acceptable excipients such as binders (for example, pregelatinized cornstarch, polyvinylpyrrolidone, , or hydroxypropylmethylcellulose); fillers (such as lactose, microcrystalline cellulose, or dibasic calcium phosphate); lubricants (such as magnesium stearate, talc, or silicon dioxide); disintegrants (such as potato starch or sodium starch glycolate) or a humectant (such as sodium lauryl sulfate). These tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional methods using pharmaceutically acceptable additives such as suspending agents (such as sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (such as lecithin or acacia); non-aqueous vehicles (such as almond oil). , oily esters, ethanol or fractionated vegetable oils) and preservatives (such as methyl paraben, propyl paraben or sorbic acid). These preparations may also contain suitable buffer salts, flavouring, coloring and sweetening agents.

Preparations for oral administration may be suitably formulated so as to provide controlled release of the active compound.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds used according to the invention are in the form of aerosol sprays packed in pressurized bags or nebulisers, using suitable propellants such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethylene Alkanes, carbon dioxide, or other appropriate gases are routinely delivered. For pressurized aerosols, the dosage unit may be determined by a valve delivering a metered amount. Capsules and cartridges of eg gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration by injection, eg, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, eg, in ampoules or in multidose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg sterile, pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds may also be formulated as depot formulations. Such long-acting formulations may be administered by implantation (eg, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated using suitable polymeric or hydrophobic materials (eg, as emulsions in oils) or ion exchange resins, or as sparingly soluble derivatives, eg, sparingly soluble salts.

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more dosage forms containing the active ingredient. For example packaging may contain metal or plastic foil, such as blister packs. The pack or dispenser device may be accompanied by instructions for administration.

Pharmaceutical compositions suitable for use in the present invention include compositions containing the active ingredient in an effective amount to achieve the intended purpose. Determination of an effective amount is within the ability of those skilled in the art.

For any compound, the therapeutically effective dose can be estimated initially in cell culture assays such as neoplastic cells or in animal models, typically mice, rabbits, dogs or pigs. Animal models can also be used to determine appropriate concentration ranges and routes of administration. A dose is determined in animal models to obtain a circulating plasma concentration range that includes the IC50 (ie, the concentration of the test compound which achieves a half-maximal inhibition of symptoms). Such information can then be used to determine useful doses and routes of administration in humans.

A therapeutically effective amount refers to the amount of active ingredient used to prevent, treat or alleviate the specified pathological condition of interest. Efficacy and toxicity can be determined by standard pharmaceutical methods in cell culture or experimental animals, eg, ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and this index can be expressed as the ratio LD50/ED50. Pharmaceutical compositions exhibiting large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to determine a range of dosage for human use. The dosage contained in such compositions lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. Dosage variations within this range are dependent upon the dosage form employed, patient sensitivity and the route of administration.

The exact dosage will be determined by the physician according to factors associated with the patient in need of treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors that may be considered include severity of the disease condition, general health of the subject, age, weight, and sex of the subject, diet, time and frequency of administration, drug combination, reaction sensitivity, and tolerance/response to treatment . Long-acting pharmaceutical compositions may be administered every 3 or 4 days, weekly or biweekly, depending on the half-life and clearance rate of the particular formulation.

Depending on the route of administration, normal doses may range from 0.1 to 100,000 micrograms, up to a total dose of about 1 g. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art employ different formulations of nucleotides than proteins or their inhibitors. Likewise, delivery of polynucleotides or polypeptides will be specific to a particular cell, condition, location, and the like. Pharmaceutical formulations suitable for oral administration of proteins are described, for example, in US Patents 5,008,114; 5,505,962; 5,641,515; 5,681,811;

The following examples further illustrate the invention and are not intended to limit the invention.

The following materials and methods were used to carry out Examples 1-5 below:

A collection of human full-length cDNA clones

We have archived and sequenced approximately 170,000 clones at the 5' end from multiple high-quality full-length cDNA libraries prepared from mRNA from 33 human tissue types. Using proprietary bioinformatics methods, we have identified all cDNA clones with ORF initiation ATG codons that were either experimentally determined or conceptually predicted and thus likely represent full-length transcript. A total of 20,702 clones in the pCMVSport6 vector (Invitrogen, Carlsbad, CA) from archived cloning series were rearranged into 384-well Genetix plates containing 60 μl LB medium (LB) using Q-bot (Genetix Limited, Hampshire, UK) middle. Based on bioinformatic analysis of the 5' sequences of these 20,702 clones, they were derived from approximately 11,000 genes for which there was strong evidence for structure and existence, although most of them were not functional, and 6,000 potential novel sequences Not yet present in public cDNA databases.

Arrayed clones are replicated to create multiple copies for archiving. One copy was used to generate miniprep DNA using a QIAGEN BioRobot 8000 (Qiagen, Valencia, CA). DNA samples were eluted into 96-well UV plates (Corning, Acton, MA) and DNA sample concentrations and yields were determined by measuring OD260 values on a SPECTRAmax 190 (Molecular Devices, Sunnyvale, CA). The resulting 20,702 DNA samples were then aliquoted in 384-well plates to generate multiple copies for archiving (80 pg/well in TE buffer) and for cell-based assays (in OPTI-MEM cell culture medium ( 50ng/well in Invitrogen).

Plates were sealed and stored at -20°C.

A genome-wide screen for activators of cyclic AMP response elements

Hela cells (ATCC, Manassas, VA) grown in 225 ml tissue culture flasks were trypsinized and diluted to ¹⁰⁵ cells/ml in DMEM medium (Invitrogen). The cell suspension was then dispensed into 384-well tissue culture plates at 30 μl/well using a Multi-drop 384 (Thermo Labsystems, Beverly, MA). After overnight incubation, a mixture consisting of 0.25 μl of Fugene 6 transfection reagent (Roche Applied Biosciences), 6 μl of OPTI-MEM medium containing 50 ng of pCRE-Luc plasmid construct (Stratagene) and 50 ng of each cDNA plasmid from the clone pool was incubated with Biomek FX A liquid handling robot (Beckman Coulter) was added to each well of a 384-well plate. Forty hours after transfection, luciferase activity in each well was assayed using the BrightGlo Luciferase Assay System (Promega, Madison, WI) in a LUMINOSKAN Ascent Luminometer (ThermoLabsystems) following the manufacturer's protocol. Raw luciferase data were processed using an in-house data processing and analysis system specifically designed to handle high-throughput gene functionalization projects. The entire analysis was performed in duplicate to generate 41,404 data points, each corresponding to a miniaturized transfection experiment using a single cDNA clone in a single well.

HTS Sampling Point Confirmation and Validation

For each group of 20,702 data points in duplicate, Z-scores (computed as fold activation divided by population standard deviation) and fold activation against population median were calculated and stored in an annotated searchable database. Potential activators were selected based on the following 2 criteria: (1) Z-score greater than 3.0 in either assay and (2) fold increase in luciferase/median greater than 8.0 in both assays. A total of 85 clones (0.4% of total clones) were identified based on the above criteria. DNA samples from these sampling points were retrieved from the clone archive and retransformed into bacterial strain XL-10Gold (Stratagene). Individual clones for each sample were picked and DNA minipreps were performed. A portion of the miniprep DNA sample was sequenced from the 5' end for clonal verification. The remaining samples were used for sampling site confirmation where they were hand-transformed with the pCRE-Luc reporter construct and the pRL-SV40 plasmid (Promega) encoding renilla (kenilla) luciferase under the control of the SV40 early promoter. Hela cells were transfected, followed by a dual luciferase assay (Promega) following the manufacturer's recommendations.

Northern blot analysis and in vitro transcription and translation analysis

The pCMVSport6 plasmid containing CREAP1 cDNA was digested with EcoRI and NotI, the insert was gel-purified using the Qiagen DNA Gel Extraction Kit and Enzo random primer labeling system was used according to the vendor's manual (Bio-11-dCTP deoxynucleotide package , Cat. No. 42723, Enzo Biochem, Farmingdale, NY) for labeling. Briefly, 200 ng of CREAP1 fragment or 100 ng of β-actin cDNA (Clontech) were denatured at 100 °C for 10 min, cooled on ice for 3-5 min, and then mixed with 5 μl of 10x hexamer random primers, 5 μl of CTP-11-Bio mix Mix with 1 μl Klenow fragments and incubate at 37°C for 4 hours. Probes were hybridized to multi-tissue mRNA Northern blot membranes (Clontech) according to the suggested protocol. Signal detection was achieved using a biotin detection kit (Ambion, Austin, TX). Expose the film to X-ray film for 10-30 seconds. After the first exposure, membranes were deprobed and rehybridized with a β-actin probe (Clontech) to normalize expression levels.

In vitro transcription and translation of CREAP1 protein was performed using TNT SP6 Quick Coupled Transcription and Translation System (Promega) according to the vendor's manual. Translation products were separated on Nupage precast gels (4-20%) (Invitrogen), transferred to nitrocellulose membranes and detected with the Transcend non-radioactive detection system (Promega) according to the manufacturer's instructions.

Analysis of CREAP1-CREB signaling pathway

For in vivo kinase assays, activation domains of CREB or ATF2 transcription factors (Stratagene, PathDetect In Vivo Signal Transduction Pathway Trans Reporter System) fused to yeast GAL4 DNA binding domain (amino acids 1-147) constructs were used. An HLR cell line containing a 5 x GAL4 DNA binding element and a TATA box driven luciferase reporter gene was used according to the manufacturer's protocol (Stratagene). Disperse ¹⁰⁴ HLR cells in each well of a 96-well tissue culture plate. After 16 hours, transfection with 100 ng of Creb-GAL4 or ATF2-GAL4 fusion construct, 30 ng of Renilla luciferase control plasmid, and 100 ng of pCMVSPORT6, pCMVSPORT-CREAP1, pFC-PKA or pFC-MEKK (Stratagene) activator plasmid, respectively cell. Transfection was performed using Fugene6 reagent (Roche Molecular Biochemicals, Basel, Switzerland) according to the manufacturer's manual. Forty hours after transfection, a Dual-Glo luciferase assay (Promega) was performed using the manufacturer's protocol.

For the dominant negative CREB assay, CREB dominant negative constructs (non-phosphorylated S133A mutant or DNA binding domain K287L mutant K-Creb) (Clontech, cat. no. K6014-1 ) were used. The transfection and luciferase assay methods above were modified with some modifications according to the manufacturer. HeLa cells, pCMVSPORT6, pCMV-CREAP1, pS133A-Creb or pK-Creb constructs were used for transfection.

Functional analysis of CREAP1 protein deletion

Amino acids 1-170, 1-356, 1-494, 1-580 and 170-650 of the CREAP1 protein were inserted into the pFlag-CMV4 expression vector (Sigma, St. Louis, MO) using a PCR strategy familiar to those skilled in the art.

Disperse 10 ⁴ Hela cells in each well of a 96-well tissue culture plate. After 16 hours, cells were transfected with 100 ng pCRE-Luc reporter construct, 30 ng Renilla luciferase control plasmid, and 100 ng pCMVSPORT6, pCMVSPORT-CREAP1 and different Flag-CREAP1 deletion fusion constructs, respectively. Transfection was performed using Fugene6 reagent (Roche Applied Biosciences) following the manufacturer's instructions. A Dual-Glo luciferase assay (Promega) was performed 40 hours after transfection. Firefly luciferase counts were normalized and plotted against Renilla luciferase.

Example 1

A genome-wide screen for cyclic AMP response element-activated genes

To identify cDNAs encoding proteins that lead to CRE activation, we screened a collection of annotated and indexed 20,702 human cDNA clones predicted to represent 11,000-16,000 clones within the miniaturized CRE-luciferase reporter gene system. full-length transcripts of a single gene. Experiments were performed in duplicate to generate 41,404 data points, each corresponding to luciferase activity from transient protein overexpression assays using approximately 3,000 pairs of cDNA clones of interest and plasmids containing the firefly luciferase gene HeLa cells were transiently transfected. Statistical analysis of the two sets of data has yielded a list of 85 clones resulting in at least an 8-fold increase in luciferase activity compared to the population median in 2 primary screening experiments in duplicate. In a subsequent second validation experiment, when individual colonies of these clones were recovered and assayed similarly but using Renilla luciferase under the control of the SV40 promoter for data normalization, 14 clones were identified (data not shown). The obtained sampling sites included a protein of so far unknown function, named KIAA0616 by the Kazusa DNA Institute (accession number: NM 025021). Based on our functional analysis of this protein, we renamed this protein as CRE-activating protein 1 or "CREAP1" based on its ability to activate CRE in the transiently overexpressed luciferase reporter gene assay system described herein.

To further define the pathway or promoter specificity of CREAP1, a panel of various promoter-luciferase constructs was tested in a similar assay system in Hela cells. These constructs enable testing of the ability of CREAP1 to activate CREB, NFAT and NFkB transcription factor binding elements and the authentic promoters of IL-8, VCAM, IL-24 and NPY. Additionally, 3 luciferase vectors are included for background testing and specificity controls. The results indicated that CREAP1 is a CRE-specific activator (data not shown).

Example 2

DNA sequence and amino acid sequence of CREAP1 gene

Both strands of the 2.4 kb cDNA insert of the active CREAP1 clone were sequenced according to conventional methods. The results showed that the coding region of the gene was 1950 nucleotides and the amino acid sequence was predicted to be 650 amino acids. Bioinformatic analysis indicated that CREAP1 does not contain conserved protein functional domains (such as kinase ATP-binding domains or transcription factor DNA-binding domains) except for a proline-rich domain at amino acids 379-448 in the middle of the molecule. The DNA and amino acid sequences are shown below.

Confirmed full-length DNA sequence of CREAP1:

CCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTG

AACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATC

CAGCCTCCGGACTCTAGCCTAGGCCGCGGGACGGATAACAATTTCACACAGGAAACAGCTATGACCAT

TAGGCCTATTTAGGTGACACTATAGAACAAGTTTGTACAAAAAAAGCAGGCTGGTACCGGTCCGGAATT

CCCGGGAGGAGGAGGAGGTGGCGGCGAGAAGATGGCGACTTCGAACAATCCGCGGAAATTCAGCGAGA

AGATCGCGCTGCACAATCAGAAGCAGGCGGAGGAGACGGCGGCCTTCGAGGAGGTCATGAAGGACCTG

AGCCTGACGCGGGCCGCGCGGCTCCAGCTCCAGAAATCCCAGTACCTGCAACTGGGCCCCAGCCGAGG

CCAGTACTATGGCGGGTCCCTGCCCAACGTGAACCAGATCGGGAGTGGCACCATGGACCTGCCCTTCC

AGCCCAGCGGATTTCTGGGGGAGGCCCTGGCAGCGGCTCCTGTCTCCTGACCCCCTTCCAATCCTCG

GGCCTGGACACCAGCCGGACCACCCGGCACCATGGGCTGGTGGACAGGGTGTACCGGGAGCGTGGCCG

GCTCGGCTCCCCACACCGCCGGCCCCTGTCAGTGGACAAACACGGACGGCAGGCCGACAGCTGCCCCT

ATGGCACCATGTACCTCTCACCACCCGCGGACACCAGCTGGAGAAGGACCAATTCTGACTCCGCCCTG

CACCAGAGCACAATGACGCCCACGCAGCCAGAATCTTTAGCAGTGGGTCCCAGGACGTGCACCAGAA

AAGAGTCTTACTGTTAACAGTCCCAGGAATGGAAGAGACCACATCAGAGGCAGACAAAAACCTTTCCA

AGCAAGCATGGGACACCAAGAAGACGGGGTCCAGGCCCAAGTCCTGTGAGGTCCCCGGAATCAACATC

TTCCCGTCTGCCGACCAGGAAAACACTACAGCCCTGATCCCCGCCACCCACAACACAGGGGGGTCCCT

GCCCGACCTGACCAACATCCACTTCCCCTCCCCGCTCCCGACCCCGCTGGACCCCGAGGAGCCCACCT

TCCCTGCACTGAGCAGCTCCAGCAGCACCGGCAACCTCGCGGCCAACCTGACGCACCTGGGCATCGGT

GGCGCCGGCCAGGGAATGAGCACACCTGGCTCCTCTCCACAGCACCGCCCAGCTGGCGTCAGCCCCCT

GTCCCTGAGCACAGAGGCAAGGCGTCACGCAGGCATCGCCCACCCTGTCCCGCTGTCACCCCATCACTC

AGGCTGTAGCCATGGACGCCCTGTCTCTGGAGCAGCAGCTGCCCTACGCCTTCTTCACCCAGGCGGGC

TCCCAGCAGCCACCGCCGCAGCCCCCAGCCCCCGCCGCCTCCTCCACCCGCGTCCCAGCAGCACCACC

CCCGCCACCCCCACAGGCGCCCGTCCGCCTGCCCCCTGGTGGCCCCCTGTTGCCCAGCGCCAGCCTGA

CTCGTGGGCCACAGCCGCCCCCGCTTGCAGTCACGGTACCGTCCTCTCTCCCCCAGTCCCCCCCAGAG

AACCCTGGCCAGCCATCGATGGGGATCGACATCGCCTCGGCGCCGGCTCTGCAGCAGTACCGCACTAG

CGCCGGCTCCCCGGCCAACCAGTCTCCCCACCTCGCCAGTCTCCAATCAAGGCTTCTCCCCAGGGAGCT

CCCCGCAACACACTTCCACCCTGGGCAGCGTGTTTGGGGACGCGTACTATGAGCAGCAGATGGCGGCC

AGGCAGGCCAATGCTCTGTCCCACCAGCTGGAGCAGTTCAACATGATGGAGAACGCCATCAGCTCCAG

CAGCCTGTACAGCCCGGGCTCCACACTCAACTACTCGCAGGCGGCCATGATGGGCCTCACGGGCAGCC

ACGGGAGCCTGCCGGACTCGCAGCAACTGGGATACGCCAGCCACAGTGGCATCCCCAACATCATCCTC

ACAGTGACAGGAGAGTCCCCCCCCCAGCCTCTCTAAAGAACTGACCAGCTCTCTGGCCGGGGTCGGCGA

CGTCAGCTTCGACTCCGACAGCCAGTTTCCCCTGGACGAACTCAAGATCGACCCCCTGACCCTCGACG

GACTGCACATGCTCAACGACCCCGACATGGTTCTGGCCGACCCAGCCACCGAGGACACCTTCCGGATG

GACCGCCTGTGAGCGGGCACGCCGGCACCCTGCCGCTCAGCCGTCCCGACGGCGCCTCCCCAGCCCGG

GGACGGCCGTGCTCCGTCCCTCGCCAACGGCCGAGCTTGTGATTCTGAGCTTGCAATGCCGCCAAGCG

CCCCCCGCCAGCCCGCCCCCGGTTGTCCACCTCCCGCGAAGCCCAATCGCGAGGCCGCGAGCCGGGCC

GTCCACCCACCCGCCCGCCCAGGGCTGGGCTGGGATCGGAGGCCGTGAGCCTCCCGCCCCTGCAGACC

CTCCCTGCACTGGCTCCCTCGCCCCCAGCCCCGGGGCCTGAGCCGTCCCCTGTAAGATGCGGGAAGTG

TCAGCTCCCGGCGTGGCGGGCAGGCTCAGGGGAGGGGCGCGCATGGTCCGCCAGGGCTGTGGGCCGTG

GCGCATTTTCCGACTGTTTGTCCAGCTCTCACTGCCTTCCTTGGTTCCCGGTCCCCCCAGCCCATCCGCGC

CATCCCCAGCCCGTGGTCAGGTAGAGAGTGAGCCCCACGCCGCCCCAGGGAGGAGGCGCCAGAGCGCG

GGGCAGACGCAAAGTGAAATAAACACTATTTTGACGGCAAAAAAAAAAAAAAAGGGCGGCCGCTCTAG

AGTATCCCTCGAGGGGCCCAAG (SEQ ID NO 1)

Predicted amino acid sequence of CREAP1 (650 amino acids):

MATSNNPRKFSEKIALHNQKQAEETAAFEEVMKDLSLTRAARLQLQKSQYLQLGPSRGQYYGGSLPNV

NQIGSGTMDLPFQPSGGLGEALAAAPVSLTPFQSSGLDTSRTTRHHGLVDRVYRERGRLGSPHRRPLS

VDKHGRQADSCPYGTMYLSPPADTSWRRTNSDSALHQSTMTPTQPESFSSGSQDVHQKRVLLLTVPGM

EETTSEADKNLSKQAWDTKKTGSRPKSCEVPGINIFPSADQENTTALIPATHNTGGSLPDLTNIHFPS

PLPTPLDPEEPTFPALSSSSSTGNLAANLTHLGIGGAGQGMSTPGSSPQHRPAGVSPLSLSTEARRQQ

ASPTLSPLSPITQAVAMDALSLEQQLPYAFFTQAGSQQPPPQPQPPPPPPPASQQPPPPPPPPQAPVRL

PPGGPLLPSASLTRGPQPPPLAVTVPSSLPQSPPENPGQPSMGIDIASAPALQQYRTSAGSPANQSPT

SPVSNQGFSPGSSPQHTSTLGSVFGDAYYEQQMAARQANALSHQLEQFNMMENAISSSSLYSPGSTLN

YSQAAMMGLTGSHGSLPDSQQLGYASHSGIPNIILTVTGESPPSLSKELTSSLAGVGDVSFDSDSQFP

LDELKIDPLTLDGLHMLNDPDMVLADPATEDTFRMDRL (SEQ ID NO 2)

Example 3

Northern blot and in vitro translation of CREAP1 protein.

To investigate the expression of the CREAP1 gene in different human tissues, we performed northern blot analysis using randomly labeled CREAP1 probes. According to Northern blot analysis, two mRNAs, 2.4Kb and 7Kb, were observed. The 2.4Kb band is consistent with the size of the coding region. The 7.0 Kb band reflects an alternative spliced form of the mRNA. Although expressed in most human tissues, CREAP1 mRNA is abundant in brain, heart, skeletal muscle and kidney (data not shown).

To test the accuracy of the predicted amino acid sequence of CREAP1, we used pCMVSPORT-CREAP1 as a template and performed in vitro transcription and translation reactions. Afterwards, the in vitro translation products were resolved in SDS-PAGE, and a single CREAP1 protein band of about 80Kd was observed, which is consistent with the view that it contains 650 amino acids (data not shown).

Example 4

CREAP1 acts through CREB

Since CREAP1 strongly activates CRE promoter transcription, we next investigated whether CREAP1 acts through the CREB pathway. To address this issue, fusion constructs consisting of the transactivation domain of CREB or ATF2 transcription factors and the GAL4 DNA-binding domain (amino acids 1–147) were used with HLRs stably integrated with the PathDetect trans-reporter plasmid (Stratagene). Cell lines were subjected to in vivo kinase assays. In this system, only those upstream regulators (presumably kinases) that activate the transactivation domain of CREB or ATF2 are able to drive the expression of the luciferase reporter gene. The results indicate that CREAP1 strongly stimulates the transactivation of the CREB-GAL4 fusion molecule on the GAL4 promoter and its activity is even stronger than that of the PKA catalytic subunit, the canonical kinase that phosphorylates CREB. Interestingly CREAP1 was unable to activate ATF2-GAL4 fusion molecules whereas MEKK (upstream kinase of the ATF2 pathway, Stratagene kit manual) was able to stimulate ATF2 fusion more than 100-fold. This result demonstrates that CREAP1 is a specific upstream activator of the CREB pathway.

To further confirm this observation, two CREB dominant negative constructs (non-phosphorylatable S133A mutant or DNA binding domain mutant K287L (Clontech)) were used in co-transfection assays. Experimental data showed that either the CREB S133A mutant or the K287L mutant could completely abolish the activation of the CRE promoter by CREAP1, indicating that CREAP1 specifically acts upstream of the CREB signal transduction pathway and that the phosphorylation and DNA binding activity of CREB are both signaled by CREAP1. needed.

Example 5

Functional Analysis of the Intramolecular Domain of CREAP1 Protein

To study the functional domains within the CREAP1 molecule, the CREAP1 protein amino acid fragments 1-170, 1-356, 1-494, 1-580, and 170-650 were subcloned into the pFlag-CMV4 vector by using a PCR-based strategy and as described above. Functionality was tested in the Dual Glo luciferase assay as described. The results suggest that an amino-terminal fragment comprising amino acids 1-170 of CREAP1 is important for its function, as K5 (aa 170-650) lacking this amino-terminus lacks almost all stimulatory activity. However, the 1-170 fragment (K1) alone is not sufficient for its function. On the other hand, the C-terminus of CREAP1 is not essential for its function, since the K4 deletion (deletion of amino acids 581-650) retained almost all wild-type activity. A comparison of the activities of K2 and K4 showed that amino acids 356-580 (with a proline-rich domain) are very important for CREAP1 function, since removal of this portion from K4 (which results in the production of K2) reduced CREAP1 functional activity to 1/ 10 (see Table 1 below). fragment Amino acids of CREAP1 active SEQ ID NO# K1K2K3K4K5 1-170, 1-356, 1-494, 1-580170-650 Inactive Inactive Partially active All active Inactive 3233343536

Table 1. Functions of CREAP fragments.

The following materials and methods were used to carry out the experiments outlined in Examples 6-9 below:

DNA construct

The pGL-2-IL-8 _P -Luc constructed in house using conventional methods (Roebuck, J. Interferon and Cytokine Res. 19: 429-438 (1999)) contains the 1.5 kb sequence containing the IL-8 promoter driven Firefly luciferase gene. pGL3B-IL-8 was constructed by ligation of 1.5 kb human IL-8 promoter DNA digested with Hind III/XhoI pGL-2-IL-8 _P -Luc and inserted into Hind III/XhoI digested pGL3Basic (Promega) _P -Luc.

The pIL-8Luc reporter was constructed by inserting the human IL-8 gene -1491 to +43 region into the pGL3Basic vector (Promega, Inc). PCR generated wild-type minimal IL-8 promoter and point mutations. Wu et al. (Wu et al., J. Biol. Chem., 272:2396-2403 (1997)) describe mutations in AP-1, C/EBP, NF-κΒ. The sequence TGACATAA of the putative CRE-like site was mutated to TCGATCAA. Hexaplex copies carrying a CRE-like response element (pCREL-Luc) or the CRE- A promoter construct with 5 copies of the TGACACAA-like element. All techniques were performed using conventional methods.

Construction of IL-8 promoter deletion and point mutation variants

Polymerase chain reaction (PCR) was used to generate IL-8 promoter variants. PCR amplification cycles were: 94°C for 2 minutes, 5×[94°C for 15 seconds, 55°C for 30 seconds and 72°C for 15 seconds] and 20×[94°C for 15 seconds, 65°C for 30 seconds and 72°C for 15 seconds]. Advantage 2 DNA polymerase (BD Biosciences) was used for all amplification steps. All variants were amplified using a common antisense primer P2.1 having the nucleotide sequence (5'-GCCC AAGCTT TGTGCTCTGCTGTCTCTGAAAG-3') (SEQ ID NO 3), which corresponds to the human IL-8 gene sequence +13-+ 43 (Roebuck' J. Interferon and Cytokine Res. 19:429-438 (1999). The BamHI restriction site (underlined) was included in all sense primers. The PCR product was gel purified and cloned using Zero Blunt TOPOPCR Kit (Invitrogen) ligation. Sequence confirmed clones were cut with HindIII/BamH I from pCR-Blunt II-TOPO and ligated into Hind III/BamH I digested pGL3Basic. Carrying a truncated minimal pIL-8p[δAP-1]-Luc of the IL-8 promoter was generated by amplification with P2.1 and S3 (5'-GCCCTGAGGGGATGGGCCATCAG-3') (SEQ ID NO 4), which were Primers used to generate a 157nt product corresponding to human IL-8 gene sequence -114-+43.

