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WO2002004644A2 - Aspartyl-protease - Google Patents

Aspartyl-protease Download PDF

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Publication number
WO2002004644A2
WO2002004644A2 PCT/EP2001/007512 EP0107512W WO0204644A2 WO 2002004644 A2 WO2002004644 A2 WO 2002004644A2 EP 0107512 W EP0107512 W EP 0107512W WO 0204644 A2 WO0204644 A2 WO 0204644A2
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WO
WIPO (PCT)
Prior art keywords
gly
phe
leu
asp
val
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Ceased
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PCT/EP2001/007512
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English (en)
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WO2002004644A3 (fr
Inventor
Christian Haass
Harald Steiner
Katja Fechteler
Marcus Kostka
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Boehringer Ingelheim Pharma GmbH and Co KG
Boehringer Ingelheim Pharmaceuticals Inc
Original Assignee
Boehringer Ingelheim Pharma GmbH and Co KG
Boehringer Ingelheim Pharmaceuticals Inc
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Priority to AU2001281926A priority Critical patent/AU2001281926A1/en
Publication of WO2002004644A2 publication Critical patent/WO2002004644A2/fr
Publication of WO2002004644A3 publication Critical patent/WO2002004644A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
    • C12N9/6413—Aspartic endopeptidases (3.4.23)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6478—Aspartic endopeptidases (3.4.23)

Definitions

  • the invention relates to proteins, peptides or aspartyl-proteases comprising specified consensus motifs and nucleic acids encoding said proteins, peptides or aspartyl-proteases.
  • the invention further relates to methods of screening for substances capable of inhibiting said aspartyl-proteases, substances identifiable with said method and pharmaceutical compositions comprising said substances.
  • the invention belongs to the field of neurodegenerative diseases and Alzheimer's disease.
  • PS proteins undergo endoproteolysis (3) and exist as complex composed of N- and C- terminal fragments [NTF and CTF; (4, 5, 6)]. Complex formation appears to be a prerequisite for PS function (7, 8). PS proteins are required to support the cleavage of membrane bound proteins either within or close to the cytoplasmic site of the membrane (9, 10).
  • substrates include members of the APP family, Notch, and Irel, and a PS1 gene ablation results in the inhibition of endoproteolysis of these proteins (11, 12, 13).
  • a Notch like phenotype is observed (14, 15), which is strongly augmented if in addition to PS1 the homologous PS2 is deleted as well (16, 17).
  • the invention relates to proteins, peptides or aspartyl-proteases comprising specified consensus motifs and nucleic acids encoding said proteins, peptides or aspartyl-proteases.
  • the invention further relates to methods of screening for substances capable of inhibiting said aspartyl- proteases, substances identifiable with said method and pharmaceutical compositions comprising said substances.
  • FIG. 1 (A) Schematic representation of PS 1. The critical aspartates in TM 6 and 7 (arrow heads) as well as the endoproteolytic cleavage site(s) (black box) and the mutations introduced at amino acid residue 384 are indicated. (B) Expression, endoproteolytic processing and replacement of the mutant PS1 derivatives. Upper panel: Cell lysates were immunoprecipitated with antibody 3027 and precipitated PS1 derivatives (CTFs and holoprotein) were visualized by immunoblotting using antibody BI.3D7. Note, that endoproteolysis of PS1 is diminished in cell lines expressing the G384K, G384D, and D385A mutant.
  • ⁇ APP-CTFs generated by ⁇ -, and ⁇ -secretase (longer exposure reveals significant amounts of ⁇ -secretase generated ⁇ APP CTFs) strongly accumulated in cells expressing the PSl G384 artificial mutants as well as in cells expressing PSl D385A as observed previously (23).
  • Figure 2 Effects of the mutant PSl derivatives on APP endoproteolysis.
  • A Upper panel (conditioned media): A ⁇ species were immunoprecipitated from conditioned media of metabolically labeled cells with antibody 3926 and separated on a previously described Tris- Bicine gel system, which allows the specific identification of A ⁇ 40 and A ⁇ 42 (53). APPs was immunoprecipitated with antibody 5313.
  • B Quantitation of A ⁇ levels by phosphorimaging.
  • a ⁇ species were immunoprecipitated as in (A) but separated on 10-20% Tris-Tricine gels. Total A ⁇ levels are expressed relative to the A ⁇ levels in PSl wt expressing cells. Bars represent the mean +/- S.E. of three independent experiments.
