MXPA06015175A - Diagnostic and screening methods and kits associated with proteolytic activity - Google Patents

Abstract

Methods and kits for assessing proteolytic enzyme activity and a modulatorâ¿¿s effect thereof employ a Ubiquitin or Ubiquitin-like Protein and a signal producing structure. The corresponding fusion polynucleotide is employed for production of transgenic cells, plants and animals that may be produced by stably transfection, optionally transforming the cell, plant or animal with a Ubiquitin-,UBL-or their C-terminal binding functional fragment-Reporter fusion polynucleotide. A method of diagnosing a disease or condition, comprises contacting or administering a sample obtained from a subject suspected of being afflicted with the disease or condition with the cell, plant or animal, detecting any signal produced by the reporter in the presence of the sample and comparing the signal to controls for 0%and 100%signals.

Description

DIAGNOSTIC AND SIZING METHODS AND EQUIPMENT ASSOCIATED WITH PROTEOLYTIC ACTIVITY BACKGROUND OF THE INVENTION Related Requests This patent claims the priority of the filing date of June 21, 2004 of the Provisional Application of the United States of America 60 / 580,900, entitled "Methods for Assessing Isopeptidasa Activity".

Field of the Invention This patent provides materials and methods for the qualitative and quantitative determination of ubiquitin and proteolytic enzyme activity similar to ubiquitin as well as the discovery of novel enzymes, for evaluating and / or screening compounds for their effects on proteolytic enzyme activity, and for detect activity in biological samples which are useful for the diagnosis of conditions and diseases associated with altered levels, amounts, sequences and / or enzyme activities. This technology is also incorporated in models of disease in the form of transgenic cells, plants and animals.

Background of the Invention Ubiquitin isopeptidates (Ub) were cloned for the first time more than a decade ago. However, up to this point, there was no adequate test for proteolytic enzymes specific for Ub or Ubiquitin-like Proteins (UBL) or for rapid screening of modulators or inhibitors of the enzyme. Most of the tests currently in use are supported by the fragmentation of linear Ub-fusions, which are produced in E. coli, for example tetra-Ub, Ub-CEP52, Ub-GSTPI, Ub-DHFR, Ub-PESTc, and the like, or chemically synthesized. In these tests the reaction products are analyzed by means of gel electrophoresis, or precipitates selectively and then analyzed by means of liquid scintillation spectrometry. These tests have significant disadvantages, for example that gel-based approaches are labor intensive and expensive. Although selective precipitation / scintillation counting provides quantitative results and allows processing of a greater number of samples than gel-based tests, centrifugation and separation of the supernatant is required. Ubiquitin-7-amido-4-metoumarin (Ub-AMC) is a fluorogenic substrate for High Productivity Screening (HTS) that is commercially available and easy to use. However, unlike the Ubiquitin C-terminal hydrolases (UCHs), most Ubiquitin-specific Proteases (USPs) do not fragment into small groups from the Ubiquitin (Ub) molecule. In addition, AMC is highly hydrophobic and, based on its own interactions with the test compounds, can give rise to false positives in screening. Other ways to detect fragmentation, for example High Pressure Liquid Chromatography (HPLC) and mass spectroscopy have also been used although they have their own disadvantages. Furthermore, none of the methods of the prior art is suitable or adaptable to high productivity screening, which requires simple tests (minimum number of stages) that can be conducted using multi-well plates, and whose endpoints are read direct from the plates.

There is therefore a need for tests and equipment that are simple and relatively inexpensive in nature while at the same time they are suitable for conducting high productivity screening for ubiquitin modulators or proteolytic enzymes of Ubiquitin-like Protein (UBL) .

BRIEF DESCRIPTION OF THE INVENTION One aspect of the present invention relates to a method for determining the activity of proteolytic enzyme, which method comprises providing a fusion polymer comprising a first polymer comprising Ubiquitin or a Ubiquitin-like Protein (UBL) or a C-terminal segment functional thereof and a second polymer comprising a free N-terminal amino acid required for detection; wherein the first and second polymers are operably linked to one another through the C-terminal UBL and the second N-terminal polymer; contacting the fusion polymer with the proteolytic enzyme that cleaves at the UBL C-terminus; detecting a signal associated with any amount or activity of fragmented polymer; and establishing a correlation between the fragmented polymer signal for the proteolytic enzyme activity. For the practice of the above method this patent provides a device for the determination of the activity of proteolytic enzyme, comprising a fusion polymer comprising a first polymer comprising Ubiquitin or a protein similar to Ubiquitin (UBL) or a C-terminal segment thereof and a second polymer comprising a polypeptide that requires a free N-terminal amino acid for detection; wherein the first and second polymers are operably linked to each other through the Ubiquitin or C-terminal UBL and the second N-terminal polymer; and the instruction for conducting the proteolytic enzyme test, detecting a signal associated with the amount or activity of the first and second polymers, and establishing a correlation of the detected signal for the proteolytic activity of the enzyme; and optionally a source of the proteolytic enzyme that cleaves in the C-terminal UBL and many other components.

Another aspect of the invention relates to a method for screening compounds for their effect on proteolytic activity, which comprises obtaining a fusion polymer comprising a first polymer comprising Ubiquitin or a protein similar to Ubiquitin (UBL) or a segment C- functional agglutination terminal thereof and a second polymer comprising a free N-terminal amino acid; wherein the first and second polymers are operatively linked to one another through the N-C-terminals; contacting the fusion polymer with a proteolytic cleavage enzyme UBL C-terminal under conditions effective for fragmentation to occur; detecting a signal associated with a fragmentation amount to obtain a fragmentation signal of 100%; repeating the contacting and detection steps in the presence of a complete inhibitor of proteolytic enzyme activity to obtain a 0% fragmentation signal; obtain a set of compounds; to separately repeat the steps of obtaining the fusion polymer, the contacting and the detection in the presence of each compound to obtain a fragmentation signal; normalize each compound fragmentation signal by reference to the fragmentation signal of 0% and 100% and assign a value of proteolytic enzyme activity to each compound.

For the practice of this second method this patent provides a screening equipment modulating the activity of proteolytic enzyme, comprising a fusion polymer comprising a first polymer comprising Ubiquitin or a protein similar to Ubiquitin (UBL) or a segment C- terminal thereof and a second polymer comprising a polypeptide that requires a free N-terminal amino acid for detection; wherein the first and second polymers are operably linked to each other through the Ubiquitin or C-terminal UBL and the second N-terminal polymer; and the instruction for conducting the proteolytic enzyme test, detecting a signal associated with the amount or activity of the first and / or second polymers, and establishing a correlation of the detected signal for the proteolytic activity of the enzyme for a plurality of modulators and controls.; and optionally a source of the proteolytic enzyme that cleaves in Ubiquitin or C-terminal UBL, and other components suitable for different modalities. This invention also relates to a transgenic cell, plant or animal, comprising reporter fusion polynucleotide of Ubiquitin- or UBL that is optionally integrated into the chromosome of the cell, plant or animal; wherein Ubiquitin or BL-specific Isopeptidase U is associated with a specific disease or condition or family thereof. The transgenic cell, plant or animal can be produced by obtaining a cell, plant or animal; obtaining a reporter-fusion fusion polynucleotide from Ubiquitin-, UBL- or its C-terminal functional fragment; obtaining a hybrid vector that transports the hybrid polynucleotide operatively linked to a vector; and stably transfecting the hybrid vector within the cell, plant or animal. A method for diagnosing a disease or condition, comprising obtaining the cell, plant or animal of the invention, or fractions or tissue thereof, wherein the Ubiquitin or UBL-specific Isopeptidase is associated with a disease is also described in this patent. or condition; contacting to administer a sample obtained from an individual who is suspected of having the disease or condition with the cell, plant or animal; detect any signal produced by the reporter in the presence of the sample; and compare the signal with the controls for 0% and 100% signals. Other objects, advantages and features of the present invention will become apparent to those skilled in the art from the following description.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES OF THE INVENTION This invention arises from a desire of the inventors to improve the prior art test methods of the proteolytic enzyme activity associated with Ubiquitin and Ubiquitin-like Proteins (UBL). By observing the inherent disadvantages of existing methods, the inventors investigated in the field in search of an opportunity to provide a method that is simple, has easy-to-determine conclusions and is suitable or adaptable for high productivity screening, automation and computerization of the Data Capture. The enzyme and equipment activity tests herein employ relatively inexpensive elements, require a small number of operations, can be conducted using multi-well plates and thus be automated, and their termination points can be read directly from the same and the data capture and analysis can be computerized. There are also features of the method and equipment herein for high productivity screening of Ubiquitin proteolytic enzyme modulators or Ubiquitin-like Protein (collectively UBL). This technology responds to the needs of research and the health care industry by providing fast, non-expensive, selective and simple methods to test the activity of UBL enzymes, which are adaptable to high productivity screening. for proteolytic enzyme modulators, and useful for the diagnosis of diseases and conditions that are associated with UBL proteolytic enzyme modulators, and useful for diagnosing diseases and conditions that are associated with UBL proteolytic enzymes, such as isopeptidases and the like, and for screening new enzymes and UBL enzyme modulators. The present invention relates to the field of enzymatic activity, its modulation and detection, and more specifically to the activity of Ubiquitin and Protein similar to proteolytic Ubiquitin (UBL), for example isopeptidase / hydrolase / protease, enzymes, its modulation and detection. The patent provides equipment and methods for the qualitative and / or quantitative determination of UBL proteolytic enzyme activity, a method for evaluating or screening compounds and agents for their effects on the activity of the enzyme, and a method for detecting activity in biological samples. . The ubiquitin-proteasomal pathway has been validated through the introduction and clinical success of Velcade® in the treatment of recurrent refractory multiple myeloma. See, Adams (2002). This trajectory is intended to regulate the cellular content and / or compartmentalisation of most proteins, and it is a promising scenario, although scarcely exploited for the discovery of drugs. See, Ciechanover (2001); Ciechanover (2003). At any given moment, it is considered that the cellular content of a protein is regulated by a combination of its synthetic and degradation indexes, with each protein having a characteristic pattern of synthesis and degradation to ensure adequate cellular function. It is considered that degradation occurs in different ways. It is considered that the extracellular or membrane-associated proteins are generally degraded in lysosomes, to which they are directed by endosomal Golgi devices. It is considered that cytoplasmic proteins are SoUBLe are degraded in a regulated manner through the ubiquitin-proteasomal pathway. The last trajectory is thought to explain the degradation of up to about 90% of all non-unfolded, abnormal proteins and most of the short-lived regulatory proteins in a cell. In addition, proteasomes are considered to be involved in the separation of most of the longer-lived proteins. It is estimated that the ubiquitin-proteasome pathway can account for 80-90% of cellular protein degradation. See, Lee and Goldberg (1998). The objectives of the ubiquitin-proteasome pathway include the cell cycle and division regulators, ion channels, tumor suppressors and transcription factors, among many others. See, Hershko and Ciechanover (1998); Vu and Sakamoto (2000); Conaway, et al. (2002). Because this represents a wide range of substrates, substrates, it is considered as an implication of the involvement of the Ub-proteasome pathway in the progression of the cell cycle, apoptosis, immune response, development, regulation of transcription, signal transduction, and receiver sub-regulation, among other functions. Because path plays a major role in cellular processes, the pathogenesis associated with several diseases, for example Parkinson's disease, cervical cancer, and von Hippel Lindau syndrome, among others, has been linked to aberrations of this trajectory. See, for example, Kitada, et al. (1998); Leroy, et al. (1998); Kato (1999); Swinney (2001). Ubiquitin is a protein of 76 amino acids that seems to be the most conserved eukaryotic structure. It has not been found to be encoded as a monomer but, instead, is expressed as a C-terminal extension protein. For example, two of the mammalian ribosomal proteins are encoded as ubiquitin fusion proteins. All eukaryotic cells are considered to contain potent C-terminal ubiquitin hydrolases, all of which fragment these fusion proteins into the terminal Ubiquitin carboxy (C-). In addition, it has been shown that artificial fusions of the ubiquitin gene product can be expressed and fragmented in eukaryotic cells. It has been shown that prokaryotes, for example E. coli, do not contain ubiquitin or the ubiquitin path. A number of proteins homologous to ubiquitin are known, for example SUMO, Nedd8, ISG15, Apg8, Apg12, FAT10, Urml, Hub, GDX, HCG-I, BMSC-UBP, and UBI, among others. The homology of these Ubiquitin-like Proteins (UBL) ubiquitin is generally limited to about 15 to 30% of their amino acid sequence, and many of them are encoded as precursor proteins and / or to contain C-terminal extensions. These "fusions" produced by eukaryotic cells are processed immediately by highly specific hydrolases, suggesting that UBL hydrolases (proteases) play an important role in the control of the level of mature UBLs. The C-terminus of ubiquitin is covalently conjugated to N-amino groups of target proteins by means of a variety of enzymes. After the binding of an individual molecule to its target protein, a chain of molecules can be generated and extended through covalent conjugation of C-terminals of ubiquitin to one of its 6 N-aminos. ubiquitin. It is considered that polyubiquitination acts as a signal for proteolysis, and it is considered that ubiquitinated proteins are recognized by the proteasome and degraded, and the ubiquitin molecule is recycled. Many UBLs are covalently conjugated to target proteins through isopeptide bonds in a manner similar to Ubiquitin. Although their real functions are not completely understood, it seems that the UBLs are conjugated to their target proteins, and de-conjugated from them, in a highly regulated manner that involves elaborating trajectories that are known in the art. See, for example, Glickman and Ciechanover (2002). It is estimated that there is an excess of 65 isopeptidase genes in the human genome. It is considered that isopeptidases play an important role in the regulation of the fate of UBUBL-modified proteins. For example, ubiquitin can be de-conjugated by means of an isopeptidase. When this occurs, the target protein can no longer be channeled to and recognized by the proteasome for degradation and, therefore, remain undegraded in the cell. Therefore, it is considered that isopeptidases play an important role in the edition of the ubiquitin function and in the cellular pathologies that can develop as a result of it. In addition to acting as a signal for proteolysis, it is considered that the mono-ubiquitination of proteins is involved in the control of several cellular activities, for example endocytosis, chromatin remodeling, signal transduction, and many others. Although the precise role of many UBL conjugations and deconjugations remains individual UBL proteolytic mapped enzymes, eg, isopeptidases and hydrolases, they are clearly involved in the process. This invention will be described generally, as well as specifically by means of examples with specific fusion polymers, for example proteins, Ubiquitin, Ubiquitin-like Proteins (UBLs), both collectively referred to as UBLs, and are all proteolytic enzymes. through phylogeny that recognizes and fragments the C-terminal UBL into a fusion protein. This UBL can be linked to report or signal molecules such as all reporting proteins, all binding pairs, and the like described below by way of examples to form inactive fusion polymers. This patent comprehensively covers all members of a genus, all members of a species, and the like, of the fusion members and proteolytic enzymes insofar as they belong to the functional category assigned and described for a molecule. It is considered that the covalent conjugation of a UBL, for example SUMO, for a Ran-gap protein, for example, controls the translocation of the protein between the nucleus and the cytosol. This type of mechanism can be exploited for therapeutic purposes. For example, SUMOylation of an inactive cystolic transcription factor translocates the active protein to the nucleus, where it can act to activate a tumor suppressor gene. The inhibition of a SUMO protease by means of an exogenous agent, for example a small molecule, in this case could stabilize the SUMO fusion and therefore, exhibit anti-carcinogenic activity. The regulation of a SUMO protease, for example the Ulp 1 function, by means of a small molecule could lead to an anti-carcinogenic therapeutic benefit. Similarly, the conjugation and de-conjugation of other UBLs, for example ISG-15, Apg8, or Nedd8, can be controlled by means of their respective proteolytic enzymes, for example hydrolases (proteases) including UBP43 /, Apg4, and hydrolase NUBI-linked C terminal / NEDPI / UCH-L1 / UCH-L3 / COP9, respectively. This could lead to a regulation of the functions of their respective protein modification trajectories. ISG15 is an important regulator of inflammatory responses to viral infection that is conjugated to key signal transduction regulators. See, alakov (2003). ISG 15 is one of the most strongly induced genes after the treatment of interferon (EFN), and is significantly induced by the influenza B virus, lipopolysaccharide (LPS), and genotoxic stress. It is processed from a 17 kDa precursor, and conjugated to specific proteins in the conserved Ubiquitin-conjugation pathway. An ISG15-specific isopeptidase, UBP43, is also suitable for use with this invention. See, Malakhov et al (2002); Malakhova et al (2002) Ubiquitin is fragmented from substrates by means of proteolytic enzymes generally referred to as ubiquitin isopeptidases, or ubiquitin hydrolases, proteases, or de-ubiquitination enzymes ("DUBs"). Isopeptidases with a family of cysteine hydrolases (proteases) that specifically fragment the ubiquitin-derived substrates of the Ub-X general structure, where X = any number of residual groups ranging from small thiols and amines to Ub and other proteins . See, for example, Dang, et al. (1998). Therefore, it is considered that proteolytic enzymes such as isopeptidases, act to reverse the modification of proteins by ubiquitin or proteins similar to Ubiquitin. See, Wilkinson (2000). Among the families of proteolytic enzyme, there are two main families of isopeptidases: Ubiquitin C-terminal hydrolases (UCH) and Ubiquitin-specific proteases (USP), which are active site proteases. In addition, a family of metalloprotease isopeptidases containing a unique JAMM (Jab 1 / MPN domain) active site isopeptidase is known. See, Lundgren, et al. (2003); Hochstrasser (2002); Verma, et al. (2002); Yao and Cohen (2002). In addition, the existence of a family of cysteine proteases referred to as otubains that appear to be highly specific ubiquitin isopeptidases is also known, although some appear to have sequence homology to known ubiquitin isopeptidases. See, Balakirev, et al. (2003). Proteases specific for ubiquitin-like proteins (UBLs) are also known. It is considered that the UCH enzyme family fragments Ub mainly when present with short C-terminal extensions. However, these proteases can be associated with larger protein substrates, including Ub precursors and Ub adducts with amines and small thiols, and are considered to be involved in cell signaling and nuclear-cytoplasmic transport. See, Layfiel, et al. (1999). The USP enzyme family does not exhibit homology to UCHs, and it can fragment ubiquitin from a range of protein substrates. See, for example, Wilkinson (1997); D'Andrea and Pellman (1998); Wilkinson (1998); Chung and Baek (1999); Yan, et al. (2000). While USPs show significant differences in size and amino acid sequences, they share several highly homologous patches around the residues required for catalytic activity. The sequencing of the human genome revealed 53 USP coding genes and 4 UCH genes. Both UCHs and USPs are potential targets for potential therapeutic intervention. The processes of mono- and p or I-ubiquitination are highly dynamic and are characterized by the rapid addition and / or removal of ubiquitin from proteins. The UCH enzyme family comprises, in general, relatively small proteins, of about 20 to 30 kDa, with some exceptions, for example, UCH37, a proteasome-linked enzyme of 37 kDa, and BAPI, an 81 kDa protein that binds to BRCAI. See, Jensen, et al. (1998). It is considered that its main substrates are Ub precursors and Aducios Ub with small molecules containing amine and thiol residues. Layfield, et al. (1999). UCHL1 and UCHL3 can hydrolyze e-linked amide bonds at the C-terminal Ub as well as a-linked peptide bonds. See, Johnston, et al. (1997). The UCH family includes YUHI yeast, mammalian UCHLI, also known as PGP9.5, UCH-L3, UCH37, Bap1, and many others as well as the corresponding enzyme families of other species. See, for example, Day, et al. (1990); Larsen, et al. (nineteen ninety six). The USP family generally comprises larger proteins, 41 kDa and more, which exhibit little or no homology to UCHs, and fragment ubiquitin from a range of protein substitutes. See, Wilkinson (1997); D'Andrea and Pellman (1998); Wilkinson (1998); Chung and Baek (1999); Yan, et al. (2000). Although most USPs can hydrolyse Ub linear fusions, for example. In the binding of a NH-peptide, it is considered that a main role is the removal of Ub molecules that are conjugated to proteins by means of the e-12-isopeptide bonds by lysine side chains. There are also isopeptidases that are associated with Proteins similar to Ubiquitin. For example, there are several yeast and human proteases, for example ULP1 and Ulp2, and SENP1 and SENP2 that can remove SUMO from the e-amino lysine groups as well as SUMO linear atificial fusions. See, Li and Hochstrasser (1999); Li and Hochstrasser (2000); Gong, et al. (2000). Examples of human isopeptidases are shown in Table 1 below. However, other proteolytic enzymes that recognize the C-terminus of Ubiquitin or a protein similar to Ubiquitin are also suitable, as are similar enzymes from other species, either prokaryotic or eukaryotic.