Use wtAP-1 (5'-CGC GGATCC GAAGTGTGAT GA CTCAGGTTTGCCCTG-3') (SEQ ID NO 5) and mAP-1 (5'-CGC GGATCC GAAGTGTGAT AT CTCAGGTTTGCCCTG-3') (SEQ ID NO 6) and P2.1 primers respectively Amplification of the minimal IL-8 promoter carrying wild-type or mutant AP-1 sites. Underlined are mutated nucleotides within the AP-1 site. Both 187nt products correspond to human IL-8 gene sequence -144-+43. The wild-type and AP-1 mutants were named pIL-8p[wtAP-1]-Luc and pIL-8p[mutAP-1]-Luc, respectively.

IL-8 minimal promoter variants carrying mutated Oct-1/C/EBP and NF-κB sites were prepared in a 2-step PCR step. In the first step PCR, SP3_NF-κBmut (5'-GCCCTGAGGGGATGGGCCATCAGTTGCAAATCGT TAAC TTTCCTCTGACATAAT-3') (SEQ ID NO 7) or SP3_Oct-1mut (5'-GCCCTGAGGGGATGGGCCATCAG C T A C G A G TCGTGGAAT-3') (SEQ ID NO 8), sense primers and P2.1 antisense primers were amplified to obtain 157nt products carrying mutated NF-κB and Oct-1/C/EBP binding sites, respectively. Underlined are the mutated nucleotides within the NF-κB and Oct-1 sites. In the second step PCR, AP-1_Bam sense primer (5'-CGC GGATCC GAAGTGTGATGACTCAGGTTTGCCCTGAGGGGATGGGC-3') (SEQ ID NO 9) and P2.1 antisense primer are used to reamplify the two products of the first step PCR reaction (100 fmol per reaction) to generate a 187nt cDNA corresponding to human IL-8 gene sequence -144-+43. NF-κB and Oct-1/C/EBP binding site mutants were named pIL-8p[mutNF-κB]-Luc and pIL-8p[mutOct-1]-Luc, respectively.

IL-8 minimal promoter variants carrying mutated CRE-like response elements were generated in a three-step PCR step. In the first step of PCR, CREmut sense primer (5'-CAGTTGCAAATCGTGGAATTTCCCTCT CGATC AATGAAAAGATG-3') (SEQ ID NO 10) and P2.1 antisense primer were used to generate a 137nt product. Underlined are mutated nucleotides within the CRE-like site. In the second step PCR, SP3_Oct-1wt sense primer (5'-GCCCTGAGGGGATGGGCCATCAGTTGCAAATCGTGGAAT-3') (SEQ ID NO 11) and P2.1 antisense primer were used to reamplify the product of the first step PCR reaction (each reaction 100 fmol) produced a 157nt product corresponding to the human IL-8 gene sequence -114-+43. Finally, in the third step PCR, the IL-8 minimal promoter variant 5 The ' end extends to the -144 nucleotide position of the human IL-8 gene. The resulting construct used in this study was named pIL-8p[mutCRE_like]-Luc. By PCR using CRElike_S (5'-CGCCTGGTACCGAGCTCTG-3') (SEQ ID NO 12) sense primer and CRelike_AS (5'-ACCCAAGATCTCGAGCCCG-3') (SEQ ID NO 13) antisense primer and template oligonucleotide (5 '-CGCCTGGTACCGAGCTC TGACATAATGACATAATGACATAAT GACATAATGACATAATGACATAA TTACGCGTGCTAGCCCGGGCTCGAGATCTTGGGT-3' (SEQ ID NO 14), (each reaction 100fmol) was amplified to prepare the promoter construct carrying the IL-8 promoter concatemer CRE-like response element. Underlined is Hexamer copy of the CRE-like response element (TGACATAA). PCR amplification parameters were as described above. The 99 nucleotide PCR product was cut with KpnI and BglII, gel purified and ligated into KpnI/BglII digested pTAL vector (BD Biosciences) pTAL-6 x [CRE_like] reporter was generated.

DNA preparation for high-throughput screening

The arrayed clones described above were replicated to generate multiple copies for archiving. One copy was used to generate miniprep DNA using a QIAGEN BioRobot 8000 (Qiagen, Valencia, CA). Briefly, for each 384-well plate, a 2 μl glycerol stock was used to inoculate Greiner 384-deep well plates containing 100 μl LB broth (Gibco BRL)-8% glycerol. Greiner plates were then covered with air vented paper (Qiagen), wrapped in saran wrap and incubated at 37°C without shaking for -22 hours. Subsequently, 5 μl of the culture was transferred from a 384-well Greiner plate into a Qiagen 96-well deep plate containing 1 ml Terrific broth (KD Medical) (+ 100 μg/ml ampicillin) per well. Four Qiagen plates were covered with air-perforated paper and incubated for -22 hours in a 37°C incubator with shaking at 250 rpm. Bacterial cells were pelleted by centrifugation at 4000 rpm for 15 min, the supernatant was decanted, and the plate was processed using a Qiagen BioRobot 8000 to generate a DNA preparation. The protocol used was based on the manufacturer's protocol 'QIAprep Turbo96 PB (1-4 plates)' with the only modification to use 96-well UV-transparent plates (Corning) as elution plates. Concentration and yield of DNA samples were determined by measuring OD260 values on a SPECTRAmax 190 (Molecular Devices). The resulting 20,702 DNA samples were then aliquoted to generate archived multiple copies (80 pg/well in TE buffer). For assays using pools of 2,368 cDNA clones, DNA aliquots were generated in 96-well PCR plates (ABGene, Rochester, NY) at 6 μl per well at a concentration of OPTI-MEM I cell culture medium (Gibco BRL, Carlsbad, CA 20ng DNA/μl in ). DNA aliquots for screening of 20,702 cDNA pooled samples were generated at a concentration of 7.5 ng plasmid/μl in OPTI-MEM at 4 μl per well in a 384-well PCR plate. Seal the plate with aluminum foil and store at -20°C.

cell culture

Trypsinized HeLa cells (ATCC, Manassas, VA) were resuspended at ¹⁰⁵ cells/ml in complete growth medium (DMEM, Invitrogen) containing 10% fetal bovine serum (GIBCOBRL Carlsbad, CA Cat# 10082-147 ) and 1× antibiotic-antimycotic reagent (GIBCO BRL Carlsbad, CA Cat# 15240-062) in Dulbecco's Modified Eagle Medium (D-MEM) (GIBCO BRL Carlsbad, CA Cat. #10317-022), and use Multidrop384 (ThermoLabsystems) was distributed in 24 white 96-well plates (Corning, Acton, MA) at 75 μl per well for 2,368 cDNA clone collection sample screening, or at 30 per well in 51 white 384-well plates ( Costar) was used for pool screening of 20,702 cDNA clones. Cells were grown overnight in a tissue culture incubator at 37 °C with 5% _CO2 .

High Throughput Transfection Procedure

For 2,368 cDNA clone collection sample screening, resuspend 330 μg pGL3B-IL-8 _P -Luc reporter plasmid in 33 ml OptiMEM I low serum medium in a 50 ml conical tube, the final amount of reporter plasmid is 100 ng per transfection. The tube was shaken and divided into 4 x 8 ml aliquots. Prior to transfection, 0.8 ml Fugene 6 transfection reagent (Roche Applied Bioscience) was added to each 8 ml aliquot (4 μl Fugene 6/μg transfection DNA). The mixed contents were pipetted up and down several times and dispensed in 96-well clean PCR plates (ABGene) at 75 μl/well. 10 μl of the [OptiMEM-reporter-Fugene 6] mixture was added to each well of each subplate containing 6 μl of pre-diluted cDNA using a BiomekFX pipetting station (Beckman Coulter, Fullerton, CA). The last row of plate #24 was used for pCMV-Sport6 empty vector aliquots as a negative control or for the pFC-MEKK expression construct (Stratagene) encoding the sequence corresponding to human MEKK1 AA360-672 as a positive control. Both plasmids were pre-diluted to 20 ng/μl and aliquoted to 6 μl per well. After incubation for 15 minutes at room temperature, 13 μl of the final mixture was transferred to a 96-well HeLa plate. Cells were incubated for 48 h at 37 °C in an atmosphere containing 5% _CO2 .

For screening of a pool of 20,702 cDNA clones, resuspend 1.65mg of pGL3B-IL-8P- _Luc reporter plasmid in 100ml OptiMEM I in a 250ml Erlenmeyer flask (Corning), the final amount of reporter plasmid is 50ng per transfection . The flask was shaken and divided into 8ml aliquots. Prior to transfection, 0.65 ml Fugene 6 transfection reagent was added to each 8 ml aliquot (3 μl Fugene/μg transfected DNA). The mixed contents were pipetted up and down several times and dispensed in 96-well clean PCR plates (ABGene) at 75 μl/well. 3 μl of the [OptiMEM-reporter-Fugene 6] mixture was added to each well of each 384-well plate containing 4 μl of pre-diluted cDNA using a BiomekFX (Beckman). After 15 minutes of incubation at room temperature, 7 μl of the mixture was transferred from each well to a 384-well tissue culture plate. Cells were incubated for 48 h at 37 °C in an atmosphere containing 5% _CO2 .

Luciferase assay

Firefly luciferase activity was measured 48 hours after transfection using the BrightGlo Luciferase Assay System (Promega, Madison, WI) according to the protocol provided by the manufacturer. Briefly, 90 μl or 40 μl of freshly reconstituted luciferase reagent was added to each well of a 96-well or 384-well tissue culture plate, respectively, using a Multidrop 384 (Thermo Labystems, Beverly, MA). After 2 minutes of incubation, luminescence was read on a LUMINOSKANA Ascent luminometer (Thermo Labsystems) using a 400 msec integration time according to the manufacturer's instructions.

Clone retrieval for sample point verification

For each primary assay, Z-scores and fold activations against the population median were routinely calculated and stored in an annotated searchable database. Potential sampling sites were selected based on two criteria: (1) Z-score greater than 3.0 and (2) fold activation greater than 10 and 5 in pooled screens of 2,368 and 20,702 cDNA clones, respectively. Clonal scores as sampling points in a primary assay of 2,368 clonal subarrays retrieved from glycerol stocks (copy 1 of the rearranged array plate). Sampling points for the primary assay of the entire 20,702 clonal pool were recovered by retransforming DNA aliquots from the pool. Transformation was performed in XL-10 Gold bacteria (Stratagene). Streak culture each clone on LB medium agarose plate + antibiotic (100 μ g/ml ampicillin) (KD Medical, Columbia, MD), grow overnight at 37 ° C and pick 3 colonies from each plate, grow in In a deep-well 96-well plate, each well contained 995 μl Terrific liquid medium (KD Medical) + 100 μg/ml ampicillin. Cover these deep-well plates with air-perforated tape and incubate overnight at 37 °C, shaking at 300 rpm. DNA minipreps were performed as described above. The total DNA preparation was then diluted to 125 ng/μl (in wells with a concentration greater than 125 ng/μl) and 8 μl was taken for DNA sequence confirmation. The remaining DNA was diluted to 25 ng/μl and 6 μl of the DNA was transferred to a sub 96-well PCR plate (ABGene) and used for a validation experiment using the transfection procedure described above. To normalize transfection efficiency, 20 ng of pRL-SV40 (Promega) encoding the Renilla luciferase gene under the control of the SV40 early promoter was included in each transfection. Firefly and Renilla luciferase activities were assayed using the DualGlo Luciferase Assay System (Promega) following the manufacturer's protocol. Briefly, 90 μl of freshly reconstituted luciferase reagent was added to each well of a 96-well tissue culture plate using a Multidrop 384 and, after 15 minutes of incubation, read on a LUMINOSKAN Ascent luminometer using a 400 ms integration time. Get the glow value. Then 90 μl of Stop-and-Glo reagent was added to each well of the 96-well tissue culture plate and, after 15 minutes of incubation, luminescence was read on a LUMINOSKAN Ascent luminometer using a 200 millisecond integration time. Different luciferase-based promoter constructs were used: pCRE-Luc, pMCS-Luc (Stratagene), pTAL-Luc (BD Biosciences), pNF-kB-Luc (BD Biosciences), pIL- _8P -Luc, pRhoBP-Luc (prepared in-house according to conventional methods/BD Biosciences) and pVCAM _P -Lue (prepared as described in Iademarcom, MF, JJMcQuillan, GDRosen and DCDean. 1992. J Biol Chem 267: 16323-9) tested the selected clones for specificity.

IL-8 ELISA in HeLa cells

HeLa cells were transfected at 100 ng per well in 96-well plates (Costar) using the protocol described above with DNA samples selected from sequences from validated and confirmed sampling sites. DNA samples designated as coactivators were co-transfected at 25 ng per well. The empty vector pCMV-Sport6 was used as a negative control. Seventy-two hours after transfection, IL-8 content in pre-diluted aliquots of cell growth medium corresponding to 1-5 μl of conditioned growth medium was assayed using an IL-8 ELISA kit (Sigma) following the protocol provided. As a positive control, growth medium was selected from cells transfected with empty vector and treated with 5 ng/ml IL-1 and 50 ng/ml TNFα (R&D Systems) respectively for 16 hours before harvesting the growth medium for IL-8 assay.

Gene Expression Profiling Using Affymetrix DNA Microarrays

Use Targeting F1 transfection reagent (Targeting Systems, Santee, CA) according to the protocol provided with the product with CREAP1 as described in the text or containing relA (Ruben SM et al., Science1991 Mar 22; 251 (5000): 1490-3), MAP3K11 (Hartkamp , J. et al, (1999).Cancer Res.59,2195-2202) or ANKRD3 (Muto, A. et al, (2002) J.Biol.Chem.277,31871-31876) expression construct transfection HeLa cell . Briefly, HeLa cells that were 70-80% confluent in T75 tissue culture flasks (Falcon) were used for transfection. The transfection mix was prepared by adding 20 μg of selected plasmid DNA to a 50 ml conical tube (Falcon) containing 8 ml Opti-MEM I and flicking the tube to mix. Both transfections were provided with pCMV-Sport6 empty vector. Vortex the Targetfect F-1 stock solution at full speed for 20 seconds and add 40 μl to each tube, mix again by flicking the tube and incubate at room temperature for 30 minutes to allow transfection complexes to form. Wash HeLa cells twice with 20 ml Opti-MEM I medium and add 12 ml of each transfection complex to each 1T75 flask. After 4 hours of incubation at 37°C, 8 ml of serum-containing growth medium was added to each flask. The medium was changed the next day. The medium was changed again 56 hours after transfection and 50 ng/ml of TNFα (R&D Systems) was added to a flask transfected with the pCMV-Sport6 plasmid and incubation was continued for 16 hours at 37°C. Cells were harvested 72 hours after transfection in 10 ml TRIzol reagent (Gibco BRL) and frozen at -80°C. Total RNA was isolated according to the method provided with TRIzol reagent. Synthetic labeling of double-stranded cDNA probes, Affymetrix gene chip (Gene-Chip) hybridization and data analysis were carried out according to conventional methods (see also Eberwine, J. et al., J.Neurosci.21, 8310-8314 and Hakak, Y. et al., (2001) Proc. Natl. Acad. Sci. U.S.A 98, 4746-4751).

Example 6

Characterization of the IL8P luciferase vector

The inducibility of a luciferase reporter gene controlled by a 1.5 kB IL-8 promoter containing a human IL-8 promoter fragment was tested by known regulators of cytokine-mediated gene expression. pNF-κB-Luc (BD Biosciences) and pGL2-IL-8P- _Luc reporter genes were co-transfected into HEK 293 cells containing an expression construct encoding β-Luc prepared according to routine methods using a proprietary clone pool. NF-κB pathway activator - truncated MEKK (AA 360-672) (Stratagene) and full-length TRAF6 cDNA. Cells co-transfected with empty pCMV-Sport6 vector were either left untreated or treated with TNFα (50 ng/ml, treated for 16 hours). Luciferase activity was measured 48 hours after transfection. The pNF-κB-Luc reporter was used as a positive control.

The data indicated that MEKK, TRAF6 and TNFα significantly activated the IL-8 promoter-reporter, increasing the activity of the reporter gene by 16, 4.9 and 4.7 times, respectively. For high-throughput functional screening of our proprietary collection of cDNA clones, the IL-8 promoter sequence was subcloned into the pGL3Basic vector (Promega), a derivative of the original pGL2 vector with improved specificity and efficiency.

Example 7

20,000 cDNA collections were screened based on IL-8 promoter function and assayed for sampling site activity. certificate

pGL3B-IL-8β- _Luc was co-transfected with 20,702 individual full-length cDNA clones into HeLa cells in 384-well plates and a single reporter assay was performed 48 hours after transfection as described above. Co-transfection of pCMV-Sport6 with the reporter served as a negative control. Luciferase activity was measured using the BrightGlo reporter assay system (Promega). The absolute value of IL-8 promoter reporter activity was determined and clones with a score 5-fold higher than the pCMV-Sport6 plasmid control were identified (data not shown). To verify the identity and activity of these sampling points, clones were recovered as described above and 3 independent colonies were isolated. Minipreps of DNA were used for sequence verification and secondary assays using the IL- _8β -Luc reporter (data not shown).

Individual clonal isolates that produced significant activation of the IL-8β- _Luc reporter in a secondary assay were tested for their ability to activate seven promoter luciferase reporter constructs: pTAL, NF-kB-Luc, IL- _8P -Luc, _RhoBP -Luc, _VCAMP -Luc (BD Biosciences) (same as pTAL with addition of 4 CRE response elements).

cDNA clones were selected based on the presence of predicted or characterized gene start codons from the single 5′ end sequences of 12,905 clones that were matched to RefSeq genes, 5,463 of which were functionally annotated. 20,704 cDNAs were co-transfected with a firefly luciferase reporter gene (pIL-8-Luc) controlled by the IL-8 promoter. 64 cDNA induced reporters greater than 5-fold. Validated active cDNA included 1-3 copies of 28 unique genes. Twenty-two non-redundant cDNAs were selected for further study. The entire pool was also screened in the assay for activation of cyclic AMP response element (CRE) or serum response element (SRE) driven reporters. The results obtained using the 22 cDNAs in the primary screen were clustered using hierarchical clustering (Eisen) to determine if any genes exhibited relevant activity in all three assays. Many genes are relatively specific for the IL-8 reporter. These genes include known inducers of NF-κB and are composed of subunit relA (p65) of NF-κB transcription factor, TNF receptor superfamily member 1A, TNF-related molecules TWEAK/TNFSF12, RIPK2 and TRAF6, newly identified NF-κB - kappa B activator ACT1 and kinase PKK representative. The second group represents AP-1 transcription factor site activators, including multiple clones of the JunD and JNK-induced MAP kinases MAP3K12 and MAP3K11. Also identified was -C/EBPβ, which is known to bind directly to the IL-8 promoter NF-IL6 site. Thus, the primary screen identified multiple inducers predicted to activate the IL-8 gene through many different pathways.

CREAP1 belongs to the obtained sampling points. The data thus indicate that CREAP1 is a strong activator of the CRE-Luc and IL- _8β -Luc constructs. In fact, this protein of hitherto unknown function was not only shown to be the strongest activator of CRE (even stronger than the two CRE-binding transactivators CRE-BRa and CREB1 (data not shown) and confirming the results disclosed in the above provided in the Examples) and also the strongest activator of the IL-8 gene found in these secondary assays.

Example 8

CREAP1 strongly activates a reporter carrying a tandem IL-8 promoter-specific CRE-like element

To determine whether the strong activator also induces the endogenous IL-8 gene, IL-8 secretion from HeLa cells was measured after transfection with the relA and MAP3K11 constructs as examples of NF-κB and AP-1 activators. accumulation of protein. MAP3K11 and relA induced small increases, but the combination of both induced levels of secreted IL-8 comparable to that observed with IL-1β, the most potent inducer of IL-8 known . This data suggests that the regulation of the endogenous IL-8 gene requires the interaction of multiple signal transduction pathways.

Several cDNAs were identified whose mechanism of action remains unclear. These include two Rho-dependent GTP-GDP enhancers (Rho-GEFs), p114 and ARHGEF1, C16orf15, thyrotropin embryonic factor 1 (TEF1), fibronectin (FN1), and the nuclear receptor family member NR2F2. C16orf15 encodes a proline-rich protein of unknown function that is expressed at high levels in the brain. TEF1 is a member of the basic leucine zipper transcription factors that act directly through TEF response elements. FN1 is a matrix glycoprotein expressed at high levels in injured tissues and it is also capable of inducing IL-1β through an AP-1-dependent mechanism. NR2F2 was a very strong activator in all assays and therefore its activity appeared to be non-specific.

Several of the strongest IL-8 activators were associated with CRE-dependent gene expression. C/EBPβ, JunD, c-jun, CRE-binding proteins CREB1, CRE-BPa and XBP1 were found to be potent inducers of CRE-driven reporters. A cDNA overlapping with the sequences recorded as KIAA0616 and MECT1 was also identified as the above-mentioned CREAP1 gene. Interestingly, except that the sequence encoding the first 44 amino acids of MECT1 is translocated to the Mastermind-like gene MAML2 in mucoepidermoid carcinoma (Tonon et al., Nat. Genet., 33:208-213 (2003)), regarding this protein Know nothing.

The observation that many of the strongest IL-8 activators are also CRE activators or bind proteins suggests that the IL-8 promoter may contain an unrecognized CRE. The implications were first tested by detecting the effect of increased cAMP levels on the IL-8 promoter using the plant diterpene forskolin (Sigma), a non-specific activator of adenylyl cyclase. Briefly, HEK 293 cells were co-transfected with pCRE-Luc or pIL-8-Luc with empty vector or CRE-BPa expression constructs using Fugene6 transfection reagent (Roche) as described above. Sixteen hours after transfection, an equal volume of growth medium containing 500 μM IBMX was added to the wells. Eight hours later, forskolin was added to a final concentration of 5 μM to cells pretreated with IBMX from a 50 μM stock prepared in growth medium. Cells were treated with forskolin for 16 hours at 37°C. Luciferase activity was determined using the Dual-Glo assay kit (Promega) and normalized as described above. Data are expressed as fold induction compared to untreated cells transfected with empty vector. The results indicated that forskolin weakly induced the IL-8 reporter. Co-transfection of the CRE-binding protein found in the screen, CRE-BPa, synergistically activates the IL-8 promoter upon forskolin treatment.

The IL-8 promoter sequence is then tested for the presence of a potential CRE sequence using standard techniques. A potential asymmetric variant CRE with the sequence 5'-TGACATAA-3' was found between -69 and -62 of the IL-8 promoter, which has been previously mentioned as an AP-1 binding sequence but its function has not been Reported (Roebuck, J. Interferon and Cytokine Res. 19:429-438 (1999)). We named this locus "CRE-like response element". Oligonucleotides with identical DNA sequences were shown to be bound well by CREB2 and poorly by CREB1 (Benbrook and Jones, Nucleic Acids Res., 22:1463-1469 (1994)). Interestingly, CREB2 has been proposed to play a dual role as a transcriptional activator/repressor. CREB2 binding to "CRE-like response elements" is thought to impair binding of activating proteins such as CREB and thus inhibit CRE-dependent transcription (Karpinski et al., Proc. Natl. Acad. Sci. U.S.A. 89:4820-4824 (1992)) . On the other hand, the binding of CREB2 to other transcription factors such as c-Rel, ATF-1 or the viral protein Tax can activate the transcription of several genes in these cases (Schoch et al., Neurochem. Int. 38:601-608 (2001)) .

The mechanism of IL-8 promoter induction by MAP3K11 and CREAP1 was investigated. To determine whether promoter elements are required to be activated by these genes, a series of promoter variants with mutations in IL-8CRE-like regulatory sites and other regulatory sites were generated and tested for induction of MAP3K11, CREAP1 or relA effect. The results indicated that mutations in the C/EBP binding site had no effect on the activation of either protein. NF-κB site mutations had little effect on the induction of MAP3K11 or CREAP1 but abolished the induction of relA. Mutations at the AP-1 site did not significantly alter the effect of relA but severely reduced the induction of MAP3K11. This is consistent with the ability of MAP3K11 to activate the JNK/SAPK pathway and AP-1. Surprisingly, the mutation also significantly reduced the activation of CREAP1. Mutation of CRE-like sites drastically reduced or abolished induction of CREAP1 and MAP3K11 (data not shown).

To determine whether "CRE-like elements" respond directly to CREAP1 or MAP3K11, the ability of these two genes to activate a minimal promoter carrying a concatemer CRE-like site (pCREL-Luc) was tested. Additionally, we investigated the effect of PMA, a known inducer of AP-1. Similar to the CRE reporter (pCRE-Luc), pCREL-Luc was strongly activated by CREAP1 but not by MAP3K11 or PMA treatment (data not shown). This data suggests that although CREAP1 and MAP3K11 both require intact CRE-like and AP-1 sites for their activity, they induce IL-8 priming by different mechanisms using the CRE-like or AP-1 sites, respectively, as their major effector elements son.

We further determined whether CREAP1 -induced pIL-8-Luc reporter activity is dependent on CREB. Co-expression of CREAP1 and the dominant negative form of CREB, KCREB (BD Biosciences), resulted in a significant reduction in CREAP1-induced IL-8 promoter activity (data not shown). In contrast, co-transfection with the constitutively active form of I-ΚBα - a potent inhibitor of the NF-ΚB pathway - did not affect the activity of CREAP1.

To determine whether the interaction of CREAP1 with the binding sites of CRE and AP-1 is associated with the same or different domains, we constructed several variants of CREAP1 carrying deletions from the N- and C-termini using conventional methods and tested these Variants affect the ability of CREAP1 or MAP3K1 to activate the pL-8-Luc reporter. A mutant (δ59) comprising a deletion of 59 N-terminal amino acids reduced wild-type CREAP1 and greatly inhibited the ability of MAP3K11 to induce IL-8 reporter expression (data not shown). Inhibition is specific since δ59 has no effect on relA activation. δ59 also blocked the activation of the AP-1-specific reporter pAP1(PMA)-Luc containing repeated AP-1 sites by PMA or MAP3K11 (data not shown). Meanwhile, δ59 could not block forskolin-stimulated pCRE-Luc reporter (data not shown). This data suggests that when CREAP1 is expressed through CRE activation in a CREB-dependent manner, this protein may interact directly or indirectly with an essential component of AP-1 activation.