  • C Independent quantitation of total A ⁇ levels by a previously described ELISA (7). Horizontal bars represent the mean of six independent experiments using conditioned media from unlabeled cells.
  • FIG. 3 Effects of the mutant PSl derivatives on Notch endoproteolysis.
  • Cell lines expressing PSl derivatives were transfected with the Notch ⁇ E cDNA (43).
  • NICD formation was analyzed in pulse chase experiments as described (22). Note the significant change of the ratio of N ⁇ E:NICD in cells expressing the G384P and G384K mutation as compared to those lines, which express wt PSl or PSl G384A. Consistent with previous results NICD formation was blocked in cell lines expressing PSl D385A (22, 29). As observed previously (21, 41, 43), N ⁇ E is not only processed to NICD but also degraded during the cold chase.
  • FIG. 4 Sequence comparison of the active site consensus sequence of PSs (A) with members of the TFPP family (B).
  • the invention provides a protein or peptide comprising the consensus motif characterized by the
  • X 1 is a Lys or a Arg
  • X 2 is a Lys, Phe, Met or Leu
  • X 3 is a He, Tyr or Val
  • X 4 is a Val, Gly, Ala
  • X 5 is a Met, Phe, Val, Met, He, Leu
  • X 6 is a Gly or Ala
  • X 7 is a Tyr, Gly, Asp, Phe or His
  • X 8 is a He, Phe, Leu, Pro, Val or Phe
  • X 9 is a Lys, Ala or He.
  • the protein or peptide comprises further amino acids and may also be glycosylated. Most preferably, so the protein or peptide is an aspartyl-protease.
  • consensus motifs are exemplified in figure 4 and comprise the active site of e.g. proteases of the ⁇ -secretase, presenilin or type 4 prepilin peptidase (TFPP) families.
  • the invention relates to a protein or peptide according to the invention comprising the consensus motif characterized by the amino acids X'-Leu-Gly-X 2 -Gly- Asp-Phe-X 3 wherein X 1 , is a Lys or a Arg; X 2 is a Lys, Phe, Met or Leu and X 3 is a He, Tyr or
  • the consensus motif is exemplified in figure 4 (upper part) and comprises the active site of e.g. proteases of the ⁇ -secretase or presenilin family.
  • the invention relates to a protein or peptide according to the invention comprising the consensus motif characterized by the amino acids X 4 -X 5 -X 6 -X 7 -Gly- Asp-X 8 -X 9 , wherein X 4 is a Val, Gly, Ala; X 5 is a Met, Phe, Val, Met, He, Leu; X 6 is a Gly or
  • X 7 is a Tyr, Gly, Asp, Phe or His;
  • X 8 is a He, Phe, Leu, Pro, Val or Phe and
  • X 9 is a Lys, Ala or He.
  • the consensus motif is exemplified in figure 4 (lower part) and comprises the active site of e.g. proteases of the TFPP family.
  • the invention relates to a peptide according to the invention consisting of the the consensus motif characterized by the amino acids X'-Leu-Gly-X 2 -
  • Gly-Asp-Phe-X 3 wherein X 1 , is a Lys or a Arg; X 2 is a Lys, Phe, Met or Leu and X 3 is a He, Tyr or Val.
  • the peptide consisting of the consensus motif of e.g. proteases of the ⁇ -secretase or presenilin family is depicted in figure 4 (upper part).
  • the invention relates to a peptide according to the invention consisting of the the consensus motif characterized by the amino acids X 4 -X 5 -X 6 -X 7 -
  • Gly-Asp-X s -X 9 wherein X 4 is a Val, Gly, Ala; X 5 is a Met, Phe, Val, Met, He, Leu; X 6 is a Gly or
  • the peptide consisting of the consensus motif of e.g. proteases of the TFPP family is depicted in figure 4 (lower part).
  • the invention relates to a peptide according to the invention consisting of the the consensus motif Lys-Leu-Gly-Leu-Gly-Asp-Phe-Ile.
  • the peptide consisting of the consensus motif of e.g. proteases of the ⁇ -secretase or presenilin family is depicted in figure 4 (upper part).
  • the invention relates to a peptide according to the invention consisting of the the consensus motif Gly-Met-Gly-Tyr-Gly-Asp-Phe-Lys.