Table 1: Examples of Human Isopeptidases Proteolytic enzymes such as isopeptidases play important roles in cell survival, proliferation and differentiation. It is considered that a mutation in the Drosophila FAF gene (facets of fat) increases the number of photoreceptor cells in each facet and has a maternal effect on embryogenesis. See, Huang, et al. (nineteen ninety five). FAFs were described to encode a USP that is required for the negative regulation of neuronal cell determination in the development of the compound eye. In addition, FAFs are considered to debunk and stabilize LQF. See, Chen, et al. (2002). The de-ubiquitination enzyme encoded by yeast DOA4 gene is considered to interact with proteasomes, and fragment Ub from conjugated proteins just before they are destroyed by the proteasome, thus recycling Ub. Compatible with this is the main biochemical phenotype of negative DOA4 cells, that is, a decreased content of free and conjugated Ub. Mutant cells have multiple defects, including slow growth and repair of abnormal DNA. See, Papa and Hochstrasser (1993; Pope, Amerik et al., 1999). It is thought that human USP-M is associated with chromosomes, phosphorylated at the onset of mitosis, and dephosphorylated during the metaphase / anaphase transition. , et al., (1999) The des-ubiquitin histone enzyme is considered to affect chromatin condensation, and appears to play an important role in apoptosis, see, Mimnaugh, et al. (2001) .DUB-1 and DUB -2 were identified during the analysis of cytokine-stimulated lymphocyte cell proliferation.The high level of expression of these genes can result in cell cycle arrest.In fact, DUB-1 and DUB-2 can regulate the degradation rate of the critical growth regulators, see Zhu, et al. (1996), Zhu, et al., (1997), USP7 seems to interact with the non-specific transcription activator Vmwl 10, and has also been described as an enzyme, HAUSP, which deubiquitin and stabilizes the p5 tumor suppressor 3. See, Everett, et al. (1997); Li, et al. (2002); Wood (2002). The Unp murine gene has been described as a proto-oncogene, and its overexpression in NIH3T3 cells resulted in transformation. See, Gupta, et al. (1994). In a study of primary human lung tumor tissue, it was shown that human PNU has high levels of gene expression and, therefore, have a causative role for this USP in the neoplasm. See, Gray, et al. (nineteen ninety five). In cell lines, it was shown that UNP protein levels are reduced, thus indicating that UNP is a tumor suppressor gene. See, Frederick, et al. (1998). It was demonstrated that the over-expression of the PTEN tumor suppressor gene over-regulates human UNP. See, Hong, et al. (2000). DUBS inhibitors have characteristic developmental expression patterns, biochemical properties, cellular localization patterns, tissue distributions, preferred targets, and cellular functions. See, Park et al. (2000); Layfield et al. (1999); Cai et al. (1999); Lin et al. (2000); Gong et al. (2000); Park et al. (2000); Hemelaar et al. (2004); Wilkinson (2000); Lin et al. (2001); Li et al. (2002); Hochstrasser (1996); Chung and Baek (1999); Weissman (2001). The following groups of USP substrates have been fully described: Ub precursors, for example fusions of natural occurrence of Ub with ribosomal proteins; Conjugated with mono-ubiquitinated proteins, i.e. proteins not intended for proteasomal degradation but, instead, conjugated by Ub to modify various biochemical properties of the protein, for example complex formation or cellular translocation; poorly ubiquitinated proteins, for example editing; poly-ubiquitinated proteins coupled to the proteasome, for example recycled from Ub; and Poly-ubiquitin chains, for example, monomer disassembly and recycling. In addition, the ubiquitin-proteasomal pathway has recently been validated for drug discovery. The recently approved proteasome inhibitor for multiple myeloma, Velcade, selectively inhibits the growth of several types of cancer cells (Almond and Cohen 2002).; Shah, Potter et al. 2002) (Adams 2002) and has achieved clinical responses (Adams 2002, Adams 2002, Adams 2002). The details of Velcade's therapeutic effect are still to be fully elucidated although they seem to induce apoptosis with selectivity towards cancer cells.

However, its efficacy will probably be limited by toxicities, which negatively impact patient compliance in clinical trials (Adams 2002). The present invention provides means for the improvement in the selection of compounds with a better therapeutic index, whose compound activities are associated with the ubiquitin-proteasome pathway, such as inhibitors USPs and UCHs. In one embodiment, the present invention directs the metabolism trajectories of ubiquitin as a more selective and effective means than the inhibition of all protein degradation as in the case of Velcade®. Proteins similar to ubiquitin and ubiquitin are increasingly implicated in or associated with diseases. For example, neurodegeneration was exacerbated by the addition of SUMO to a pathogenic fragment of the pathogenic protein Huntingtin (Htt) Httex. See, Steffan et al. (2004). The inventors have concluded based on these and other data that a SUMO hydrolase enzyme activator will have therapeutic utility in Huntington's disease.

Methods of the invention The test described in this patent employs any agent or "reporter", for example enzyme, protein, and the like, which requires a free N-terminal amino acid residue in order to produce a signal, for example activity. This agent is inactivated by fusion through its N-terminus to the C-terminus of another protein. By way of example, protease enzymes such as the trypsin family, e.g., Factor X, require a free N-terminal lysine to participate in active site peptide fragmentation. The test of the invention also involves the proteolytic enzyme, for example a UBL hydrolase, and forms a UBL-reporter fusion protein, which can be fragmented by the proteolytic enzyme. This fragmentation of the fusion protein releases Ubiquitin and the Ubiquitin-like Protein (collectively referred to as UBL) or a functional binding fragment thereof that has a free C-terminal, and the "reporter" with an N- free terminal. In different embodiments of the present assay both the UBL and the reporter now in its active form can be detected by a variety of means known in the art, for example with the help of radioactive, chromogenic, fluorescent, phosphorescent, chemiluminant labels and / or substrate. , and others. The test can be conducted with the help of microtiter plates in which the reaction takes place. In another embodiment of the method of the invention designed for screening proteolytic enzyme modulators each compound can be added to a microtiter well, preferably before the other compounds of the reaction mixture. When compared to a control where the fragmentation is complete, a positive sifting or "impact" will be recognized by a signal loss, eg color or fluorescence, indicating that less UBL or reporter has been released. In an embodiment wherein the fusion polymer is a fusion protein, the N-terminus of the reporter protein can be fused to the C-terminus of any of a variety of UBLs or fragment thereof, which can be recognized and fragmented by the proteolytic enzyme, for example hydrolase such as a protease or des-ubiquitinase. In another embodiment, the test of this invention can be conducted with different sources of proteolytic enzyme, for example purified hydrolases, cellular or extracts from which the activity of the enzyme must be purified. In another embodiment, the method of this invention can be applied to the discovery of new proteolytic enzymes from a variety of organisms, by replacing different putative proteolytic enzyme sources and testing their effect on the fusion polymer, for example fusion protein. In a further embodiment of the test of the invention, the agent or reporter may be an inactive precursor protein that is fused to a UBL or active fragment thereof. In this embodiment, the fragmentation of the fusion protein resulting in activation of the protein, which could generate a positive signal based on the activity of the protein, leading to the production of a signal associated with a terminal point, for example a chromogenic terminal point. Examples of such precursors are zymogenes for example fibrinogen and plasminogen, coagulation factors for example prothrombin, and viral polyproteins for example human rhinovirus and poliovirus, among others. In the last example an isopeptidase mediates the fragmentation of a giant polyprotein containing poliovirus RNA-dependent DNA polymerase (3Dpol) in E. coli. This is possible because the RNA-dependent RNA polymerase requires a free N-terminus for activity, and this activity can be easily tested or detected. Accordingly, the poliovirus system can be employed in the present test to determine the activity of ubiquitin isopeptidase because the activities of RdRp and isopeptidase can be coupled. While a cell encodes the proteolytic enzyme as required by the present invention, for example an isopeptidase or a hydrolase (protease), among others known or to be described in the art. These enzymes specifically recognize the UBL sequence and fragment at the junction between the C-terminal UBL and the N-terminal reporter to generate a free N-terminal reporter, the reporter is or can be activated in this manner, which will result in a recordable or detectable signal. Any and all reporter enzymes that meet the requirements set forth above are suitable for use in the test of this invention. Examples of reporter enzymes are provided in Table 2 below for illustrative purposes only.

Table 2: Enzymes Requiring a Free NH2-terminal for Activity In general, the simple peptide bonds differ from the 'isopeptide bonds', whereas the straight peptide bonds have a recurrent -N H-CR-CO-N H-CR'CO- structure, where the carbon COOH is in a position a with respect to the NH-carrier carbon The isopeptide bonds generally have a greater distance between the carbon atom carrying the amino group and the carboxy function, eg isopeptide bonds are generated when at least one of the amino acids involved has the amine group in a non-a position, for example β, y, d, e, and the like, with respect to the carboxyl group, Examples of the latter being amino acids such as an aspartic acid (β-position) ), glutamic acid (position?), and lysine (position e) Many peptidases and isopeptidases are capable of fragmenting linear UBL fusions In another embodiment, the method of the invention that activates a reporter by exposing its N-terminal can and be applied to cells in the construction of a genetic screen. The enzyme Glutamine phosphoribosylpyrophosphate amidotransferase (GPATase) catalyzes the initial stage of purine nucleotide biosynthesis, and is the main pathway regulating enzyme. The GPATase gene is encoded by the purF site of E. coli. See Mei and Zalkin (1990). The elimination of this gene retards the growth of E. coli. When the exogenous purine is added to the medium (adenine) cell growth is restored. In example number 2, it has been demonstrated that the GPATase enzyme can be used as an excellent reporter to monitor the activity of Ub or UBL isopeptidase since the generation of the free N-terminal Cyc in GPATase is essential for its activity. Similarly, other N-terminal nucleophilic hydrolases (Ntn) (See, Table 12) for example asparagine synthetase (See, Andrulis I et al (1989)) and glutamate synthetase (See, Oliver et al (1987)) can also be used. with this invention since its N-terminus is also required for its biological activity. A biological selection method is included herein when by means of Ub-GP ATase or UBL-GP ATase the fusion proteins are transferred to a cell line lacking the purF site. These strains can be grown in media containing adenine. The same strain can also be transformed with a plasmid (s) expressing a Ub protease, for example isopeptidase, or a UBL protease, for example isopeptidase. Cells containing dual plasmids can be cultured in synthetic media, independent of aggregated adenine. Therefore cell growth is regulated by the production of active GPATase by means of Ub or UBL proteases. If a Ub or mutant UBL protease is transformed to a GPATase containing the E. coli strain, the cells will not grow in the absence of adenine, or glutamate or asparagine as is the case for asparagine synthetase and glutamate synthetase respectively. This selection system can be used to clone novel proteases that will fragment Ub-GP ATase or UBL-GP ATase. Similarly, the system can also be used to select an enzyme, for example the best enzyme, from an error-prone PCR library that fragments Ub or UBL fusion proteins to restore growth by generating an active GPATase or other enzyme from choice. Another embodiment of the process uses the fusion protein UB / UBL- for the determination of the effect that the N-terminal amino acids can have on the function of the protein and / or the phenotype. See, Bachmair (1968); Bachmair (1989). The synthetic N-terminal ubiquitin fusion proteins undergo rapid fragmentation in vivo in eukaryotes, in order to produce proteins with designated N-terminal residues. In general, as established by the N-terminal rule, proteins with certain N-terminal residues will be more susceptible to subsequent ubiquitous-mediated degradation. See, Varshavsky (1996). In this particular embodiment, the N-terminus of a fusion protein (s) is modified to vary the stability of the protein and generate different levels of protein in vivo and, in this way, regulate its phenotypes, for example when a cellular function is affected by the level of a protein, for example in yeast, Ardí for sensitivity factor-alpha or Ura3 for survival in deficient medium of uracil. See, Park et al (1992). The present method is applied to affect protein levels in vivo and phenotypes by means of fusions with N-terminal SUMO, ISG15, or NEDD8. In these examples the fusion proteins will be fragmented in vivo and the N-terminal protein residue released will determine the level of that protein and therefore the phenotype associated with the protein (instability as established by the N-terminal rule) . The specific protein residues may be selected to designate fusion constructs according to this invention that will generate a desired level of protein (s), for example an arginine for destabilization, a methionine for stabilization, or other appropriate residue for the desired level of protein. stability in the type of organism used. Yet another embodiment of the method of this invention employs a fusion protein that includes a reporter suitable for optical imaging associated with the proteolytic enzyme in vivo, for example isopeptidase activity. By way of example, deubiquitination enzymes are employed herein as a tool in the detection of tumor using near infrared optical imaging (near IR) of protease activity with the aid of contrast agents that fluoresce only after the interaction with specific enzymes as described by Weissleder (1999), the relevant text of which describes the process and the contact agents that are incorporated in their entirety in the present. Such a proteinase, Cathepsin D, upon fragmentation of a latent or inactive fluorochrome, can release and, therefore, activate a fluorochrome by separating the intramolecular optical fluorescence quenching. See, Ching-Hsuan Tung et al. (1999). Ubiquitin and / or UBLs, therefore serve to reinforce intramolecular optical fluorescence extinction from fluorochromes in a detection probe that can be used to test in vitro, ex vivo and / or in vivo deubiquitination enzymes and activated precise activity and / or inhibited. The use of fluorescent probes of Ubiquitin and UBL is important for early tumor detection and as a follow-up test to map their treatment efficacy because different proteolytic enzymes, for example isopeptidases, have been associated with specific diseases as described below . The UBLs exhibit significant conservation of their structural folds similar to ubiquitin. Its globular structure can be separated into halves, that is, a C-terminal segment and an N-terminal segment. The SUMO molecule, for example, has been separated. When a reporter protein was fused with the C-terminal half SUMO (CTHS) it was not fragmented by a SUMO protease. However, when the CTHS-reporter fusion protein was mixed with an N-terminal half SUMO (NTHS) the enzyme fragments the reporter fusion. Therefore the reporter signal is only observed when the two halves of SUMO are able to associate. When they are associated, the structure is recognized by the enzyme protease, which is then able to fragment and generate an active reporter. In a specific application, the SUMO mode separate from the present test can be used to detect molecules, for example small molecules, such as metabolites, hormones and drugs that bind to specific receptors. Proteases that have fragmentation specificity for ubiquitin and UBLs are also suitable for use as switches or sensors, for example, when a receptor molecule bound to ubiquitin or a UBL CTHS reporter is contacted and bound to a hormone, drug or ligand, among others (collectively referred to as ligands) in the NTHS, and the protein-protein interaction or protein-small molecule leads to rapid fragmentation of the active receptor. This embodiment preferably provides two conditions for a ubiquitin and UBL protease to be an effective switch for the ligand (receptor sensor). The ubiquitin or UBL-receptor is preferably fragmented by a protease when the receptor is linked by its ligand, for example hormone. The linkage of the ligand preferably promotes a change in ubiquitin or the UBL structure that promotes the fragmentation of ubiquitin or UBL by its protease. The binding domain of the estrogen receptor ligand (ER-LBD) is an example of such an application. This modality, although widely applicable, is exemplified herein by reference to the estrogen receptor. The estrogen receptor (ER) interacts with high affinity with a co-activating molecule. This interaction, however, is completely dependent on the binding of the estrogen hormone to the ligand binding domain (LBD) of the estrogen receptor (ER). In this embodiment of the method herein, the binding domain of the ER ligand is expressed as a fusion polymer (protein) with the N-terminal half of SUMO (NTHS) or any other UBL, and the CTHS is fused to a co-activating portion of the protein that has high affinity for the ER. The co-activating reporter fusion protein-CTHS and the ER-LBD-NTHS fusion proteins can be expressed in a cellular system, for example E. coli, and optionally purified. The protein mixture can then be incubated with a test substance in the presence of the SUMO protease. When the estrogen hormone binds to its receptor in the fusion ER-LBD-NTHS promotes interaction with the co-activating molecule. The resulting complex leads to the fragmentation of the reporter, which produces or may cause a signal to occur for the reporter activity. The ability to amplify the reporter signal by means of an enzymatic reaction is the underpinning of the development of this ER-estrogen pair sensor. Since the signal is amplified enzymatically, the sensors that work by complementing separate UBLs, for example SUMO and other UBLs, will detect not only estrogen, but also other hormones and metabolites with greater sensitivity than that achieved by the equipment. Traditional ELISA Co-activators that are dependent on molecular pairs, for example hormone-dependent co-activators, and ligand-ligand receptor pairs, for example hormone and hormone receptor pairs or can be used as sensors for a variety of receptors humans, for example nuclear receptors, to detect extremely small amounts, for example picomolar, of ligands such as estrogen, androgen, thyroid hormone, or 1, 25-dihydroxy vitamin D. For the same reason, all UBLs that are separated into segments anywhere within the cycle between the alpha-1 helix and beta 3 strains, for example halves, which have affinity with one another can be used to construct the sensors with the help of their respective proteolytic enzymes, for example hydrolases. The sensor mode of the present test relies on the production of signal through the generation of a free N-terminal or its coupling to a signal producing event. Isopeptidase enzymes exist in the plant and animal kingdoms, which are considered suitable for use with this invention. The SUMO protease enzyme of yeast (ULPI), for example, is particularly resistant and reliable in its fragmentation properties, and has been used in most of the illustrative description in this patent. See, for example, Malakhov (2004). This modality of the present test was validated using other known isopeptidases for several UBLs, a requirement that depends absolutely on a given signal in the generation of a free non-fused reporter amino terminal, which in this example is generated by the enzyme SUMO protease of isopeptidase (ULPI). Any inhibition of the isopeptidase activity achieved by a modulator, for example screened compounds, results in the attenuation of the enzymatic activity. Other examples of combinations of elements for carrying out the test of the invention mentioned herein are Poliovirus 3D RNA-dependent RNA polymerase fused to SUMO and fragmented with ULP 1, Glutamine phosphoribosyl pyrophosphate amido-transferase (GPAT) fused to SUMO and fragmented with ULPI , Tríptasa fused for SUMO and fragmented with ULPI, and Phospholipase A2 fused for yeast and human SUMO, Ubiquitin, Nedd8, Ruby, and ISG15 and fragmented with ULPI, Senp2, USP2, Den1, and several cell extracts. These are only examples of the numerous combinations suitable for use in the test and equipment of the invention. Therefore, the present invention is provided in this patent in the form of several principal embodiments, which include applications of UBL-reporter fusion polymers to monitor the activities of Ubiquitin and Ubiquitin hydrolases-like protein, eg proteases, polymers UBL-reporter fusion, and reporter-free reporter structures as probes to monitor proteolytic enzymes such as UBL hydrolases, for example proteases, and their activities in man and other mammals and other animals, cells, tissue and cell fractions. Primordial among these is the use of UBL-reporter fusion polymers, and the determination of proteolytic enzyme activity, such as UBL hydrolase, for example protease, activities as selectable markers for eukaryotic and prokaryotic organisms. Another important modality is based on UBL-reporter molecules, for example enzymes, and their chimeric structures as sensors enabled by fragmentation with appropriate proteolytic enzymes, for example hydrolases and proteases, to detect the protein-protein interaction and small molecule-protein interactions. In another modality, UBL-reporter enzymes are used in the elaboration of equipment to monitor the activities and to test the proteolytic enzymes, for example UBL hydrolases and proteases. Some of the most preferred embodiments of the present invention use a variety of "reporter" structures, such as enzymes, polymerases, proteases, lipases, acylases among others, which require specific N-terminal residues for their report activity. Other modalities are based on the expression of fusion polymers, where the C-terminal of the UBL is linked to the N-terminal of the reporter in a fusion polymer that transports an inactive form of the reporter, and where a reporter structure is generated Free N-terminal by means of the action of the proteolytic enzyme, for example UBL hydrolase or protease to provide the reporter activity. The reporter can be enzymes, or other signal producing entities either by themselves or through interaction with other entities.