Example 9

Gene expression profiles in HeLa cells transiently transfected with CREAP1

To determine whether CREAP1 regulates the expression of bona fide CREB targets, cellular gene expression was measured using DNA microarrays after overexpression of CREAP1. Briefly, HeLa cells were transiently transfected with pCMV-Sport6, CREAP1 using Targetfect F1 reagent (Targeting Systems). Half of the pCMV-Sport6 transfected cells were left untreated and used as a negative control. Total RNA isolation, labeled probe preparation, and DNA microchip hybridization protocols were performed as described above.

Interestingly, the results showed that the gene expression profile in HeLa cells upon CREAP1 transfection was distinct from that of other activators in that genes known to be dependent on the cAMP/CREB pathway were particularly enriched. Specifically, CREAP1 transfection induced a 10-fold increase in seven genes (see Table 2). Other genes include well-known targets of CREB and cAMP, including TSHα, enolpyruvate phosphate carboxykinase (PEPCK), crystallin α-B, and the EGF-like molecule amphiregulin. CREAP1 also activates CREM, another gene known to be induced when cAMP levels rise, to a lesser extent. Induction of c-Jun and MAP3K11 of PAI-2, a known target of AP-1, did not affect this group of genes (Arts et al., 1996, Eur. J. Biochem 241:393-402). Thus, CREAP1 is a bona fide inducer of CREB target genes.

Each of the activators identified in the screen also activated the endogenous IL-8 gene to a relatively small extent (2-5 fold). The weak activation of the endogenous IL-8 gene compared to the strong activation observed with the artificial reporter construct may be due to the need for activation through multiple pathways as described above. We also analyzed several sets of genes differentially regulated by CREAP1 or protein kinase A (PKA) catalytic subunit overexpression in HEK293 cells using a hierarchical clustering algorithm. We also found that although both proteins act through CREB, the groups of up- and down-regulated genes do not completely overlap. The data suggest that CREAP1 may provide an alternative mechanism to the well-known phosphorylation-dependent mechanism for activating transcription. Gene Affymetrix ID Activation multiple IL-8 1369_s_at 2.5 KIAA0467 41458_at 12 Exodus-1 40385_at 15 CAPL protein 38088_r_at 19 Amphiregulin 34898_at 19 DKFZp566K192 32242_at 32 PEPCK 33702_f_at 32 TS Ha 39352_at 57

Table 2: Induction of cAMP-responsive genes by CREAP1. The fold increase in mRNA levels of the genes most strongly induced by CREAP1 as detected by Affymetrix gene chips is shown. A comparison of IL-8 transcript induction levels is also shown. These genes were the only ones that were >10-fold induced by CREAP1 and were all found in replicate experiments. The fold increase compared to the expression level observed after transfection with the control pCMV-Sport6 vector was calculated.

The two genes most strongly induced by CREAP1 are known targets of cAMP, enolpyruvate phosphate carboxykinase (PEPCK or PCK1) (Roesler, W. J. Mol. Cell Endocrinol. 162: 1-7 (2000)) and thyroid-stimulating hormone alpha (TSHα) genes (Kim, D.S et al. Mol Endocrinol. 8:528-36 (1994)). Expression of a third highly regulated gene, amphiregulin, has been reported to be dependent on PKA in some cancer cell lines (Bianco, C.G. et al., Clin. Cancerres. 3:439-48 (1997)) and we have identified The fully conserved proximal amphiregulin promoter in the mouse and human genes shared the CRE site (data not shown).

The two endogenous genes most highly induced by CREAP1 were not known targets of cAMP or CREB proteins. The first is CAPL; the second is the chemokine Exodus-1 (also known as CCL27, MIP-3α or LARC). Interestingly, the Exodus-1 gene is also a chemokine and is regulated in a very similar manner to the IL-8 gene, as the proximal promoter is reported to contain NF-kB, AP-1 and NF-IL6/C/EBP loci point. CREAP1 also induces the Exodus-1 gene to a greater extent than the endogenous IL-8 gene. It was also noted that CREAP1 is a stronger inducer of Exodus-1 than TNF-α or NF-κB (data not shown). It is not known whether the Exodus-1 promoter contains an unrecognized CRE or whether CREAP1 functions through the variant AP-1 site in question. However, activation of Exodus-1 expression by CREAP1 suggests that the Exodus-1 gene will be regulated through cAMP or other CREB-induced pathways.

The promoters of the CAPL, KIAA0467 and DKFZp566K192 genes have not been described. We examined potential CREAP1-responsive elements of the CAPL promoter, for which no apparent CRE was reported. A gene named CRE-like 2 with the sequence 5'-TGACACAA-3' was found in the PEPCK promoter (nucleotides -249 to -256) and the CAPL promoter (nucleotides -385 to -392). sequence. The CRE-like 2 element was placed upstream of the minimal promoter and tested for CREAP1 induction. This element is sufficient to mediate the induction of CREAP1. Similar to the IL-8 promoter, both IL-8 CRE-like and CRE-like 2 sequences are moderately activated by elevated cAMP and are synergistically activated by cAMP and CRE-BPa. Thus, CREAP1 response elements could be activated by the cAMP pathway but not by CREB1, since the CRE-like element found in the IL-8 promoter and the CRE-like 2 elements found in the CAPL and PEPCK promoters are unlikely to be recognized by CREB1.

CREAPs are attractive targets for drug discovery. It is especially true that CREAP is an attractive target for drug discovery if the function of CREAP is to regulate a specific subset of CREB-regulated genes through the interaction of CREB with other transcription factors. Due to the large number of CREB responsive genes, any antagonist or agonist that directly affects CREB will likely have multiple effects. On the other hand, modulators of CREAP function may have the ability to block specific subsets of genes, such as blocking chemokines such as IL-8 and Exodus-1 for the treatment of autoimmune and inflammatory diseases, blocking amphiregulin for Treatment of proliferative diseases and blockade of PEPCK for the treatment of diabetes since all of these genes are highly induced by CREAP1.

Example 10

Identification of CREAP2

The complete amino acid sequence of CREAP1 was used in a BLASTP search of the public NCBI database. Two common domain cDNAs (XM_117201 and FLJ00364) with significant homology to the CREAP1 coding region were initially identified. The nucleotide sequence of XM_117201 was used in a BLASTN search of the proprietary cDNA library EST database (Altschul SF et al., Nucleic Acids Res. 25:3389-3402 (1997)) and four clones representing the public sequence of XM_117201 were identified. Four clones were functionally tested after co-transfection with the CRE-Luc and IL-8p-Luc reporters originally found to be induced by CREAP using methods similar to those published above. Briefly, trypsinized HeLa cells were resuspended in complete growth medium at 6×10 ⁴ cells/ml and distributed in a volume of 100 μl per well in white 96-well plates (Costar). Cells were grown overnight in a tissue culture incubator at 37 °C with 5% _CO2 . The pGL3B-IL-8β- _Luc reporter plasmid or CRE-Luc reporter (BD Biosciences) and the tested cDNA were resuspended at 25 ng/ml in OptiMEM I low serum medium (GIBCO BRL). The reporter plasmid and cDNA were then distributed in 96-well clean PCR plates (ABGene) at 4 μl/well and 3 μl/well, respectively. A mixture containing 1.5 μl of Fugene 6 reagent (Roche Applied Bioscience) per transfection and 20 ng of pRL-SV40 (Promega) plasmid per transfection was added in a volume of 10 μl per well to a 96-well PCR plate containing pre-diluted cDNA. The contents of each well were mixed by pipette and left at room temperature for 10 minutes. Transfer 15 μl of the transfection mixture from each well to a 96-well tissue culture plate. Cells were incubated for 48 hours.

Firefly and Renilla luciferase activities were assayed using the DualGlo Luciferase Assay System (Promega) following the protocol provided by the manufacturer. Briefly, 115 μl of freshly prepared luciferase reagent was added to each well of a 96-well tissue culture plate using a Multidrop 384 and, after 15 minutes of incubation, integrated at 400 milliseconds on a LUMINOSKAN Ascent luminometer (Thermo Labsystems) Time to read the luminescence value. Subsequently, 115 μl of Stop-and-Glo reagent was added to each well of a 96-well tissue culture plate and, after 15 minutes of incubation, luminescence was read on a LUMINOSKAN Ascent luminometer (Thermo Labsystems) with a 200 millisecond integration time. The activity of each cDNA tested was determined as the ratio of the corresponding firefly and Renilla luciferase activities.

One of the 4 clones was shown to be active. The insert of this clone was fully sequenced in one orientation and revealed to encode an ORF of 586 amino acids that completely overlapped with the predicted common domain protein XP_117201 from the XM_117201 cDNA. This clone was annotated as CREAP2 and encoded a predicted protein of 693 amino acids with a start codon at nucleotide 177 and a TGA-encoded stop codon at 2256. Although a search of the literature indicated the existence of a cDNA encoding part of CREAP2, no CDMA containing the complete sequence of human CREAP2 was provided nor the function of the protein.

The nucleotide sequence of human CREAP2 is shown below. The start codon is located at nucleotide 177 and the TGA-encoded stop codon is located at 2256, in italics:

ANTTTTTGTACANAAAAAGCAGGCTGTTACCGGTCCGGATTCCCGGGATCTAGGCTGGGGC 60

CGGGTTCGCGGTGCTCGCTGAGGCGGCGGTGGCTACGGCTGGAGGAGCCGGGCCGAGGCC 120

GCGGCGGAGGCCGCGGCTGGTACTGGGAGGGTGGCAGGGAGGGACGGGGAAGGAAGATGG 180

CGACGTCGGGGGCGAACGGGCCTGGTTCGGCCACGGCCTCGGCTTCCAATCCGCGCAAAT 240

TTAGTGAGAAGATTGCGCTGCAGAAGCAGCGTCAGGCCGAGGAGACGGCGGCCTTCGAGG 300

AGGTGATGATGGACATCGGCTCCACCCGGTTACAGGCCCAAAAACTGCGACTGGCATACA 360

CAAGGAGCTCTCATTATGGTGGGTCTCTGCCCAATGTTAACCAGATTGGCTCTGGCCTGG 420

CCGAGTTCCAGAGCCCCCTCCACTCACCTTTGGATTCATCTCGGAGCACTCGGCACCATG 480

GGCTGGTGGAACGGGTGCAGCGAGATCCTCGAAGAATGGTGTCCCCACTTCGCCGATACA 540

CCCGCCACATTGACAGCTCTCCCCTATAGTCCTGCCTACTTATCTCTCCCCCAGAGTCTA 600

GCTGGCGAAGGACGATGGCCTGGGGCAATTTCCCTGCAGAGAAGGGGCAGTTGTTTCGAC 660

TACCATCTGCACTTAACAGGACAAGCTCTGACTCTGCCCTTCATACAAGTGTGATGAACC 720

CCAGTCCCCAGGATACCTACCCAGGCCCCACACCTCCCAGCATCCTGCCCAGCCGACGTG 780

GGGGTATTCTGGATGGTGAAATGGACCCCAAAGTACCTGCTATTGAGGAGAACTTGCTAG 840

ATGACAAGCATTTGCTGAAGCCATGGGATGCTAAGAAGCTATCCTCATCCTCTTCCCGAC 900

CTCGGTCCTGTGAAGTCCCTGGAATTAACATCTTTTCCATCTCCTGACCAGCCTGCCAATG 960

TGCCTGTCCTCCCACCTGCCATGAACACGGGGGGCTCCCTACCTGACCTCACCAACCTGC 1020

ACTTTCCCCCACCACTGCCCACCCCCCTGGACCCTGAAGAGACAGCCTACCCTAGCCTGA 1080

GTGGGGGCAACAGTACCTCCAATTTGACCCACACCATGACTCACCTGGGCATCAGCAGGG 1140

GGCATGGGCCTGGGCCCGGCTATGATGCACCAGGACTTCATTCACCTCTCAGCCACCCAT 1200

CCCTGCAGTCCTCCCTAAGCAATCCCAACCTCCAGGCTTCCCTGAGCAGTCCTCAGCCCC 1260

AGCTTCAGGGCTCCCACAGCCACCCCTCTCTGCCTGCCTCCTCCTTGGCCTGCCATGTAC 1320

TGCCCACCCACCTCCCTGGGCCACCCCACTCAGTGCTCCGGCTCTCTCCCTCCTCCTCTT 1380

CCTCCTCCTCCACTTCATCTCCTGTTTTGGGCGCCCCCCTCTTACCCTGCTTCTACCCCTG 1440

GGGCCTCCCCCCACCACCGCCGTGTGCCCCTCAGCCCCCTGAGTTTGCTCGCGGGCCCAG 1500

CCGACGCCAGAAGGTCCCAACAGCAGCTGCCCAAACAGTTTTCGCCAACAATGTCACCCA 1560

CCTTGTCTTCCATCACTCAGGGCGTCCCCCTGGATACCAGTAAACTGTCCACTGACCAGC 1620

GGTTACCCCCCTACCCATACAGCTCCCCCAAGTCTGGTTCTGCCTACCCAGCCCCACACCC 1680

CAAAGTCTCTACAGCAGCCAGGGCTGCCCTCTCAGTCTTGTTCAGTGCAGTCCTCAGGTG 1740

GGCAGCCCCCAGGCAGGCAGTCTCATTATGGGACACCGTACCCACCTGGGCCCAGTGGGC 1800

ATGGGCAACAGTCTTACCACCGGCCAATGAGTGACTTCAACCTGGGGAATCTGGAGCAGT 1860

TCAGCATGGAGAGCCCATCAGCCAGCCTGGTGCTGGATCCCCCTGGCTTTTCTGAAGGGC 1920

CTGGATTTTTAGGGGGTGAGGGGCCAATGGGTGGCCCCCAGGATCCCCACACCTTCAACC 1980

ACCAGAACTTGACCCACTGTTCCCGCCATGGCTCAGGGCCTAACATCATCCTCACAGGGG 2040

ACTCCTCTCTCAGGTTTCTCTAAGGAGATTGCAGCAGCCCTGGCCGGAGTGCCTGGCTTTG 2100

AGGTGTCAGCAGCTGGATTGGAGCTAGGGCTTGGGCTAGAAGATGAGCTGCGCATGGAGC 2160

CACTGGGCCTGGAAGGGCTAAACATGCTGAGTGACCCCCTGTGCCCTGCTGCCTGATCCTG 2220

CTGTGGAGGAGTCATTCCGCAGTGACCGGCTCCAATGAGGGCACCTCATCACCATCCCTC 2280

TTCTTGGCCCCATCCCCCACCACCATTCCTTTCCTCCCTTCCCCCTGGCAGGTAGAGACT 2340

CTACTCTCTGTCCCCAGATCCCTCTTTCTAGCATGAATGAAGGATGCCAAGAATGAGAAAA 2400

AGCAAGGGGTTTGTCCAGGTGGCCCCTGAANTCTGCGCAAGGGATGGGCCTGNGGGGAAC 2460

CTCANGGNNAGGGCCCAANGGCCACTTNNAANCTTTGAACCGTCNGTCTGGNANGGTCNN 2520

(SEQ ID NO 15)

The predicted amino acid sequence of human CREAP2 is shown below:

MATSGANGPGSATASASNPRKFSEKIALQKQRQAEETAAFEEVMMDIGSTRLQAQKLRL

AYTRSSHYGGSLPNVNQIGSGLAEFQSPLHSPLDSSRSTRHHGLVERVQRDPRRMVSPL

RRYTRHIDSSPYSPAYLSPPPESSWRRTMAWGNFPAEKGQLFRLPSALNRTSSSDSALHT

SVMNPSPQDTYPGPTPPSILPSRRGGILDGEMDPKVPAIEENLLDDKHLLKPWDAKKLS

SSSSRPRSCEVPGINIFPSPDQPANVPVLPPAMNTGGSLPDLTNLHFPPLPTPLDPEE

TAYPSLSGGNSTSNLTTHTMTHLGISRGHGPGPGYDAPGLHSPLSHPSLQSSLSNPNLQA

SLSSPQPQLQGSHHSHPSLPASSLACHVLPTTSLGHPSLSAPALSSSSSSSSTSSPVLGA

PSYPASTPGASPHHRRVPLSPLSLLAGPADARRSQQQLPKQFSPTMSPTLSSITQGVPL

DTSKLSTDQRLPPYPYSSPSLVLPTQPHTPKSLQQPGLPSQSCSVQSSGGQPPGRQSHY

GTPYPPGPSGHGQQSYHRPMSDFNLGNLEQFSMESPSASLVLDPPGFSEGPGFLGGEGP

MGGPQDPHTFNHQNLTHCSRHGSGPNIILTGDSSPGFSKEIAAALAGVPGFEVSAAGLE

LGLGLEDELRMEPLGLEGLNMLSDPCALLPDPAVEESFRSDRLQ (SEQ ID NO 16)

Example 11

Identification of CREAP3

Using methods similar to those published above, a clone was found in our proprietary cDNA library EST database by comparison to the sequence of the public domain clone cDNAFLJ00364. The predicted protein encoded by FLJ00364 lacks the start codon ATG and has an N-terminal sequence non-homologous to CREAP1. Comparison of public domain clone sequences with similar clones in our database shows that our proprietary clone sequence contains an extra C in the sequence CCGTCATTTCAC C AAGC (SEQ ID NO 17), where the extra C is underlined . This additional C was confirmed by comparison with the genome sequence. This change resulted in the deletion of the first 63 amino acids predicted by FLJ00364 cDNA, and the replacement of another 81 amino acids in frame at the beginning of the highly conserved amino acid sequence EETRAFE (SEQ ID NO 18) with the CREAP1 predicted protein sequence E ²³ ETAAFE (SEQ ID NO 18) ID NO 19).

Three consecutive rounds of polymerase chain reaction (PCR) were performed to obtain the complete ORF of the proprietary clone. The PCR amplification cycles were: 94°C for 2 minutes, 23x [94°C for 15 seconds, 68°C for 30 seconds and 72°C for 15 seconds] and 72°C for 2 minutes. All amplification steps were performed using Advantage 2 DNA polymerase (BD Biosciences). All three PCR products were amplified using a common sense primer KIAAhS3_R1 having a nucleotide sequence corresponding to the start of the ORF (5'-CCGGAATTCGCCATGGCCGCCTCGCCGGGCTCGGG-3') (SEQ ID NO 20). An EcoRI restriction site was included in the 5' end sequence of the primers using conventional methods. For the initial PCR, human genomic DNA (BD Biosciences) was used as template (2 mg per reaction) and the antisense primer was KIAAhAS2 (5'-CCGCGACAGGGTGAGGTCGGTCATGAGCTGCTCGAAGGCCCGCG-3') (SEQ ID NO 21). The 142 nt PCR product was extracted using a phenol-chloroform mixture and precipitated with isopropanol. The pellet was washed with ice-cold 70% ethanol and resuspended in TE buffer. Get 5ng product as the template of the second round of PCR reaction, use KIAAhS3_R1 sense primer and KIAAhAS3 (5'-GAAGCTTCTGAAATTGAACCCGCGACAGGGTGAGGTCGGTCATG-3') (SEQ ID NO 22) antisense primer. The 161nt PCR product was treated similarly to the original PCR product and 5ng of resuspended DNA was used as the template for the final round of PCR, using KIAAhS3_R1 sense and KIAAhAS4(5'-TGGTAAGGATCCTCCATGGTACTGTGTAAGGCGCAGTTGCTGAAGCTTCTGAAATTGAACCCG-3')(SEQ3) ID NO 2 primers. All primers were obtained from SIGMA-Aldrich Company (Saint Louis, MO, USA) or prepared according to conventional methods.

The 202nt product was gel purified and digested with EcoRI and BamHI and inserted into the EcoRI/BamHI digested plasmid of the proprietary clone. Sixteen different clones of the reconstituted full-length FLJ00364 cDNA were sequence verified and functionally tested using the CRE-Luc and IL-8p-Luc reporters as described above. Clone #5, free of PCR-introduced mismatches and strongly activating both reporters, was used for DNA and protein alignment and has been annotated as CREAP3.

The nucleotide sequence of CREAP3 is provided below. Italics show the start codon at nucleotide 46 and the TGA stop codon at 1905. Note that the bold underlined residue 288 C has been added due to comparison of the genomic sequence and the proprietary cloned sequence. The underlined CGAGG sequence indicates the 5'-end of the proprietary clone. Nucleotide sequences upstream of this sequence were amplified by PCR using genomic DNA as a template and reinserted into proprietary clones as described above.

Nucleotide sequence of CREAP3:

NTTTTTTGTACANAAAAAGCAGGCTGTTACCGGTCCGGAATTCGCCATGGCCGCCTCGCCG 60

GGCTCGGGCAGCGCCAACCCGCGGAAGTTCAGTGAGAAGATCGCGCTGCACACGCAGAGA 120

CAGGC CGAGG AGACGCGGGCCTTCGAGCAGCTCATGACCGACCTCACCCCTGTCGCGGGTT 180

CAATTTCAGAAGCTTCAGCAACTGCGCCTTACACAGTACCATGGAGGATCCTTACCAAAT 240

GTGAGCCAGCTGCGGAGCAATGCGTCAGAGTTTCAGCCGTCATTTCA C CAAGCTGATAAT 300

GTTCGGGGAACCCGCCATCACGGGCTGGTGGAGAGGCCATCCAGGAACCGCTTCCACCCC 360

CTCCACCGAAGGTCTGGGGACAAGCCAGGGCGACAATTTGATGGTAGTGCTTTTGGAGCC 420

AATTATTCCTCCACAGCCTCTGGATGAGAGTTGGCCAAGGCAGCAGCCTCCTTGGAAAGAC 480

GAAAAGCATCCTGGGTTCAGGCTGACATCTGCACTTAACAGGACCAATTCTGATTCTGCT 540

CTTCACACGAGTGCTCTGAGTACCAAGCCCCAGGACCCCTATGGAGGAGGGGGCCAGTCG 600

GCCTGGCCTGCCCCATACATGGGGTTTTGTGATGGTGAGAATAATGGACATGGGGAAGTA 660

GCATCTTTCCCTGGCCCATTGAAAGAAGAGAATCTGTTAAATGTTCCTAAGCCACTGCCA 720

AAACAACTGTGGGAGACCAAGGAGATTCAGTCCCTGTCAGGACGCCCTCGATCCTGTGAT 780

GTTGGAGGTGGCAATGCTTTTCCACATAATGGTCAAAAACCTAGGCCTCTCACCCTTCTTG 840

GGGACTTTGAACACTGGAGGGTCATTGCCAGATCTAACCAACCTCCACTACTCGACACCC 900

CTGCCAGCCTCCCTGGACACCACCGACCACCACTTTGGCAGTATGAGTGTGGGGAATAGT 960

GTGAACAACATCCCAGCTGCTATGACCCACCTGGGTATAAGAAGCTCCTCTGGTCTCCAG 1020

AGTTCTCGGAGTAACCCTCTCATCCAAGCCACGCTCAATAAGACTGTGCTTTCCTCTTCC 1080

TTAAATAACCACCCACAGACATCTGTTCCCAACGCATCTGCTCTTCACCCTTCGCTCCGT 1140

CTGTTTTTCCCTTAGCAACCCATCTCTTTCCACCACAAAACCTGAGCGGCCCGTCTCGCCGT 1200

CGGCAGCCTCCCGTCAGCCTCTCACGCTTTCTCCTGGCCCTGAAGCACATCAAGGTTTC 1260

AGCAGACAGCTGTCTTCAACCAGCCCACTGGCCCCATATCCTACCCTCCCAGATGGTGTCC 1320

TCAGACCGAAGCCAACTTTTCCTTTCTGCCCACAGAAGCTCAAGCCCAGGTGTCGCCGCCA 1380

CCCCCTTACCCTGCACCCCAGGAGCTCACCCAGCCCCTCCTGCAGCAGCCCCGCGCCCCT 1440

GAGGCCCCTGCCCAGCAGCCCCAGGCAGCCTCTCACTGCCACAGTCAGACTTTCAGCTT 1500

CTCCCGGCCCAGGGCTCATCTTTGACCAACTTCTTCCCAGATGTGGGTTTTGACCAGCAG 1560

TCCATGAGGCCAGGCCCTGCCTTTCCTCAACAGGTGCCTCTGGTGCAACAAGGTTCCCGA 1620

GAACTGCAGGACTCTTTTCATTTGAGACCAAGCCCGTATTCCAACTGCGGGAGTCTCCCG 1680

AACACCATCCTGCCAGAAGACTCCAGGCACCAGCCTGTTCAAAGACCTCAACAGTGCGCTG 1740

GCAGGCCTGCCTGAGGTCAGCCTGAACGTGGACACTCCATTTCCACTGGAAGAGGAGCTG 1800

CAGATTGAACCCCTGAGCCTGGATGGACTCAACATGTTAAGTGACTCCAGCATGGGCCTG 1860

CTGGACCCCCTCTGTTGAAGAGACGTTTCGAGCTGACAGACTGTGAACAGAAGGCAGTGGA 1920

ACAGAAGAATGTTTTTCTGCAACAGCCAAATAGAATGGAATAGAATGAAGCCAGCTGAT 1980

ACCACGGGCTTTCGTTATTCTTGACATAGAAGGAAGCAGTGCCACGGCTCCAGGGTTTCAG 2040

ATGAGATCCCATCTCAGACACTGTGGCTTCCTCCAGATCACACAGCTTTGTACTGCCTCT 2100

CCCGCCTGTGGCCAAAGTCGTGTTGCAGCAGGCAGGCTGCTTGGAGCTTCCCATGAACTG 2160

GAAAGCTCACCTCCACTGCATCTTTTTACTGGCCATCCAGTCAGCCGATGTGTAAGAGTA 2220

GGAAATACTGTGTCACTGGAGGCCCTCCGTAGCATTGGG 2259

(SEQ ID NO 24)

The CREAP3 cDNA encodes a predicted protein of 619 amino acids shown below with a start codon at nucleotide 46 and a TGA-encoded stop codon at 1905. The alternative correct amino acid sequence encoded by CREAP3 that differs from the predicted sequence of public clone FLJ00364 is underlined. Glutamate and alanine at amino acid positions 551 and 616 are shown in bold.

MAASPGSGSANPRKFSEKIALHTQRQAEETRAFEQLMTDLTLSRVQFQKLQ

QLRLTQYHGGSLPNVSQLRSNASEFQPSFH QADNVRGTRHHGLVERPSRNR

FHPLHRRSGDKPGRQFDGSAFGANYSSQPLDESWPRQQPPWKDEKHPGFRL

TSALNRTNSDSALHTSALSTKPQDPYGGGGQSAWPAPYMGFCDGENNGHGE

VASFPGPLKEENLLNVPKPLPKQLWETKEIQSLSGRPRSCDVGGGNAFPHN

GQNLGLSPFLGTLNTGGSLPDLTNLHYSTPLPASLDTTDHHFGSMSVGNSV

NNIPAAMTHLGIRSSSGLQSSRSNPSIQATLNKTVLSSSLNNHPQTSVPNA

SALHPSLRLFSLSNPSLSTTNLSGPSRRRQPPVSPLTLSPGPEAHQGFSRQ

LSSTSPLAPYPTSQMVSSDRSQLSFLPTEAQAQVSPPPPYPAPQELTQPLL

QQPRAPEAPAQQPQAASSLPQSDFQLLPAQGSSLTNFFPDVGFDQQSMRPG

PAFPQQVPLVQQGSRELQDSFHLRPSPYSNCGSLPNTILPEDSSTSLFKDL

NSALAGLPEVSLNVDTPFPLEEELQIEPLSLDGLNMLSDSSMGLLDPSVEE

TFRADRL

(SEQ ID NO 25)

Due to the presence of the aforementioned extra C at position 288, the first 81 amino acids differ between FLJ00364 and the predicted polypeptide from the correct proprietary clone.