Another preferred embodiment employs reporter UBL fusion polymers, for example proteins, obtained or expressed in and purified from any source, for example E. coli, and fragmented, for example in vitro, by means of the proteolytic enzyme such as a UBL hydrolase, protease or isopeptidase to generate the active signal produced by the active reporter or enzyme. The signal thus produced and / or the reporter activity of the enzyme can be used as a measurable output of the proteolytic enzyme, for example UBL hydrolase or protease, the activity in a high productivity screening method and equipment to identify modulators of activity of the enzyme, such as small molecules that inhibit or activate the proteolytic enzyme, for example hydrolase or protease. In a further embodiment, proteolytic enzymes such as UBL-hydrolases or proteases can be used to generate active reporter signals, for example signal-producing enzymes, in vivo and to develop cell-based assays for specific proteolytic enzymes such as UBL hydrolases and proteases. In a more preferred embodiment, the U BL-reporter fusion polymers can be used as integrated transgenic constructs within the cell chromosomes for use as sensors in order to track the activity of specific proteolytic enzymes, for example hydrolases and proteases, in various organisms , cells, bacteria, fungi, and animal and plant tissues and cell fractions. Another embodiment uses UBL-pro-reporter structures, such as pro-enzymes, as affinity agents or matrices for various proteolytic enzymes, such as UBL hydrolases and proteases. In addition, the UBL-reporter fusion structures can be used in the present test and equipment as tools for discovering novel proteolytic enzyme structures, for example UBL domain structures. The method and equipment herein may also be used with the aid of mutant or modified Ubiquitin or UBL structures, or linker functional segments thereof, and UBL mutant reporter polymers to discover and invent improved novel proteolytic enzymes, for example UBL hydrolases and proteases. In an extremely preferred embodiment of the invention, the test and kit are based on UBL-reporter fusion structures employed as selectable markers for eukaryotic and prokaryotic growth and / or phenotype detection. A further embodiment provides a method and equipment employing UBL-reporter fusion structures as probes for screening novel UBL-reporter enzymes. These equipment will include the UBL-fusions along with other reagents to test the activity of proteolytic enzymes, such as UBL hydrolases and proteases. In a different embodiment, this patent provides a method and equipment that employs UBL-reporter fusion structures as tools to produce better signaling reporters, for example enzymes. Another important embodiment provides a method and equipment of the invention that uses a reporter that has a special requirement for its free N-terminal, as is the case of an enzyme, for example a tautomerase, which requires proline as the active N-terminal. . In this case the methods and equipment used by the reporter in the UBL-N-Proline reporter fusion polymers to discover novel proteolytic enzymes, for example hydrolases and proteases that are specific for UBL-proline fusion bonds. This patent provides a clear improvement over the prior art technology in the form of a test and equipment to identify and test "reporter (s)" enzyme (s) that are (are) inactive in their fused ubiquitin configuration or UBL if it is fused (n) through its N-terminus, and activated (s) to fragmentation by means of a ubiquitin or UBL protease due to the release of its N-termini. Table 2 above lists examples of several classes of reporter enzymes that require a free N-terminus for biological activity. In many cases, the N-terminal residue is part of the catalytic site or is essential for the catalytic mechanism. For these reasons, the fusion of its N-terminal to ubiquitin or UBLs inactivates the enzymes in a reversible manner, while the removal of ubiquitin or UBLs rapidly restores its activity. The generation of an active reporter enzyme is, therefore, a direct function of the respective protease. In a further embodiment, the Ubiquitin and UBL-reporter fusion genes can be transferred to any organism through known means, and optionally integrated into the host chromosome, to thereby generate transgenic plants and animals. Since many of the enzymes listed in Table 2 have unique substrates, they can be employed in a signal production test, for example a fluorescence or chromogenic signal, in tissues or cells where said enzymes are present. Therefore, the UBL-reporters coupled with easily detected substitutes serve as novel biochemical and genetic markers, reporting unique activities of proteolytic enzymes, for example isopeptidases and proteases, in situ. The Ubiquitin and UBL fusion genes of this patent are also useful as selectable markers. By way of example, an N-terminal fusion of the E. coli glutamine-PRPP-amidotransferase gene (GPAT) that is essential for purine biosynthesis for a Ubiquitin or UBL fusion gene may be linked to a vector, for example a plasmid, and used to transfect cells whose chromatomal GPAT gene is deleted or mutated. The GPAT enzyme requires a free N-terminal to be active, isopeptidase fragmentation of the fusion protein, therefore, it would be required to restore the biosynthetic pathway of novo purine. Cells containing said UBL marker gene would allow the selection of plasmids carrying the appropriate isopeptidase or protease genes. Therefore, the protease gene would act as a switch to activate the protein essential for cell viability. The ubiquitin proteins and inactive purified UBL-reporter fusion proteins can be used, for example, for in vitro screening of novel proteolytic enzymes, such as proteases and isopeptidases. The test herein is also suitable for monitoring the activities of a mixture of proteases exhibiting selective fragmentation of ubiquitin or one or more members of the UBL family. A SUMO-X reporter, for example, may be an excellent substrate for ULPI (SUMO protease) whereas a SUMO-Y reporter may be an excellent substrate for Ulp2, a second, different SUMO protease. More specifically, this patent provides in a first aspect of the invention a method for determining proteolytic enzyme activity comprising providing a fusion polymer comprising a first polymer comprising Ubiquitin or a Ubiquitin-like Protein (UBL) or a C segment. - functional terminal thereof and a second polymer comprising a free N-terminal amino acid required for detection; wherein the first and second polymers are operably linked to each other through the C-terminal UBL and the second N-terminal polymer; contacting the fusion polymer with the proteolytic enzyme or a sample comprising or presumed to comprise the enzyme that is fragmented in the C-terminal UBL; detecting a signal associated with any amount or activity of fragmented polymer; and establishing a correlation between the fragmented polymer signal for the proteolytic enzyme activity. In one embodiment, the method described above can be implemented in a variety of ways and for different purposes.

The method may also include the normalization of each enzyme or sample fragmentation signal by reference to 0% and 100% fragmentation signals and assigning a proteolytic enzyme activity value to each enzyme or sample thereof; wherein, in the case where the enzyme activity obtained is below a cut-off value it can be said that the enzyme or sample is inactive and when it is above the cut-off value it is active. Fragmentation signals of 0% and 100% can be obtained through methods known in the art, one being the repetition of the contacting, detection and establishment steps for complete fragmentation and complete inhibition of proteolytic enzyme activity at order to obtain the fragmentation signals of 100% and 0%. The normalization step is commonly conducted through the normalization of each enzyme or sample fragmentation signal by reference to an activity curve of the enzyme fragmentation values and allocating a proteolytic enzyme activity to each enzyme or sample thereof.; wherein, when the activity of the obtained enzyme is below a cut-off value it can be said that enzyme or sample is inactive and when it is above the cut-off value it is active. However, other means of normalization for a space and / or control, and the like, are also considered. In another preferred embodiment, the first polymer may comprise the proteins ubiquitin, SUMO, Nedd8, ISG15, Apg8, Apgl2, FAT10, Urml, Hub, UBi, Ruby, ISG15, among many others, or a functional C-terminal segment they comprise an amino acid within the UBL cycle that binds its a-helix 1 and β-3 to the C-terminal strain. In another embodiment the second polymer can comprise a variety of different constructs, a reporter protein being most preferred. enzyme, the transcription factor or functional signaling fragment thereof, and / or the first polymer may comprise a UBL, i.e. Ubiquitin or a Ubiquitin-like Protein, or a functional C-terminal segment thereof which comprises a amino acid within the UBL cycle that binds its a-helix 1 and β-3 to the C-terminal strain. In one form of the method either the first or second polymer, or both, becomes (are) detectable (s) fragmentation of the proteolytic enzyme. The first and / or second polymer (s) can be made detectable through the activation of a signal that carries (n) and / or through the link to the generation of a detectable signal. Commonly, the signals are a radioactive, fluorescent, phosphorescent, chromogenic, sonogenic, or chemiluminescent signal. However, any other type is equally within the confines of this patent. In a particularly preferred embodiment, the method may also include obtaining an N-terminal UBL segment comprising an amino acid within the UBL cycle that binds its a-helix 1 and β-3 strain to the C-terminus, or the remaining amino acid to form a UBL with the C-terminal UBL segment; the N-terminal UBL segment that is operatively linked to one of the first and second link pairs; and get the second link pair. In this form the fusion polymer is commonly fragmented to the bond of the first and second binding pairs, which preferably comprise a receptor and a receptor binding agent. Other receptor binding agents may comprise a drug, hormone, antigen, ligand, or receptor binding functional fragment thereof; as the corresponding receptor a drug receptor, hormone receptor, antibody, ligand binding receptor, or functional binding fragment thereof. In this modality the binding of UBL N- and C-terminals allows the recognition of a UBL conformation and polymer that is fragmented in the C-terminal by means of the proteolytic enzyme. Of particular importance is a form of the method in which the first and second polymers are covalently linked to one another. In another the first and second polymers are commonly linked operatively to each other through a linker, which can comprise at least one amino acid. A more preferred embodiment of the method the fusion polymer comprises a fusion protein, and the proteolytic enzyme commonly comprises an isopeptidase or a fragment functional fragment thereof. Examples of proteolytic enzymes are a C-terminally hydrolase ubiquitin and a ubiquitin-specific protease or functional fragment fragment thereof. Other examples among the many constructs comprising one or more functional fragmentation enzymes such as ULP1, ULP2, SENP1, SENP2, yeast YUHI, mammalian UCHL1, UCH-L3, UCH37, Bapl, USP-M, DUB-1, DUB- 2, USP7, UNP, CYLD, CYLDI, KIAA0849, USP9X, DFFRX, USP9, FAFX, USP9Y, DFFRY, USPIO, FAFY, OTUBI, OTBI, OTUI, HSPC263, OTUB2, C14orfl37, OTB2, OTU2, USPIO, KIAA0190, USP11, UHXI5 USP12, UBHIm, USP12L1, USP13, ISOT3, USP14, TGP, USP15, KIAA0529, USP16, UBPM, USP18, UBP43, USP19, KIAA0891, ZMYND9, USP20, KIAA1003, LSFR3A, USP21, USP23, NEDD8-specific protease, USP22, KIAA1063, USP24, KIAA1057, USP25, USP26, USP28, USP28, USP29, USP32, USP32, USP32, USP32, KIAA1097, VDUI, USP35, KIAA1372, USP34, USP36, KIAA1453, USP37, KIAA1594, USP38, KIAAI 891, USP40, USP42, USP44 , USP46, USP49, USP51. UBPI, USPI, UBP2, USP2, UBP41, UBP3, USP3, UBP4, USP4, UNP, UNPH, UBP5, USP5, ISOT, UBP6, USP6, TRE2, UBP7, USP7, HAUSP, UBP8, USP8, KIAA0055, UBPY, VCIP, VCIP135, KIAAI 850, Cezannel, Cezanne2, A20, UCH-LI, Park5, UCH-L3, UCH-L5, UCH-37, ATXN3, ATX3, MJD, MJDI, SCA3, POHI, PSMD14, CSN5, COPS5, JABI, SENPI , SENP2, SENP3, SSP3, SUSP3, SENP5, FKSG45, SENP6, FKSG6, KIAA0797, SSPI, SUSPI, SENP7, KIAA1707, SSP2, SUSP2, SENP8, VCIP, VCIP135, KIAAI 850, A20, UCH-LI, Park5, UCH- L3, UCH-L5, UCH-37, ATXN3, ATX3, MJD, MJDI, SCA3, POHI, PSMD14, CSN5, C0PS5, JABI, SENP2, SENP2, SENP3, SSP3, SUSP3, SENP5, FKSG45, SENP6, FKSG6, KIAA0797, SSPI, SUSPI, SENP7, KIAA1707, SSP2, SUSP2, SENP8, FKSG8, PRSC2, DUBI, DUB2, DUB3, or DUB4, or functional fragments of fragmentation thereof. Although several examples have been provided, many others known in the art, and which will be discovered, are also suitable insofar as they exhibit the required characteristics. The method of this invention can be practiced with a second polymer comprising a "reporter" or signal producing construct comprising an enzyme such as serine protease, prohormone precursor, subtilisin / prohormone convertase similar to kexin, carboxypeptidase, a DNA-like disintegrin-metalloprotease domain (reprolysin type) with TromboSpondin type I motif (ADAMTS), a Desintegrin and metalloprotease (ADAM) domain, cysteine aspartase, aspartic proteinase, matrix metalloproteinase (MMP), RNA polymerase RNA -dependant, N-terminal nucleophile (Ntn) hydrolase, 4-oxalocrotonate tautomerase, corismate synthase, β-lactam acylase, reverse transcriptase, phospholipase, transcription factor, or a functional fragment of binding and signaling thereof. Other examples are those in which the second polymer comprises a viral reverse transcriptase, sigma transcription factor, Glutaminphosforibosylpyrophosphate (PRPP) amidotransferase (GPATase), coagulation factor Xa, 3Dpol RNA-dependent RNA polymerase, glutamine 5-phosphoribosyl-l-pyrophosphate amidotransferase, penicillin acylase, reverse transcriptase, corismato synthase, tryptase, chymase, enterokinase, transcription factor s ?, thrombin, dipeptidyl peptidase, HtrA2, neurophysin, vasopressin, furin, carboxypeptidase B, carboxypeptidase Y, vW F-protease fragmentation / ADAMTS 13, ADAM 1, ADAM 2, caspase, pepsin, renin, cathepsin D, virus proteinaza of monkey Mason-Pf izer, MMP20, MMP26, glycosilasparginase, 20S proteasome ß subunit, glutamine PRPP amidotransferase, YdcE, YwhB, cephalosporin acylase, reverse transcriptase CaMV, phospholipase A2, or a fragment of binding and signaling thereof. The method of this invention can also be practiced by additionally expressing a polynucleotide that encodes the fusion protein under conditions effective for expression thereof as a means to provide the fusion protein in situ. Said polynucleotide can be expressed in a prokaryotic cell as well as eukaryotic or in a tissue or reaction or extract thereof. In this way, the fusion protein thus obtained can be isolated, and optionally purified before the enzyme test. In addition, the method may further comprise repeating the steps of contacting, detecting and establishing in the presence of a sample that is assumed to comprise a modulator of proteolytic enzyme activity, and determining a value for the effect of the sample on enzyme activity. proteolytic by reference of the sample signal for the corresponding activity of the enzyme signal obtained in the absence of the sample. The sample subjected to the test commonly comprises a physiological fluid, a tissue sample, cell, cell fraction, or extracts or fractions thereof. Clearly, one or more of the steps can be conducted in vitro, in vivo, ex vivo, in cell or tissue culture, in cell or tissue reaction extracts, among others. Although several signals and detection methods can be employed, detection of cell growth, or the use of chromogenic, radioactive, fluorescent, phosphorescent or chemiluminescent signals are common. The steps of contacting, detecting, establishing and determining can be conducted separately for a plurality of samples that are assumed to comprise a modulator of proteolytic enzyme activity to obtain a value for the effect of the sample on the activity of proteolytic enzyme. , and the method can even be automated. In the latter mode, the collection, processing and generation of the information obtained for each sample and controls can be computerized. The texts of the provisional application of the United States of America 60 / 580,900 and of the publications WO 03/057174 A2 and WO 2005/003313 A2 are incorporated in their entirety in this patent in order to provide sources, methods of preparation, examples and other conditions and elements suitable for the activation of products, their parts and processes used in the present invention. As described above, the method of this invention can be applied to determine the presence, or discovery, of a variety of proteolytic enzymes, modulators, and other regulators in vivo in a plant or animal model. For this purpose, the inventors have designated a transgenic cell, plant or animal, comprising a Ubiquitin- or BL-reporter fusion gene that is optionally integrated into the chromosome of the cell, plant or animal. In other embodiments, the fusion polynucleotide may comprise a polynucleic acid encoding a C-terminal segment of Ubiquitin or a UBL having the characteristics described above. In another embodiment, the "switch and sensor" mode of the invention may also be incorporated in this aspect. The fusion gene can be constructed either as described in this patent or through the various methods known in the art. See, Sambrook, et al. (1989). It can then be cloned into a vector, for example a plasmid and transfected into a cell, plant or animal. Also provided in this patent is a device for determining the activity of proteolytic enzyme, whose equipment in its basic form comprises a fusion polymer comprising a first polymer comprising Ubiquitin or a protein similar to Ubiquitin (UBL) or a C-terminal segment thereof and a second polymer comprising a polypeptide that requires a free N-terminal amino acid for detection; wherein the first and second polymers are operably linked to each other through the Ubiquitin or C-terminal UBL and the second N-terminal polymer; and instruction to conduct the proteolytic enzyme test, detecting a signal associated with the amount or activity of the first and / or second polymers, and establishing a correlation of the detected signal for the proteolytic activity of the enzyme. The kit can optionally include a source of the proteolytic enzyme that is cleaved in the C-terminal UBL, or the enzyme can be acquired separately. Similarly, another characteristic of the equipment may be the incorporation of plastic material, reagents, and the like, for the implementation of the invention. The equipment may additionally include one or more first and second link pairs, wherein the first pair may be operably linked to an N-terminal UBL segment, and wherein when the first and second link pairs are linked one to the another the C-terminal UBL segment is linked to the N-terminal UBL segment allowing a UBL conformation, the proteolytic enzyme fragments the fusion polymer; reagents for conducting the enzyme fragmentation step; means for conducting the detection step; and means for correlating the detectable signal for proteolytic enzyme activity or change thereof. In this form of the kit the fusion polymer commonly includes a fusion protein, or alternatively a fusion polynucleotide that encodes the fusion protein, and optionally one or more reagents to express the polynucleotide and / or a cell (s) or fraction or extract thereof to express the polynucleotide when it is used in place of the fusion protein. A more complete form of the equipment may contain means for separately containing a plurality of samples, and instructions for conducting the test automatically. In this way the team incorporates means to automatically process data of each sample, and instructions for its use. Another application of the present invention is a method for screening compounds for their effect on proteolytic activity, and the method can be practiced by obtaining a fusion polymer comprising a first polymer comprising Ubiquitin or a protein similar to Ubiquitin. (UBL) or a functional C-terminal segment thereof and a second polymer comprising a free N-terminal amino acid; wherein the first and second polymers are operably linked to each other through N-C-terminals; contacting the fusion polymer with a proteolytic cleavage enzyme UBL C-terminal under conditions effective for fragmentation to occur; detecting a signal associated with a fragmentation amount to obtain a fragmentation signal of 100%; repeating the contacting and detection steps in the presence of a total inhibitor of proteolytic enzyme activity to obtain a 0% fragmentation signal; obtain a set of compounds; repeating separately the steps of obtaining the fusion polymer, contacting and detecting in the presence of each compound to obtain a fragmentation signal; normalize each compound fragmentation signal by reference to the fragmentation signal of 0% and 100% and assign a value of proteolytic enzyme activity to each compound. The above-described method can be practiced by conducting the contacting and detecting steps with a known proteolytic enzyme activity modulator to obtain a point fragmentation control signal in place of the 0% fragmentation signals and 100%; determining a value of proteolytic enzyme activity for the modulator by reference to the activity value of the corresponding enzyme obtained in the absence of the modulator; and normalizing each fragmentation signal of the compound by reference to the control fragmentation signal and assigning a value of proteolytic enzyme activity to each compound. Said modulator commonly comprises an activator or inhibitor of proteolytic enzyme activity. In this form of the method one or more of the in vitro stages can be conducted, in vivo, ex vivo, in cell or tissue culture, in cells or in tissue fractions or extracts. Some of the parameters that can be observed for detection include cell growth, or chromogenic, radioactive, fluorescent, phosphorescent, sonogenic, or chemiluminescent detection signals. In one embodiment of this method the step of one embodiment of this method the normalization step can be conducted by normalizing each fragmentation signal of the compound by reference to a curve of the fragmentation values of the activity of the enzyme and assigning an activity of proteolytic enzyme to each compound; wherein, when the enzyme activity obtained is below a cut-off value, it can be said that the compound is inactive and when it is above the cut-off value it is active. In this mode, the cut-off value can be selected to be 50% of proteolytic enzyme activity, and when the enzyme activity in the presence of the compound is decreased by at least 50% it can be said that the compound is an inhibitor , and when the enzyme activity is improved by at least 50%, the compound is an enhancer. In addition, the method can include the determination of a concentration of the compound that inhibits (IC50) and / or improves (EC50) the enzyme activity by 50%, and comparing the IC50 and / or EC50 of the compound to determine its activity of the enzyme resistance as an inhibitor and / or enhancer. This form of the method can be applied to a library of compounds, and all IC50 and / or EC50 compared to determine the relative strength of the compounds with respect to each other. As described above, the first polymer generally comprises ubiquitin, SUMO, Nedd8, ISG15, Apg8, Apg12, FAT10, Urm1, Hub, UBi, Ruby, ISG15, or a functional C-terminal bond segment comprising an amino acid within the UBL cycle that binds its a-helix 1 and β-3 to the C-terminal strain. The latter is particularly useful in the "switch and sensor" mode of the invention, also employing the N-terminal segment of the proteins, which can be linked in a common manner to one of a link pair, the second member of the pair of link that could be linked to a co-activator that facilitates the libration of the N-terminal segment to link to the C-terminal segment corresponding to the link of the members of the pair with each other as described in this patent. The remaining elements for practicing this form of the method of this invention are as described above and need not be repeated for this particular application. In this way, the method also includes obtaining a functional UBL N-terminal segment of linkage comprising an amino acid within the UBL cycle that links its a-helix 1 and β-3 to the N-terminal thereof, where, when the N-terminal segment and the C-terminal segment are linked together form a complete UBL, and the N-terminal UBL segment which is operatively linked to one of the first and second link pairs and, as described, the obtaining of the second link pair; wherein the fusion polymer, for example protein, is fragmented in a manner common to the binding of the first and second binding pairs, whether or not aided by a co-activator. As previously described, this form of the test can also be carried out by obtaining the fusion protein through the expression of a polynucleotide that encodes it in situ, either in a prokaryotic cell model or eukaryotic or a transgenic plant or animal transformed with the fusion polynucleotide described above. The fusion protein thus obtained can be isolated, and optionally purified. This form of the method can also be automated and the collection, processing and generation of report on the information obtained for each modulator and controls is executed in a computerized form by means of the appropriate software that is commercially available, or it can be designed without greater complexity. The above-described method can be carried out with the aid of a proteolytic enzyme activity modulating screening apparatus, which can comprise a fusion polymer comprising a first polymer comprising Ubiquitin or a protein similar to Ubiquitin (UBL) or a C-terminal segment thereof and a second polymer comprising a polypeptide that requires a free N-terminal amino acid for detection; wherein the first and second polymers are operably linked to each other through the Ubiquitin or C-terminal UBL and the second N-terminal polymer; and instruction to conduct the proteolytic enzyme test, detecting a signal associated with the amount or activity of the first and / or second polymers, and establishing a correlation of the detected signal for the proteolytic activity of the enzyme for a plurality of modulators and controls; and optionally a source of the proteolytic enzyme that is fragmented in the Ubiquitin or C-terminal UBL. This form of the equipment may also be provided with one or more of the first and second link pairs, wherein the first pair may be operably linked to an N-terminal UBL segment, and wherein, when the first and second link pairs are link to each other the UBL C-terminal segment links to the N-terminal UBL segment allowing a UBL conformation, the proteolytic enzyme fragments the fusion polymer; reagents for conducting the cleavage of C-terminal UBL enzyme from the fusion polymer; means for detecting a signal (s) emitted by the first and / or second fragmented polymers; and means for correlating the detectable signal (s) for proteolytic enzyme activity or changing it by reference to a control. A preferred form of the kit is that when the fusion polymer comprises, or is, a fusion protein, with the kit further comprising a fusion polynucleotide that encodes it and is replaced by the fusion polymer, and optionally one or more reagents for expression of the polynucleotide, and / or a cell (s) or fraction or extract thereof to express the polynucleotide when it is used in place of the fusion protein. A particularly important form of the equipment also incorporates means for separately containing a plurality of samples, and instructions for conducting the test automatically, may also contain means for automatically processing the data for each sample; and instructions for its use. This patent also provides a transgenic cell, plant or animal, comprising Ubiquitin- or UBL-reporter fusion polynucleotide that is optionally integrated within the chromosome of the cell, plant or animal; wherein Ubiquitin or Ubiquitin-specific proteolytic enzyme, for example isopeptidase, is associated with a specific disease or condition or family thereof. The transgenic cell, plant or animal is capable of expressing a Ubiquitin- or UBL-Reporter fusion protein associated with a specific disease or condition or family thereof. It can be obtained by forming a hybrid vector by cloning the fusion polynucleotide into a vector, and transfecting the cell, plant or animal with the hybrid vector. In one preferred embodiment the vector comprises a plasmid, and the cell comprises a eukaryotic cell. However, other vectors and types of cells are also suitable, including prokaryotic cells. The transgenic cell, plant or animal of this invention can be modified to serve as a cell, plant or animal model for a disease or condition. In specific modalities suitable for the diagnosis of a specific disease or condition Ubiquitin or U BL-isopeptidase is associated with an auto-immune, neoplastic, metabolic, vascular, neurodegenerative or other genetic disease or condition, although this is not a fully inclusive list . All other known diseases or to be determined to interact with a UBL-specific Ubiquitin or Isopeptidase in order to produce a signal are also included in the present application. Specific examples of the diseases and conditions are cancer, for example breast cancers, prostate, and cancers associated with von Hippel-Lindau disease which predisposes to a number of cancers such as hemangioblastomas, pheochromocytomas, and cystadenomas as well as other diseases such as such as lupus, diabetes, IBD, Parkinson's disease and cardiovascular disease. Examples of isopeptidase / deubiquitination enzymes associated with disease include the following: VDU1 / 2 v Cancer Von Hippel-Lindau disease is a hereditary carcinogenic syndrome caused by mutations of the VHL gene germination line. See, Sims (2001). It predisposes those with the disease to several tumors, including hemangioblastomas in the CNS and retina, clear cell renal carcinomas, adrenal pheochromocytomas, pancreatic tumors, cystdenomas of the epididymis, and tumors of the inner ear. See, Li et al (2002); Maher and Kaelin (1997). The VHL protein (pVHL) is associated with elongin C, elongin B, and cullin-2 to form a complex, VCB-CUL2, which acts as a ubiquitin E3 ligase. See, Lisztwan et al (1999). Because mutated pVHL is associated with malignancies, ligase can be considered to be a tumor suppressor and its substrates potential oncogenic molecules. The hypoxia-inducible factor (HIF-a), known to be a substrate of VCB-CUL2, plays a role in the development of hemangioblastomas, and similarly in tumor angiogenesis in general, through VEGA induction, see Ohh et al (2000); Tyers et al (1999) and Benjamín et al (1997). Also among its substrates is a ubiquitin isopeptidase, VDUI, found by hybrid screening of yeast 2 to interact with pVHL. A highly homologous protease, VDU2, is also known; Although it has not been studied in terms of association of pVHL, VDU2 has physiological substrates in common with VDUI. See, Curcio-M or re 11 et al (2003). The ß-domain region of pVHL, site of naturally occurring mutations, is the site of VDUI interaction, and VDUI can be co-immunoprecipitated in the VCB-CUL2 complex. The ubiquitination and degradation of VDUI by means of a pVHL-dependent pathway is eliminated by VHL mutations that alter the interactions with VDUI. Therefore, targeted degradation of VDU 1 by pVHL is important in the suppression of tumor formation and / or maintenance, and VDUI may have oncogenic activity that is discovered in the absence of functional ligase. Therefore, VDUI is important in neoplastic diseases characterized by mutated pVHL (100% of patients with VHL (autosomal dominant disease), and 50-80% of the largest number of patients with sporadic renal cell carcinoma. , Stolle et al (1998), Gnarraet al (1994) Inhibition of VDUI functionality mimics the activity of the wild-type tumor suppressor pVHL.

USP7, USP2a and Cancer Deubiquitination enzymes can serve to restore certain proteins, or at least prolong their cell life time by removing the initial ubiquitin tag, thus preventing proteasomal degradation. In said isopeptidase, USP7 also known as HAUSP, it is known to stabilize the p53 tumor suppressor. See, Li et al (2002). Another isopeptidase, USP2a, has been implicated in the regulation of fatty acid synthase (FAS), a molecular identification of prostate cancer. See, Rossi et al (2003); Agostini et al (2004); Graner et al (2004). USP2a is regulated by androgen and is expressed in prostate cancer, and is therefore an oncogenic protein. Therefore, depending on the roles of their substrates, the deubiquitination enzymes can be activated or inhibited to achieve the therapeutic effect.

Isopeptidase T and Cardiovascular Disease The de-ubiquitination enzyme Isopeptidase T is sub-regulated in patients with chromosome 22 elimination syndrome q I 1, which encompasses a variety of heart failures. See, Yamagishi et al (1999). Together with UFD 1, isopeptidase T is sub-regulated in myocytes from patients with heart failure. See, Kostin et al (2003). This isopeptidase is known to remove polyubiquitin chains from ubiquitin-protein conjugates and stimulates protein degradation, and its absence results in the accumulation of polyubiquitinated proteins and an alteration of the degradation pathway of ubiquitin-proteasome, leading to this way to autophagic cell death. See, Hadari et al (1992); Johnson et al (1995); Stefanis et al (2001).

AMSH Isopeptidase Motive JAMM and Lung Disease and Cancer A protein containing the JAMM domain is linked to the signal of the transduction signal associated with the endosomal classification, that is, the transfer between the membrane and the ensdosomal / lysosomal compartments of the EGF receptor (EGFR). This protein, AMSH (Molecule associated with the SH3 domain of STAM, a protein that regulates the classification of receptor in the endosome). See, McCullough et al (2004); Clague and Urbe (2001). The EGFR regulates numerous cellular functions by initiating signal transduction cascades. See, Lockhart and Berlin (2005); von Ahsen and Bomer (2005); Le Roy and Wrana (2005); Spano et al (2005). During the cellular life of the EGFR, it is recycled from the membrane to the initial endosome (classification), before finally being selected to classify the endosome and lysosome ends, where it is degraded by acidic proteases. EGFR participates in signal transduction both in the membrane and in the initial endosome compartment. While much of the signaling is involved with the regulation of cell growth and other functions, a component of signal transduction regulates the transfer of EGFR. The E3 ligase Cbl mediates the ubiquitination of phosphorylated EGFR. Subsequent signaling events result in receptor degradation in the final endosomes / lysosomes. Ub-EGFR is recognized by the Hrs protein on the endosomal surface, and additional interactions with the endosomal-associated complex required for transport (ESCRT) mediated by ubiquitin results in translocation to internal vesicles of the multi-vesicular body (MVB), compromising the EFGR for the degradation of the protease in the lysosome. The degradation, the final result of Cbl-mediated ubiquitination of EGFR, can be eliminated by a ubiquitin isopeptidase, AMSH, for example ablation of AMSH activity by incubation of cells with if RNA leads to increased EGFR degradation; Purified AMSH des-ubiquitin EGFR-Ub in vitro. See, McCullough et al (2004). GFR kinase inhibitors and receptor binding antagonists are currently in clinical trial for several cancers. See, Ciardiello and Tortora (2001); LoRusso et al (2003). Other disease areas with critical needs not covered are also associated with EGFR activity, with an airway inflammation and mucosal hypersecretion associated with bronchial asthma. While asthma is a multifactorial disease that damages the bronchial epithelium associated with leukocyte infiltration and increased airway response, these characteristics are consistent. See, Puddicombe et al (2000). The EFGR system has been postulated to play important roles in the growth and differentiation of the cellular types of epithelial and connective tissue in the lung. EGFR and its ligands are elevated during the pathogenesis of asthma, and the induction of this system correlates with goblet cell hyperplasia in asthmatic areas. See, Takeyama et al (2001). Any intended repair of epithelial cell damage leads to hyperproliferation and differentiation responses that are linked to EGFR and EGFR activation. See, Bonner (2002). Asthmatics appear to develop chronically elevated levels of EGFR even in undamaged epithelium. This maintains a constant inflammatory condition, and leads to fibrosis and mucosal hypersecretion associated with airway obstruction, morbidity and lethality in asthma, COPD, and other lung diseases.

UCHL1 v Parkinson's disease UCHLI, or ubiquitin carboxy terminal hydrolase, is genetically associated with PD Parkinson's disease. See, Chung et al (2003); Toda et al (2003); Maraganore et al (2004). Mutations in UCHLI cause autosomal dominant PD, consistent with the notion that disorders in the ubiquitin proteasomal pathway play important roles in the demise of dopamine neurons in PD. Other proteolytic enzymes are associated with other diseases as is known in the art. Several examples are included in Table 3 shown below.

Table 3: Deubiquitination Enzymes Associated with Physiology, Disease & USP2a Enzyme Physiology Ap-UCH prosthetic cancer essential for long-term memory in Aplysia BAP1 tumor suppressor (associated with BRCA1) CYLD1 tumor suppressor DUB-1 cytokine-inducible, selective B-cell DUB-2 cytokine-inducible, selective T-cell D-ubp-64E Drosophila inhibitor of position effect variation FAF (fat facets) Drosophila eye development FAM embryo development of pre-implantation mouse HAUSP (USP7) tumor suppressor (p53 stabilization) Tre-2 (USP6) oncoprotein Ubp3 inhibitor of transcriptional silencing in yeast UBP41 apoptosis, bone formation UBP43 negative regulator of IFN signaling, hematopoiesis UBP45 myogenesis UBP69 myogenesis UbpB (Dictyostelium) synchronization of development and formation of spatial pattern UBP-M (USP16) control cell cycle (chromatin condensation) UBPY cell cycle / cell growth USP14 (ataxia) UCH-L1 synaptic function (PGP9.5) Parkinson's disease, gracile axonal dystrophy VDU1 / VDU2 tumorigenesis (associated with von Piel-Lindau protein) The hybrid cell, plant or animal of the invention described above can be produced through several methods, including obtaining a cell, plant or animal; obtain a Ubiquitin-, UBL- or its functional fragment of C-terminal bond-reporter fusion nucleotide; obtain a hybrid vector that transports the hybrid polynucleotide operably linked to a vector; and stably transfecting the hybrid vector within the cell, plant or animal. In one embodiment, the fusion polynucleotide is integrated into the chromosome of the cell, plant or animal, and becomes completely stabilized. In another embodiment, the fusion polynucleotide comprises a fusion deoxyribonucleotide. The cell, plant or animal of the present invention can be used in the diagnosis of a disease or condition, for example by obtaining the cell, plant or animal of claim 71, or fractions or tissue thereof, wherein the reporter is associated with a disease or condition; contacting or administering a sample obtained from a subject that is assumed to be affected by the disease or condition with the cell, plant or animal; detect any signal produced by the reporter in the presence of the sample; and compare the signal with the controls for the 0% and 100% signals. Having generally described this invention, it will be better understood by reference to certain specific examples, which are included herein for purposes of illustration only and are not intended to limit the invention or any modality thereof, unless so stated. specify EXAMPLES The following examples are illustrative of the present invention and are not intended to be limitations thereof. Unless otherwise indicated, all percentages are based on 100% by weight of the final composition.

Example 1: Poliovirus Protease The following example illustrates a preferred isopeptidase test according to the present invention. The reporter enzyme RNA-dependent RNA polymerase (3Dpd) requires a free N-terminal for activity. See, Gohara, et al. (1999). The polymerase was fused with Smt3, thus blocking its N-terminus. When this fusion protein was treated with SUMO isopeptidase a free N-terminal 3Dpo1 was generated. Ibid. Gohara, et al. (1999). Subsequent to fusion fragmentation, 3Dpol activity can be quantified using the polymerase test as described below. Therefore, the isopeptidase-mediated fragmentation of poliovirus RNA-dependent RNA polymerase is required in vitro for polymerase activity, and the poliovirus activity RdRp is a surrogate measurement of isopeptidase activity.

Construction of Plasmid. Expression v Purification A Smt3-3Dpt fusion > 1 (Mahoney strain), was elaborated, expressed and purified in a manner similar to that described by Malakhov et al. (2004, No.7). Briefly, a segment of the 3Dpo1 gene of the poliovirus (Mahoney strain) was PCR amplified from the plasmid pET26b-Ub-3D-GSSG-6H previously described by Gohara et al. (1999) using synthetic primers that incorporate a Bsal site at the 5 'end and a BamHI site at the 3' end to facilitate cloning into the pET24-6H-SUMO vector. The sequences of the primers were as follows: '-GCAGGTCTCAAGGTGGTG AAATCCAGTGG ATG AG-3 '5'-GCAGGATCCCTAGTGGTGGTGGTG-3' The structure of pET24-6H-SUMO-3Dpol, the final construct was verified by DNA sequencing.