We believe that the shown amino acid sequence encoded by CREAP3 is correct as it shows extensive homology to CREAP1 and CREAP2. Briefly, CREAP gene family sequences were compared using ClustalW. Amino acid identity was determined using Align, version 2.0 (Myers E.W. and Miller W., Bull. Math Biol 51:5-37 (1989)) and the Blosum 50 scoring matrix (CITE). Genomic sequence alignments were performed using BlastN and the Celera CHD database (Celera Genomics, Rockville, MD) and searches were performed using masked consensus sequences.

(File: CHGD_masked_assembly_500k-i)

The amino acid sequences predicted by the proprietary clone and the FLJ00364 cDNA differ in two other regions. The proprietary clone contained an additional GAA triplet as shown above, which resulted in the addition of a glutamic acid at amino acid 551. Finally, a single nucleotide A/G change in the CREAP3 cDNA resulted in a threonine/alanine change at amino acid 616 as shown above.

Example 12

Identification of CREAP genes from other species

Identification of the Drosophila CREAP gene dCREAP:

BLASTP searches of the Genebank protein and DNA sequence databases using the CREAP1 and CREAP3 coding regions followed conventional methods and identified a single predicted Drosophila gene, CG6064. This gene has been named dCREAP and its amino acid sequence is shown below. This sequence was discovered from sequencing of the Drosophila melanogaster genome as a predicted gene of unknown function: GenBank entry|7293954|gb|AAF49313.1|CG6064-PA[Drosophilamelanogaster] (Adams et al. : 2185-2195 (2000)). The dCREAP gene CG6064 contains no insert and is a predicted 797 amino acid protein, which is slightly larger than human CREAP.

dCREAP DNA sequence

ATGGCCAATCCGCGCAAGTTCAGCGAGAAGATCGCTCTGCAGAAGCAGAAGCAGGCGGAGGGCACAGCGG

AATTCGAGCGGATCATGAAGGAGGTGTATGCCACGAAGAGGGATGAGCCGCCTGCGAATCAGAAGATCCT

AGACGGCCTTGTCGGCGGTCAGGAGGTAAGCCAATCCTCGCCAGGCGCAGGCAATGGGACGGGCGGAGGT

GGCAGTGGTTCCGGCAGTGGAGCCAGCGGCGGAGGAGCCTCACCAGATGGCCTGGGAGGCGGCGGTGGTT

CTCCGACGGCTTATCGAGAATCCCGAGGGCGCAGCGTAGGTGTGGGTCCCATGCGAAGACCGTCGGAGCG

CAAGCAGGATCGTTCGCCCTACGGCAGCAGCAGTACGCAACAAACCTTAGACAACGGCCAGCTAAATCCG

CATCTTCTTGGTCCACCTACGGCGGAGAGTTTGTGGCGGCGGTCCAGCTCCGATTCGGCGCTGCACCAAA

GTGCGCTGGTGGCGGGCTTCAATAGCGACGTGAACTCGATGGGCGCCAACTATCAGCAGCAGCAACATCA

GCAACAACAGCAACCGGGCCAGCCAAGATCTCACTCGCCGCACCATGGTATAAACAGGACCATGAGTCCG

CAGGCGCAACGGAGGAAGTCGCCGCTACTGCAGCCCCATCAGCTGCAGTTGCAGCAACTGCAACAGCAGC

AGCAACAGATGCAACATCAGCATCAGCTGCACCAGCAGCTCCAAATGCAGCAGCTGCAACAGCACCAGCA

GCAACACCAGCAGCAGCAGCAACAACAGAACACGCCATACAACAACGCCAAATTCACGAATCCTGTGTTC

CGGCCGCTGCAGGATCAGGTCAACTTTGCCAACACCGGCTCCCTGCCCGATCTCACGGCCCTTCAAAACT

ATGGACCCCAGCAGCAGCAGCAGCAATCCCAGCAACAGCCGTCGCAGCAACAACAGCAGTTGCAGCAAAC

CCTGTCGCCAGTCATGTCTCCGCACAATCACCGCCGCGAACGGGATCAGTCGCCCAGTCCGTTTAGTCCG

GCGGGTGGAGGAGGGGGAGCAGGTCCCGGGTCGCCCTATCAGCAGCAACAGCACTCGCCCACCGGAAACA

CGCAACAGCAGCAGCAGCAGCACCAACAGCCCAGCAACTCGCCGCACCTGTCCTTTACCAATCTGGCCAC

CACGCAGGCAGCTGTTACCACATTTAACCCGCTCCCCACGCTGGGTCCGCACAATGCCACCGACTACCGC

CAGCCACCGAATCCTCCTAGTCCACGCTCTTCGCCCGGCTTGCTGAGCAGCGTATCGGCCACGGATCTGC

ACTCCAGTGCACCGGCCAGTCCCATACGCCAGCAGCAACAGGCCCATCAGCAGCAACAGCAGCAGCAACA

GGCGCAGCAACAACAGCAACAGTTTGATAACTCCTACAACAGTCTGAATACCTCGTTTCACAAATCAGTTT

GAGATTTTCTCGCTGGGCGACAGCAATTCCTCGCCGGAACAGCAGGGCTTTGCAAATAATTTCGTGGCCC

TCGACTTTGACGACCTGAGTGGCGGCGGAGGTGGTGGCCCAAGCGGGGGCGGCGGCAGCAATGGAGGAGG

TCTGACCAACGGTTACAACAAGCCGGAGATGTTGGACTTCAGCGAGCTGAGCGGCAGCCCGGAGGCGAGT

GGGAACAACAACCACATGCGGCGAGGAGTGAGCAACCTGAACAACAACGGGTTGAGCAATGGTGTGGTGG

GATCCACGCACAACGGCAGCACAAATCTAAATGGAGCGGGAAACAACAATAGCAGTAGTGGAGGTGGCAC

GGCGCAGGATCCTTTGGGAATAACCACTTCGCCTGTGCCCTCACCCTTGGGCTGCCCCAGTTCACCGCTG

CCGATACCGATTCCGATGTCGGCGCAAAGCTCGCCACAGCAGCAGCACCACCATCATCAGCAGCAGCAAC

AACAGCATCATCAGCAGCAACACCATCAGCAGCAGCAATTATCATTTATCTCTGCACCATTCGCCGCATCA

TTCGCCAATGCATTCGCCGCACCATGGGAATTCACCGCTTTCAAGCAGCTCGCCAGTGAGTCACAATGCC

TGCTCCAACTCCAACGTGGTGATGAACCACCAGCAGCAGCAGCAACAACATCACCACCAGCAACACCATC

ATCAGGGCTCCTCGCAAAGTCACACGCCGACCACAGCGAATATACCCTCTATTATCTTTAGTGATTACTC

CTCCAACGCGGATTATACCAGGGAGATCTTCGACTCCCTCGATCTGGATCTGGGACAGATGGACGTAGCC

GGTTTGCAGATGCTGTCCGACCAGAACCCCATCATGATCGCCGATCCCAACATCGAGGATAGTTTTCGAC

GCGACCTCAACTGATACTATGAGGAGGCTGTTGCGGCCATTGAGAGCGGAGTGCTGCTGGAGGAGGACTA

CCAGGCGCTGCTCGGATCAGAGGCGCTGGCGGATGAACAGGTGGTCACAGTCGAGGCCGCCGGAGCCGCA

GCAGCAGTAGTAACAGTTGAAGAGGCAGCCACAGTTAGCGAGAAGGACAAAAAAGATTTGGAAGTTGTGG

AACTTCTGGTGTCCGGTGTTATGGATGACCTGGTGGACTCCAGTGACCTGGACGAGGAAGTGCGCAATTT

CTTTTTTTAGGCAGCCAGCAAGTCATTTTTGTCGTTAACACAACTGATGGAATTTTCGTTTTTAACACAG

ATGAGGAAGTGAATTACGTTTTTTAAACGCATTCACTTGCCATTTCTCGATTAAATGCCATATTACTTAA

GCTCAGGATTTACAAGCTTAATGCGAATTAAGTTAATTTCGGAAATGCTGACGAGAGTGATTGCAAAGTT

CAAAATTGATACAAATTCACTTCCGCAAATTCATGCTGAAACTGAAAGTTTTTCTAACAGTCCTCAATATT

GTTATCTCGTTATCGTCCGTGCTTTCGTAGCTAGCTCTACAACAAAAATAC

(SEQ ID NO 26)

The predicted amino acid sequence of dCREAP is shown below:

MANPRKFSEKIALQKQKQAEGTAEFERIMKEVYATKRDEPPANQKILDGLVGGQEVSQSSPGAGNGTG

GGGSGSGSGASGGGASPDGLGGGGGSPTAYRESRGRSVGVGPMRRPSERKQDRSPYGSSSTQQTLDNG

QLNPHLLGPPTAESLWRRSSSSDSALHQSALVAGFNSDVNSMGANYQQQQHQQQQQPGQPRSHSPHHGI

NRTMSPQAQRRKSPLLQPHQLQLQQLQQQQQQMQHQHQLHQQLQMQQLQQHQQQHQQQQQQQNTPYNN

AKFTNPVFRPLQDQVNFANTGSLPDLTALQNYGPQQQQQQSQQQPSQQQQQLQQTLSPVMSPHNHRRE

RDQSPSPFSPAGGGGGAGPGSPYQQQQHSPTGNTQQQQQQHQQPSNSPHLSFTNLATTQAAVTTFNPL

PTLGPHNATDYRQPPNPPSPRSSPGLLSSVSATDLHSSAPASPIRQQQQAHQQQQQQQQAQQQQQQFD

NSYNSLNTSFHNQFEIFSLGDSNSSPEQQGFANNFVALDFDDLSGGGGGGPSGGGGSNGGGLTNGYNK

PEMLDFSELSGSPEASGNNNHMRRGVSNLNNNGLSNGVVGSTHNGSTNLNGAGNNNSSSGGGTAQDPL

GITTSPVPSPLGCPSSPLPIPIPMSAQSSPQQQHHHHQQQQQQHHQQQHHQQQQLSLSLHHSPHHSPM

HSPHHGNSPLSSSSPVSHNACSNSNVVMNHQQQQQQHHHQQQHHHQGSSQSHTPTTANIPSIIFSDYSS

NADYTREIFDSLDLDLDLGQMDVAGLQMLSDQNPIMIADPNIEDSFRRDLN

(SEQ ID NO 27)

The activity of dCREAP was analyzed as follows:

A 2.3 kb cDNA encoding the dCREAP open reading frame was amplified by PCR using sense (SEQ ID 37) and antisense (SEQ ID 38) primers.

The sense primer that is used to amplify dCREAP ORF cDNA: (the underlined part is fruit fly Kozak sequence CAAC) CAAC ATGGCCAATCCGCGCAAGTTCAGCGAG (SEQ ID 37) The antisense primer that is used to amplify dCREAP ORF cDNA: TCAGTTGAGGTCGCGTCGAAAACTATCCTC (SEQ ID 38) will amplify The amplification product was inserted into the Drosophila P-element transformation vector pUAST (Brand and Perrimon, Development 118:401-415 (1993)). The final construct pUAS-dCREAP was used for transfection experiments in Drosophila melanogaster Schneider cells (S2). A firefly luciferase reporter gene was established comprising 4 copies of the Drosophila CRE enhancer element (SEQ ID 39) (Eresh, S. et al. EMBO J.16:2014-2022 (1997)) and after the reporter gene Is the hsp70 minimal promoter. The oligonucleotide sequence contains 4 copies of the Drosophila Cre. The underlined part is the sequence of the CRE element: GGAGCC TGGCGTCA GAGAGCC TGGCGTCA GAGAGCC TGGCGTCA GAGAGCC TGGCGTCA GAG (SEQ ID 39)

_S2 cells were transfected by the CaPO4 method (Bunch, T. and Goldstein, L. Nucleic Acids Res. 17:9761-9782 (1989)) in 6-well plates (Costar). A total of 25 μg of DNA was transfected into 6-well plates containing 4 ml of cells (˜1×10 ⁶ cells/ml). The transfection mixture was removed after 18 hours and the luciferase assay was performed after 48 hours. The UAS-transgene was activated by co-transfection using the actin promoter-Gal4 plasmid provided by Dr. Norbert Perrimon. Transfection efficiency was normalized by co-transfection with hsp ^min Renilla luciferase driven by a minimal heat shock promoter (prepared according to conventional methods). Luciferase activity was determined using a dual-luciferase assay kit (Promega). As a negative control, S2 cells were co-transfected with the CRE-hsp-Luc reporter and an empty pUAST vector. Data are calculated as fold induction compared to reporter gene activity determined in cells designated as negative controls.

The results showed (see Table 3) that, like human CREAPS, dCREAP is also able to regulate CRE in Drosophila because it efficiently induces the activity of the CRE-hsp-Luc reporter when the CRE element is present.

Activate

Multiple STDEV

pUAST/CRE-hsp-Luc 1.02 0.28

dCREAP/hsp-Luc 0.96 0.15

dCREAP/CRE-hsp-Luc 136.04 37.13

Table 3: dCREAP efficiently induces the activity of the CRE-hsp-Luc reporter. S2 cells were co-transfected with empty pUAST vector or pUAST-dCREAP construct (dCREAP) and hsp-Luc reporter or hsp-Luc reporter carrying 4 copies of Drosophila CRE (CRE-hsp-Luc). Luciferase activity was measured 48 hours after transfection.

Identification of the mouse CREAP1 (mCREAP1) gene:

The mouse CREAP1 protein was also identified using conventional methods. Briefly, mCREAP1 cDNA was assembled in the following order:

Nucleotides 1-483 are from mouse EST BY752080 (GenBank accession numbers here and below);

Nucleotides 484-891 are from mouse EST BM950955;

Nucleotides 892-909 from mouse genomic DNA sequence Celera clone

Nucleotides 910-981 are from mouse EST CA326891.

Nucleotides 982-1610 are from mouse EST BM935820.

Nucleotides 1611-2416 are from mouse EST BI453510.

The resulting mCREAP1 nucleotide sequence:

GGGACGAAGAGTAGGAGTAGGAGGAGGCGGCGAGAAGATGGCGACTTCGAACAATCCGCGGAAATTTA

GCGAGAAGATCGCACTGCACAACCAGAAGCAGGCGGAGGAGACGGCGGCCTTCGAGGAGGTCATGAAG

GACCTGAGCCTGACGCGGGCCGCGCGGCTTCAGCTGCAGAAGTCCCAGTACCTGCAGCTGGGCCCCAG

CCGTGGCCAGTACTACGGTGGGTCCCTGCCCAACGTGAACCAGATTGGAAGCAGCAGCGTGGACCTGG

CCTTCCAGACCCCATTTCAGTCCTCAGGCCTGGACACGAGTCGGACCACACGACATCATGGGCTTGTG

GACAGAGTATATCGTGAGCGTGGCAGACTTGGCTCCCCGCACCGTCGACCCCTGTCAGTAGACAAGCA

TGGGCGACAGGCTGACAGCTGCCCCTATGGCACCGTGTACCTCTCGCCTCCTGCGGACACCAGCTGGA

GGAGGACCAACTCTGACTCTGCCCTGCACCAGAGCACAATGACACCCAGCCAGGCAGAGTCTCTTCACA

GGCGGGTCCCAGGATGCGCACCAGAAGAGAGTCTTACTGCTAACTGTCCCAGGAATGGAGGACACCGG

GGCTGAGACAGACAAGACCCTTTCTAAGCAGTCATGGGACTCAAAGAAGGCGGGTTCCAGGCCCAAGT

CCTGTGAGGTCCCCGGAATCAACATCTTTCCGTCTGCAGACCAGGAGAACACAACAGCCCTGATCCCT

GCCACCCACAACACAGGGGGCTCCCTTCCTGACCTCACCAACATCCACTTCGCCTCCCCACTCCCGAC

ACCACTGGACCCTGAGGAGCCTCCGTTCCCTGCTCTCACCAGCTCCAGCAGCACCGGCAGCCTTGCAC

ATCTGGGCGTTGGCGGCGCAGGCGGTATGAACACCCCCAGCTCTTCTCCAGCACCGGCCAGCAGTC

GTCAGCCCCCTGTCCCTGAGCACAGAGGCCAGGCGGCAGCAGGCCCAGCAGGTGTCACCCCACCCTGTC

TCCGTTGTCACCCCATCACTCAGGCCGTGGCTATGGATGCCCTGTCCTTGGAGCAGCAGCTGCCCTATG

CCTTTCTTCACCCAGACTGGCTCCCAGCAGCCTCCCCCACAGCCCCAGCCACCGCCTCCACCTCCACCG

GTATCCCAGCAGCAGCCACCACCTCCACAGGTGTCTGTGGGCCTCCCCCAGGGTGGTCCACTGCTGCC

CAGTGCCAGCCTGACTCGGGGGCCCCAGCTGCCACCACTCTCAGTTACTGTACCATCCACTTCTTCCCC

AGTCCCTACAGAGAACCCAGGCCAGTCACCAATGGGGATCGATGCCACTTCGGCACCAGCTCTGCAG

TACCGCACGAGTGCAGGGTCACCTGCCACCCAGTCTCCCACCTCTCCGGTCTCCAACCAAGGCTTCTC

CCCTGGAAGCTCCCCACAGCACACGTCCACCCTGGGCAGCGTGTTTGGGGATGCGTACTATGAGCAGC

AGATGACAGCCAGGCAGGCCAATGCTCTGTCNCGCCAGCTGGAGCAGTTCAACATGATGGAGAACGCC

ATCAGCTCCAGCAGCCTATACAACCCGGGCTCCACACTCAACTATTCCCAGGCTGCCATGATGGGTCT

GAGCGGGAGCCACGGGGGCCTACAGGACCCGCAGCAGCTCGGCTACACAGGCCACGGTGGAATCCCCA

ACATCATCCTCACGGTGACAGGAGAGTCACCACCGAGCCTCTCTAAGGAACTGAGCAGCACACTGGCA

GGAGTCAGTGATGTCAGCTTTGATTCGGACCATCAGTTCCACTGGACGAGCTGAAGATTGACCCTCT

GACCCTGGACGGACTCCATATGTTGAATGACCCAGACATGGTTTTAGCCGACCCAGCCACCGAGGACA

CCTTCCGAATGGACCGCCTGTGAGTGGCTGTGCCCACCAGCCGCCGCTGGTCAGTCTCCAACGGCGCT

GCCCCAAACCTGGGGACGGCAATGGCGTCCCCCCTTTGCCAACGGCCAAGCTTGTGGTTCTGAGCTTGC

AATGCTGCCCAGTGCCCCTGCCAGCCCCCCGCCACCCCGGTCGTTCACCTCCCATGATGCCTGGCGTG

CGTGAGGCCGCTGTGTACTAGGCTGGCTATCTGTCTGTCCATCCATCTACCTGGGGTCAGGCTGATGG

CCGAGGCTGTGAGTGCCTGGCCCCCATGGATGTTCCCCGTGCTCGCTCCCTCACCCTCACTGGGGAT

GTGAGAGCCCTCATCAGATACCCAAAGTGTCACTCACTTCCAGCATGTGCTGTGCAACGGAGGGCCGG

GGCGTGGGTGTGGAGCGCCCAGAGGCTTAGGTGCGCCATCCATTCGACTGTTGTCAGCTGTCACTGCC

TTCCCTCCATCCTGTCCCCCGTCCCCACCGCCATCCCT

(SEQ ID NO.28)

The open reading frame encoding the mCREAP1 protein sequence is encoded by nucleotides 25-1914.

Protein sequence of mCREAP1:

MATSNNPRKFSEKIALHNQKQAEETAAFEEVMKDLSLTRAARLQLQKSQYLQLGPSRGQYYGGSLPNV

NQIGSSSVDLAFQTPFQSSGLDTSRTTRHHGLVDRVYRERGRLGSPHRRRPLSVDKHGRQADSCPYGTV

YLSPPADTSWRRTNSDSALHQSTMTPSQAESFTGGSQDAHQKRVLLLTPGMEDTGAETDKTLSKQSW

DSKKAGSRPKSCEVPGINIFPSADQENTTALIPATHNTGGSLPDLTNIHFASPLTPLDPEEPFPAL

TSSSSTGSLAHLGVGGAGGMNTPSSSPQHRPAVVSPLSLSTEARRQQAQQVSPTLSPLSPITQAVAMD

ALSLEQQLPYAFFTQTGSQQPPPQPQPPPPPPPVSQQQPPPPQVSVGLPQGGPLLPSASLTRGPQLPP

LSVTVPSTLPQSPTENPGQSPMGIDATSAPALQYRTSAGSPATQSPTSPVSNQGFSPGSSPQHTSTLG

SVFGDAYYEQQMTARQANALSRQLEQFNMMENAISSSSLYNPGSTLNYSQAAMMGLSGSHGGLQDPQQ

LGYTGHGGIPNIILTVTGESPPSLSKELSSTLAGVSDVSFDSDHQFPLDELKIDPLTLDGLHMLNDPD

MVLADPATEDTFRMDRL (SEQ ID NO: 29)

Identification of Pufferfish CREAP1:

CREAP1 was identified in the redfin oriental porpoise. The sequence was identified by alignment of the human CREAP1 protein sequence to the pufferfish genome (version 3) using TBLASTN. Highly homologous regions were found by comparison. The found sequences were further manually edited.

Puffer fish CREAP1 amino acid sequence:

MASSSNNPRKFSEKIALHNQKQAEETAAFEEVMKDLNVTRAARLQLQKTQYLQLGQNRGQYYGGSLPNV

NQIGNGNIDLPFQVSNSVLDTSRTTRHHGLVERVYRDRNRISSPHRRPLSVDKHGRQRTNSDSALHQS

AMNPKPHEVFAGGSQELQPKRLLLTVPGTEKSESNADKDSQEQSWDDKKSIFPSPDQELNPSVLPAAH

NTGGSLPDLTNIQFPPPLSTPLDPEDTVTFPSLSSSNSTGSLTTNLTHLGISVASHGNNGEKNIFFLK

TCTSCEDVYDFYFVGIPTSSQTTMTATAQRRQPPVVPLTLTSDLTLQQSPQQLSPTLSSPINITQSMK

LSASSSLQQYRNQTGSPATQSPTSPVSNQGFSPGSSPQPQHIPVVGSIFGDSFYDQQLALRQTNALSHQ

VCEDGRRLEITHVRLSRLHAELCFCFSQLEQFNMIENPISSTSLYNQCSTLNYTQAAMMGLTGSSLQD

SQQLGYGNHGNIPNIILTISVTGESPPSLSKELTNSLAGVGDVSFDPDTQFPLDELKIDPLTLDGLHM

LNDPDMVLADPATEDTFRMDRL (SEQ ID NO. 30)

Puffer fish CREAP1 DNA sequence:

ATGGCGTCCTCTAACAATCCTCGCAAATTTAGCGAAAAAATCGCACTGCATAACCAGAAACAAGCAGA

GGAGACTGCTGCGTTCGAAGAAGTGATGAAGGACCTGAACGTCACAAGGGCTGCCCGGGTAAGACAGC

TGCAGTTACAGAAGACCCAGTATTTGCAACTAGGGCAGAATCGTGGACAGTACTATGGAGGCTCACTG

CCCAATGTCAAATCAGATTGGAAATGGCAACATTGACCTGCCTTTTCAGGTGAGCAGGACAAACTCAGA

CTCAGCTTTACATCAGAGTGCCATGAATCCAAAGCCCCACGAAGTGTTTGCTGGGGGGTCGCAGGAGC

TGCAGCCCAAACGACTGCTGCTAACAGTGCCTGGAACCGAAAAATCGGAATCAAACGCAGACAAAGAT

TCGCAGGAGCAGTCGTGGGATGACAAAAAGAGTATTTTTCCATCACCAGACCAGGAGTTAAACCCCTC

CGTGCTTCCAGCCGCGCACAACACCGGCGGTTCGCTCCCCGACCTGACCAACATCCAGTTCCCTCCTC

CACTGTCCACCCCACTGGACCCCGAGGACACCGTCACCTTTCCCCTCCCTCAGCTCCTCTAACAGCACA

GGCAGTCTGACTACCAACCTCACCCACCTGGGCATCAGTGTGGCCAGCCATGGTAATAACGGAGAGAA

AAATATATTTTTTTTAAAAACATGCACTTCATGCGAGGATGTTAAATAATATTACGACTTTTATTTTG

TAGGGATTCCCACTTCCTCTCAAAACCACCATGACAGCAACAGCACAGCGGCGGCAACCACCCGTGGTC

CCCCTCACCCTCACCTCTGACCTGACTCTTCAACAGTCCCCCCAGCTTTTCACCCACCCTCTCCTCTC

ACCCATTAACATCACACAGAGCATGAAGCTTAGTGCTAGCTAACATTCTTCCCTCCAACAGTACCGCA

ATCAGACTGGCTCACCAGCCACTCAGTCTCCAACCTCCCCAGTCTCCAATCAAGGCTTCTCCCCCGGC

AGCTCGCCTCAACCACAGCACATTCCTGTGGTGGGCAGTATATTTGGGGACTCCTTCTATGATCAGCA

GTTGGCTCTGAGGCAGACCAATGCCCTTTCTCATCAGGTGTGTGAGGACGGCCGCAGGTTAGAAATAA

CACACGTACGTCTCTCACGACTTCACGCCGAGCTTTGTTTTTGTTTTTCTCAGCTGGAGCAGTTCAAT

ATGATAGAGAACCCCATCAGCTCCACCAGCCTGTACAATCAGTGCTCCACCCTTAATTACACACAGGC

AGCCATGATGGGCCTCACCGGGAGCAGCCTGCAGGACTCGCAGCAGCTCGGCTACGGCAATCACGGCA

ACATCCCCAAACATCATACTGACAATTTCAGTCACAGGGGAGTCTCCGCCGAGCCTCTCCAAAGAGCTG

ACCAACTCATTGGCCGGCGTCGGCGACGTCAGCTTTGATCCAGACACGCAGTTTCCTCTGGACGAGCT

GAAGATCGACCCGCTGACCTTGGACGGCCTGCACATGCTCAACGACCCAGACATGGTGCTGGCAGACC

CCGCCACAGAGGACACGTTCAGGATGGACAGGCTGTAA (SEQ ID NO. 31)

Example 13

Comparison of the human CREAP coding region with other CREAP proteins from other species

These sequences were compared by a global alignment of the coding regions of all three CREAP sequences as shown in Figure 1 . Each protein is similar in size, with CREAP2 being slightly larger (693 amino acids compared to 650 and 619 amino acids for CREAP1 and 3, respectively). These proteins can be roughly divided into three structural domains based on conservation. The first is the one-third conserved amino terminus, which shares high identity with CREAP1 up to amino acid 267 (ie, amino acids 1-267). This region is approximately 33% identical across all three CREAPs. The second domain is the middle region spanning amino acids 289-538 of CREAP1, which is highly abundant in proline, glycine and serine residues. This region corresponds to amino acids 289-529, 376-606 and 235-533 of CREAP1, CREAP2 and CREAP3, respectively. This region has little amino acid identity, but is similar in amino acid composition. Finally, the carboxy-terminal third of the protein (approximately corresponding to the last 78 amino acids of CREAP1 (amino acids 575-650 of CREAP1)) is again highly conserved, with 38% amino acid identity between all three proteins.