Fragmentation Using Yeast Enzyme ULP1 A C-terminal His-tagged protease SUMO 1, ULPI (403-621) p was expressed from pET24d on Rosetta- (DE3) pLysS (Novagen), and purified by means of Ni resin -NTA See, Li and Hochstrasser (1999) and Mossessova and Lima (2000). The Ulp and fragmentation reactions were completed under standard conditions as described by Malakhov et al. (2004, No.7). the reaction was terminated and the samples boiled for 5 minutes and subjected to SDS-PAGE. The activity was determined by quantification of the fragmented fusion by means of SDS-PAGE. The gels obtained were scanned using the Scion Image software to determine the activity quantitatively. 3Dpol Activity Test (Polymerase) The polymerase activity of 3Dpol and its fusion derivative were tested by nucleotide incorporation or primer extension. See, Arnold and Cameron (1999). The nucleotide incorporation test was conducted with radionucleotide with the following reaction mixture: 50 mM HEPES pH 7.5, 10 mM ß-mercaptoethanol, 5 mM MgCl2, 60 μ? ZnCl2, 500 μ? UTP, 0.4 Ci / pL [a-32P] UTP, 1.8 μ? dTI 5 / 0.15 μ? poly (rA) 4oo primer / template and 3Dpol. All reactions were performed in a total volume of 25 μ? using 250 ng of purified 3Dpol at 300 ° C for 5 minutes. The reactions were quenched by the addition of 0.5 M EDTA to 83 mM, and 10 μl of the quenched reaction were marked on DE81 filter paper discs, and dried completely. The discs were then washed 3 times for 10 minutes with 250 mL of 5% sodium phosphate dibasic and rinsed with absolute ethanol. The radioactivity bound to each filter was quantified by means of liquid scintillation spectrometry in 5 mL of scintillation fluid. The primer extension reaction contained 50 mM HEPES (pH 7.5), 10 mM ß - ≥5 mM MgCl2, 500 μ? ATP, 1 μ? sym / sub-U, 0.14 Mg / μ? of the enzyme 3D and +/- 1 μ? ULPI (25 pL total reaction volume).

In vitro fragmentation by ULPI The Smt3-3Dpol Ulp-I fusion was incubated under standard conditions for 1, 2, 4, 10, 20, 30, and 60 minutes, and resulted in 100% fragmentation of 3Dpol as confirmed by SDS-PAGE and staining of Coomassie Blue. The activity of 3Dpol was tested by incorporation of radionucleotide using the test described above. The results obtained are shown in Table 4 below.

Table 4: 3Dpo1 activity in the Radionucleotide Incorporation Test As seen in Table 4, the 3Dpo 'activity was elevated by coincubation of the fusion with UI p 1 and performing the radionucleotide test and further improved by preincubation of the Ulpl fusion and subsequently performing the radionucleotide incorporation test. . It is evident from Table X that the treatment of 3Dp0 'fusion with Ulpl SUMO yeast protease activates 3Dpo'.

Example 2: Glutamine Phosphoribosylpyrrophosphate Amidotransferase (GPATase) This example illustrates a preferred isopeptidase test according to the present invention employing GPATase. The enzyme Glutamine phosphoribosylpyrophosphate amidotransferase (GPATase) catalyzes the initial stage of purine nucleotide biosynthesis, and is the main regulatory enzyme in the path. GPATase transfers the nitrogen from glutamine amide (free NH3) to phosphoribosylpyrophosphate (PRPP), producing phosphor ribosyl amine, pyrophosphate, and glutamate. GPATase belongs to a family of 16 glutamine amido transferases involved in the utilization of glutamine amide nitrogen for biosynthetic purposes. See, Zalkin (1993); Tso et al. (1982). E. coli GPATase can be in the form of a tetramer or trimer formed of identical subunits and does not contain iron unlike birds, mammals, or B subtilis GPATase. See, Mantsala and Zalkin (1976). An active site cistern is required for the transfer of glutamine, a critical stage of the catalytic mechanism. Since this cysteine and also the N-terminal residue of the mature GPATase is clear that the enzyme requires a free N-terminal for the catalytic activity. See, Tso et al. (1982). The reaction is coupled to the reaction NAD + - >; NADH, which allows the determination of GPATase activity through the measurement of the absorption of the reaction product NADH a? = 363 nm as shown below.

Methods: Plasmid Construction, Expression and Purification of Smt3-GPATase A Smt3-GPATase fusion was elaborated, expressed and purified in a manner similar to that described by Malakhov et al (2004, # 7). The E. coli purF gene encoding the glutamine phosphor ribosyl pyrophosphate amidotransferase (GPATase) was PCR-amplified using the pETpurF plasmid described by Bera et al., J.B.C. 275, 7975-7979 (2000) using the following synthetic primers: Dirfecta: 5'-GTCAGGTCTCAAGGTTGCGGTATTGTCGGTATCGC-3 'Reverse: 5'-GTCAGG ATCCTCATCCTTCGTTATGCATTT-3' These primer sequences incorporate a Bsal site at the 5 'end, and a BamHI site at the 3' end of the purF sequence to facilitate cloning into the pET24-6H-SUMO vector. This construct was designed to direct the synthesis of a fusion protein in which the amino acid sequence SUMO C-terminal -Gly-Gly is directly linked to the mature N-terminal GPATase, which is a residue of cistern in the amino acid position 2. The structure of the final construct, pET24-6H-SUMO-GPATase, was verified through DNA sequencing.

Fragmentation of SUMO-GPATase Using the Yeast Enzyme ULP1 The SUMO protein was fragmented from a SUMO-GPATase fusion protein by treatment with the ULPI yeast protein (SUMO protease catalytic domain). Approximately 5 pg of SUMO-GPATase were incubated at 300 ° C for 3 hours with increasing concentrations of the enzyme ULPI enzyme in a reaction mixture containing 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 10 mM DTT. The reaction was quenched by addition of the SDS-PAGE sample pH regulator, heated and loaded directly onto a 12% SDS-polyacrylamide gel, and subjected to electrophoresis. After electrophoresis, the gel was stained to visualize the proteins. The SDS-PAGE analysis demonstrated the fragmentation of the fusion with only .009 units of Ulp 1; however, fragmentation was not fully achieved with 78.3 Units. This inability of the ULPI enzyme to cut the fusion protein may reflect the occurrence of steric hindrance around the fragmentation site, or some partial modification of the binding sequence, for example oxidation of the Cysteine residue at the amino-terminal end of the GPATasa.

GPATase In Vitro Activity Test GPATase, a glutaminase enzyme, hydrolyzes 5-phospho ribosyl pyrophosphate (PRPP) producing glutamine, 5-phosphoribosyl- (b) l-amine (PRA) and pyrophosphate (PPj). This glutaminase reaction was monitored through the measurement of glutamate production by means of a coupled glutamate dehydrogenase (GDH) test under standard reaction conditions described by Messenger and Zalkin (1979). When the fusion protein was incubated for 30 minutes with Ulp 1, and the reaction added to the GDH substrate, there was an increase in absorbance at the OD 363 reading, indicative of an active Glutamate, which in turn indicates GPATase activity. In the absence of Ulp 1, this increase in absorbance did not occur.

In addition, when the increasing concentration of the Smt3-GPATase fusion increased, it was increasing. Additional additional quantified examples are described below and establish this difference between fragmented SUMP-GPATase ULP1 and untreated fusion protein. The coupled GDH test that was used to determine GPATase activity was examined for accuracy. Increasing amounts of the purified SUMO-GPATase fusion protein, either untreated or fragmented by means of the ULP1 SUMO protease, were incubated in the presence of glutamine and PRPP for a fixed period, and the completed reaction was then used as a substrate in a GDH test. The results show that there is a linear relationship between the GPATase enzyme concentration and the GDH reaction absorbance read at 363. This is particularly at enzyme concentrations of 1 pg or less. Therefore, the amount of reduced NAD + in the GDH reaction correlates directly with the amount of active GPATase enzyme in the initial reaction. In addition, treatment with ULP1 results in at least a 10-fold increase in the glutaminase activity of the GPATase, despite the fact that only the partial fragmentation of the SUMO-GPATase has been observed so far. Example 3: Tryptase test The following example illustrates a preferred isopeptidase test that is provided according to the present invention.

Triptases are neutral serine proatases with a molecular weight of 134 kDa. The enzyme is formed by 4 subunits of non-covalent bond and each subunit has a single active site. Mainly there are two members in this family, a-triptase and ß-tryptase with approximately 90% sequence identity between the two. Triptases are synthesized as inactive precursors, and are stored in secretory granules as active enzymes along with other proteinaces. Also, there is also a constitutively expressed and secreted version, called a-protriptase. The activation of β-tryptase is a double-proteolytic process by means of which the initial fragmentation is an autocatalytic intermolecular fragmentation that results in the formation of a monomer, with a di-peptide in the N-terminus that inhibits the formation of the required tetramer . The role of dipeptidyl peptidase I is to fragment the dipeptide from the N-terminus allowing the formation of the mature tryptase structure and increasing the activity 50-fold.

Expression of Triptase gene fusions in insect cells Recombinant human tryptase was expressed in a baculovirus (autographa californica nuclear polyhidrosis virus (AcNPV)) of insect cell (Spodoptera frugiperda system (Sf9)) as described by O'Reilly (1992). The tryptase cDNA encoding human tryptase was fused in-frame at the 3"end of the ubiquitin sequence coding (Ub) or a SUMO, which carries a six histidine tag residue (6 x His) in the N- This hybrid gene was then inserted immediately downstream of the signal sequence for the envelope glycoprotein secreted by gp67 baculovirus.Expression of the gene fusion was controlled by the polyhedrin pi 0 or basic protein gene promoter AcNPV After the cloning of the gene fusion within the baculovirus transfer vector, the recombinant plasmid transporting the Ub / UBL-tryptase construct was co-transfected with AcNPV baculovirus DNA into insect cells. recombinants that arise by means of homologous recombination of the transfer vector and the deoxyrubonucleic acid AcNPV (DNA) were selected, purified by plate and amp The insect cells infected with the purified recombinant baculovirus are capable of producing milligram quantities of the fusion protein per liter of culture. Ub / SUMO-tryptase protein was screened into the medium as an intact fusion protein without the gp67 signal sequence. The presence of the N-termminal xHis tag facilitated the subsequent purification of the protein in Ni-NTA Agarose (metal affinity chromatography) and the size validated by SDS-PAGE. Fragmentation with the appropriate isopeptidase resulted in adequate fall bands.

Triptase Enzymatic Activity Test Human tryptase activity was measured using either a chromogenic or a fluorometric enzyme test or both. The purified fusion protein was fragmented through the appropriate UBL hydrolase / protease, which activated the tryptase. Tryptase activity was tested using a chromogenic peptidyl substrate whose hydrolysis was monitored through the measurement of changes in absorption at 410 nm. See, Schechter et al. (1998 1998). Upon fragmentation of Smt3-tryptase purified with Ulp 1, the increased tryptase activity was measured following the protocol of Schechter and colleagues. Before fragmentation by means of isopeptidase, neither Ubiquitin-tryptase nor SU MO-tryptase had any tryptase activity. Only the fragmentation by means of isopeptidase was observed the activity of tryptase. Therefore, tryptase fusion is a useful tool to identify the activity of isopeptidase.

Example 4 Phospholipase A2 Test The following example illustrates a preferred mode of the test of the invention employing phospholipase A2. Phospholipases are a family of enzymes that were initially identified in viper venom, and later found to be conserved through higher organisms. Phospholipases are grouped into subfamilies according to their size, expression pattern and dependence on co-factors. The subfamily of secreted phospholipase A2 enzyme (sPLA2) can be differentiated from other PLA2 subgroups, such as cytosolic intracellular members, and PLA2 Ca2 + isoforms -independent in that they are 14-16 kDa disulfide rich proteins that require millimolar concentrations of Ca2 + for catalysis. See, Gelb (1995). In mammalian systems there is an excess of eleven enzymes sPLA2 enzymes, for example IB, NA, HC, IID, HE, IEF, III, V, X, X 11 A and XIIB. sPLA2 possess a broad specificity for phospholipids with different polar upper groups and fatty acyl chains. Enzymes of the PLA2 family catalyze the fragmentation of phospholipid at the sn-2 position to produce free fatty acids and lysophospholipids. See, Dennis (1994). The sPLA2 enzymes are generated as proenzymes that are catalytically inactive. See Dijkstra (1981). Upon secretion and fragmentation when processing proteases, such as trypsin, the N-terminal propeptide is fragmented to produce the active enzyme with the desired N-terminus. See, Cupíllard (1997). It is the free N-terminal that is required for catalytic activity since it is involved in the hydrogen bond and the interfacial bond. See, Dijkstra (1984); Yuan, (1999); Grataroli et al (1982).

Construction of Ubiquitin / UBL-PLA Plasmid? MX All plasmid constructs for expression in E. coli were derived from the expression vector pET24d (+) (Novagen). The expression vectors of Ubiquitin / U BL-fusion were constructed as detailed by Malakov et al (2004). The Mouse X PLA2 group was selected as an example of the entire PLA2 enzyme group. The fusion constructs were made by PCR amplification of the murine gene PLA2 Group X with the designated primers that only amplified the active processed group X PLA2 enzyme form. Included within the 5 'and 3' primers were the unique Bsal and BamHI restriction sites, respectively. This allowed downstream insertion of the Ubiquitin / UBL gene, and thus in the translation of the structure of the fusion protein. The primers used were as follows: Direct: 5'-G ATCGGTCTCAAGGTGG ACTCCTGG AGCTGGCAGGG-3 'Reverse: 5'-G ATCGGATCCTCAATTGCACTTGGGAGAGTC-3' Finally Ubiquitin-PLA2mX, (yeast SUMO) Smt3-PLA2mX, human SUM03-PLA2mX, ISGI 5- PLA2mX, human Nedd8-PLA2mX and yeast fusions Rubl-PLA2mX were created using the same protocol detailed above. Prior to expression, the expression plasmid was verified in sequence to be the correct one in the structure translation products.

Expression and Purification of Ubiquitin / UBL-PLA? MX in E. coli Bacterial expression of Ubiquitin / UBL fusion proteins was performed after transformation into BL21 (DE3) or Rosetta (DE3), two E. coli host strains . The SoUBLe and insoUBLe fractions were deposited, subjected to electrophoresis on a gel SDS-PAGE gel, and stained with Coomassie Blue to verify the size and expression of each fusion. Then the Ubiquitin / UBL-PLA2mX was purified from the insoUBLe fraction by chromatography on NiNTA resin (Qiagen), and dialyzed for 48 hours with pH regulator exchange. All fractions were collected, and then subjected to electrophoresis by SDS-PAGE and stained with Coomassie Blue. Each construction expressed the appropriate sized band as expected, as shown in Table 5 below.

Test PLA? Phosphididylcholine was used with a fluorophore conjugate at the sn-2 position of the lipid as a substrate to test the activity of PLA2. The fluorophore released at the cleavage of the sn-2 acyl bond by means of active PLA2 was detected at its specific excitation and emission wavelengths. Two fluorophores were used: NDB (ex: 460nm / em: 534nm) and BODIPY FL (ex: 503nm / em: 512nm) (Molecular Probes / In Vitro gene). The lipid substrate was diluted in PLA2 test buffer (10 mM Tris, pH 8, 100 mM KCI, and 2 mM Ca2 +) for a final concentration of 5 μm. Fragmentation reactions were performed on a 96-well plate or 1.5 ml Eppendorf tube, and then a lipid substrate was added. Upon addition of the fragmentation product or ubiquitin / UBL-isopeptidase, readings of 400 milliseconds were recorded at 15 second intervals for a total of 30 minutes, in another protocol employed a fragmentation reaction was assembled on a 96 well plate by the collectively adding the PLA2 fusion construct, either as a cell extract or as an isopeptidase, and substrate. Successive readings were taken up to a desired time point, such as 30 minutes. The fragmentation of the fusions was detected through an increase in the fluorescence of the fragmented lipid substrate.

Fragmentation of Ubiqutin / UBL-PLA? MX with Complete Cell and Plant Extracts The fragmentation of several Ubiquitin / UBL-PLA2 fusions was carried out with the help of complete cell extracts. It was determined that this fragmentation resulted in appropriate or expected size fall bands. Two bands were observed on the gels stained with Coomassie Blue: a 14 kDa constant band PLA2mX, and the Ubiquitin / UBL fusion pair sized band, whose size depends on the UBL fusion pair. The results obtained are shown below in Table 5.

Table 5: Ubiquitin Intact Sizes / UBL-Fusions & Fragmentation Products Each of the fragmentation reactions contained 5 pg of fusion protein, and a similar amount of Insect Cells, Rabbit Reticulocyte Fraction II, U20S human osteosarcoma cells, DLDI and HCTI 16 colon cancer cell lines, H460 cells of human non-small cell lung cancer, human embryonic kidney 293T cells, murine T helper lymphocyte clone, or wheat germ cell extract. The reaction mixture samples were incubated overnight, removed, electrophoresed on a 15% SDS-PAGE gel, and stained with Coomassie Blue for fragmentation analysis. The results are shown below in Table 6.

Table 6: Fragmentation Activity Profile of Cell and Plant Extracts (+ /. = < 10%, +: 10% - 25%, ++ = 25% -50%, + + + = 50% - 75%, 4 + + + = 75% -100%) In addition, the PLA2 test was performed as described above to measure PLA2 activity using the labeled BODIPY FL lipid substrate described above and represented as an activity relationship where the extracts were added to the fusion protein on the fusion protein. In herself. The results are shown below in Table 7.

Table 7: Ratio of PLA2 Activity from Fragments Fragmented by Cell Extracts & Plant As can be seen from the data provided in Table 7 above, fragmentation of Ubiquitin / UBL-PLA2mX fusion protein correlated well with PLA2 activity. For the ISG15-PLA2mX activity, whole cell extracts were used from HEK293T cells that were transfected to express UBP43, the ISG15-specific isopeptidase. In this test, the ISG15 fusion was incubated with transfected UBP43 extracts and non-transfected control extracts and the fragmentation and fluorescence were monitored in real time. There was fluorescent activity in both conditions, although the PLA2 activity was present within the UBP43 transfected cell extracts before those of the non-transfected cells. This could be attributed to constitutively expressed processing proteases, endogenous, since ISG15 is initially a 17 kDa precursor that is processed at 15 kDa. This processing protease may have some affinity for the ISG15-PLA2 fusion, although at a much lower affinity than that of UBP43 for ISG15, therefore, the fluorescent readings delayed in the PLA2 assay. However, the test was able to detect UBP activity directed towards ISG15 mergers. In the presence of UBP43, there is a 4-fold increase in the PLA2 activity index, demonstrating the utility of the ISG15-PLA2 fusion to detect the activity of ISG 15-isopeptidase.

Fragmentation of Ubiquitin / UBL-PLA? MX using Ubiguitin / UBL-specific lsopeptidase that produces PLA activity.