Interestingly, the most conserved part of the protein is the amino terminus. A region of 80% identity over 24 amino acids is present in all three proteins. This region is also conserved in Drosophila and is essential for CREAP function and may represent a key region regulating CREAP function. The conservation of the amino terminus of CREAP suggests that this region is critical for its function. This notion is supported by data showing that deletion of the amino-terminal 250 amino acids abolishes CREAP1 activity (see Table 1 above).

To further identify whether the vast majority of amino-terminal residues are critical, a deletion of the most N-terminal 59 amino acids was made in CREAP1. CREAP1 cDNA was excised from the original pCMV-SPORT6 plasmid with ScaI/XhoI restriction enzymes (Roche Applied Science, Indianapolis, IN, USA). A ScaI digested 2382nt CREAP1 cDNA fragment lacking 177 nucleotides of the CREAP1 ORF was gel purified and subcloned into EcoRV/SalI digested pFLAG-CMV6B vector (BD Biosciences). Correct clones were isolated and sequence verified by routine methods. This protein (delta 59) was tested in a promoter-reporter assay. Consistent with their conservation, deletion of these residues resulted in an 80-90% loss of CREAP activity (data not shown).

The similarity between human CREAP1 and homologues from other species is shown in Figure 1 . Taken together, the three domains described for human CREAP are also contained in other CREAP sequences. The amino terminus is highly conserved. Notably, the most amino-terminal conserved amino acid of human CREAP is also highly conserved in these proteins.

The human CREAP1 cDNA identified in this study encodes a predicted 650 amino acid protein. This cDNA partially overlaps with many cDNAs annotated KIAA0616 but differs at the predicted c-terminus of the encoded protein. We were able to identify an N-terminal coil-coil domain (amino acids 8-54), a serine/glutamine-rich domain (amino acids 289-559) and a strongly negatively charged C-terminal domain ( amino acids 602-643) (data not shown). As shown, along with human CREAP2 and CREAP3, genes encoding proteins highly similar to CREAP1 were found in the mouse and pufferfish genomes. All human and mouse CREAP1 genes are 90% identical. The predicted fugu protein is 566 amino acids long and 66% identical to human CREAP1.

We also identified a predicted CREAP1-like gene in the Drosophila genome. Although the mammalian and fish CREAP1 genes share only about 20 percent identity with the Drosophila sequence, the Drosophila sequence shares a similar organization with other CREAP1 proteins. Each protein contains highly conserved amino- and carboxy-terminal regions and a central domain rich in proline, glutamine and serine residues. We have named the Drosophila predictor gene dCREAP. The first 22 of the 28 amino acids of dCREAP are identical to human CREAP1. The amino terminus has an absolutely conserved consensus PKA or PKC consensus phosphorylation site (RKFS) similar to the phosphorylation site in the CREB protein. Phosphorylation of this serine in CREB (serine 133 in CREB1) is required for the induction of CREB-dependent gene expression by cAMP.

The first 32 amino acids of DCREAP are 69% and 84% identical and similar to human CREAP1, respectively, which again supports the idea that the amino terminus of CREAP is critical for its function. Additionally, the central domain of dCREAP is a region of lower complexity with little homology. Although the predicted dCREAP coding region does have some glycine and proline rich regions, it is unique in being highly rich in glutamine residues. In addition, similar to human CREAP, the most carboxyl terminus of this protein is highly conserved with human CREAP1 (more than 30% of the last 30 amino acids).

The correlation of the CREAP gene is shown in Table 4. Overall, human CREAP genes were more related to each other than dCREAP. CREAP2 is slightly more similar to CREAP1 and CREAP3 than between CREAP1 and CREAP3, but all share 34-39% identity between them. All human CREAP was found to be about 20% identical to the predicted dCREAP gene, although as shown above the similarity is more due to the highly conserved amino and carboxy termini of the protein. It should be noted that all three CREAP genes are highly conserved in the mouse and human genomes (data not shown). This suggests that the different isoforms have unique and critical functions. The evolutionary conservation of CREAP supports the notion that CREAP is a key regulator of CRE activity. HCREAP1 HCREAP2 HCREAP3 MCREAP1 FCREAP1 dCREAP HCREAP1HCREAP2HCREAP3 32 3233 893430 633160 191815 MCREAP1FCREAP1dCREAP 60 2120

Table 4: Amino acid similarity of CREAP genes from different species. Data shown represent percent amino acid identity across protein coding regions and were calculated as described above. Percent identities are based on automated alignments using ClustalW V1.74.

Example 14

Activity of CREAP2 and CREAP3

The homology between the human CREAP genes suggests that they are functionally related. To investigate this, the ability of CREAP2 and CREAP3 to activate gene expression driven by the IL-8 promoter and the CRE-dependent promoter was tested in a co-transfection assay as disclosed above. Briefly, after co-transfection with an empty pCMV-SPORT6 expression vector or the same vectors carrying CREAP1, CREAP2, or CREAP3 cDNA, the expression of fireflies driven by the IL-8 promoter or a minimal promoter linked to four copies of CRE was determined. Expression levels of the luciferase reporter. The results showed that co-transfection of CREAP1 with either the IL-8-dependent promoter or the -CRE-dependent firefly luciferase gene resulted in a dramatic increase in luciferase activity (see Table 5). Transfection of CREAP2 or CREAP3 also produced similar activation of both reporters. Additional experiments indicated that this activity was dependent on the integrity of CRE or CRE-like sites present in the IL-8 promoter (data not shown). Interestingly, CREAP3 consistently showed 2-4 fold higher levels of induction of gene expression compared to CREAP1 and CREAP2. Thus, CREAP2 and CREAP3 are potent activators of CRE-driven gene expression and the CREAP family represents a family of conserved sequences and activities. Furthermore, all three CREAP family members showed the ability to activate the CREB-GAL4 fusion protein when overexpressed in the HLR-CREB cell line (Stratagene), which carries a genome-integrated UAS-Luc reporter, which This supports evidence that CREAP proteins can induce gene expression by interacting with promoter-bound CREB proteins (data not shown). CRE IL-8 control 1 1 CREAP1 28.6 175.8571 CREAP2 38.6 126.8571 CREAP3 71.4 574.5714

Table 5. Induction of CREAP gene family on CRE driven promoter or interleukin-8 promoter. Luciferase expression constructs driven by a minimal or interleukin-8 promoter linked to multiple copies of CREAP were co-transfected with empty vector (control) or expression vectors encoding the three CREAP genes. The expression level of luciferase is expressed relative to the level obtained by co-transfection with empty vector.

Example 15

The CREAP1 protein is a transcriptional activator

Several observations suggest that CREAP1 is a transcriptional coactivator. First, although we have not yet been able to identify any DNA-binding activity in CREAP1, each CREAP protein contains a predicted N-terminal coiled-coil domain (hCREAP1 residues 8-54), a serine/glutamine-rich domain (hCREAP1 residues 289-559) and a negatively charged carboxyl terminus.

To determine whether CREAP proteins could function as transcriptional activators, different regions of all three CREAP homologues (amino acids 300-650 of CREAP1, amino acids 296-694 of CREAP2, and amino acids 335-635 of CREAP3) were bound to DNA expressed as GAL4 Domains were expressed as fusion proteins and tested for their ability to activate the expression of a reporter gene (UAS-Luc (pFR-Luc reporter) linked to the GAL4 protein binding sequence. Briefly, the indicated CREAP1, CREAP2 and CREAP3 region and subcloned into the pCMV-BD vector (Stratagene) in the reading frame encoding the GAL4 DNA binding domain. The selected plasmid and the empty vector (pCMV-SPORT6 ) into HEK 293 cells. Co-transfected with pFR-Lucreporter (Stratagene) encoding the firefly luciferase gene driven by a minimal promoter linked to the quintuplex GAL4 binding site (UAS) at 100 ng/well As a positive control, the reporter was also co-transfected with a plasmid encoding the GAL4-CREB fusion protein (Stratagene) alone or in the presence of the expression construct pFC-PKA (Stratagene) encoding the protein kinase A catalytic subunit to activate CREB kinase Inducible activation domain. The fold induction was compared with the reporter activity measured in cells transfected with only the expression vector pCMV-BD carrying the GAL4 DNA-binding domain. Although the three full-length CREAP proteins did not significantly affect reporter activity, but fusion proteins containing the carboxy-terminal part of CREAP1-3 effectively induced the expression of UAS-Luc, see Table 6. Induction multiple STDEV Vector CREAP1CREAP2CREAP3GAL4-CREAP1.1GAL4-CREAP2.1GAL4-CREAP3-1GAL4-CREBGAL4-CREB/PKA 1.002.4675312.471.582692.601373.881364.177.66351.4352 0.211.4788080.410.74556.19222.52263.620.3411.52481

Table 6: Demonstration that the CREAP1 protein is an activator of transcription. Expression constructs encoding full-length CREAP1, CREAP2, and CREAP3 and the Gal4 DNA-binding domain alone or fused to C-terminal portions of CREAP1, CREAP2, and CREAP3 were tested to induce fluorescence controlled by a minimal promoter linked to the GAL4 DNA-binding site The ability to express the luciferase gene (pFRLuciferase). Values shown are normalized to those obtained with pCMV-BD vector co-transfected with pFR-Luc reporter.

To determine whether CREAP proteins can directly activate CREB1 proteins, the CREAP1, CREAP2, and CREAP3 expression constructs were transfected alone or together with the GAL4-CREB plasmid (Stratagene) into an HLR cell line (Stratagene) harboring integrated pFR-Luc Genomic DNA copied by reporter. Briefly, HLR cells were maintained according to the manufacturer's instructions. Transfections were performed with selected plasmids and empty vector (pCMV-BD) or GAL4-CREB plasmid at 75 ng/well using Fugene6 transfection reagent (Roche) as described above. As a positive control, the expression construct pFC-PKA (Stratagene) encoding the catalytic subunit of protein kinase A was also co-transfected with GAL4-CREB. Fold activation was compared to reporter activity determined in cells transfected with empty vector. Although the three full-length CREAP proteins did not significantly affect reporter activity, the activity of the GA4L-CREB fusion protein was upregulated when co-transfected with the three full-length CREAP proteins, suggesting that CREB and CREAP proteins interact to form an active transcriptional complex. See Table 7 below. Induction multiple STDEV pCMV-SPORT6GAL4-CREBGAL4-CREB/PKACREAP1GAL4-CREB/CREAP1CREAP2GAL4-CREB/CREAP2CREAP3GAL4-CREB/CREAP3 175.54687245676.35317566.122284386233.14302922.435298629177.55398542.457796272447.635808 1.732050814.424213916.864978483.116913233.13457372.0595979323.06787722.4245262436.439389

Table 7: CREAP1 functions by activating CREB. The ability of full-length CREAP1, CREAP2 and CREAP3 to induce the activity of the GAL4-CREB fusion protein (Stratagene) was tested. The data presented are normalized to the values obtained using the pCMV-BD vector. All CREAP and PKA significantly induce GAL4-CREB-mediated activation. Note that the fold induction obtained with the positive control is lower when compared with the data from Table 6, which was obtained by transient transfection with all plasmids containing the reporter.

To determine whether CREAP1 can directly interact with CREB, the K1 and K5 variants of CREAP1 (see Table 1) were transfected into 100 mm dishes (Falcon) using Fugene6 reagent (Roche Applied Science) following the protocol provided by the manufacturer. HEK293 cells. 40 hours after transfection, cells were scraped from the plate, placed in PBS and lysed in 800 μl low stringency buffer containing: 10 mM HEPES pH 7.6, 250 mM NaCl, 5 mM EDTA, 1 mM DTT, 0.1% NP-40 and freshly dissolved protease inhibitors. Immunoprecipitation was performed using M2-Sepharose beads (Sigma). Precipitated proteins were separated on 4-20% SDS-PAGE (Invitrogen) and transferred to nitrocellulose membranes (Invitrogen). Western blotting was performed using an antibody against CREB (Cell Signaling Technology). An expression construct encoding FLAG-tagged human histone deacetylase (HDAC1) was used as a negative control. We found that a fragment of the N-terminal 170 amino acids of CREAP1 containing a highly conserved coiled-coil domain binds endogenous CREB1 in vivo. The data shown in Table 1 demonstrate that this region is absolutely required for CREAP-mediated CRE activation.

In addition to the recently identified LIM-only protein family (Fimia, G. et al. 2000, Mol Cell Biol 20, 8613-8622), the CREAP family may represent an evolutionarily conserved branch of CREB coactivators. Interestingly, while LIM-only proteins bind CREM, a known CRE inhibitor, and provide phosphorylation- and CBP-independent activation functions, our data suggest that CREAP can bind to canonical CRE sites. CREB1 and CREB2 that bind to CRE-like elements not recognized by CREB1 interact and thus activate the expression of diverse target gene repertoires. Furthermore, CREAP1 appears to allow for synergy between proteins that surface bind to the CRE and AP-1 binding sites. Elucidation of the role of CREAP1 will explain the mechanisms governing tissue-selective responses to CREB activators.

The experiments described here raise obvious questions about the importance of CRE-like loci in regulating IL-8 expression during disease. Although previously demonstrated that no CRE or CRE-like sites exist on the IL-8 promoter, β ₂ -adrenergic receptor agonists (β ₂ -AR), which act to increase intracellular cAMP IL-8 secretion is induced in cells (Kavelaars A. et al. J. Neuroimmunol. 1997 Aug;77(2):211-6). This is especially important since the use of _β2 -AR agonists as bronchodilators can exacerbate asthma and should be combined with anti-inflammatory steroids (Cockcroft, D. et al., 1993; Lancet 342:833-837; Knox, AJ 2002; Curr. Pharm Des. 1863-1869; Vathenen et al., 1988 Lancet 1:554-558)). The data presented suggest that this effect may be directly due to the activation of IL-8 transcription via CRE-like sites and perhaps CREAP1.

Sequence Listing

<110> Novatis Corporation

<120> Cyclic AMP Response Element Activation Protein and Related Uses

<130>4-32999P2

<150>60/463,934

<151>2003-04-18

<160>39

<170>FastSEQ for Windows version 4.0

<210>1

<211>2878

<212>DNA

<213> people

<400>1

ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc 60

gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa 120

gacaccggga ccgatccagc ctccggactc tagcctaggc cgcgggacgg ataacaattt 180

cacacaggaa acagctatga ccattaggcc tattaggtg acactataga acaagtttgt 240

acaaaaaagc aggctggtac cggtccggaa ttcccggggag gaggaggagg tggcggcgag 300

aagatggcga cttcgaacaa tccgcggaaa ttcagcgaga agatcgcgct gcacaatcag 360

aagcaggcgg aggagacggc ggccttcgag gaggtcatga aggacctgag cctgacgcgg 420

gccgcgcggc tccagctcca gaaatccccag tacctgcaac tgggccccag ccgaggccag 480

tactatggcg ggtccctgcc caacgtgaac cagatcggga gtggcaccat ggacctgccc 540

ttccagccca gcggatttct gggggaggccc tggcagcgg ctcctgtctc tctgacccccc 600

ttccaatcct cgggcctgga caccagccgg accacccggc accatgggct ggtggacagg 660

gtgtaccggg agcgtggccg gctcggctcc ccacaccgcc ggcccctgtc agtggacaaa 720

cacggacggc aggccgacag ctgcccctat ggcaccatgt acctctcacc acccgcggac 780

accagctgga gaaggaccaa ttctgactcc gccctgcacc agagcacaat gacgcccacg 840

cagccagaat cctttagcag tgggtcccag gacgtgcacc agaaaagagt cttactgtta 900

acagtccccag gaatggaaga gaccacatca gaggcagaca aaaacctttc caagcaagca 960

tgggacacca agaagacggg gtccaggccc aagtcctgtg aggtccccgg aatcaacatc 1020

ttcccgtctg ccgaccagga aaacactaca gccctgatcc ccgccaccca caacacaggg 1080

gggtccctgc ccgacctgac caacatccac ttcccctccc cgctcccgac cccgctggac 1140

cccgaggagc ccaccttccc tgcactgagc agctccagca gcaccggcaa cctcgcggcc 1200

aacctgacgc acctgggcat cggtggcgcc ggccagggaa tgagcacacc tggctcctct 1260

ccacagcacc gcccagctgg cgtcagcccc ctgtccctga gcacagaggc aaggcgtcag 1320

caggcatcgc ccaccctgtc cccgctgtca cccatcactc aggctgtagc catggacgcc 1380

ctgtctctgg agcagcagct gccctacgcc ttcttcaccc aggcgggctc ccagcagcca 1440

ccgccgcagc cccagccccc gccgcctcct ccacccgcgt cccagcagcc accacccccg 1500

ccaccccccac aggcgcccgt ccgcctgccc cctggtggcc ccctgttgcc cagcgccagc 1560

ctgactcgtg ggccacagcc gcccccgctt gcagtcacgg taccgtcctc tctcccccag 1620

tcccccccag agaaccctgg ccagccatcg atggggatcg acatcgcctc ggcgccggct 1680

ctgcagcagt accgcactag cgccggctcc ccggccaacc agtctcccac ctcgccagtc 1740

tccaatcaag gcttctcccc agggagctcc ccgcaacaca cttccaccct gggcagcgtg 1800

tttggggacg cgtactatga gcagcagatg gcggccaggc aggccaatgc tctgtccccac 1860

cagctggagc agttcaacat gatggagaac gccatcagct ccagcagcct gtacagcccg 1920

ggctccacac tcaactactc gcaggcggcc atgatgggcc tcacgggcag ccacgggagc 1980

ctgccggact cgcagcaact gggatacgcc agccacagtg gcatccccaa catcatcctc 2040

acagtgacag gagagtcccc ccccagcctc tctaaagaac tgaccagctc tctggccggg 2100

gtcggcgacg tcagcttcga ctccgacagc cagtttcccc tggacgaact caagatcgac 2160

cccctgaccc tcgacggact gcacatgctc aacgaccccg acatggttct ggccgaccca 2220

gccaccgagg acaccttccg gatggaccgc ctgtgagcgg gcacgccggc accctgccgc 2280

tcagccgtcc cgacggcgcc tccccagccc ggggacggcc gtgctccgtc cctcgccaac 2340

ggccgagctt gtgattctga gcttgcaatg ccgccaagcg ccccccgcca gcccgccccc 2400

ggttgtccac ctcccgcgaa gcccaatcgc gaggccgcga gccgggccgt ccaccccaccc 2460

gcccgcccag ggctgggctg ggatcggagg ccgtgagcct cccgcccctg cagaccctcc 2520

ctgcactggc tccctcgccc ccagccccgg ggcctgagcc gtcccctgta agatgcggga 2580

agtgtcagct cccggcgtgg cgggcaggct caggggaggg gcgcgcatgg tccgccaggg 2640

ctgtgggccg tggcgcattt tccgactgtt tgtccagctc tcactgcctt ccttggttcc 2700

cggtccccca gcccatccgc catccccagc ccgtggtcag gtagagagtg agccccacgc 2760

cgccccaggg aggaggcgcc agagcgcggg gcagacgcaa agtgaaataa acactatttt 2820

gacggcaaaa aaaaaaaaaa agggcggccg ctctagagta tccctcgagg ggcccaag 2878

<210>2

<211>650

<212>PRT

<213> people

<400>2

Met Ala Thr Ser Asn Asn Pro Arg Lys Phe Ser Glu Lys Ile Ala Leu

1 5 10 15

His Asn Gln Lys Gln Ala Glu Glu Thr Ala Ala Phe Glu Glu Val Met

20 25 30

Lys Asp Leu Ser Leu Thr Arg Ala Ala Arg Leu Gln Leu Gln Lys Ser

35 40 45

Gln Tyr Leu Gln Leu Gly Pro Ser Arg Gly Gln Tyr Tyr Gly Gly Ser

50 55 60

Leu Pro Asn Val Asn Gln Ile Gly Ser Gly Thr Met Asp Leu Pro Phe

65 70 75 80

Gln Pro Ser Gly Phe Leu Gly Glu Ala Leu Ala Ala Ala Pro Val Ser

85 90 95

Leu Thr Pro Phe Gln Ser Ser Gly Leu Asp Thr Ser Arg Thr Thr Arg

100 105 110

His His Gly Leu Val Asp Arg Val Tyr Arg Glu Arg Gly Arg Leu Gly

115 120 125

Ser Pro His Arg Arg Pro Leu Ser Val Asp Lys His Gly Arg Gln Ala

130 135 140

Asp Ser Cys Pro Tyr Gly Thr Met Tyr Leu Ser Pro Pro Ala Asp Thr

145 150 155 160

Ser Trp Arg Arg Thr Asn Ser Asp Ser Ala Leu His Gln Ser Thr Met

165 170 175

Thr Pro Thr Gln Pro Glu Ser Phe Ser Ser Ser Gly Ser Gln Asp Val His

180 185 190

Gln Lys Arg Val Leu Leu Leu Thr Val Pro Gly Met Glu Glu Thr Thr

195 200 205

Ser Glu Ala Asp Lys Asn Leu Ser Lys Gln Ala Trp Asp Thr Lys Lys

210 215 220

Thr Gly Ser Arg Pro Lys Ser Cys Glu Val Pro Gly Ile Asn Ile Phe

225 230 235 240

Pro Ser Ala Asp Gln Glu Asn Thr Thr Ala Leu Ile Pro Ala Thr His

245 250 255

Asn Thr Gly Gly Ser Leu Pro Asp Leu Thr Asn Ile His Phe Pro Ser

260 265 270

Pro Leu Pro Thr Pro Leu Asp Pro Glu Glu Pro Thr Phe Pro Ala Leu

275 280 285

Ser Ser Ser Ser Ser Ser Thr Gly Asn Leu Ala Ala Asn Leu Thr His Leu

290 295 300

Gly Ile Gly Gly Ala Gly Gln Gly Met Ser Thr Pro Gly Ser Ser Pro

305 310 315 320

Gln His Arg Pro Ala Gly Val Ser Pro Leu Ser Leu Ser Thr Glu Ala

325 330 335

Arg Arg Gln Gln Ala Ser Pro Thr Leu Ser Pro Leu Ser Pro Ile Thr

340 345 350

Gln Ala Val Ala Met Asp Ala Leu Ser Leu Glu Gln Gln Leu Pro Tyr

355 360 365

Ala Phe Phe Thr Gln Ala Gly Ser Gln Gln Pro Pro Pro Pro Gln Pro Gln

370 375 380

Pro Pro Pro Pro Pro Pro Pro Pro Ala Ser Gln Gln Pro Pro Pro Pro Pro Pro

385 390 395 400

Pro Pro Gln Ala Pro Val Arg Leu Pro Pro Gly Gly Pro Leu Leu Pro

405 410 415

Ser Ala Ser Leu Thr Arg Gly Pro Gln Pro Pro Pro Leu Ala Val Thr

420 425 430

Val Pro Ser Ser Leu Pro Gln Ser Pro Pro Glu Asn Pro Gly Gln Pro

435 440 445

Ser Met Gly Ile Asp Ile Ala Ser Ala Pro Ala Leu Gln Gln Tyr Arg

450 455 460

Thr Ser Ala Gly Ser Pro Ala Asn Gln Ser Pro Thr Ser Pro Val Ser

465 470 475 480

Asn Gln Gly Phe Ser Pro Gly Ser Ser Pro Gln His Thr Ser Thr Leu

485 490 495

Gly Ser Val Phe Gly Asp Ala Tyr Tyr Glu Gln Gln Met Ala Ala Arg

500 505 510

Gln Ala Asn Ala Leu Ser His Gln Leu Glu Gln Phe Asn Met Met Glu

515 520 525

Asn Ala Ile Ser Ser Ser Ser Ser Leu Tyr Ser Pro Gly Ser Thr Leu Asn

530 535 540

Tyr Ser Gln Ala Ala Met Met Gly Leu Thr Gly Ser His Gly Ser Leu

545 550 555 560

Pro Asp Ser Gln Gln Leu Gly Tyr Ala Ser His Ser Gly Ile Pro Asn

565 570 575

Ile Ile Leu Thr Val Thr Gly Glu Ser Pro Pro Ser Leu Ser Lys Glu

580 585 590

Leu Thr Ser Ser Leu Ala Gly Val Gly Asp Val Ser Phe Asp Ser Asp

595 600 605

Ser Gln Phe Pro Leu Asp Glu Leu Lys Ile Asp Pro Leu Thr Leu Asp

610 615 620

Gly Leu His Met Leu Asn Asp Pro Asp Met Val Leu Ala Asp Pro Ala

625 630 635 640

Thr Glu Asp Thr Phe Arg Met Asp Arg Leu

645 650

<210>3

<211>32

<212>DNA

<213> Artificial sequence

<220>

<223> Primer

<400>3

gcccaagctt tgtgctctgc tgtctctgaa ag 32

<210>4

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> Primer

<400>4

gccctgaggg gatgggccat cag 23

<210>5

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> Primer

<400>5

cgcggatccg aagtgtgatg actcaggttt gccctg 36

<210>6

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> Primer

<400>6

cgcggatccg aagtgtgata tctcaggttt gccctg 36

<210>7

<211>54

<212>DNA

<213> Artificial sequence

<220>

<223> Primer

<400>7

gccctgaggg gatgggccat cagttgcaaa tcgttaactt tcctctgaca taat 54

<210>8

<211>39

<212>DNA

<213> Artificial sequence

<220>

<223> Primer

<400>8

gccctgaggg gatgggccat cagctacgag tcgtggaat 39

<210>9

<211>47

<212>DNA

<213> Artificial sequence

<220>

<223> Primer

<400>9

cgcggatccg aagtgtgatg actcaggttt gccctgaggg gatgggc 47

<210>10

<211>43

<212>DNA

<213> Artificial sequence

<220>

<223> Primer

<400>10

cagttgcaaa tcgtggaatt tcctctcgat caatgaaaag atg 43

<210>11

<211>39

<212>DNA

<213> Artificial sequence

<220>

<223> Primer

<400>11

gccctgaggg gatgggccat cagttgcaaa tcgtggaat 39

<210>12

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> Primer

<400>12

cgcctggtac cgagctctg 19

<210>13

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>13

acccaagatc tcgagcccg 19

<210>14

<211>99

<212>DNA

<213> Artificial sequence

<220>

<223> Primer

<400>14

cgcctggtac cgagctctga cataatgaca taatgacata atgacataat gacataatga 60

cataattacg cgtgctagcc cgggctcgag atcttgggt 99

<210>15

<211>2520

<212>DNA

<213> people

<220>

<221> Modified bases

<222>(0)...(0)

<221>misc feature

<222> 2, 13, 2431, 2453, 2465, 2468, 2469, 2479, 2488, 2489, 2492,

2505, 2512, 2514, 2519, 2520

<223>n=A, T, C or G

<400>15

antttttgta canaaaagca ggctgttacc ggtccggatt cccgggatct aggctggggc 60

cgggttcgcg gtgctcgctg aggcggcggt ggctacggct ggaggagccg ggccgaggcc 120

gcggcgggagg ccgcggctgg tactgggagg gtggcaggga gggacggggga aggaagatgg 180

cgacgtcggg ggcgaacggg cctggttcgg ccacggcctc ggcttccaat ccgcgcaaat 240

ttagtgagaa gattgcgctg cagaagcagc gtcaggccga ggagacggcg gccttcgagg 300

aggtgatgat ggacatcggc tccacccggt tacaggccca aaaactgcga ctggcataca 360

caaggagctc tcattatggt gggtctctgc ccaatgttaa ccagattggc tctggcctgg 420

ccgagttcca gagccccctc cactcacctt tggattcatc tcggagcact cggcaccatg 480

ggctggtgga acgggtgcag cgagatcctc gaagaatggt gtccccactt cgccgataca 540

cccgccacat tgacagctct ccctatagtc ctgcctactt atctcctccc ccagagtcta 600

gctggcgaag gacgatggcc tggggcaatt tccctgcaga gaaggggcag ttgtttcgac 660

taccatctgc acttaacagg acaagctctg actctgccct tcatacaagt gtgatgaacc 720

ccagtcccca ggatacctac ccaggcccca cacctcccag catcctgccc agccgacgtg 780

ggggtattct ggatggtgaa atggaccccca aagtacctgc tattgaggag aacttgctag 840

atgacaagca tttgctgaag ccatgggatg ctaagaagct atcctcatcc tcttcccgac 900

ctcggtcctg tgaagtccct ggaattaaca tctttccatc tcctgaccag cctgccaatg 960

tgcctgtcct cccacctgcc atgaacacgg ggggctccct acctgacctc accaacctgc 1020

actttccccc accactgccc accccctgg accctgaaga gacagcctac cctagcctga 1080

gtgggggcaa cagtacctcc aatttgaccc acaccatgac tcacctgggc atcagcaggg 1140

ggcatgggcc tgggcccggc tatgatgcac caggacttca ttcacctctc agccacccat 1200

ccctgcagtc ctccctaagc aatcccaacc tccaggcttc cctgagcagt cctcagcccc 1260

agcttcaggg ctcccacagc cacccctctc tgcctgcctc ctccttggcc tgccatgtac 1320

tgcccaccac ctccctgggc cacccctcac tcagtgctcc ggctctctcc tcctcctctt 1380

cctcctcctc cacttcatct cctgttttgg gcgccccctc ttaccctgct tctacccctg 1440

gggcctcccc ccaccaccgc cgtgtgcccc tcagccccct gagtttgctc gcgggcccag 1500

ccgacgccag aaggtcccaa cagcagctgc ccaaacagtt ttcgccaaca atgtcaccca 1560

ccttgtcttc catcactcag ggcgtccccc tggataccag taaactgtcc actgaccagc 1620

ggttacccccc ctacccatac agctccccaa gtctggttct gcctacccag ccccacaccc 1680

caaagtctct acagcagcca gggctgccct ctcagtcttg ttcagtgcag tcctcaggtg 1740

ggcagccccc aggcaggcag tctcattatg ggacaccgta cccacctggg cccagtgggc 1800

atgggcaaca gtcttaccac cggccaatga gtgacttcaa cctggggaat ctggagcagt 1860

tcagcatgga gagcccatca gccagcctgg tgctggatcc ccctggcttt tctgaagggc 1920

ctggattttt agggggtgag gggccaatgg gtggccccca ggatccccac accttcaacc 1980

accagaactt gacccactgt tcccgccatg gctcagggcc taacatcatc ctcacagggg 2040

actcctctcc aggtttctct aaggagattg cagcagccct ggccggagtg cctggctttg 2100

aggtgtcagc agctggattg gagctagggc ttgggctaga agatgagctg cgcatggagc 2160

cactgggcct ggaagggcta aacatgctga gtgacccctg tgccctgctg cctgatcctg 2220

ctgtggagga gtcattccgc agtgaccggc tccaatgagg gcacctcatc accatccctc 2280

ttcttggccc catcccccac caccatcct ttcctccctt ccccctggca ggtagagact 2340

ctactctctg tccccagatc ctctttctag catgaatgaa ggatgccaag aatgagaaaa 2400

agcaaggggt ttgtccaggt ggcccctgaa ntctgcgcaa gggatggggcc tgggggaac 2460

ctcanggnna gggcccaang gccacttnna anctttgaac cgtcngtctg gnagggtcnn 2520

<210>16

<211>693

<212>PRT

<213> people

<400>16

Met Ala Thr Ser Gly Ala Asn Gly Pro Gly Ser Ala Thr Ala Ser Ala

1 5 10 15

Ser Asn Pro Arg Lys Phe Ser Glu Lys Ile Ala Leu Gln Lys Gln Arg

20 25 30

Gln Ala Glu Glu Thr Ala Ala Phe Glu Glu Val Met Met Asp Ile Gly

35 40 45

Ser Thr Arg Leu Gln Ala Gln Lys Leu Arg Leu Ala Tyr Thr Arg Ser

50 55 60

Ser His Tyr Gly Gly Ser Leu Pro Asn Val Asn Gln Ile Gly Ser Gly

65 70 75 80

Leu Ala Glu Phe Gln Ser Pro Leu His Ser Pro Leu Asp Ser Ser Arg

85 90 95

Ser Thr Arg His His Gly Leu Val Glu Arg Val Gln Arg Asp Pro Arg

100 105 110

Arg Met Val Ser Pro Leu Arg Arg Tyr Thr Arg His Ile Asp Ser Ser

115 120 125

Pro Tyr Ser Pro Ala Tyr Leu Ser Pro Pro Pro Glu Ser Ser Trp Arg

130 135 140

Arg Thr Met Ala Trp Gly Asn Phe Pro Ala Glu Lys Gly Gln Leu Phe

145 150 155 160

Arg Leu Pro Ser Ala Leu Asn Arg Thr Ser Ser Asp Ser Ala Leu His

165 170 175

Thr Ser Val Met Asn Pro Ser Pro Gln Asp Thr Tyr Pro Gly Pro Thr

180 185 190

Pro Pro Ser Ile Leu Pro Ser Arg Arg Gly Gly Ile Leu Asp Gly Glu

195 200 205

Met Asp Pro Lys Val Pro Ala Ile Glu Glu Asn Leu Leu Asp Asp Lys

210 215 220

His Leu Leu Lys Pro Trp Asp Ala Lys Lys Leu Ser Ser Ser Ser Ser

225 230 235 240

Arg Pro Arg Ser Cys Glu Val Pro Gly Ile Asn Ile Phe Pro Ser Pro

245 250 255

Asp Gln Pro Ala Asn Val Pro Val Leu Pro Pro Ala Met Asn Thr Gly

260 265 270

Gly Ser Leu Pro Asp Leu Thr Asn Leu His Phe Pro Pro Pro Leu Pro

275 280 285

Thr Pro Leu Asp Pro Glu Glu Thr Ala Tyr Pro Ser Leu Ser Gly Gly

290 295 300

Asn Ser Thr Ser Asn Leu Thr His Thr Met Thr His Leu Gly Ile Ser

305 310 315 320

Arg Gly His Gly Pro Gly Pro Gly Tyr Asp Ala Pro Gly Leu His Ser

325 330 335

Pro Leu Ser His Pro Ser Leu Gln Ser Ser Leu Ser Asn Pro Asn Leu

340 345 350

Gln Ala Ser Leu Ser Ser Pro Gln Pro Gln Leu Gln Gly Ser His Ser

355 360 365

His Pro Ser Leu Pro Ala Ser Ser Leu Ala Cys His Val Leu Pro Thr

370 375 380

Thr Ser Leu Gly His Pro Ser Leu Ser Ala Pro Ala Leu Ser Ser Ser Ser

385 390 395 400

Ser Ser Ser Ser Ser Ser Thr Ser Ser Ser Pro Val Leu Gly Ala Pro Ser Tyr

405 410 415

Pro Ala Ser Thr Pro Gly Ala Ser Pro His His Arg Arg Val Pro Leu

420 425 430

Ser Pro Leu Ser Leu Leu Ala Gly Pro Ala Asp Ala Arg Arg Ser Gln

435 440 445

Gln Gln Leu Pro Lys Gln Phe Ser Pro Thr Mer Ser Pro Thr Leu Ser

450 455 460

Ser Ile Thr Gln Gly Val Pro Leu Asp Thr Ser Lys Leu Ser Thr Asp

465 470 475 480

Gln Arg Leu Pro Pro Tyr Pro Tyr Ser Ser Pro Ser Leu Val Leu Pro

485 490 495

Thr Gln Pro His Thr Pro Lys Ser Leu Gln Gln Pro Gly Leu Pro Ser

500 505 510

Gln Ser Cys Ser Val Gln Ser Ser Gly Gly Gln Pro Pro Gly Arg Gln

515 520 525

Ser His Tyr Gly Thr Pro Tyr Pro Pro Gly Pro Ser Gly His Gly Gln

530 535 540

Gln Ser Tyr His Arg Pro Met Ser Asp Phe Asn Leu Gly Asn Leu Glu

545 550 555 560

Gln Phe Ser Met Glu Ser Pro Ser Ala Ser Leu Val Leu Asp Pro Pro

565 570 575

Gly Phe Ser Glu Gly Pro Gly Phe Leu Gly Gly Glu Gly Pro Met Gly

580 585 590

Gly Pro Gln Asp Pro His Thr Phe Asn His Gln Asn Leu Thr His Cys

595 600 605

Ser Arg His Gly Ser Gly Pro Asn Ile Ile Leu Thr Gly Asp Ser Ser

610 615 620

Pro Gly Phe Ser Lys Glu Ile Ala Ala Ala Leu Ala Gly Val Pro Gly

625 630 635 640

Phe Glu Val Ser Ala Ala Gly Leu Glu Leu Gly Leu Gly Leu Glu Asp

645 650 655

Glu Leu Arg Met Glu Pro Leu Gly Leu Glu Gly Leu Asn Met Leu Ser

660 665 670

Asp Pro Cys Ala Leu Leu Pro Asp Pro Ala Val Glu Glu Ser Phe Arg

675 680 685

Ser Asp Arg Leu Gln

690

<210>17

<211>17

<212>DNA

<213> people

<400>17

ccgtcatttc accaagc 17

<210>18

<211>7

<212>PRT

<213> people

<400>18

Glu Glu Thr Arg Ala Phe Glu

1 5

<210>19

<211>7

<212>PRT

<213> unknown

<220>

<223> predicted protein

<400>19

Glu Glu Thr Ala Ala Phe Glu

1 5

<210>20

<211>34

<212>DNA

<213> Artificial sequence

<220>

<223> Primer

<400>20

ccggaattcg ccatggccgc ctcgccgggc tcgg 34

<210>2l

<211>44

<212>DNA

<213> Artificial sequence

<220>

<223> Primer

<400>21

ccgcgacagg gtgaggtcgg tcatgagctg ctcgaaggcc cgcg 44

<210>22

<211>44

<212>DNA

<213> Artificial sequence

<220>

<223> Primer

<400>22

gaagcttctg aaattgaacc cgcgacaggg tgaggtcggt catg 44

<210>23

<211>63

<212>DNA

<213> Artificial sequence

<220>

<223> Primer

<400>23

tggtaaggat cctccatggt actgtgtaag gcgcagttgc tgaagcttc tgaaattgaac 60

ccg 63

<210>24

<211>2259

<212>DNA

<213> people

<220>

<221>misc_feature

<222>1,13

<223>n=A, T, C or G

<400>24

nttttttgta canaaaagca ggctgttacc ggtccggaat tcgccatggc cgcctcgccg 60

ggctcgggca gcgccaaccc gcggaagttc agtgagaaga tcgcgctgca cacgcagaga 120

caggccgagg agacgcgggc cttcgagcag ctcatgaccg acctcaccct gtcgcgggtt 180

caatttcaga agcttcagca actgcgcctt acacagtacc atggaggatc cttaccaaat 240

gtgagccagc tgcggagcaa tgcgtcagag tttcagccgt catttcacca agctgataat 300

gttcggggaa cccgccatca cgggctggtg gagaggccat ccaggaaccg cttccacccc 360

ctccaccgaa ggtctgggga caagccaggg cgacaatttg atggtagtgc ttttggagcc 420

aattattcct cacagcctct ggatgagagt tggccaaggc agcagcctcc ttggaaagac 480

gaaaagcatc ctgggttcag gctgacatct gcacttaaca ggaccaattc tgattctgct 540

cttcacacga gtgctctgag taccaagccc caggacccct atggaggagg gggccagtcg 600

gcctggcctg ccccatacat ggggttttgt gatggtgaga ataatggaca tggggaagta 660

gcatctttcc ctggcccatt gaaagaagag aatctgttaa atgttcctaa gccactgcca 720

aaacaactgt gggagaccaa ggagattcag tccctgtcag gacgccctcg atcctgtgat 780

gttggaggtg gcaatgcttt tccacataat ggtcaaaacc taggcctctc acccttcttg 840

gggactttga acactggagg gtcattgcca gatctaacca acctccacta ctcgacaccc 900

ctgccagcct ccctggacac caccgaccac cactttggca gtatgagtgt ggggaatagt 960

gtgaacaaca tcccagctgc tatgacccac ctgggtataa gaagctcctc tggtctccag 1020

agttctcgga gtaacccctc catccaagcc acgctcaata agactgtgct ttcctcttcc 1080

ttaaataacc accccacagac atctgttccc aacgcatctg ctcttcaccc ttcgctccgt 1140

ctgttttccc ttagcaaccc atctctttcc accacaaacc tgagcggccc gtctcgccgt 1200

cggcagcctc ccgtcagccc tctcacgctt tctcctggcc ctgaagcaca tcaaggtttc 1260

agcagacagc tgtcttcaac cagcccactg gccccatatc ctacctccca gatggtgtcc 1320

tcagaccgaa gccaactttc ctttctgccc acagaagctc aagcccaggt gtcgccgcca 1380

ccccccttacc ctgcacccca ggagctcacc cagcccctcc tgcagcagcc ccgcgcccct 1440

gaggcccctg cccagcagcc ccaggcagcc tcctcactgc cacagtcaga ctttcagctt 1500

ctcccggccc agggctcatc tttgaccaac ttcttcccag atgtgggttt tgaccagcag 1560

tccatgaggc caggccctgc ctttcctcaa caggtgcctc tggtgcaaca aggttcccga 1620

gaactgcagg actcttttca tttgagacca agcccgtatt ccaactgcgg gagtctcccg 1680

aacaccatcc tgccagaaga ctccagcacc agcctgttca aagacctcaa cagtgcgctg 1740

gcaggcctgc ctgaggtcag cctgaacgtg gacactccat ttccactgga agaggagctg 1800

cagattgaac ccctgagcct ggatggactc aacatgttaa gtgactccag catgggcctg 1860

ctggacccct ctgttgaaga gacgtttcga gctgacagac tgtgaacaga aggcagtgga 1920

acagaagaat gtttttctgc aacagccaaa atagaatgga atagaatgaa gccagctgat 1980

accacgggct ttcgttatct tgacatagaa ggaagcagtg ccacggctcc agggtttcag 2040

atgagatccc atctcagaca ctgtggcttc ctccagatca cacagctttg tactgcctct 2100

cccgcctgtg gccaaagtcg tgttgcagca ggcaggctgc ttggagcttc ccatgaactg 2160

gaaagctcac ctccactgca tctttttact ggccatccag tcagccgatg tgtaagagta 2220

ggaaatactg tgtcactgga ggccctccgt agcattggg 2259

<210>25

<211>619

<212>PRT

<213> people

<400>25

Met Ala Ala Ser Pro Gly Ser Gly Ser Ala Asn Pro Arg Lys Phe Ser

1 5 10 15

Glu Lys Ile Ala Leu His Thr Gln Arg Gln Ala Glu Glu Thr Arg Ala

20 25 30

Phe Glu Gln Leu Met Thr Asp Leu Thr Leu Ser Arg Val Gln Phe Gln

35 40 45

Lys Leu Gln Gln Leu Arg Leu Thr Gln Tyr His Gly Gly Ser Leu Pro

50 55 60

Asn Val Ser Gln Leu Arg Ser Asn Ala Ser Glu Phe Gln Pro Ser Phe

65 70 75 80

His Gln Ala Asp Asn Val Arg Gly Thr Arg His His Gly Leu Val Glu

85 90 95

Arg Pro Ser Arg Asn Arg Phe His Pro Leu His Arg Arg Ser Gly Asp

100 105 110

Lys Pro Gly Arg Gln Phe Asp Gly Ser Ala Phe Gly Ala Asn Tyr Ser

115 120 125

Ser Gln Pro Leu Asp Glu Ser Trp Pro Arg Gln Gln Pro Pro Trp Lys

130 135 140

Asp Glu Lys His Pro Gly Phe Arg Leu Thr Ser Ala Leu Asn Arg Thr

145 150 155 160

Asn Ser Asp Ser Ala Leu His Thr Ser Ala Leu Ser Thr Lys Pro Gln

165 170 175

Asp Pro Tyr Gly Gly Gly Gly Gln Ser Ala Trp Pro Ala Pro Tyr Met

180 185 190

Gly Phe Cys Asp Gly Glu Asn Asn Gly His Gly Glu Val Ala Ser Phe

195 200 205

Pro Gly Pro Leu Lys Glu Glu Asn Leu Leu Asn Val Pro Lys Pro Leu

210 215 220

Pro Lys Gln Leu Trp Glu Thr Lys Glu Ile Gln Ser Leu Ser Gly Arg

225 230 235 240

Pro Arg Ser Cys Asp Val Gly Gly Gly Asn Ala Phe Pro His Asn Gly

245 250 255

Gln Asn Leu Gly Leu Ser Pro Phe Leu Gly Thr Leu Asn Thr Gly Gly

260 265 270

Ser Leu Pro Asp Leu Thr Asn Leu His Tyr Ser Thr Pro Leu Pro Ala

275 280 285

Ser Leu Asp Thr Thr Asp His His Phe Gly Ser Met Ser Val Gly Asn

290 295 300

Ser Val Asn Asn Ile Pro Ala Ala Met Thr His Leu Gly Ile Arg Ser

305 310 315 320

Ser Ser Gly Leu Gln Ser Ser Arg Ser Asn Pro Ser Ile Gln Ala Thr

325 330 335

Leu Asn Lys Thr Val Leu Ser Ser Ser Leu Asn Asn His Pro Gln Thr

340 345 350

Ser Val Pro Asn Ala Ser Ala Leu His Pro Ser Leu Arg Leu Phe Ser

355 360 365

Leu Ser Asn Pro Ser Leu Ser Thr Thr Asn Leu Ser Gly Pro Ser Arg

370 375 380

Arg Arg Gln Pro Pro Val Ser Pro Leu Thr Leu Ser Pro Gly Pro Glu

385 390 395 400

Ala His Gln Gly Phe Ser Arg Gln Leu Ser Ser Thr Ser Pro Leu Ala

405 410 415

Pro Tyr Pro Thr Ser Gln Met Val Ser Ser Asp Arg Ser Gln Leu Ser

420 425 430

Phe Leu Pro Thr Glu Ala Gln Ala Gln Val Ser Pro Pro Pro Pro Tyr

435 440 445

Pro Ala Pro Gln Glu Leu Thr Gln Pro Leu Leu Gln Gln Pro Arg Ala

450 455 460

Pro Glu Ala Pro Ala Gln Gln Pro Gln Ala Ala Ser Ser Leu Pro Gln

465 470 475 480

Ser Asp Phe Gln Leu Leu Pro Ala Gln Gly Ser Ser Leu Thr Asn Phe

485 490 495

Phe Pro Asp Val Gly Phe Asp Gln Gln Ser Met Arg Pro Gly Pro Ala

500 505 510

Phe Pro Gln Gln Val Pro Leu Val Gln Gln Gly Ser Arg Glu Leu Gln

515 520 525

Asp Ser Phe His Leu Arg Pro Ser Pro Tyr Ser Asn Cys Gly Ser Leu

530 535 540

Pro Asn Thr Ile Leu Pro Glu Asp Ser Ser Thr Ser Leu Phe Lys Asp

545 550 555 560

Leu Asn Ser Ala Leu Ala Gly Leu Pro Glu Val Ser Leu Asn Val Asp

565 570 575

Thr Pro Phe Pro Leu Glu Glu Glu Leu Gln Ile Glu Pro Leu Ser Leu

580 585 590

Asp Gly Leu Asn Met Leu Ser Asp Ser Ser Met Gly Leu Leu Asp Pro

595 600 605

Ser Val Glu Glu Thr Phe Arg Ala Asp Arg Leu

610 615

<210>26

<211>2992

<212>DNA

<213> Drosophila melanogaster

<400>26

atggccaatc cgcgcaagtt cagcgagaag atcgctctgc agaagcagaa gcaggcggag 60

ggcacagcgg aattcgagcg gatcatgaag gaggtgtatg ccacgaagag ggatgagccg 120

cctgcgaatc agaagatcct agacggcctt gtcggcggtc aggaggtaag ccaatcctcg 180