In these experiments reporter fusion proteins were incubated in the presence or absence of sopeptidases that target specific portions of Ubiquitin or UBL, and tested for isopeptidase activity by monitoring fusion fragmentation by SDS-PAGE or by detection of PLA2 activity. Since the yeast Ulp 1 isopeptidase has specificity for the yeast gene product Smt3 was used for the fragmentation of a yeast fusion protein Smt3. Similarly, SENP2 isopeptidase was used for fragmentation of the human SUM03 fusion protein, and the shared core enzyme domain of the bound USP2 product, either USP2a or USP2b. See, Lin et. to the. (2000). It was used for fragmentation of a Ubiquitin fusion protein. And finally, Den1, a Nedd8-specific isopeptidase was used to fragment a Nedd8-PLA2mX fusion protein. See, Gan-Erdene (2003). 10 pg of yeast Smt3-PLA2mX were incubated with 1 pg Ulp-1 for one hour and analyzed by means of SDS-PAGE. Analysis of the gel showed complete fragmentation of the reporter fusion protein to produce the separate components, Smt3 (at 21 kDa) and 14 kDa PLA2mX. When the same experiment was conducted in the absence of ULP1 there was no auto-catalytic activity. Only intact 32 kDa fusion was observed. The yeast enzyme ULP1 did not fragment the Ubiquitin-PLA2mX fusion protein in a demonstration of specificity for the SUMO fusion protein. When 10 pg hSUM03-PLA2mX were incubated with 1 pg SENP2, and the samples monitored by SDS-PAGE the appropriately sized hSUM03 (21 kDa) and PLA2mX (14 kDa) bands were observed, indicating complete fragmentation of the fusion protein. The SENP2 enzyme did not fragment the Ubiqutin-PLA2mX fusion protein, as previously observed with ULP1, demonstrating specificity of isopeptidase activity for SUMO.

When 10 pg of the Ubiquitin-PLA2mX fusion were incubated with 3 pg of the USP2 core domain, the complete fragmentation of Ub-PLA2mX fused was observed. No parent band (fusion protein) 24 kDa was observed while the 14 kDa band PLA2mX and the 9 kDa band Ubiquitin appeared. When the Nedd8-PLA2mX fusion protein was incubated with Den1 Nedd8-specific isopeptidase, the Nedd8 fusion protein fragmented and the 14 kDa PLA2mX and 9 kDa Nedd8 bands appear. A PLA2 test was run in vitro to determine the PLA2mX activity for the fragmentation of the Ubiquitin / UBL fusion protein by means of specific isopeptidases. In all cases where the co-incubation of the Ubiquitin / UBL-PLA2mX fusion and the specific isopeptidase lead to the fragmentation and generation of the 14 kDa PLA2mX band, there is elevated PLA2 activity, as visualized by means of the intensity increase of fluorescence. In all the cases described above, in which the UBL-specific Isopeptidase fragmented the fusion, PLA2 activity was also monitored and the ratio of PLA2 activity from the Fusions incubated with and without UBL-specific Isopeptidase are shown below in Table 8 Table 8: Ratio of PLA2 Activity from UBL-specific Isopeptidase Activity This example shows the usefulness of fusion proteins Ubiquitin / UBL-PLA2 to detect proteolytic activity, for example of isopeptidase. In the absence of any isopeptidase activity, the fusion protein alone has no signal generated. In the presence of cell extracts containing isopeptidase activity or more specifically, purified recombinant isopeptidases, the fusion protein is fragmented and produces a quantifiable level of PLA2 activity. A large number of enzymes are known that are capable of cleaving ubiquitin or UBLs from their target proteins or linear fusion proteins. Genome sequencing through phylogeny is producing other examples. Until now there was no direct functional test suitable for rapid screening, precise and selective of these enzymes or for the screening of compounds that modulate their activities. Currently available tests are difficult and require many steps in order to produce isopeptide-linked substrates containing ubiquitin or UBLs to conveniently test proteolytic enzyme activities, for example isopeptidase or hydrolase (protease). However, the removal of ubiquitin or UBLs from an objective protein according to this invention can be monitored through Western blotting.; However, the prior art tests are characterized by low sensitivity and total efficiency. An UBL such as ubiquitin and UBLs can be fused to a protein domain such as GST, which can subsequently be immobilized on a solid and fragmented support, and one of the products filtered or tested by a modified total high throughput ELISA. However, prior art methods achieve relatively low sensitivity, contain multiple steps, and are expensive because they require ELISA reagents. Further, if, for example, GST fusions or fusions with any other enzyme were employed in the prior art tests they would also be prone to artifacts if the protein were recognized by the antibodies and / or the enzyme remains active even in a state UBL-merged. Having thus described the invention, it will be obvious that it can be modified or varied in many ways. Said modifications and variations will not be considered as a separation of the spirit and scope of the invention and all such modifications and variations are intended to be included within the scope of the following claims. The following references are cited as indicative of the general state of the art, and as an aid to understanding the subject matter in question in its own context. The citation of a reference in this application will not be considered as an admission of material for patentability of the inventive subject in question, nor as an admission that any reference is prior art; the material references will be cited in a Statement of Information Description. All references cited herein and below are incorporated by reference in their entirety herein.

REFERENCES Adams, J. (2002). "Development of the proteasome inhibitor PS-341." Oncologist 7 (1): 9-16. Adams J. (2002) "Preclin / lin eval of proteasome inhibited PS-341 for cancer." Curr Opin Chem Biol 6 (4): 493-500., Adams, J. (2002). "Proteasome inhibition: a novel approach to cancer ther" Trends Mol Med 8 (4 Suppl): S49-54. Almond, J. B. and G. M. Cohen (2002). "The proteasome: target for cancer chemother" Leukemia 16 (4): 433-43. Agostini, M., S. D. Silva, et al. (2004). "Fatty acid synthase required for prolif of human oral squamous carcinoma cells" Oral Oncol 40 (7): 728-35. Andrulis, I. Shotwell, M, Evans-Blackler S. et al (1989) Fine Structure analysis of the Chimney hamster AS gene encoding asparagine synthetase. Gene 80: 75-85 Arnold, JJ., And CE. Cameron (1999). "Poliovirus RNA-dependent RNA polymerase (3Dpol) is sufficient for TEMPLE switching in vitro." J Biol Chem 274 (5): 2706-16 Baek, S. H. , K. S. Choi, et al. (1997). "Molecular cloning of a novel ubiquitin-specific protease, UBP41, with isopeptidase activity in chick skeletal muscle." J Biol Chem 272 (41): 25560-5. Baek, S.H., K.C. Park, et al. (1998). "A novel family of ubiquitin-specific proteases in chick skeletal muscle with distinct -and C-terminal extensions." Biochem J 334 (Pt 3): 677-84. Bachmair, A., D. Finley, and A. Varshavsky (1986) In vivo half-life of a protein is a function of its amino-terminal residue. Science 234: 179-186. Bachmair, A., and A. Varshavsky (1989) The degradation signal in a short-lived protein. Cell 56: 1019-1032. Baker, R.T., S.A. Smith, et al. (1994). "Protein expression using cotranslational fusion and cleavage of ubiquitin, Mutag of glutathione-binding site of human Pi glutathione S-transf J Biol Chem 269 (41): 25381-6 Baker, RT, JW Tobias, et al. (1992). "Ubiquitin-specific proteases of Saccharomyces cerevisiae. Cloning of UBP2 and UBP3, and functional analysis of the UBP gene family. "J Biol Chem 267 (32): 23364-75, Balakirev, MY, SO Tcherniuk, et al. (2003)." Ouabains: a new family of cysteine proteases in the ubiquitin pathway. "EMBO Rep 4 (5): 517-22.

Brain and Williams (1988) "Subst P reg vasodil act of calcitonin gene-related peptide" Nature 335 (6185): 73-5. Benjamín, L. E. and E. Keshet (1997). "Conditional switching of vascular endothelial growth factor (VEGF) expression in tumors: induction of endothelial cell shedding and regression of hemangioblastoma-like vessels by VEGF withdrawal." Proc Nati Acad Sci U S A 94 (16): 8761-6. Bonner, J. C. (2002). "The epidermal growth factor receptor at the crossroads of airway remodeling." Am J Physiol Lung Cell Mol Physiol 283 (3): L528-30. Buchanan, J. M. (1973). "The aminotransferases." Adv Enzymol Relat Areas Mol Biol 39: 91-183. Cai, S. Y., R. W. Babbitt, et al. (1999). "A mutant deubiquitinating enzyme (Ubp-M) associates with mitotic chromosomes and blocks cell division." Proc Nati Acad Sci U S A 96 (6): 2828-33. Chen, X., B. Zhang, et al. (2002). "A specific protein substrate for a deubiquitinating enzyme: Liquid facets is the substrate of Fat facets." Genes Dev 16 (3): 289-94. Chung, C. H. and S. H. Baek (1999). "Deubiquitinating enzymes: their diversity and emerging roles." Biochem Biophys Res Commun 266 (3): 633-40. Chung, K.K., V.L. Dawson, et al. (2003). "New insights into Parkinson's disease." J Neurol 250 Suppl 3: 11115-24. Ciardiello, F. and G. Tortora (2001). "A novel approach in the treatment of cancer: targeting the epidemic growth receptor factor." Clin Cancer Res 7 (10): 2958-70. Ciechanover, A. (2001). "Ubiquitin-mediated degradation of cellular proteins: why is it essential for construction, and how did it get from the test tube to the patient's bed." Isr Med Assoc J 3 (5): 319-27. Ciechanover, A. (2003). "The ubiquitin proteolytic system and pathogenesis of human diseases: a novel platform for mechanism-based drug targeting." Biochem Soc Trans 31 (2): 474-81. Conaway, R. C, C. S. Brower, et al. (2002). "Emerging roles of ubiquitin in transcription regulation." Science 296 (5571): 1254-8. Clague and Urbe (2001). "The interface of receptor trafficking and signaling." J Cell Sci 114 (Pt 17): 3075-81. Cupillard, L, et al. (1997) Cloning, Chromosomal Mapping, and Expression of a Novel Human Secretory Phospholipase A2. J. Biol. Chem. 272 (25): p. 15745-15752. Curcio-Morelli, C, A.M. Zavacki, et al. (2003). "Deubiquitination of type 2 iodothyronine deiodinase by von Hippel-Lindau prot-interacting deubiquit enzymes reg thyroid hormone activation." J Clin Invest 112 (2): 189-96. D'Andrea and Pellman (1998) "Deubiquit enz: new class of biol reg" Crit Rev Biochem Mol Biol 33 (5): 337-52. Dang, L. C, F. D. Melandri, et al. (1998). "Kinetic and mechanistic studies on the hydrolysis of ubiquitin C-terminal 7-amido4-methylcoumarin by deubiquitinating enzymes." Biochemistry 37 (7): 1868-79. Day, I. N. , L. J. Hinks, et al. (1990). "The structure of the human gene encoding protein gene product 9.5 (PGP9.5), a neuron-specific ubiquitin C-terminal hydrolase." Biochem J 268 (2): 521-4. Dennis (1994) Div group types, reg and funct of phospholipase A2 J Biol Chem 269 (18): 13057-13060. Dijkstra, Drenth & Kalk, (1981) Active site and catal mech of phospholipase A2. Nature.289 (5798): 604-6. Dijkstra et al. (1984). "Role of the N-terminal in the interaction of pancreatic phospholipase A2 with aggregated substrates, Properties and crystal structure of transaminated phospholipase A2." Biochemistry 23 (12): 2759-66. Dubiel and Gordon (1999). "Ubiquitin pathway: link in the polyubiquitin chain?" Curr Biol 9 (15): R554-7. Everett, R. D., M. Meredith, et al. (1997). "A novel ubiquitin-specific protease is dynamically associated with the PML nuclear domain and binds to a herpesvirus regulatory protein." Embo J 16 (3): 566-77. Frederick et al. (1998) "The human UNP locus at 3p2 .31 Jan 2 tissue-select, cytopl isoforms with deubiquit activity that have reduced expression in small cell lung carcinoma cell lines." Oncogene 16 (2): 153-65. Gan-Erdene et al (2003) Ident & charact of DENI, a deneddylase of ULP fam JBiolChem 278 (31): 28892-900 Gelb, M.H., et al., (1995) Interfacial Enzymology of Glycerolipid Hydrolases: Lessons from Secreted Phospholipases A2. Annual Review of Biochemistry 64 (1): p. 653-688. Glickman & Ciechanover (2002) The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction Physiol Rev. Apr; 82 (2): 373-428. Gnarra et al. (1994). "Mutations of the VHL tumor suppressor gene in renal carcinoma." Nat Genet 7 (1): 85-90. Gohara, D. W., C. S. Ha, et al. (1999). "Production of" authentic "poliovirus RNA-dependent RNA polymerase (3D (pol)) and ubiquitin-protease-mediated cleavage in Escherichia c or I i." Protein Expr Purif 17 (1): 128-38. Gong, L., T. Kamitani, et al. (2000). "Identification of a novel isopeptidase with dual specificity for ubiquitin- and NEDDó-conjugated proteins." J Biol Chem 275 (19): 14212-6. Gong et al. (2000) "Diff reg of sentrinized prot by novel sentrin-specifíc protease." J Biol Chem 275 (5): 3355-9. Graner et al (2004) "lsopeptidase USP2a reg stab of fatty acid synthase in prostate cancer" Cancer Cell 5 (3): 253-61. Grataroli, R., et al., Studies on prophospholipase A2 and its enzyme from human pancreatic juice. Catalytic properties and sequence of the N-terminal region. Eur J Biochem, 1982. 122 (1): p. 111-7. Gray, D.A., J. Inazawa, et al. (nineteen ninety five). "Elevated expression of Unph, a proto-oncogene at 3p21.3, in human lung tumors." Oncogene 10 (11): 2179-83.

Gupta, K., et al. (1994). "The Unp proto-oncogene encodes to nuclear protein." Oncogene 9 (6): 1729-31. Hadari, T., J.V. Warms, et al. (1992). "A ubiquitin C-termnal isopeptidase that acts on polyubiquitin chains. Role in protein degradation." J Biol Chem 267 (2): 719-27. Hansen et al. (1997). "Structure of the RN A-dependent RNA polymerase of poliovirus." Structure 5 (8): 1109-22. Hemelaar, J., A. Borodovsky, et al. (2004). "Specific and covalent targeting of conjugating and deconjugating enzymes of ubiquitin-like proteins." Mol Cell Biol 24 (1): 84-95. Hendfilamento, J. M., Schmid, J., and Amrheim, N (1995). "Only the Mature Form of the Platidic Chorismate Synthase Is Enzymatically Active." Plant Physiol 108: 1127-1132. Henriksen, R. A., C.K. Dunham, et al. (1998). "Prothrombin Greenville, Arg517 ~> Gln, identified in an individual heterozygous for dysprothrombinemia." Blood 91 (6): 2026-31. Hershko, A. and A. Ciechanover (1998). "The ubiquitin system." Annu Rev Biochem 67: 425-79. Hicke, L. (2001). "Protein regulation by monoubiquitin." Nat Rev Mol Cell Biol 2 (3): 195-201. Hochstrasser, M. (1996). "Ubiquitin-dependent protein degradation." Annu Rev Genet 30: 405-39. Hochstrasser, M. (2002). "Molecular biology, New proteases in a ubiquitin stew." Science 298 (5593): 549-52. Hofmann & Pickart (2001) "In vitro assemb / recognizing of Lys-63 polyubiq chains" JBiolChem 276 (30): 27936-43. Hong, T.M., P.C. Yang, et al. (2000). "Profiling the downstream genes of tumor suppressor PTEN in lung cancer cells by complementary DNA microarray." Am J Respir Cell Mol Biol 23 (3): 355-63. Huang, Y., R. T. Baker, et al. (nineteen ninety five). "Control of cell fate by a deubiquitinating enzyme encoded by the fat facets gene." Science 270 (5243): 1828-31. Jensen, D. E., M. Proctor, et al. (1998). "BAPI: a novel ubiquitin hydrolase which binds to the BRCAI RING finger and enhancers BRCAI -mediated cell growth suppression." Oncogene 16 (9): 1097-112. Johnson, E.S., P.C. Ma, et al. (nineteen ninety five). "A proteolytic pathway that recognizes ubiquitin as a degradation signal." J Biol Chem 270 (29): 17442-56. Johnston, S. C, C. N. Larsen, et al. (1997). "Crystal structure of a deubiquitinating enzyme (human UCH-L3) at 1.8 A resolution." Embo J 16 (13): 3787-96. Kato, G. J. (1999). "Human genetic diseases of proteolysis." Hum Mutat 13 (2): 87-98. Kawakami, T., T. Suzuki, et al. (1999). "Isolation and characterization of cytosolic and membrane-bound deubiquitinylating enzymes from bovine brain." J Biochem (Tokyo) 126 (3): 612-23. Kitada et al (1998) "Mutat in parkin gene cause autos recess juvenile parkinsonism" Nature 392 (6676): 605-8.

Kostin et al (2003). "Myocytes die by multiple mechanisms in failing human hearts." CircRes 92 (7): 715-24. Larsen, C. N. , J. S. Price, et al. (nineteen ninety six). "Substrate binding and catalysis by ubiquitin C-terminal hydrolases: Identification of two active site residues." Biochemistry 35 (21): 6735-44. LaVallie, E. R., A. Rehemtulla, et al. (1993). "Cloning and functional expression of acDNA encoding the catalytic subunit of bovine enterokinase." J Biol Chem 268 (31): 23311-7. Layfiel, R., K. Franklin, et al. (1999). "Chemically synthesized ubiquitin extension proteins detect distinct catalytic capacities of deubiquitinating enzymes." Anal Biochem 274 (1): 40-9. Le Roy (2005) "Clathrin- and non-clathrin-med endocyt reg of cell signaling" NatRevMolCelIBiol 6 (2): 112-26 Lee (1998) "Proteasome inhibitors: valuable new tools for cell biologists." Trends Cell Biol 8 (10): 397-403. Leroy, E., R. Boyer, et al. (1998). "The ubiquitin pathway in Parkinson's disease." Nature 395 (6701): 451-2. Li et al (2002) "Deubiquit of p53 by HAUSP is pathway for p53 stabil" Nature 416 (6881): 648-53. Li & Hochstrasser (1999) "New protease req for cell-cycle progression in yeast." Nature 398 (6724): 246-51. Li, S. J. and M. Hochstrasser (2000). "The yeast ULP2 (SMT4) gene encodes a novel protease specific for the ubiquitin-like Smt3 protein." Mol Cell Biol 20 (7): 2367-77. Li, Z., X. Na, et al. (2002). "Ubiquitination of a novel deubiquitinating enzyme requires direct binding to von Hippel-Lindau tumor suppressor protein." J Biol Chem 277 (7): 4656-62. Lin, H., A. Keriel, et al. (2000). "Divergent N-terminal sequences target an inducible testis deubiquitinating enzyme to distinct subcellular structures." Mol Cell Biol 20 (17): 6568-78. Lin, H., L. Yin, et al. (2001). "Divergent N-terminal sequences of a deubiquitinating enzyme modulate substrate specificity." J Biol Chem 276 (23): 20357-63. Lisztwan, J., G. Imbert, et al. (1999). "The von Hippel-Lindau tumor suppressor protein is a component of an E3 ubiquitin-protein ligase activity." Genes Dev 13 (14): 1822-33. [0183] Lockhart (2005) "Epid growth factor rec target for colorectal cancer therapy" Semin Oncol 32 (1): 52-60. LoRusso, P. M., R. S. Herbst, et al. (2003). "Improvements in quality of life and disease-related symptoms in phase I trials of the selective oral epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 in non-small cell lung cancer and other solid tumors." Clin Cancer Res 9 (6): 2040-8. Lu, S., R. Halberg, et al. (1990). "Processing of the mother-cell sigma factor, sigmaK, may depend on events occurring in the forest during Bacillus subtilis development." Proc Nati Acad Sci U S A 87 (24): 9722-6. Lundgren, J1, P. Masson, et al. (2003). "Use of RNA interference and complementation to study the function of the Drosophila and human 26S proteasome subunit S13." Mol Cell Biol 23 (15): 5320-30. Maher, E. R. and W. G. Kaelin, Jr. (1997). "vonHippel-Lindau disease." Medicine (Baltimore) 76 (6): 381-91. Malakhov, M. P., Kim, K. L5 Malakhova, O. A., Jacobs, B.S., Borden, E.C5 Zhang, D.-E. (2003). High-throughput Immunoblotting. Ubiquitine-like Protein ISGI 5 Modifies Key Regulators of Signal Transduction, J. Biol. Chem. 278: 16608-16613 Malakhova et al. (2002) Lipopolys Act Expr of I SG 15-specif ic Protease UBP43 via Interferon Regulatory Factor 3. J. Biol. Chem. 277: 14703-14711 Malakhov et al (2002) UBP43 (USP18) Spec Removes ISG15 from Conj Prot J. Biol. Chem. 277: 9976-9981 Maraganore et al. (2004). "UCHLI is a Parkinson's disease susceptibility gene." Ann Neurol 55 (4): 512-21. Mei & Zalkin (1990) Amino-term defines glutamine amide transfer domain in glutamine phosphoribosylpyrophosphate amidotransferase & PurF-type amidotransferases. J Bacteriol. 172: 3512-3514 Malakhov et al (2004). "SUMO fusion and SUMO-specific proteases for efficient expression and purification of proteins." J. Structural and Functional Genomics (in press). Mantsala &; Zalkin (1976) "Glutamate synth Prop of glutamine-dependent act" J Biol Chem 251 (11): 3294-9. Matsuka et al. (1999). "Fibrinogen cleavage by the Streptococcus pyogenes extracellular cysteine protease and generation of antibodies that inhibit enzyme proteolytic activity." Infect Immun 67 (9): 4326-33. McCullough etal. (2004). "AMSH is an endosome-assoc ubiquitin isopeptidase." J Cell Biol 166 (4): 487-92. Messenger, L.J., and H. Zalkin (1979) "Glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli., Purification and properties." J Biol Chem 254 (9): 3382-92 Mimnaugh, E. G. , G. Kayastha, et al. (2001). "Caspase-dependent deubiquitination of monoubiquitinated nucleosomal histone H2A induced by diverse apoptogenic stimuli." Cell Death Differ 8 (12): 1182-96. Mossessova, E. and C. D. Lima (2000). "Ulp 1 -SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast." Mol Cell 5 (5): 865-76. Muchmore, C. R., J. M. Krahn, et al. (1998). "Crystal structure of glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli." Protein Sci 7 (1): 39-51. Ohh, M., C. W. Park, et al. (2000). "Ubiquitination of hypoxia-inducible factor requires direct binding to the beta-domain of the von Hippel-Lindau protein." Nat Cell Biol 2 (7): 423-7. Oliver, G., Gosset G ,. Sanchez-Pescadore, E. et al (1987) Determination of the sequence for the glutamate synthetase structural gene of E. coli K-12. Gene 60: 1-11 O'Reilly et al (1992). Chapters 11, 12. Baculovirus expression vectors: a laboratory manual. NY, Freeman. Padmanabhan et al (1993) "Struct of human des (l-45) factor Xa at 2.2 A resolution." J Mol Biol 232 (3): 947-66. Papa et al (1999) "Inter of Doa4 deubiquitinating enz with yeast 26S proteasome." Mol Biol Cell 10 (3): 741-56. Papa, F. R. and M. Hochstrasser (1993). "The yeast DOA4 gene encodes to deubiquitinating enzyme related to a product of the human tre-2 oncogene." Nature 366 (6453): 313-9. Park, E.G., D. Finley, and J. W. Szostak (1992) A strategy for the generation of conditional mutations by protein destabilization. Proc. Nati Acad. Sci. USA 89: 1249-1252. Park et al (2000) "Tissue-specif, funct charact &subcellular local of rat ubiquitin-specific process protease, UBP 109, whose mRNA expression is developmentally regulated." Biochem J 349 (Pt 2): 443-53. Piccinini, M., A. Merighi, et al. (nineteen ninety six). "Affinity purification and characterization of protein gene product 9.5 (PGP9.5) from retina." Biochem J 318 (Pt 2): 711-6. Pickart, C. M. (2001). "Mechanisms underlying ubiquitination." Annu Rev Biochem 70: 503-33. Puddicombe, S.M., R. Polosa, et al. (2000). "Involvement of the epidermal growth factor receptor in epithelial repair in asthma." Faseb J 14 (10): 1362-74. Rossi, S., E. Graner, et al. (2003). "Fatty acid synthase expression defines distinct molecular signatures in prostate cancer." Mol Cancer Res 1 (10): 707-15. Sims, K. B. (2001). "Von Hippel-Lindau disease: gene to bedside." CurrOpin Neurol 14 (6): 695-703. Sambook, J., E. Fritsch, and T. Maniatis (1989) Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Sakai, K., S. Ren, etal. (nineteen ninety six). "A novel heparin-dependent processing pathway for human tryptase. Autocatalysis followed by activation with dipeptidyl peptidase I." J Clin Invest 97 (4): 988-95. Schechter, N.M., J. L. Sprows, et al. (1989). "Reaction of human skin chymotrypsin-like proteinase chymase with plasma proteinase inhibitors." J Biol Chem 264 (35): 21308-15. Shah et al. (2002). "Ubiquitin proteasome inhibition and cancer therapy." Surgery 131 (6): 595-600. Sizmann, D., C. Keilmann, et al. (1990). "Primary structure requirements for the maturation in vivo of penicillin acylase from Escherichia coli ATCC 11105." Eur J Biochem 192 (1): 143-51. Smyth, M.J., M.D. O'Connor, et al. (nineteen ninety six). "Granzymes: a variety of serine protease specif icities encoded by genetically dístinct subfamilies." J Leukoc Biol 60 (5): 555-62. Sommerhoff (1989) "Mast cell chymase, secretagogue for airway gland serous cells" Jlmmunoll42 (7): 2450-6. Spano, J. P., R. Fagard, et al. (2005). "Epidermal growth factor receptor signaling in colorectal cancer: preclinical data and therapeutic perspectives." Ann Oncol 16 (2): 189-94.

Stefanis, L., K. E. Larsen, et al. (2001). "Expression of A53T mutant but not wild-type alpha-synuclein in PC 12 cells induces alterations of the ubiquitin-dependent degradation system, loss of dopamine relase, and autophagic cell death." J Neurosci 21 (24): 9549-60. Steffan et al (2004) "SUMO mod of Huntingtin &Huntington's disease pathology." Science 304 (5667): 100-4. Stein et al (1995) "Kinetic st of isopeptidase T: modul of peptidase act by ubiquitin" Biochem 34 (39): 12616-23. Stolle, C, G. Glenn, et al. (1998). "Improved detection of germline mutations in the von Hippel- Lindau disease tumor suppressor gene." Hum Mutat 12 (6): 417-23. Stroud et al. (1977). "Mechanisms of zymogen activation." Annu Rev Biophys Bioeng 6: 177-93. Swinney, D. C. (2001). "Targeting protein ubiquitination for drug discovery.What is in the drug discovery toolbox?" Drug Discov Today 6 (5): 244-250. Takatsuji, H., H. Yamauchi, et al. (1992). "Cauliflower mosaic virus reverse transcriptase Activation by proteolytic processing and functional alteration by terminal deletion." J Biol Chem 267 (16): 11579-85. Takeyama, K., J.V. Fahy, et al. (2001). "Relationship of epidermal growth factor receptors to goblet cell production in human bronchi." Am J Respir Crit Care Med 163 (2): 511-6. Tobias, J. W. and A. Varshavsky (1991). "Cloning and functional analysis of the ubiquitin-specific protease gene UBPI of Saccharomyces cerevisiae." J Biol Chem 266 (18): 12021-8. Toda et al (2003) "ldentif of susceptibility genes for sporadic Parkinson's disease." JNeurol 250 Suppl 3: III40-3. Tung, C.H., et al. , (1999) Preparation of a cathepsin D-sensitive near-infrared fluorescence probé for imaging. Bioconjug Chem 10 (5): p. 892-6. Tyers, M. and R. Rottapel (1999). "VHL: a very hip ligase." Proc Nati Acad Sci U S A 96 (22): 12230-2. Tso, J. Y., M. A. Hermodson, et al. (1982). "Glutamine phosphoribosylpyrophosphate amidotransferase from cloned Escherichia coli purF. NH2-termnal amino acid sequence, Identification of the glutamine site, and trace metal analysis. "J Biol Chem 257 (7): 3532-6. Varshavsky A. (1996) The N-end rule: functions, mysteries, uses. Proc. Nati Acad. Sci USA 93: 12142-12149. Venkateswarlu, D., L. Perera, et al. (2002). "Structure and dynamics of zymogen human blood coagulation factor X." Biophys J 82 (3): 1190-206. Verma, R., L. Aravind, et al. (2002). "Role of Rpnl 1 metalloprotease in deubiquitination and degradation by the 26S proteasome." Science 298 (5593): 611-5. Von Ahsen & Bomer (2005). "High-Throughput Screening for Kinase Inhibitors." Chembiochem 6 (3): 481-490. Vu & Sakamoto (2000) "Ubiquitin-mediated proteolysis and human disease." Mol Genet Metab 71 (1-2): 261-6. Walls (1998) Mast cell proteases ¡n asthma. Inflamm mech in asthma Holgate, Busse, WW. NY, Marcel Dekker. Wang, Q. M., R. B. Johnson, et al. (1998). "Enzymatic characterization of refolded human rhinovirus type 14 2A protease expressed in Escherichia coli." J Virol 72 (2): 1683-7. Wang, Q. M., R.B. Johnson, et al. (1997). "A continuous colorimetric assay for rhinovirus-14 3C protease using peptide p-nitroanilides as substrates." Anal Biochem 252 (2): 238-45. Wang, Q. M., R.B. Johnson, et al. (1998). "Dual inhibition ofhuman rhinovirus 2A and 3 C proteases by homophthalimides." Antimicrob Agents Chemother 42 (4): 916-20. Wang Z., M. Walter, T, Selwood, H. Rubin and N.M. Schechter (1998) "Recombinant Expression of human mast cell proteases chymase and tryptase." Biol Chem 379 (2): 167-74. Weissleder, R., et al., (1999) In vivo imaging of tumors with protease-activated near-infrared fluorescent probes. Nat Biotechnol, 17 (4): p. 375-8. Weissman, A. M. (2001). "Themes and variations on ubiquitylation." Nat Rev Mol Cell Biol 2 (3): 169-78. Wilkinson (1997). "Reg of ubiquitin-dep processes by deubiquitinating enzymes." Faseb J 11 (14): 1245-56. Wilkinson, K. D. (2000). "Ubiquitination and deubiquitination: targeting of proteins for degradation by the proteasome." Semin Cell Dev Biol 11 (3): 141-8.

Wilkinson, K. D. , M. J. Cox, et al. (1986). "Synthesis and characterization of ubiquitin ethyl ester, a new substrate for ubiquitin carboxyl-terminal hydrolase." Biochemistry 25 (21): 6644-9. Wilkinson, K. D. a. H., M. (1998). Ubiquitin and the biology of the cell. H. J. R. Peters J.M., Finley D. New York, Plenum Press: 99-125. Wong, S., T. H. Morales, et al. (1990). "Ubiquitin-EP52 fusion protein homologs from Trypanosoma brucei." Nucleic Acids Res 18 (23): 7181. Woo (1995) "Multiple ubiquitin C-term hydrolases from chick skeletal muscle" JBiolChem 270 (32): 18766-73. Wood (2002) "Dubble or nothing? Is HAUSP deubiquit enz arbiter of p53 levéis?" Sci STKE 2002 (143): PE34. Yamagishi, H., V. Garg, et al. (1999). "A molecular pathway revealing a genetic base for human cardiac and craniofacial defects. "Science 283 (5405): 1158-61, Yan, N., JH Doelling, et al., (2000)." The ubiquitin-specific protease family from Arabidopsis. AtUBPI and 2 are required The resistance to the amino acid analog canavanine. "Plant Physiol 124 (4): 1828-43, Yao, T. and RE Cohen (2002)." A cryptic protease couples deubiquitination and degradation by the proteasome. "Nature 419 (6905) : 403-7 Yuan, C, et al. (1999) Structural analysis of phospholipase A2 from functional perspective 1. Functionally relevant solution structure and roles of the hydrogen-bonding network Biochemistry.38 (10): p.2909- 18. Zalkin, H. (1993). "The amidotransferases." Adv Enzymol Relat Areas Mol Biol 66: 203-309, Zhu, Y., M. Carroll, et al. (1996). "DUB-I, a deubiquitinating enzyme with g rowth-suppressing activity. "Proc Nati Acad Sci U S A 93 (8): 3275-9. Zhu, Y., K. Lambert, et al. (1997). "DUB-2 is a member of a novel family of cytokine-inducible deubiquitinating enzymes." J Biol Chem 272 (1): 51-7.

Claims (85)

1. A method for determining proteolytic enzyme activity, comprising providing a fusion polymer comprising a first polymer comprising Ubiquitin or a Ubiquitin-like Protein (UBL) or a C-terminal functional segment thereof and a second polymer comprising a free N-terminal amino acid required for detection; wherein the first and second polymers are operably linked to each other through the C-terminal UBL and the second N-terminal polymer; contacting the fusion polymer with the proteolytic enzyme that is fragmented in the C-terminal UBL; detecting a signal associated with any amount or activity of the fragmented polymer, or a sample that is assumed to comprise the enzyme; and establishing a correlation between the fragmented polymer signal for proteolytic enzyme activity.

The method according to claim 1, further comprising normalizing each sample fragmentation enzyme or signal by reference to the 0% and 100% fragmentation signals and assigning a proteolytic enzyme activity value to each enzyme or sample Of the same; wherein, when the enzyme activity obtained is below a cut-off value it can be said that the enzyme or sample is inactive and when it is above the cut-off value it is active.

3. The method according to claim 2, characterized in that the fragmentation signals of 0% and 100% are obtained by repeating the contacting, detection and establishment steps for complete fragmentation and complete inhibition of the enzyme activity. proteolytic to obtain the fragmentation signals of 100% and 0%.

4. The method according to claim 2, characterized in that the normalization step is carried out by means of the normalization of each enzyme or sample fragmentation signal by reference to a curve of fragmentation values of enzyme activity and assigning a Proteolytic enzyme activity to each enzyme or sample thereof; wherein, when the enzyme activity obtained is below a cut-off value it can be said that the enzyme or sample is inactive and when it is above the cut-off value it is active.

The method according to claim 1, characterized in that the first polymer comprises ubiquitin, SUMO, Nedd8, ISG15, Apg8, Apg12, FAT10, Urml, Hub, UBi, Ruby, ISG15, or a functional segment of C-terminal bond thereof comprising an amino acid within the UBL cycle linking its a-helix 1 and β-filament 3 to the C-terminus.

The method according to claim 1, characterized in that the second polymer comprises a reporter protein, antigen, transcription factor or functional fragment thereof; and / or the first polymer comprises a UBL or a functional C-terminal bond segment comprising an amino acid within the UBL cycle linking its a-helix 1 and β-filament 3 to the C-terminus.

The method according to claim 1, characterized in that the first and / or second polymer (s) are made detectable (s) to the fragmentation of the proteolytic enzyme.

8. The method according to claim 1, characterized in that the first and / or second polymer (s) are made detectable by activating a signal that carries (n) and / or by means of a link ao generation of a detectable signal.

The method according to claim 1, characterized in that the detectable signal comprises a radioactive, fluorescent, phosphorescent, chromogenic, sonogenic, or chemiluminescent signal.

The method according to claim 6, further comprising obtaining an N-terminal UBL segment comprising an amino acid within the UBL cycle linking its a-helix 1 and β-strand 3 to the C-terminus, or the segment remaining amino acid to form a UBL with the C-terminal UBL segment; the N-terminal UBL segment that is operatively linked to one of a first and second link pairs; and obtain the second link pair; and wherein the fusion polymer is fragmented to the bond of the first and second binding pairs.

The method according to claim 10, characterized in that the binding pairs comprise a receiver and a binding agent of the receiver.

The method according to claim 11, characterized in that the receptor binding agent comprises a drug, hormone, antigen, ligand, or functional binding fragment of the receptor thereof; and the receptor comprises a drug receptor, hormone receptor, antibody, ligand binding receptor, or functional binding fragment thereof.

13. The method according to claim 10, characterized in that the binding of the UBL N- and C-terminal allows the recognition of a UBL conformation and the fragmentation of the polymer in the C-terminal by means of the proteolytic enzyme.

The method according to claim 1, characterized in that the first and second polymers are covalently bound to each other.

15. The method according to claim 1, characterized in that the first and second polymers are operably linked through a linker.

16. The method according to claim 15, characterized in that the linker comprises at least one amino acid.

17. The method according to claim 1, characterized in that the fusion polymer comprises a fusion protein.

18. The method according to claim 15, characterized in that the proteolytic enzyme comprises an isopeptidase or functional fragment thereof. The method according to claim 1, characterized in that the proteolytic enzyme comprises a C-terminal ubiquitin hydrolase, ubiquitin-specific protease or functional fragment thereof. The method according to claim 15, characterized in that the proteolytic enzyme comprises ULP1, ULP2, SENP1, SENP2, yeast YUH1, mammal UCHU, UCH-L3, UCH37, Bapl, USP-M, DUB-I, DUB-2 , USP7, NPU, CYLD, CYLDI, KIAA0849, USP9X, DFFRX, USP9, FAFX, USP9Y, DFFRY, USPIO, fafy, OTUBI, OTBI, lower urinary tract obstruction, HSPC263, OTUB2, C14orfl37, OTB2, OTU2, USPIO, KIAA0190, USPI 1 UHXI, USP12, UBHIm, USP12L1, USP13, ISOT3, USP14, TGT, USP15, KLAA0529, USP16, UBPM, USP18, UBP43, USP

19, KIAA0891, ZMYND9, USP

20, KIAA1003, LSFR3A, USP

21, USP23, NEDD8-specific protease, USP22 , KIAA1063, USP24, KIAA1057, USP25, USP25, USP28, USP28, USP29, USP30, USP32, USP32, USP32, KIAA1097, VDUI, USP35, KIAA1372, USP34, USP36, KIAA1453, USP37, KIAA1594, USP38, KIAA1891, USP40, USP42, USP44, USP46 , USP49, USP51, UBPI, USPI, UBP2, USP2, UBP41, UBP3, USP3, UBP4, USP4, NPU, UNPH, UBP5, USP5, ISOT, UBP6, USP6, TRE2, UBP7, USP7, HAUSP, UBP8, USP8, KIAA0055 , UBPY, VCIP, VCIP135, KIAAI 850, Cezannel, Cezanne2, A20, UCH-LI, Park5, UCH-L3, UCH-L5, UCH-37, ATXN3, ATX3, MJD, IDYM, SCA3, pohi, PSMD14, CSN5, COPS5, JABI, Senpi, SENP2, SENP3, SSP3, SUSP3, SENP5, FKSG45, SENP6, FKSG6, KIAA0797, SSPI, SUSPI, SENP7, KIAA1707, SSP2, SUSP2, SENP8, VCEP, VCEP135, KIAA1850, A20, UCH-LI, Park5, UCH-L3, UCH-L5, UCH-37, ATXN3, ATX3, MJD, IDYM, SCA3, pohi, PSMD14, CSN5, COPS5, JABI, Senpi, SENP2, SENP3, SSP3, SUSP3, SENP5, FKSG45, SENP6, F SG6, KIAA0797, SSPI, SUSPI5 SENP7, KIAA1707, SSP2, SUSP2, SENP8, FKSG8, PRSC2, DUBI, DUB2, DUB3, DUB4, or a functional fragment thereof. The method according to claim 1, characterized in that the second polymer comprises a serine protease, precursor of pro-hormone, subtilisin / convertase prohormone similar to kexin, carboxypeptidase, a domain similar to Disintegrin and metalloprotease (reprolysin type) with TromboSpondin type motif I (ADAMTS) a disintegrin and metalloprotease (ADAM) domain, cysteine aspartase, aspartic proteinase, Metaloproteínaza (MMP), RNA-dependent RNA polymerase, N-terminal hydrolase nucleophile (Ntn), tautomerase 4-oxalocrotonato, chorismate synthase, β-lactam acylase, reverse transcriptase, phospholipase, transcription factor, or a functional fragment thereof.