ccaggcgcag gcaatgggac gggcggaggt ggcagtggtt ccggcagtgg agccagcggc 240

ggaggagcct caccagatgg cctgggaggc ggcggtggtt ctccgacggc ttatcgagaa 300

tcccgagggc gcagcgtagg tgtgggtccc atgcgaagac cgtcggagcg caagcaggat 360

cgttcgccct acggcagcag cagtacgcaa caaaccttag acaacggcca gctaaatccg 420

catcttcttg gtccacctac ggcggagagt ttgtggcggc ggtccagctc cgattcggcg 480

ctgcaccaaa gtgcgctggt ggcgggcttc aatagcgacg tgaactcgat gggcgccaac 540

tatcagcagc agcaacatca gcaacaacag caaccgggcc agccaagatc tcactcgccg 600

caccatggta taaacaggac catgagtccg caggcgcaac ggaggaagtc gccgctactg 660

cagccccatc agctgcagtt gcagcaactg caacagcagc agcaacagat gcaacatcag 720

catcagctgc accagcagct ccaaatgcag cagctgcaac agcaccagca gcaacaccag 780

cagcagcagc aacaacagaa cacgccatac aacaacgcca aattcacgaa tcctgtgttc 840

cggccgctgc aggatcaggt caactttgcc aacaccggct ccctgcccga tctcacggcc 900

cttcaaaact atggaccccca gcagcagcag cagcaatccc agcaacagcc gtcgcagcaa 960

caacagcagt tgcagcaaac cctgtcgcca gtcatgtctc cgcacaatca ccgccgcgaa 1020

cgggatcagt cgcccagtcc gtttagtccg gcgggtggag gaggggggagc aggtcccggg 1080

tcgccctatc agcagcaaca gcactcgccc accggaaaca cgcaacagca gcagcagcag 1140

caccaacagc ccagcaactc gccgcacctg tcctttacca atctggccac cacgcaggca 1200

gctgttacca catttaaccc gctccccacg ctgggtccgc acaatgccac cgactaccgc 1260

cagccaccga atcctcctag tccacgctct tcgcccggct tgctgagcag cgtatcggcc 1320

acggatctgc actccagtgc accggccagt cccatacgcc agcagcaaca ggcccatcag 1380

cagcaacagc agcagcaaca ggcgcagcaa caacagcaac agtttgataa ctcctacaac 1440

agtctgaata cctcgtttca caatcagttt gagattttct cgctgggcga cagcaattcc 1500

tcgccggaac agcagggctt tgcaaataat ttcgtggccc tcgactttga cgacctgagt 1560

ggcggcggag gtggtggccc aagcgggggc ggcggcagca atggaggagg tctgaccaac 1620

ggttacaaca agccggagat gttggacttc agcgagctga gcggcagccc ggaggcgagt 1680

gggaacaaca accacatgcg gcgaggagtg agcaacctga acaacaacgg gttgagcaat 1740

ggtgtggtgg gatccacgca caacggcagc acaaatctaa atggagcggg aaacaacaat 1800

agcagtagtg gaggtggcac ggcgcaggat cctttgggaa taaccacttc gcctgtgccc 1860

tcacccttgg gctgccccag ttcaccgctg ccgataccga ttccgatgtc ggcgcaaagc 1920

tcgccacagc agcagcacca ccatcatcag cagcagcaac aacagcatca tcagcagcaa 1980

caccatcagc agcagcaatt atcattatct ctgcaccat cgccgcatca ttcgccaatg 2040

cattcgccgc accatgggaa ttcaccgctt tcaagcagct cgccagtgag tcacaatgcc 2100

tgctccaact ccaacgtggt gatgaaccac cagcagcagc agcaacaaca tcaccaccag 2160

caacaccatc atcagggctc ctcgcaaagt cacacgccga ccacagcgaa tataccctct 2220

attatcttta gtgattactc ctccaacgcg gattatacca gggagatctt cgactccctc 2280

gatctggatc tgggacagat ggacgtagcc ggtttgcaga tgctgtccga ccagaaccccc 2340

atcatgatcg ccgatcccaa catcgaggat agttttcgac gcgacctcaa ctgatactat 2400

gaggaggctg ttgcggccat tgagagcgga gtgctgctgg aggaggacta ccaggcgctg 2460

ctcggatcag aggcgctggc ggatgaacag gtggtcacag tcgaggccgc cggagccgca 2520

gcagcagtag taacagttga agaggcagcc acagttagcg agaaggacaa aaaagatttg 2580

gaagttgtgg aacttctggt gtccggtgtt atggatgacc tggtggactc cagtgacctg 2640

gacgaggaag tgcgcaattt ctttttttag gcagccagca agtcattttt gtcgttaaca 2700

caactgatgg aattttcgtt tttaacacag atgaggaagt gaattacgtt ttttaaacgc 2760

attcacttgc catttctcga ttaaatgcca tattacttaa gctcaggatt tacaagctta 2820

atgcgaatta agttaatttc ggaaatgctg acgagagtga ttgcaaagtt caaaattgat 2880

acaaattcac ttccgcaaat tcatgctgaa actgaaagtt ttctaacagt cctcaatatt 2940

gttatctcgt tatcgtccgt gctttcgtag ctagctccta caacaaaaat ac 2992

<210>27

<211>797

<212>PRT

<213> Drosophila melanogaster

<400>27

Met Ala Asn Pro Arg Lys Phe Ser Glu Lys Ile Ala Leu Gln Lys Gln

1 5 10 15

Lys Gln Ala Glu Gly Thr Ala Glu Phe Glu Arg Ile Met Lys Glu Val

20 25 30

Tyr Ala Thr Lys Arg Asp Glu Pro Pro Ala Asn Gln Lys Ile Leu Asp

35 40 45

Gly Leu Val Gly Gly Gly Gln Glu Val Ser Gln Ser Ser Pro Gly Ala Gly

50 55 60

Asn Gly Thr Gly Gly Gly Gly Ser Gly Ser Gly Ser Gly Ala Ser Gly

65 70 75 80

Gly Gly Ala Ser Pro Asp Gly Leu Gly Gly Gly Gly Gly Ser Pro Thr

85 90 95

Ala Tyr Arg Glu Ser Arg Gly Arg Ser Val Gly Val Gly Pro Met Arg

100 105 110

Arg Pro Ser Glu Arg Lys Gln Asp Arg Ser Pro Tyr Gly Ser Ser Ser

115 120 125

Thr Gln Gln Thr Leu Asp Asn Gly Gln Leu Asn Pro His Leu Leu Gly

130 135 140

Pro Pro Thr Ala Glu Ser Leu Trp Arg Arg Ser Ser Ser Asp Ser Ala

145 150 155 160

Leu His Gln Ser Ala Leu Val Ala Gly Phe Asn Ser Asp Val Asn Ser

165 170 175

Met Gly Ala Asn Tyr Gln Gln Gln Gln His Gln Gln Gln Gln Gln Pro

180 185 190

Gly Gln Pro Arg Ser His Ser Pro His His Gly Ile Asn Arg Thr Met

195 200 205

Ser Pro Gln Ala Gln Arg Arg Lys Ser Pro Leu Leu Gln Pro His Gln

210 215 220

Leu Gln Leu Gln Gln Leu Gln Gln Gln Gln Gln Gln Gln Met Gln His Gln

225 230 235 240

His Gln Leu His Gln Gln Leu Gln Met Gln Gln Leu Gln Gln His Gln

245 250 255

Gln Gln His Gln Gln Gln Gln Gln Gln Gln Asn Thr Pro Tyr Asn Asn

260 265 270

Ala Lys Phe Thr Asn Pro Val Phe Arg Pro Leu Gln Asp Gln Val Asn

275 280 285

Phe Ala Asn Thr Gly Ser Leu Pro Asp Leu Thr Ala Leu Gln Asn Tyr

290 295 300

Gly Pro Gln Gln Gln Gln Gln Gln Ser Gln Gln Gln Pro Ser Gln Gln

305 310 315 320

Gln Gln Gln Leu Gln Gln Thr Leu Ser Pro Val Met Ser Pro His Asn

325 330 335

His Arg Arg Glu Arg Asp Gln Ser Pro Ser Pro Phe Ser Pro Ala Gly

340 345 350

Gly Gly Gly Gly Ala Gly Pro Gly Ser Pro Tyr Gln Gln Gln Gln His

355 360 365

Ser Pro Thr Gly Asn Thr Gln Gln Gln Gln Gln Gln Gln His Gln Gln Pro

370 375 380

Ser Asn Ser Pro His Leu Ser Phe Thr Asn Leu Ala Thr Thr Gln Ala

385 390 395 400

Ala Val Thr Thr Phe Asn Pro Leu Pro Thr Leu Gly Pro His Asn Ala

405 410 415

Thr Asp Tyr Arg Gln Pro Pro Asn Pro Pro Ser Pro Arg Ser Ser Pro

420 425 430

Gly Leu Leu Ser Ser Val Ser Ala Thr Asp Leu His Ser Ser Ala Pro

435 440 445

Ala Ser Pro Ile Arg Gln Gln Gln Gln Ala His Gln Gln Gln Gln Gln

450 455 460

Gln Gln Gln Ala Gln Gln Gln Gln Gln Gln Phe Asp Asn Ser Tyr Asn

465 470 475 480

Ser Leu Asn Thr Ser Phe His Asn Gln Phe Glu Ile Phe Ser Leu Gly

485 490 495

Asp Ser Asn Ser Ser Pro Glu Gln Gln Gly Phe Ala Asn Asn Phe Val

500 505 510

Ala Leu Asp Phe Asp Asp Leu Ser Gly Gly Gly Gly Gly Gly Pro Ser

515 520 525

Gly Gly Gly Gly Ser Asn Gly Gly Gly Leu Thr Asn Gly Tyr Asn Lys

530 535 540

Pro Glu Met Leu Asp Phe Ser Glu Leu Ser Gly Ser Pro Glu Ala Ser

545 550 555 560

Gly Asn Asn Asn His Met Arg Arg Gly Val Ser Asn Leu Asn Asn Asn

565 570 575

Gly Leu Ser Asn Gly Val Val Gly Ser Thr His Asn Gly Ser Thr Asn

580 585 590

Leu Asn Gly Ala Gly Asn Asn Asn Ser Ser Ser Gly Gly Gly Thr Ala

595 600 605

Gln Asp Pro Leu Gly Ile Thr Thr Ser Pro Val Pro Ser Pro Leu Gly

610 615 620

Cys Pro Ser Ser Pro Leu Pro Ile Pro Ile Pro Met Ser Ala Gln Ser

625 630 635 640

Ser Pro Gln Gln Gln His His His His Gln Gln Gln Gln Gln Gln His

645 650 655

His Gln Gln Gln His His His Gln Gln Gln Gln Gln Leu Ser Leu Ser Leu His

660 665 670

His Ser Pro His His Ser Pro Met His Ser Pro His His Gly Asn Ser

675 680 685

Pro Leu Ser Ser Ser Ser Pro Val Ser His Asn Ala Cys Ser Asn Ser

690 695 700

Asn Val Val Met Asn His Gln Gln Gln Gln Gln Gln His His His Gln

705 710 715 720

Gln His His His Gln Gly Ser Ser Gln Ser His Thr Pro Thr Thr Ala

725 730 735

Asn Ile Pro Ser Ile Ile Phe Ser Asp Tyr Ser Ser Asn Ala Asp Tyr

740 745 750

Thr Arg Glu Ile Phe Asp Ser Leu Asp Leu Asp Leu Gly Gln Met Asp

755 760 765

Val Ala Gly Leu Gln Met Leu Ser Asp Gln Asn Pro Ile Met Ile Ala

770 775 780

Asp Pro Asn Ile Glu Asp Ser Phe Arg Arg Asp Leu Asn

785 790 795

<210>28

<211>2416

<212>DNA

<213> mouse

<220>

<221>misc feature

<222>1528

<223>n = A, T, C or G

<400>28

gggacgaaga gtaggagtag gaggaggcgg cgagaagatg gcgacttcga acaatccgcg 60

gaaatttagc gagaagatcg cactgcacaa ccagaagcag gcggaggaga cggcggcctt 120

cgaggaggtc atgaaggacc tgagcctgac gcgggccgcg cggcttcagc tgcagaagtc 180

ccagtacctg cagctgggcc ccagccgtgg ccagtactac ggtgggtccc tgcccaacgt 240

gaaccagatt ggaagcagca gcgtggacct ggccttccag accccatttc agtcctcagg 300

cctggacacg agtcggacca cacgacatca tgggcttgtg gacagagtat atcgtgagcg 360

tggcagactt ggctccccgc accgtcgacc cctgtcagta gacaagcatg ggcgacaggc 420

tgacagctgc ccctatggca ccgtgtacct ctcgcctcct gcggaccacca gctggaggag 480

gaccaactct gactctgccc tgcaccagag cacaatgaca cccagccagg cagagtcctt 540

cacaggcggg tcccaggatg cgcaccagaa gagagtctta ctgctaactg tcccaggaat 600

ggaggacacc ggggctgaga cagacaagac cctttctaag cagtcatggg actcaaagaa 660

ggcgggttcc aggcccaagt cctgtgaggt ccccggaatc aacatctttc cgtctgcaga 720

ccaggagaac acaacagccc tgatccctgc cacccacaac acaggggggct cccttcctga 780

cctcaccaac atccacttcg cctccccact cccgacacca ctggaccctg aggagcctcc 840

gttccctgct ctcaccagct ccagcagcac cggcagcctt gcacatctgg gcgttggcgg 900

cgcaggcggt atgaacaccc ccagctcttc tccacagcac cggccagcag tcgtcagccc 960

cctgtccctg agcacagagg ccaggcggca gcaggcccag caggtgtcac ccaccctgtc 1020

tccgttgtca cccatcactc aggccgtggc tatggatgcc ctgtccttgg agcagcagct 1080

gccctatgcc ttcttcaccc agactggctc ccagcagcct cccccacagc cccagccacc 1140

gcctccacct ccaccggtat cccagcagca gccaccacct ccacagggtgt ctgtggggcct 1200

cccccagggt ggtccactgc tgcccagtgc cagcctgact cgggggcccc agctgccacc 1260

actctcagtt actgtaccat ccactcttcc ccagtcccct acagagaacc caggccagtc 1320

accaatgggg atcgatgcca cttcggcacc agctctgcag taccgcacga gtgcagggtc 1380

acctgccacc cagtctccca cctctccggt ctccaaccaa ggcttctccc ctggaagctc 1440

cccacagcac acgtccaccc tgggcagcgt gtttggggat gcgtactatg agcagcagat 1500

gacagccagg caggccaatg ctctgtcncg ccagctggag cagttcaaca tgatggagaa 1560

cgccatcagc tccagcagcc tatacaaccc gggctccaca ctcaactatt cccaggctgc 1620

catgatgggt ctgagcggga gccacggggg cctacaggac ccgcagcagc tcggctacac 1680

aggccacggt ggaatcccca acatcatcct cacggtgaca ggagagtcac caccgagcct 1740

ctctaaggaa ctgagcagca cactggcagg agtcagtgat gtcagctttg attcggacca 1800

tcagtttcca ctggacgagc tgaagattga ccctctgacc ctggacggac tccatatgtt 1860

gaatgaccca gacatggttt tagccgaccc agccaccgag gaacaccttcc gaatggaccg 1920

cctgtgagtg gctgtgccca ccagccgccg ctggtcagtc tccaacggcg ctgccccaaa 1980

cctggggacg gcaatggcgt ccccctttgc caacggccaa gcttgtggtt ctgagcttgc 2040

aatgctgccc agtgcccctg ccagcccccc gccaccccgg tcgttcacct cccatgatgc 2100

ctggcgtgcg tgaggccgct gtgtactagg ctggctatct gtctgtccat ccatctacct 2160

gggtcaggc tgatggccga ggctgtgagt gcctggcccc catggatgtt ccccgtgctc 2220

gctccctcac ccctcactgg ggatgtgaga gccctcatca gatacccaaa gtgtcactca 2280

cttccagcat gtgctgtgca acggagggcc ggggcgtggg tgtggagcgc ccagaggctt 2340

aggtgcgcca tccattcgac tgttgtcagc tgtcactgcc ttcctccatc ctgtcccccg 2400

tcccaccgcc atccct 2416

<210>29

<211>629

<212>PRT

<213> mouse

<400>29

Met Ala Thr Ser Asn Asn Pro Arg Lys Phe Ser Glu Lys Ile Ala Leu

1 5 10 15

His Asn Gln Lys Gln Ala Glu Glu Thr Ala Ala Phe Glu Glu Val Met

20 25 30

Lys Asp Leu Ser Leu Thr Arg Ala Ala Arg Leu Gln Leu Gln Lys Ser

35 40 45

Gln Tyr Leu Gln Leu Gly Pro Ser Arg Gly Gln Tyr Tyr Gly Gly Ser

50 55 60

Leu Pro Asn Val Asn Gln Ile Gly Ser Ser Ser Val Asp Leu Ala Phe

65 70 75 80

Gln Thr Pro Phe Gln Ser Ser Gly Leu Asp Thr Ser Arg Thr Thr Arg

85 90 95

His His Gly Leu Val Asp Arg Val Tyr Arg Glu Arg Gly Arg Leu Gly

100 105 110

Ser Pro His Arg Arg Pro Leu Ser Val Asp Lys His Gly Arg Gln Ala

115 120 125

Asp Ser Cys Pro Tyr Gly Thr Val Tyr Leu Ser Pro Pro Ala Asp Thr

130 135 140

Ser Trp Arg Arg Thr Asn Ser Asp Ser Ala Leu His Gln Ser Thr Met

145 150 155 160

Thr Pro Ser Gln Ala Glu Ser Phe Thr Gly Gly Ser Gln Asp Ala His

165 170 175

Gln Lys Arg Val Leu Leu Leu Thr Val Pro Gly Met Glu Asp Thr Gly

180 185 190

Ala Glu Thr Asp Lys Thr Leu Ser Lys Gln Ser Trp Asp Ser Lys Lys

195 200 205

Ala Gly Ser Arg Pro Lys Ser Cys Glu Val Pro Gly Ile Asn Ile Phe

210 215 220

Pro Ser Ala Asp Gln Glu Asn Thr Thr Ala Leu Ile Pro Ala Thr His

225 230 235 240

Asn Thr Gly Gly Ser Leu Pro Asp Leu Thr Asn Ile His Phe Ala Ser

245 250 255

Pro Leu Pro Thr Pro Leu Asp Pro Glu Glu Pro Pro Phe Pro Ala Leu

260 265 270

Thr Ser Ser Ser Ser Ser Thr Gly Ser Leu Ala His Leu Gly Val Gly Gly

275 280 285

Ala Gly Gly Met Asn Thr Pro Ser Ser Ser Pro Gln His Arg Pro Ala

290 295 300

Val Val Ser Pro Leu Ser Leu Ser Thr Glu Ala Arg Arg Gln Gln Ala

305 310 315 320

Gln Gln Val Ser Pro Thr Leu Ser Pro Leu Ser Pro Ile Thr Gln Ala

325 330 335

Val Ala Met Asp Ala Leu Ser LeuGlu Gln Gln Leu Pro Tyr Ala Phe

340 345 350

Phe Thr Gln Thr Gly Ser Gln Gln Pro Pro Pro Gln Pro Gln Pro Pro

355 360 365

Pro Pro Pro Pro Pro Val Ser Gln Gln Gln Pro Pro Pro Pro Pro Gln Val

370 375 380

Ser Val Gly Leu Pro Gln Gly Gly Pro Leu Leu Pro Ser Ala Ser Leu

385 390 395 400

Thr Arg Gly Pro Gln Leu Pro Pro Leu Ser Val Thr Val Pro Ser Thr

405 410 415

Leu Pro Gln Ser Pro Thr Glu Asn Pro Gly Gln Ser Pro Met Gly Ile

420 425 430

Asp Ala Thr Ser Ala Pro Ala Leu Gln Tyr Arg Thr Ser Ala Gly Ser

435 440 445

Pro Ala Thr Gln Ser Pro Thr Ser Pro Val Ser Asn Gln Gly Phe Ser

450 455 460

Pro Gly Ser Ser Pro Gln His Thr Ser Thr Leu Gly Ser Val Phe Gly

465 470 475 480

Asp Ala Tyr Tyr Glu Gln Gln Met Thr Ala Arg Gln Ala Asn Ala Leu

485 490 495

Ser Arg Gln Leu Glu Gln Phe Asn Met Met Glu Asn Ala Ile Ser Ser

500 505 510

Ser Ser Leu Tyr Asn Pro Gly Ser Thr Leu Asn Tyr Ser Gln Ala Ala

515 520 525

Met Met Gly Leu Ser Gly Ser His Gly Gly Leu Gln Asp Pro Gln Gln

530 535 540

Leu Gly Tyr Thr Gly His Gly Gly Ile Pro Asn Ile Ile Leu Thr Val

545 550 555 560

Thr Gly Glu Ser Pro Pro Ser Leu Ser Lys Glu Leu Ser Ser Thr Leu

565 570 575

Ala Gly Val Ser Asp Val Ser Phe Asp Ser Asp His Gln Phe Pro Leu

580 585 590

Asp Glu Leu Lys Ile Asp Pro Leu Thr Leu Asp Gly Leu His Met Leu

595 600 605

Asn Asp Pro Asp Met Val Leu Ala Asp Pro Ala Thr Glu Asp Thr Phe

610 615 620

Arg Met Asp Arg Leu

625

<210>30

<211>566

<212>PRT

<213>Red-finned oriental dolphin (fugu rubripres)