22. The method according to claim 1, characterized in that the second polymer comprises a viral reverse transcriptase, sigma transcription factor, glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase (GPATase), coagulation factor Xa, 3Dpol RNA-dependent RNA polymerase, glutamine 5- phosphoribosyl-l-pyrophosphate amidotransferase, penicillin acylase, reverse transcriptase, corismato synthase, tryptase, chymase, enterokinase, transcription factor s * ", thrombin, dipeptidyl peptidase, HtrA2, neurophysin, vasopressin, furin, carboxypeptidase B, carboxypeptidase Y, vWF- protein protease / AD AMTS 13, ADAM 1, ADAM 2, caspase, pepsin, renin, cathepsin D, monkey virus protein Mason-Pf izer, MMP20, MMP26, glycosilasparginase, 20S proteasome ß subunit, glutamine PRPP amidotransferase, YdcE, YwhB, cephalosporin acylase, reverse transcipatase CaMV, phospholipase A2, or a functional fragment thereof

23. The method of compliance with the indication 1, which further comprises expressing a polynucleotide that encodes the fusion protein under conditions effective for expression thereof.

24. The method according to claim 23, characterized in that the polynucleotide is expressed in a eukaryotic cell, or fraction or extract thereof.

25. The method according to claim 23, further comprising isolating the fusion protein thus obtained; and optionally purifying the fusion protein.

26. The method according to claim 1, further comprising repeating the steps of contacting, detecting and establishing in the presence of a sample that is assumed to comprise a modulator of proteolytic enzyme activity; and determining a value for the effect of the sample on the proteolytic enzyme activity by reference of the sample signal to the activity signal of the corresponding enzyme obtained in the absence of the sample.

27. The method according to claim 25, characterized in that the sample comprises a physiological fluid, a tissue sample, cell, or cell fraction.

28. The method according to claim 1, characterized in that one or more of the steps are conducted in vitro, in vivo, ex vivo, in cell or tissue culture, in cell or tissue extracts.

29. The method according to claim 1, characterized in that the detection step comprises the detection of cell growth, or of a chromogenic, radioactive, fluorescent, phosphorescent or chemiluminescent signal.

30. The method according to claim 26, characterized in that the steps of contacting, detecting, establishing and determining are conducted separately for a plurality of samples that are assumed to comprise a modulator of proteolytic enzyme activity to obtain a value for the effect of the sample on the activity of proteolytic enzyme.

31. The method according to claim 30, characterized in that it is automated.

32. The method according to claim 30, characterized in that it collects, processes and reports on the information obtained for each sample and controls.

33. A kit for determining proteolytic enzyme activity, comprising a fusion polymer comprising a first polymer comprising Ubiquitin or a Ubiquitin-like protein (UBL) or a C-terminal segment thereof and a second polymer comprising a polypeptide that requires a free N-terminal amino acid for detection; wherein the first and second polymers are operably linked to each other through the Ubiquitin or C-terminal UBL and the second N-terminal polymer; and instruction to conduct the proteolytic enzyme test, detecting a signal associated with the amount or activity of the first and / or second polymers, and establishing a correlation of the detected signal for the proteolytic activity of the enzyme; and optionally a source of the proteolytic enzyme that cleaves in the C-terminal UBL.

34. The equipment according to claim 33, which further comprises one or more first and second link pairs, wherein the first pair can be operatively linked to an N-terminal UBL segment, and wherein, when the first and second link pairs are linked together the segment UBL C-terminal is linked to the N-terminal UBL segment allowing a UBL conformation, the proteolytic enzyme fragments the fusion polymer; reagents for conducting the enzyme fragmentation step; means for conducting the detection step; and means for correlating the detectable signal for proteolytic enzyme activity or change thereof.

35. The equipment according to claim 33, characterized in that the fusion polymer comprises a fusion protein; the kit further comprising a fusion polynucleotide encoding them is replaced by the fusion polymer; and optionally one or more reagents for expressing the polynucleotide; and / or a cell (s) or fraction or extract thereof to express the polynucleotide when it is used in place of the fusion protein.

36. The equipment according to claim 33, further comprising means for separately containing a plurality of samples; and instructions to conduct the test automatically.

37. The equipment according to claim 33, further comprising Means for automatically processing the data of each sample; and instructions for its use.

38. A method for screening compounds for their effect on proteolytic activity, which comprises obtaining a fusion polymer comprising a first polymer comprising Ubiquitin or a Ubiquitin-like protein (UBL) or a functional C-terminal bonding segment of the same and a second polymer comprising a free N-terminal amino acid; wherein the first and second polymers are operatively linked to one another through the N-C-terminals; contacting the fusion polymer with a proteolytic enzyme that cleaves C-terminal UBL under conditions effective for fragmentation to occur; detecting a signal associated with a fragmentation amount to obtain a fragmentation signal of 100%; repeating the contacting and detection steps in the presence of a total inhibitor of proteolytic enzyme activity to obtain a 0% fragmentation signal; obtain a set of compounds; repeating separately the steps of obtaining the fusion polymer, contacting and detecting in the presence of each compound to obtain a fragmentation signal; normalize each compound fragmentation signal by reference to the fragmentation signal of 0% and 100% and assign a value of proteolytic enzyme activity to each compound.

39. The method according to claim 38, comprising conducting the contacting and detecting steps with a known modulator of proteolytic enzyme activity to obtain a fragmentation control signal from a point instead of the 0% fragmentation signals and 100%; determining a value of proteolytic enzyme activity for the modulator by reference to the activity value of the corresponding enzyme obtained in the absence of the modulator; and normalizing each compound fragmentation signal by reference to the control fragmentation signal and assigning a proteolytic enzyme activity value to each compound.

40. The method according to claim 38, characterized in that the normalization step is conducted by normalizing each compound fragmentation signal by reference to a fragmentation value curve of the activity of the enzyme and assigning an activity of proteolytic enzyme to each compound; wherein, when the enzyme activity obtained is below a cut-off value, it can be said that the compound is inactive and when it is above the cut-off value it is active.

41. The method according to claim 37, characterized in that the cut-off value is 50% of the proteolytic enzyme activity, and when the enzyme activity in the presence of the compound decreases by at least 50% it can be said that the compound it is an inhibitor, and when the enzyme activity is increased by at least 50% the compound is an enhancer.

42. The method according to claim 38, further comprising determining a concentration of compound that inhibits (IC50) and / or improves (EC50) enzyme activity by 50%; and comparing the IC50 and / or EC50 of the compound to determine its resistance to enzyme activity as an inhibitor and / or enhancer.

43. The method according to claim 38, characterized in that the modulator comprises an activator of proteolytic enzyme activity.

44. The method according to claim 38, characterized in that the modulator comprises an inhibitor of proteolytic enzyme activity.

45. The method according to claim 38, characterized in that one or more of the steps are conducted in vitro, in vivo, ex vivo, in cell or tissue culture, in cell fractions or tissue extracts.

46. The method according to claim 38, characterized in that the detection step comprises detection of cell growth, chromogenic, radioactive, fluorescent, phosphorescent, sonogenic, chemiluminescent.

47. The method according to claim 38, characterized in that the first polymer comprises ubiquitin, SUMO, Nedd8, ISG15, Apg8, Apg12, FAT10, Urml, Hub, UB, Ruby, ISG15, or a functional segment of C-bond. terminal comprising an amino acid within the UBL cycle linking its a-helix 1 and β-filament 3 to the C-terminus.

48. The method according to claim 38, characterized in that the second polymer comprises a reporter protein, or a functional segment of C-terminal bond thereof; and / or the first polymer comprises a functional C-terminal bond segment comprising an amino acid within the UBL cycle linking its a-helix 1 and β-filament 3 to the C-terminus.

49. The method according to claim 38, characterized in that the first and / or second polymer (s) becomes (are) detectable (s) to the fragmentation of the proteolytic enzyme.

50. The method according to claim 38, characterized in that the first and / or second polymer (s) is (are) detectable by means of the activation of a signal that transports (n) and / or by link ao generation of a detectable signal.

51. The method according to claim 38, characterized in that the detectable signal comprises a radioactive, fluorescent, phosphorescent, chromogenic, sonogenic signal.

52. The method according to claim 45, further comprising obtaining a functional U-terminal N-terminal bond segment comprising an amino acid within the UBL cycle linking its a-helix 1 and β-filament 3 to the N-terminus thereof, wherein, when the N-terminal segment and the C-terminal segment are linked to each other form a complete UBL, and the N-terminal UBL segment that is operatively linked to one of a first and second link pairs; and obtain the second link pair; and wherein the fusion polymer is fragmented to the bond of the first and second binding pairs.

53. The method according to claim 52, characterized in that the binding pairs comprise a receiver and a binding agent of the receiver.

54. The method according to claim 52, characterized in that the receptor binding agent comprises a drug, hormone, antigen, ligand, or functional fragment thereof; and the receptor comprises a drug receptor, a hormone receptor, an antibody, a ligand binding receptor, or a functional fragment thereof.

55. The method according to claim 52, characterized in that the binding of UBL N- and C-terminals allows the recognition of a UBL conformation and polymer fragmentation by means of the proteolytic enzyme.

56. The method according to claim 38, characterized in that the first and second polymers are linked to each other covalently.

57. The method according to claim 38, characterized in that the first and second polymers are operatively linked through a linker.

58. The method according to claim 57, characterized in that the linker comprises at least one amino acid.

59. The method according to claim 38, characterized in that the fusion polymer comprises a fusion protein.

60. The method according to claim 38, characterized in that the proteolytic enzyme comprises an isopeptidase or functional fragment thereof.

61. The method according to claim 38, characterized in that the proteolytic enzyme comprises a C-terminal ubiquitin hydrolase, ubiquitin-specific protease or functional fragment thereof.

62. The method according to claim 38, characterized in that the proteolytic enzyme comprises ULP 1, ULP2, SENP 1, SENP2, yeast YUHI, mammal UCHU, UCH-L3, UCH37, Bapl, USP-M, DUB-I, DUB -2, USP7, UNP, CYLD, CYLDI, KIAA0849, USP9X, DFFRX, USP9, FAFX, USP9Y, DFFRY, USPIO, FAFY, OTUBI, OTBI, OTUI, HSPC263, OTUB2, C14orfl37, OTB2, OTU2, USPIO, KIAA0190, USPI 1, UHXI, USP12, UBHIm, USP12L1, USP13, ISOT3, USP14, TGP, USP15, KIAA0529, USP16, UBPM, USP18, UBP43, USP19, KIAA0891, ZMYND9, USP20, KIAA1003, LSFR3A, USP21, USP23, NEDD8-specific protease Ca, USP22, KIAA1063, USP24, KIAA1057, USP25, USP26, USP28, USP29, USP30, USP32, USP32, USP32, KIAA1097, VDUI, USP35, KIAA1372, USP34, USP36, KIAA1453, USP37, KIAA1594, USP38, KIAA1891, USP40, USP42 , USP44, USP46, USP49, USP51, USP51, UBPI, USPI, UBP2, USP2, UBP41, UBP3, USP4, USP4, USP4, USP4, UNP, UBP5, USP5, ISP, UBP6, USP6, TRE2, UBP7, USP7, HAUSP, UBP8 , USP8, KIAA0055, UBPY, VCIP, VCIP135, KIAA1850, Cezanne 1, Cezanne2, A20, UCH- Ll, Park5, UCH-L3, UCH-L5, UCH-37, ATXN3, ATX3, JD, MJDI, SCA3, POHI, PSMD14, CSN5, COPS5, JABI, SENPI, SENP2, SENP3, SSP3, SUSP3, SENP5, FKSG45, SENP6, FKSG6, KIAA0797, SSPI, SUSPI, SENP7, KIAA1707, SSP2, SUSP2, SENP8, VCIP, VCIP135, KJAA1850, A20, UCH-LI, Park5, UCH-L3, UCH-L5, UCH-37, ATXN3, ATX3, MJD, MJDI, SCA3, POHI, PSMD14, CSN5, C0PS5, JABI, SENPI, SENP2, SENP3, SSP3, SUSP3, SENP5, FKSG45, SENP6, FKSG6, KIAA0797, SSPI, SUSPI, SENP7, KIAA1707, SSP2, SUSP2, SENP8, FKSG8, PRSC2, DUBI, DUB2, DIJB3, DUB4, or a functional fragment thereof.

63. The method according to claim 38, characterized in that the second polymer comprises a serine protease, precursor of pro-hormoneSubtilisin / kexin-like hormone convertase, carboxypeptidase, a disintegrin-like domain and metalloprotease (reprolysin-like type) with type I TromboSpondin (ADAMTS) motif, a Disintegrin and metalloprotease (ADAM) domain, cysteine aspartase, aspartic proteinase, Matrix metalloproteinase (MMP), RNA-dependent RNA polymerase, N-terminal nucleophile (Ntn) hydrolase, 4-oxalocrotonate tautomerase, corismato synthase, β-lactam acylase, reverse transcriptase, phospholipase, transcription factor or a functional fragment thereof .

64. The method according to claim 38, characterized in that the second polymer comprises a viral reverse transcriptase, sigma transcription factor, glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase (GPATase), coagulation factor Xa, 3Dpol RNA-dependent RNA polymerase, glutamine 5-phosphoribosyl-l-pyrophosphate amidotransferase, penicillin acylase, reverse transcriptase, corismate synthase, tryptase, chymase, enterokinase, transcription factor, thrombin, dipeptidyl peptidase, HtrA2, neurophysin, vasopressin, furin, carboxypeptidase B, carboxypeptidase Y, vW F-fragmentation protease / ADAMTS 13, ADAM 1, ADAM 2, caspase, pepsin, renin, cathepsin D, monkey virus protein Mason-Pf izer, MMP20, MMP26, glycosilasparginase, 20S proteasome ß subunit, glutamine PRPP amidotransferase, YdcE , YwhB, cephalosporin acylase, CaMV reverse transcriptase, phospholipase A2, or a functional fragment thereof.

65. The method according to claim 59, characterized in that the fusion protein is obtained by expression of a polynucleotide that encodes the fusion protein.

66. The method according to claim 65, characterized in that the polynucleotide is expressed in a eukaryotic cell or fraction or extract thereof.

67. The method according to claim 65, further comprising isolating the fusion protein thus obtained; and optionally purifying the fusion protein.

68. The method according to claim 38, characterized in that it is automated.

69. The method according to claim 68, characterized in that it collects, processes and reports the information obtained for each modulator and controls.

70. An enzyme proteolytic activity modulator screening machine, comprising a fusion polymer comprising a first polymer comprising Ubiquitin or a Ubiquitin-like protein (UBL) or a C-terminal segment thereof and a second polymer comprising polypeptide that requires a free N-terminal amino acid for detection; wherein the first and second polymers are operably linked to each other through the Ubiquitin or C-terminal UBL and the second N-terminal polymer; and instruction to conduct the proteolytic enzyme test, detecting a signal associated with the amount or activity of the first and / or second polymers, and establishing a correlation of the detected signal for the proteolytic activity of the enzyme for a plurality of modulators and controls; and optionally a source of the proteolytic enzyme that is fragmented in the Ubiquitin or C-terminal UBL.

71. The equipment according to claim 70, further comprising one or more first and second link pairs, wherein the first pair can be operably linked to an N-terminal UBL segment, and wherein, when the first and second second binding pairs are linked to each other the C-terminal UBL segment is linked to the N-terminal UBL segment allowing a UBL conformation, the proteolytic enzyme fragments the fusion polymer; reagents for conducting the cleavage of C-terminal UBL enzyme from the fusion polymer; means for detecting a signal (s) emitted by the first and / or second fragmented polymers; and means for correlating the detectable signal (s) for proteolytic enzyme activity or changing it by reference to a control.

72. The equipment according to claim 70, characterized in that the fusion polymer comprises a fusion protein; and the kit further comprises a fusion polynucleotide encoding it is replaced by the fusion polymer; and optionally one or more reagents for polynucleotide expression; and / or a cell (s) or fraction or extract thereof to express the polynucleotide when it is used in place of the fusion protein.

73. The equipment according to claim 70, further comprising means for separately containing a plurality of samples; and instructions to conduct the test automatically.

74. The equipment according to claim 70, further comprising means for automatically processing the data for each sample; and instructions for its use.

75. A transgenic cell, plant or animal, comprising a Ubiquitin- or U BL-reporter fusion polynucleotide that is optionally integrated within the chromosome of the cell, plant or animal; where the reporter is associated with a specific disease or condition or family of the same.

76. The transgenic cell, plant or animal according to claim 75, characterized in that it is capable of expressing a Ubiquitin- or UBL-reporter fusion protein.

77. The transgenic cell, plant or animal according to claim 76, characterized in that it is obtained by means of the formation of a hybrid vector by cloning the fusion polynucleotide within a vector, and transfecting the cell, plant or animal with the hybrid vector.

78. The transgenic cell, plant or animal according to claim 77, characterized in that the vector comprises a plasmid, and the cell comprises a eukaryotic cell.

79. The transgenic cell, plant or animal according to claim 70, characterized in that it is further modified to serve as a cell, plant or animal model for a disease or condition.

80. The transgenic cell, plant or animal according to claim 79, characterized in that the isopeptidase is associated with an auto-immune, neoplastic, genetic mutation, metabolic, cardiovascular or neurodegenerative disease or condition.

81. The transgenic cell, plant or animal according to claim 80, characterized in that the disease or condition comprises cancer, lupus, diabetes, IBD, cardiovascular disease, neurodegeneration, or inflammatory condition.

82. A method for producing the transgenic cell, plant or animal according to claim 75, comprising obtaining a cell, plant or animal; obtain a Ubiquitin-, UBL- fusion polynucleotide or its functional C-terminal-reporter linkage fragment; obtain a hybrid vector that transports the hybrid polynucleotide operably linked to a vector; and stably transfecting the hybrid vector within the cell, plant or animal.

83. The method according to claim 82, characterized in that the fusion polynucleotide is integrated into a cell, plant or animal chromosome.

84. The method according to claim 82, characterized in that the fusion polynucleotide comprises a fusion deoxyribonucleotide.

85. A method for diagnosing a disease or condition, comprising obtaining the cell, plant or animal of claim 75, or fractions or tissue thereof, wherein the reporter is associated with a disease or condition; contacting or administering a sample obtained from a subject that is supposed to be suffering from the disease or condition with the cell, plant or animal; detect any signal produced by the reporter in the presence of the sample; and compare the signal with the controls for the 0% and 100% signals.