<400>30

Met Ala Ser Ser Asn Asn Pro Arg Lys Phe Ser Glu Lys Ile Ala Leu

1 5 10 15

His Asn Gln Lys Gln Ala Glu Glu Thr Ala Ala Phe Glu Glu Val Met

20 25 30

Lys Asp Leu Asn Val Thr Arg Ala Ala Arg Leu Gln Leu Gln Lys Thr

35 40 45

Gln Tyr Leu Gln Leu Gly Gln Asn Arg Gly Gln Tyr Tyr Gly Gly Ser

50 55 60

Leu Pro Asn Val Asn Gln Ile Gly Asn Gly Asn Ile Asp Leu Pro Phe

65 70 75 80

Gln Val Ser Asn Ser Val Leu Asp Thr Ser Arg Thr Thr Arg His His

85 90 95

Gly Leu Val Glu Arg Val Tyr Arg Asp Arg Asn Arg Ile Ser Ser Pro

100 105 110

His Arg Arg Pro Leu Ser Val Asp Lys His Gly Arg Gln Arg Thr Asn

115 120 125

Ser Asp Ser Ala Leu His Gln Ser Ala Met Asn Pro Lys Pro His Glu

130 135 140

Val Phe Ala Gly Gly Ser Gln Glu Leu Gln Pro Lys Arg Leu Leu Leu

145 150 155 160

Thr Val Pro Gly Thr Glu Lys Ser Glu Ser Asn Ala Asp Lys Asp Ser

165 170 175

Gln Glu Gln Ser Trp Asp Asp Lys Lys Ser Ile Phe Pro Ser Pro Asp

180 185 190

Gln Glu Leu Asn Pro Ser Val Leu Pro Ala Ala His Asn Thr Gly Gly

195 200 205

Ser Leu Pro Asp Leu Thr Asn Ile Gln Phe Pro Pro Pro Leu Ser Thr

210 215 220

Pro Leu Asp Pro Glu Asp Thr Val Thr Phe Pro Ser Leu Ser Ser Ser Ser

225 230 235 240

Asn Ser Thr Gly Ser Leu Thr Thr Asn Leu Thr His Leu Gly Ile Ser

245 250 255

Val Ala Ser His Gly Asn Asn Gly Glu Lys Asn Ile Phe Phe Leu Lys

260 265 270

Thr Cys Thr Ser Cys Glu Asp Val Tyr Asp Phe Tyr Phe Val Gly Ile

275 280 285

Pro Thr Ser Ser Gln Thr Thr Met Thr Ala Thr Ala Gln Arg Arg Gln

290 295 300

Pro Pro Val Val Pro Leu Thr Leu Thr Ser Asp Leu Thr Leu Gln Gln

305 310 315 320

Ser Pro Gln Gln Leu Ser Pro Thr Leu Ser Ser Pro Ile Asn Ile Thr

325 330 335

Gln Ser Met Lys Leu Ser Ala Ser Ser Ser Leu Gln Gln Tyr Arg Asn Gln

340 345 350

Thr Gly Ser Pro Ala Thr Gln Ser Pro Thr Ser Pro Val Ser Asn Gln

355 360 365

Gly Phe Ser Pro Gly Ser Ser Ser Pro Gln Pro Gln His Ile Pro Val Val

370 375 380

Gly Ser Ile Phe Gly Asp Ser Phe Tyr Asp Gln Gln Leu Ala Leu Arg

385 390 395 400

Gln Thr Asn Ala Leu Ser His Gln Val Cys Glu Asp Gly Arg Arg Leu

405 410 415

Glu Ile Thr His Val Arg Leu Ser Arg Leu His Ala Glu Leu Cys Phe

420 425 430

Cys Phe Ser Gln Leu Glu Gln Phe Asn Met Ile Glu Asn Pro Ile Ser

435 440 445

Ser Thr Ser Leu Tyr Asn Gln Cys Ser Thr Leu Asn Tyr Thr Gln Ala

450 455 460

Ala Met Met Gly Leu Thr Gly Ser Ser Leu Gln Asp Ser Gln Gln Leu

465 470 475 480

Gly Tyr Gly Asn His Gly Asn Ile Pro Asn Ile Ile Leu Thr Ile Ser

485 490 495

Val Thr Gly Glu Ser Pro Pro Ser Leu Ser Lys Glu Leu Thr Asn Ser

500 505 510

Leu Ala Gly Val Gly Asp Val Ser Phe Asp Pro Asp Thr Gln Phe Pro

515 520 525

Leu Asp Glu Leu Lys Ile Asp Pro Leu Thr Leu Asp Gly Leu His Met

530 535 540

Leu Asn Asp Pro Asp Met Val Leu Ala Asp Pro Ala Thr Glu Asp Thr

545 550 555 560

Phe Arg Met Asp Arg Leu

565

<210>31

<211>1602

<212>DNA

<213> Red-finned oriental dolphin

<400>31

atggcgtcct ctaacaatcc tcgcaaattt agcgaaaaaa tcgcac tgca taaccagaaa 60

caagcagagg agactgctgc gttcgaagaa gtgatgaagg acctgaacgt cacaagggct 120

gcccgggtaa gacagctgca gttacagaag accccagtatt tgcaactagg gcagaatcgt 180

ggacagtact atggaggctc actgcccaat gtcaatcaga ttggaaatgg caacattgac 240

ctgccttttc aggtgagcag gacaaactca gactcagctt tacatcagag tgccatgaat 300

ccaaagcccc acgaagtgtt tgctgggggg tcgcaggagc tgcagcccaa acgactgctg 360

ctaacagtgc ctggaaccga aaaatcggaa tcaaacgcag acaaagattc gcaggagcag 420

tcgtgggatg acaaaaagag tatttttcca tcaccagacc aggagttaaa cccctccgtg 480

cttccagccg cgcacaacac cggcggttcg ctccccgacc tgaccaacat ccagttccct 540

cctccactgt ccaccccact ggaccccgag gacaccgtca ccttcccctc cctcagctcc 600

tctaacagca caggcagtct gactaccaac ctcacccacc tgggcatcag tgtggccagc 660

catggtaata acggagagaa aaatatattt tttttaaaaa catgcacttc atgcgaggat 720

gttaaataat attacgactt ttattttgta gggattccca cttcctctca aaccaccatg 780

acagcaacag cacagcggcg gcaaccaccc gtggtccccc tcaccctcac ctctgacctg 840

actcttcaac agtcccccca gcagctttca cccaccctct cctcacccat taacatcaca 900

cagagcatga agcttagtgc tagctaacat tcttccctcc aacagtaccg caatcagact 960

ggctcaccag ccactcagtc tccaacctcc ccagtctcca atcaaggctt ctcccccggc 1020

agctcgcctc aaccacagca cattcctgtg gtgggcagta tatttgggga ctccttctat 1080

gatcagcagt tggctctgag gcagaccaat gccctttctc atcaggtgtg tgaggacggc 1140

cgcaggttag aaataacaca cgtacgtctc tcacgacttc acgccgagct ttgtttttgt 1200

ttttctcagc tggagcagtt caatatgata gagaacccca tcagctccac cagcctgtac 1260

aatcagtgct ccacccttaa ttacacacag gcagccatga tgggcctcac cgggagcagc 1320

ctgcaggact cgcagcagct cggctacggc aatcacggca acatccccaa catcatactg 1380

acaatttcag tcacaggggga gtctccgccg agcctctcca aagagctgac caactcattg 1440

gccggcgtcg gcgacgtcag ctttgatcca gacacgcagt ttcctctgga cgagctgaag 1500

atcgacccgc tgaccttgga cggcctgcac atgctcaacg accccagacat ggtgctggca 1560

gaccccgcca cagaggacac gttcaggatg gacaggctgt aa 1602

<210>32

<211>170

<212>PRT

<213> people

<400>32

Met Ala Thr Ser Asn Asn Pro Arg Lys Phe Ser Glu Lys Ile Ala Leu

1 5 10 15

His Asn Gln Lys Gln Ala Glu Glu Thr Ala Ala Phe Glu Glu Val Met

20 25 30

Lys Asp Leu Ser Leu Thr Arg Ala Ala Arg Leu Gln Leu Gln Lys Ser

35 40 45

Gln Tyr Leu Gln Leu Gly Pro Ser Arg Gly Gln Tyr Tyr Gly Gly Ser

50 55 60

Leu Pro Asn Val Asn Gln Ile Gly Ser Gly Thr Met Asp Leu Pro Phe

65 70 75 80

Gln Pro Ser Gly Phe Leu Gly Glu Ala Leu Ala Ala Ala Pro Val Ser

85 90 95

Leu Thr Pro Phe Gln Ser Ser Gly Leu Asp Thr Ser Arg Thr Thr Arg

100 105 110

His His Gly Leu Val Asp Arg Val Tyr Arg Glu Arg Gly Arg Leu Gly

115 120 125

Ser Pro His Arg Arg Pro Leu Ser Val Asp Lys His Gly Arg Gln Ala

130 135 140

Asp Ser Cys Pro Tyr Gly Thr Met Tyr Leu Ser Pro Pro Ala Asp Thr

145 150 155 160

Ser Trp Arg Arg Thr Asn Ser Asp Ser Ala

165 170

<210>33

<211>356

<212>PRT

<213> people

<400>33

Met Ala Thr Ser Asn Asn Pro Arg Lys Phe Ser Glu Lys Ile Ala Leu

1 5 10 15

His Asn Gln Lys Gln Ala Glu Glu Thr Ala Ala Phe Glu Glu Val Met

20 25 30

Lys Asp Leu Ser Leu Thr Arg Ala Ala Arg Leu Gln Leu Gln Lys Ser

35 40 45

Gln Tyr Leu Gln Leu Gly Pro Ser Arg Gly Gln Tyr Tyr Gly Gly Ser

50 55 60

Leu Pro Asn Val Asn Gln Ile Gly Ser Gly Thr Met Asp Leu Pro Phe

65 70 75 80

Gln Pro Ser Gly Phe Leu Gly Glu Ala Leu Ala Ala Ala Pro Val Ser

85 90 95

Leu Thr Pro Phe Gln Ser Ser Gly Leu Asp Thr Ser Arg Thr Thr Arg

100 105 110

His His Gly Leu Val Asp Arg Val Tyr Arg Glu Arg Gly Arg Leu Gly

115 120 125

Ser Pro His Arg Arg Pro Leu Ser Val Asp Lys His Gly Arg Gln Ala

130 135 140

Asp Ser Cys Pro Tyr Gly Thr Met Tyr Leu Ser Pro Pro Ala Asp Thr

145 150 155 160

Ser Trp Arg Arg Thr Asn Ser Asp Ser Ala Leu His Gln Ser Thr Met

165 170 175

Thr Pro Thr Gln Pro Glu Ser Phe Ser Ser Ser Gly Ser Gln Asp Val His

180 185 190

Gln Lys Arg Val Leu Leu Leu Thr Val Pro Gly Met Glu Glu Thr Thr

195 200 205

Ser Glu Ala Asp Lys Asn Leu Ser Lys Gln Ala Trp Asp Thr Lys Lys

210 215 220

Thr Gly Ser Arg Pro Lys Ser Cys Glu Val Pro Gly Ile Asn Ile Phe

225 230 235 240

Pro Ser Ala Asp Gln Glu Asn Thr Thr Ala Leu Ile Pro Ala Thr His

245 250 255

Asn Thr Gly Gly Ser Leu Pro Asp Leu Thr Asn Ile His Phe Pro Ser

260 265 270

Pro Leu Pro Thr Pro Leu Asp Pro Glu Glu Pro Thr Phe Pro Ala Leu

275 280 285

Ser Ser Ser Ser Ser Ser Thr Gly Asn Leu Ala Ala Asn Leu Thr His Leu

290 295 300

Gly Ile Gly Gly Ala Gly Gln Gly Met Ser Thr Pro Gly Ser Ser Pro

305 310 315 320

Gln His Arg Pro Ala Gly Val Ser Pro Leu Ser Leu Ser Thr Glu Ala

325 330 335

Arg Arg Gln Gln Ala Ser Pro Thr Leu Ser Pro Leu Ser Pro Ile Thr

340 345 350

Gln Ala Val Ala

355

<210>34

<211>494

<212>PRT

<213> people

<400>34

Met Ala Thr Ser Asn Asn Pro Arg Lys Phe Ser Glu Lys Ile Ala Leu

1 5 10 15

His Asn Gln Lys Gln Ala Glu Glu Thr Ala Ala Phe Glu Glu Val Met

20 25 30

Lys Asp Leu Ser Leu Thr Arg Ala Ala Arg Leu Gln Leu Gln Lys Ser

35 40 45

Gln Tyr Leu Gln Leu Gly Pro Ser Arg Gly Gln Tyr Tyr Gly Gly Ser

50 55 60

Leu Pro Asn Val Asn Gln Ile Gly Ser Gly Thr Met Asp Leu Pro Phe

65 70 75 80

Gln Pro Ser Gly Phe Leu Gly Glu Ala Leu Ala Ala Ala Pro Val Ser

85 90 95

Leu Thr Pro Phe Gln Ser Ser Gly Leu Asp Thr Ser Arg Thr Thr Arg

100 105 110

His His Gly Leu Val Asp Arg Val Tyr Arg Glu Arg Gly Arg Leu Gly

115 120 125

Ser Pro His Arg Arg Pro Leu Ser Val Asp Lys His Gly Arg Gln Ala

130 135 140

Asp Ser Cys Pro Tyr Gly Thr Met Tyr Leu Ser Pro Pro Ala Asp Thr

145 150 155 160

Ser Trp Arg Arg Thr Asn Ser Asp Ser Ala Leu His Gln Ser Thr Met

165 170 175

Thr Pro Thr Gln Pro Glu Ser Phe Ser Ser Ser Gly Ser Gln Asp Val His

180 185 190

Gln Lys Arg Val Leu Leu Leu Thr Val Pro Gly Met Glu Glu Thr Thr

195 200 205

Ser Glu Ala Asp Lys Asn Leu Ser Lys Gln Ala Trp Asp Thr Lys Lys

210 215 220

Thr Gly Ser Arg Pro Lys Ser Cys Glu Val Pro Gly Ile Asn Ile Phe

225 230 235 240

Pro Ser Ala Asp Gln Glu Asn Thr Thr Ala Leu Ile Pro Ala Thr His

245 250 255

Asn Thr Gly Gly Ser Leu Pro Asp Leu Thr Asn Ile His Phe Pro Ser

260 265 270

Pro Leu Pro Thr Pro Leu Asp Pro Glu Glu Pro Thr Phe Pro Ala Leu

275 280 285

Ser Ser Ser Ser Ser Ser Thr Gly Asn Leu Ala Ala Asn Leu Thr His Leu

290 295 300

Gly Ile Gly Gly Ala Gly Gln Gly Met Ser Thr Pro Gly Ser Ser Pro

305 310 315 320

Gln His Arg Pro Ala Gly Val Ser Pro Leu Ser Leu Ser Thr Glu Ala

325 330 335

Arg Arg Gln Gln Ala Ser Pro Thr Leu Ser Pro Leu Ser Pro Ile Thr

340 345 350

Gln Ala Val Ala Met Asp Ala Leu Ser Leu Glu Gln Gln Leu Pro Tyr

355 360 365

Ala Phe Phe Thr Gln Ala Gly Ser Gln Gln Pro Pro Pro Pro Gln Pro Gln

370 375 380

Pro Pro Pro Pro Pro Pro Pro Pro Ala Ser Gln Gln Pro Pro Pro Pro Pro Pro

385 390 395 400

Pro Pro Gln Ala Pro Val Arg Leu Pro Pro Gly Gly Pro Leu Leu Pro

405 410 415

Ser Ala Ser Leu Thr Arg Gly Pro Gln Pro Pro Pro Leu Ala Val Thr

420 425 430

Val Pro Ser Ser Leu Pro Gln Ser Pro Pro Glu Asn Pro Gly Gln Pro

435 440 445

Ser Met Gly Ile Asp Ile Ala Ser Ala Pro Ala Leu Gln Gln Tyr Arg

450 455 460

Thr Ser Ala Gly Ser Pro Ala Asn Gln Ser Pro Thr Ser Pro Val Ser

465 470 475 480

Asn Gln Gly Phe Ser Pro Gly Ser Ser Pro Gln His Thr Ser

485 490

<210>35

<211>580

<212>PRT

<213> people

<400>35

Met Ala Thr Ser Asn Asn Pro Arg Lys Phe Ser Glu Lys Ile Ala Leu

1 5 10 15

His Asn Gln Lys Gln Ala Glu Glu Thr Ala Ala Phe Glu Glu Val Met

20 25 30

Lys Asp Leu Ser Leu Thr Arg Ala Ala Arg Leu Gln Leu Gln Lys Ser

35 40 45

Gln Tyr Leu Gln Leu Gly Pro Ser Arg Gly Gln Tyr Tyr Gly Gly Ser

50 55 60

Leu Pro Asn Val Asn Gln Ile Gly Ser Gly Thr Met Asp Leu Pro Phe

65 70 75 80

Gln Pro Ser Gly Phe Leu Gly Glu Ala Leu Ala Ala Ala Pro Val Ser

85 90 95

Leu Thr Pro Phe Gln Ser Ser Gly Leu Asp Thr Ser Arg Thr Thr Arg

100 105 110

His His Gly Leu Val Asp Arg Val Tyr Arg Glu Arg Gly Arg Leu Gly

115 120 125

Ser Pro His Arg Arg Pro Leu Ser Val Asp Lys His Gly Arg Gln Ala

130 135 140

Asp Ser Cys Pro Tyr Gly Thr Met Tyr Leu Ser Pro Pro Ala Asp Thr

145 150 155 160

Ser Trp Arg Arg Thr Asn Ser Asp Ser Ala Leu His Gln Ser Thr Met

165 170 175

Thr Pro Thr Gln Pro Glu Ser Phe Ser Ser Ser Gly Ser Gln Asp Val His

180 185 190

Gln Lys Arg Val Leu Leu Leu Thr Val Pro Gly Met Glu Glu Thr Thr

195 200 205

Ser Glu Ala Asp Lys Asn Leu Ser Lys Gln Ala Trp Asp Thr Lys Lys

210 215 220

Thr Gly Ser Arg Pro Lys Ser Cys Glu Val Pro Gly Ile Asn Ile Phe

225 230 235 240

Pro Ser Ala Asp Gln Glu Asn Thr Thr Ala Leu Ile Pro Ala Thr His

245 250 255

Asn Thr Gly Gly Ser Leu Pro Asp Leu Thr Asn Ile His Phe Pro Ser

260 265 270

Pro Leu Pro Thr Pro Leu Asp Pro Glu Glu Pro Thr Phe Pro Ala Leu

275 280 285

Ser Ser Ser Ser Ser Ser Thr Gly Asn Leu Ala Ala Asn Leu Thr His Leu

290 295 300

Gly Ile Gly Gly Ala Gly Gln Gly Met Ser Thr Pro Gly Ser Ser Pro

305 310 315 320

Gln His Arg Pro Ala Gly Val Ser Pro Leu Ser Leu Ser Thr Glu Ala

325 330 335

Arg Arg Gln Gln Ala Ser Pro Thr Leu Ser Pro Leu Ser Pro Ile Thr

340 345 350

Gln Ala Val Ala Met Asp Ala Leu Ser Leu Glu Gln Gln Leu Pro Tyr

355 360 365

Ala Phe Phe Thr Gln Ala Gly Ser Gln Gln Pro Pro Pro Pro Gln Pro Gln

370 375 380

Pro Pro Pro Pro Pro Pro Pro Pro Ala Ser Gln Gln Pro Pro Pro Pro Pro Pro

385 390 395 400

Pro Pro Gln Ala Pro Val Arg Leu Pro Pro Gly Gly Pro Leu Leu Pro

405 410 415

Ser Ala Ser Leu Thr Arg Gly Pro Gln Pro Pro Pro Leu Ala Val Thr

420 425 430

Val Pro Ser Ser Leu Pro Gln Ser Pro Pro Glu Asn Pro Gly Gln Pro

435 440 445

Ser Met Gly Ile Asp Ile Ala Ser Ala Pro Ala Leu Gln Gln Tyr Arg

450 455 460

Thr Ser Ala Gly Ser Pro Ala Asn Gln Ser Pro Thr Ser Pro Val Ser

465 470 475 480

Asn Gln Gly Phe Ser Pro Gly Ser Ser Pro Gln His Thr Ser Thr Leu

485 490 495

Gly Ser Val Phe Gly Asp Ala Tyr Tyr Glu Gln Gln Met Ala Ala Arg

500 505 510

Gln Ala Asn Ala Leu Ser His Gln Leu Glu Gln Phe Asn Met Met Glu

515 520 525

Asn Ala Ile Ser Ser Ser Ser Ser Leu Tyr Ser Pro Gly Ser Thr Leu Asn

530 535 540

Tyr Ser Gln Ala Ala Met Met Gly Leu Thr Gly Ser His Gly Ser Leu

545 550 555 560

Pro Asp Ser Gln Gln Leu Gly Tyr Ala Ser His Ser Gly Ile Pro Asn

565 570 575

Ile Ile Leu Thr

580

<210>36

<211>481

<212>PRT

<213> people

<400>36

Ala Leu His Gln Ser Thr Met Thr Pro Thr Gln Pro Glu Ser Phe Ser

1 5 10 15

Ser Gly Ser Gln Asp Val His Gln Lys Arg Val Leu Leu Leu Thr Val

20 25 30

Pro Gly Met Glu Glu Thr Thr Ser Glu Ala Asp Lys Asn Leu Ser Lys

35 40 45

Gln Ala Trp Asp Thr Lys Lys Thr Gly Ser Arg Pro Lys Ser Cys Glu

50 55 60

Val Pro Gly Ile Asn Ile Phe Pro Ser Ala Asp Gln Glu Asn Thr Thr

65 70 75 80

Ala Leu Ile Pro Ala Thr His Asn Thr Gly Gly Ser Leu Pro Asp Leu

85 90 95

Thr Asn Ile His Phe Pro Ser Pro Leu Pro Thr Pro Leu Asp Pro Glu

100 105 110

Glu Pro Thr Phe Pro Ala Leu Ser Ser Ser Ser Ser Ser Thr Gly Asn Leu

115 120 125

Ala Ala Asn Leu Thr His Leu Gly Ile Gly Gly Ala Gly Gln Gly Met

130 135 140

Ser Thr Pro Gly Ser Ser Pro Gln His Arg Pro Ala Gly Val Ser Pro

145 150 155 160

Leu Ser Leu Ser Thr Glu Ala Arg Arg Gln Gln Ala Ser Pro Thr Leu

165 170 175

Ser Pro Leu Ser Pro Ile Thr Gln Ala Val Ala Met Asp Ala Leu Ser

180 185 190

Leu Glu Gln Gln Leu Pro Tyr Ala Phe Phe Thr Gln Ala Gly Ser Gln

195 200 205

Gln Pro Pro Pro Gln Pro Gln Pro Pro Pro Pro Pro Pro Pro Pro Ala Ser

210 215 220

Gln Gln Pro Pro Pro Pro Pro Pro Pro Pro Gln Ala Pro Va lArg Leu Pro

225 230 235 240

Pro Gly Gly Pro Leu Leu Pro Ser Ala Ser Leu Thr Arg Gly Pro Gln

245 250 255

Pro Pro Pro Leu Ala Val Thr Val Pro Ser Ser Leu Pro Gln Ser Pro

260 265 270

Pro Glu Asn Pro Gly Gln Pro Ser Met Gly Ile Asp Ile Ala Ser Ala

275 280 285

Pro Ala Leu Gln Gln Tyr Arg Thr Ser Ala Gly Ser Pro Ala Asn Gln

290 295 300

Ser Pro Thr Ser Pro Val Ser Asn Gln Gly Phe Ser Pro Gly Ser Ser

305 310 315 320

Pro Gln His Thr Ser Thr Leu Gly Ser Val Phe Gly Asp Ala Tyr Tyr

325 330 335

Glu Gln Gln Met Ala Ala Arg Gln Ala Asn Ala Leu Ser His Gln Leu

340 345 350

Glu Gln Phe Asn Met Met Glu Asn Ala Ile Ser Ser Ser Ser Leu Tyr

355 360 365

Ser Pro Gly Ser Thr Leu Asn Tyr Ser Gln Ala Ala Met Met Gly Leu

370 375 380

Thr Gly Ser His Gly Ser Leu Pro Asp Ser Gln Gln Leu Gly Tyr Ala

385 390 395 400

Ser His Ser Gly Ile Pro Asn Ile Ile Leu Thr Val Thr Gly Glu Ser

405 410 415

Pro Pro Ser Leu Ser Lys Glu Leu Thr Ser Ser Leu Ala Gly Val Gly

420 425 430

Asp Val Ser Phe Asp Ser Asp Ser Gln Phe Pro Leu Asp Glu Leu Lys

435 440 445

Ile Asp Pro Leu Thr Leu Asp Gly Leu His Met Leu Asn Asp Pro Asp

450 455 460

Met Val Leu Ala Asp Pro Ala Thr Glu Asp Thr Phe Arg Met Asp Arg

465 470 475 480

Leu

<210>37

<211>30

<212>DNA

<213> Artificial sequence

<220>

<223> Primer

<400>37

caacatggcc aatccgcgca agttcagcga 30

<210>38

<211>29

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400>38

tcagttgagg tcgcgtcgaa aactatcct 29

<210>39

<211>62

<212>DNA

<213> Drosophila melanogaster

<400>39

ggagcctggc gtcagagagc ctggcgtcag agagcctggc gtcagagagc ctggcgtcag 60

ag 62

Claims (75)

1. the method for the pathological condition that prevention, treatment or alleviation are relevant with activation of CRE-dependent gene abnormal expression or chemokine abnormal activation, it comprises that the CREAP regulon with significant quantity is applied to the experimenter who needs it.

2. the process of claim 1 wherein that described pathological condition is a neurodegenerative disease.

3. the process of claim 1 wherein that described pathological condition is an autoimmune disease.

4. the process of claim 1 wherein that described pathological condition is an inflammatory diseases.

5. the process of claim 1 wherein that described pathological condition is selected from Alzheimer, Parkinson's disease, Huntington Chorea, osteoarthritis, psoriatic, asthma, COPD, rheumatoid arthritis, cancer, diabetes, hypertension and chronic pain.

6. the process of claim 1 wherein that described CREAP regulon suppresses the CREAP activity of proteins of any one or the multiple CREAP1 of being selected from, CREAP2 or CREAP3.

7. the method for claim 6, wherein said CREAP regulon comprises one or more at the proteinic antibody of CREAP or its fragment, wherein said antibody or its fragment can suppress described CREAP activity of proteins.

8. the method for claim 6, wherein said regulon comprises proteinic one or more peptide mimicses of CREAP, wherein said peptide mimics can suppress described CREAP activity of proteins.

9. the process of claim 1 wherein that described CREAP regulon suppresses to be selected from any one or the multiple CREAP protein expression of CREAP1, CREAP2 or CREAP3.

10. the method for claim 9, wherein said CREAP regulon comprises that one or more are selected from antisense oligonucleotide, triple helix DNA, ribozyme, RNA is fit and the material of double-stranded or single stranded RNA, wherein said agents design is for suppressing the CREAP protein expression.

11. prevention, treatment or alleviation activate with CRE-dependent gene abnormal expression or the method for the pathological condition that the chemokine abnormal activation is relevant, it comprises that the pharmaceutical composition that will comprise significant quantity CREAP regulon is applied to the experimenter who needs it.

12. the method for claim 11, wherein said pathological condition is a neurodegenerative disease.

13. the method for claim 11, wherein said pathological condition is an autoimmune disease.

14. the method for claim 11, wherein said pathological condition is an inflammatory diseases.

15. the method for claim 11, wherein said pathological condition is selected from Alzheimer, Parkinson's disease, Huntington Chorea, osteoarthritis, psoriatic, asthma, COPD, rheumatoid arthritis, cancer, diabetes, hypertension and chronic pain.

16. the method for claim 11, wherein said CREAP regulon suppress the CREAP activity of proteins of any one or the multiple CREAP1 of being selected from, CREAP2 or CREAP3.

17. the method for claim 11, wherein said CREAP regulon comprise one or more at the proteinic antibody of CREAP or its fragment, wherein said antibody or its fragment can suppress described CREAP activity of proteins.

18. the method for claim 11, wherein said CREAP regulon comprise proteinic one or more peptide mimicses of CREAP, wherein said peptide mimics can suppress described CREAP activity of proteins.

19. the method for claim 11, wherein said CREAP regulon suppress to be selected from any one or the multiple CREAP protein expression of CREAP1, CREAP2 or CREAP3.

20. the method for claim 19, wherein said CREAP regulon comprises that one or more are selected from antisense oligonucleotide, triple helix DNA, ribozyme, RNA is fit and the material of double-stranded or single stranded RNA, and wherein said agents design is for suppressing the CREAP protein expression.

21. evaluation is used to prevent, treat or alleviation activates with CRE-dependent gene abnormal expression or the method for the regulon of the pathological condition that the chemokine abnormal activation is relevant, it comprises the ability that candidate's regulon suppresses the CREAP protein active of measuring.

22. the method for claim 21, wherein said CREAP protein is selected from CREAP1, CREAP2 or CREAP3.

23. the method for claim 21, wherein said method comprise that further CREAP that mensuration identifies suppresses regulon and reverses in the body of external, the earlier external back of described pathological condition or in the body inner model and/or suffer from the ability of viewed pathologic effect in experimenter's the clinical study of described pathological condition.

24. the method for claim 21, wherein said pathological condition is a neurodegenerative disease.

25. the method for claim 21, wherein said pathological condition is an autoimmune disease.

26. the method for claim 21, wherein said pathological condition is an inflammatory diseases.

27. the method for claim 21, wherein said pathological condition is selected from Alzheimer, Parkinson's disease, Huntington Chorea, osteoarthritis, psoriatic, asthma, COPD, rheumatoid arthritis, cancer, diabetes, hypertension and chronic pain.

28. evaluation is used to prevent, treat or alleviation activates with CRE-dependent gene abnormal expression or the method for the regulon of the pathological condition that unusual chemokine activation is relevant, it comprises the ability of measuring candidate's regulon inhibition CREAP protein expression.

29. the method for claim 28, wherein said CREAP protein is selected from CREAP1, CREAP2 or CREAP3.

30. the method for claim 28, wherein said method comprise that further CREAP that mensuration identifies suppresses regulon and reverses in the body of external, the earlier external back of described pathological condition or in the body inner model and/or suffer from the ability of viewed pathologic effect in experimenter's the clinical study of described pathological condition.

31. the method for claim 28, wherein said pathological condition is a neurodegenerative disease.

32. the method for claim 28, wherein said pathological condition is an autoimmune disease.

33. the method for claim 28, wherein said pathological condition is an inflammatory diseases.

34. the method for claim 28, wherein said pathological condition is selected from Alzheimer, Parkinson's disease, Huntington Chorea, osteoarthritis, psoriatic, asthma, COPD, rheumatoid arthritis, cancer, diabetes, hypertension and chronic pain.

35. pharmaceutical composition, its one or more CREAP regulon that comprise significant quantity activate or the relevant pathological condition of unusual chemokine activation with CRE-dependent gene abnormal expression to prevent, to treat or to alleviate among the patient who needs it.

36. according to the pharmaceutical composition of claim 35, wherein said pathological condition is a neurodegenerative disease.

37. according to the pharmaceutical composition of claim 35, wherein said pathological condition is an autoimmune disease.

38. according to the pharmaceutical composition of claim 35, wherein said pathological condition is an inflammatory diseases.

39. according to the pharmaceutical composition of claim 35, wherein said pathological condition is selected from Alzheimer, Parkinson's disease, Huntington Chorea, osteoarthritis, psoriatic, asthma, COPD, rheumatoid arthritis, cancer, diabetes, hypertension and chronic pain.

40. according to the pharmaceutical composition of claim 35, wherein said CREAP regulon suppresses to be selected from any one or the multiple CREAP activity of proteins of CREAP1, CREAP2 or CREAP3.

41. the pharmaceutical composition of claim 40, wherein said CREAP regulon comprise one or more at the proteinic antibody of CREAP or its fragment, wherein said antibody or its fragment can suppress described CREAP activity of proteins.

42. the pharmaceutical composition of claim 40, wherein said CREAP regulon comprise proteinic one or more peptide mimicses of CREAP, wherein said peptide mimics can suppress described CREAP activity of proteins.

43. according to the pharmaceutical composition of claim 35, wherein said CREAP regulon suppresses to be selected from any one or the multiple CREAP protein expression of CREAP1, CREAP2 or CREAP3.

44. the pharmaceutical composition of claim 43, wherein said CREAP regulon comprises that one or more are selected from antisense oligonucleotide, triple helix DNA, ribozyme, RNA is fit and the material of double-stranded or single stranded RNA, and wherein said agents design is for suppressing CREAP genetic expression.

45. diagnosis suffers from and activates with CRE-dependent gene abnormal expression or unusual chemokine activates the related pathologies situation and may be to use the experimenter's of the suitable candidate that the CREAP regulon treats method, it comprises mensuration from the proteinic mRNA level of CREAP in described experimenter's the biological sample, and the experimenter who wherein has the mRNA level of increase compared with the control is the suitable candidate that carries out the treatment of CREAP regulon.

46. the method for claim 45, wherein said CREAP protein is selected from CREAP1, CREAP2 or CREAP3.

47. suffering from, diagnosis activates with CRE-dependent gene abnormal expression or chemokine abnormal activation related pathologies situation and may be to use the experimenter's of the suitable candidate that the CREAP regulon treats method, this method comprises detection from the proteinic level of CREAP in described experimenter's the biological sample, and the experimenter who wherein has elevated levels compared with the control is the suitable candidate that carries out the treatment of CREAP regulon.

48. the method for claim 47, wherein said CREAP protein is selected from CREAP1, CREAP2 or CREAP3.

49. prevention, treatment or alleviation and CRE-dependent gene abnormal expression activate or the method for chemokine abnormal activation related pathologies situation, it comprises:

(a) measure CREAP mRNA and/or protein level in the subject; With,

(b) will be enough to prevent, treat or the CREAP regulon of alleviating described pathological condition is applied to the CREAP mRNA that has elevated levels compared with the control and/or the experimenter of protein level.

50. the method for claim 49, wherein said pathological condition is a neurodegenerative disease.

51. the method for claim 49, wherein said pathological condition is an autoimmune disease.

52. the method for claim 49, wherein said pathological condition is an inflammatory diseases.

53. the method for claim 49, wherein said pathological condition is selected from Alzheimer, Parkinson's disease, Huntington Chorea, osteoarthritis, psoriatic, asthma, COPD, rheumatoid arthritis, cancer, diabetes, hypertension and chronic pain.

54. be used for the diagnostic kit of proteinic mRNA level of detection of biological sample CREAP and/or protein level, described test kit comprises:

(a) polynucleotide of CREAP or its fragment;

(b) with (a) in polynucleotide complementary nucleotide sequence;

(c) CREAP polypeptide, or its fragment;

(d) at the antibody of CREAP polypeptide; Or

(e) the proteinic peptide mimics of CREAP

Wherein component (a) and (b), (c), (d) or (e) can comprise etc. congruent.

55. the method for claim 54, wherein said CREAP protein is selected from CREAP1, CREAP2 or CREAP3.

56. isolated polypeptide, it comprises and is selected from SEQ ID NOs:2,16 and 25 CREAP aminoacid sequence.

57. isolated nucleic acid sequences, it comprises the nucleotide sequence of the polypeptide of the claim 56 of encoding.

58. isolated polypeptide, its by be selected from SEQ ID NOs:2,16 and 25 CREAP aminoacid sequence is formed.

59. isolated nucleic acid sequences, it comprises the nucleotide sequence of the polypeptide of the claim 58 of encoding.

60. isolating CREAP polypeptide, it is by the CREAP genes encoding of organism.

61. separated DNA, it comprises the nucleotide sequence of the CREAP polypeptide of the claim 60 of encoding.

62. carrier molecule comprises the fragment according to the isolating nucleic acid of claim 57.

63. according to the carrier molecule of claim 62, it comprises any one or multiple transcriptional control sequence.

64. host cell, it comprises the carrier molecule according to claim 63.

65. antibody or its fragment, described antibody or its fragment specific combination comprise the fragment of the polypeptide or the described polypeptide of the aminoacid sequence that is shown in claim 56.

66. according to the antibody fragment of claim 65, it is Fab or F (ab ') 2 parts.

67. according to the antibody of claim 65, it is a polyclonal antibody.

68. according to the antibody of claim 65, it is a monoclonal antibody.

69. produce method as defined polypeptide in the claim 56, it is included under the condition that is enough to the following polypeptide of expression in host cell and cultivates the wherein host cell of integrating expression vector, this expression vector comprises the polynucleotide in external source source, this polynucleotide encoding comprises the polypeptide that is selected from SEQ ID NOs:2,16 and 25 aminoacid sequence, thereby causes producing expressed polypeptide.

70. according to the method for claim 69, described method further comprises the polypeptide that reclaims described cell generation.

71. according to the method for claim 69, the polynucleotide in wherein said external source source comprise and are selected from SEQID NOs:1,15 and 24 nucleotide sequence.

72. produce method as defined polypeptide in the claim 56, it is included under the condition that is enough to the following polypeptide of expression in host cell and cultivates the wherein host cell of integrating expression vector, this expression vector comprises the polynucleotide in external source source, this polynucleotide encoding comprises the polypeptide that is selected from SEQ ID NOs:2,16 and 25 aminoacid sequence, thereby causes producing expressed polypeptide.

73. according to the method for claim 72, described method further comprises the polypeptide that reclaims described cell generation.

74. according to the method for claim 72, the polynucleotide in wherein said external source source comprise and are selected from SEQID NOs:1,15 and 24 nucleotide sequence.

75. carrier molecule, it comprises the nucleotide sequence that is selected from coding people CREAP protein fragments amino acid region 1-267,289-538,356-580 and 575-650.