  • the peptide consisting of the consensus motif of e.g. proteases of the TFPP family is depicted in figure 4
  • the invention relates to a protein according to the invention characterized in that it is an aspartyl-protease.
  • the invention relates to a aspartyl-protease according to the invention comprising the consensus motif Lys-Leu-Gly-Leu-Gly-Asp-Phe-Ile.
  • the consensus motif is exemplified in figure 4 (upper part) and comprises the active site of e.g. aspartyl-proteases of the ⁇ -secretase or presenilin family.
  • the invention relates to a aspartyl-protease according to the invention comprising the consensus motif Gly-Met-Gly-Tyr-Gly-Asp-Phe-Lys.
  • the consensus motif is exemplified in figure 4 (lower part) and comprises the active site of e.g. aspartyl-proteases of the TFPP family.
  • the invention relates to a aspartyl-protease comprising the consensus motif characterized by the amino acids Gly-X'-X ⁇ Gly-Asp-X 3 ; wherein X 1 is Ala or no amino acid (Ala is an optional amino acid, i.e. no amino acid means either Ala is at this position or the sequence continues with X 2 ); X 2 is any amino acid and X 3 is
  • This aspartyl-protease comprises e.g. the consensus motif for any aspartyl- protease comprising the ⁇ -secretase, presenilin and TFPP families (see example 1).
  • the invention relates to a peptide consisting of the consensus motif characterized by the amino acids Gly-X'-X 2 -Gly-Asp-X 3 ; wherein X 1 is Ala or no amino acid; X 2 is any amino acid and X 3 is Phe, He, Val or Leu.
  • This peptide consists of e.g. the consensus motif for any aspartyl-protease comprising the ⁇ -secretase, presenilin and TFPP families (see example 1).
  • a further aspect of the present invention is a nucleic acid encoding any one of the proteins or peptides according to the invention.
  • Yet another important embodiment of the present invention is a method of screening for substances capable of inhibiting an aspartyl-protease according to the invention as disclosed above, characterized in that a) cells expressing the aspartyl-protease according to the invention (see above) and a membrane-associated fusion protein comprising the substrate with the specific cleavage site of said aspartyl-protease and a reporter, are cultured b) said cells are incubated with a test substance, s c) the amount of cleaved-off reporter is measured and d) the value obtained is compared to the value obtained in the absence of the test compound.
  • Membrane-associated according to the invention means, that the substrate is bound to the surface of the membrane or to integrated membrane proteins.
  • substrates also include substrates which interact with the hydrophobic part of the membrane via chemical groups which were o added by post-translational modifications.
  • Membrane-associated substrates further include substrates which interact with the hydrophobic part of the membrane via amino acid side chains, even though less than integrated membrane proteins.
  • Substrate relates to peptides and proteins which at least contain one cleavage site of said protease. Said substrates may also be modified, e.g. glycosylated (Sambrook and Maniatis (1989). Molecular Cloning:A Laboratory Manual, s Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.). According to the invention the substrate is fused to a reporter which is used to detect the activity of the aspartyl-protease (cleavage).
  • the fusion proteins according to the invention can be generated by standard gentechnology methods (Sambrock and Maniatis (1989, see above).
  • the DNA encoding a reporter protein is commercially available, e.g. Clontech, Heidelberg and can be inserted by 0 standard methods into suitable vectors for expression.
  • the DNA encoding the substrate may be derived by standard methods and is available from suitable gene banks.
  • Test substances may be any substance , e.g. a protein or chemical compound known to the artisan or new substances. Said test substances are available e.g. from commercial substance libraries.
  • the specifity of the method can be controlled. By using such a control, the person skilled in the art can recognize the substances which inhibit the aspartyl- protease according to the invention.
  • a suitable cell or cell line, preferably an eukaryotic cell or a cell line, to be transformed with nucleic acid constructs to express said the aspartyl-protease according to the invention may be o any cell or cell line known to the expert in the field, in particular cells or cell lines used in neurological and neurobiology research.
  • Examples of such cells or cell lines useful for producing the transformed cell lines of the invention include mammalian cells or cell lines (e.g.
  • HEK human embryonic kidney
  • BHK BHK
  • CHO Ltk "
  • fibroblasts myelomas
  • neuroblastomas hybridomas
  • oocytes embryonic stem cells
  • insect cell lines e.g., using baculovirus vectors such as pPbac or pMbac (Stratagene, La Jolla, USA)
  • yeast e.g., Pichia pastoris or using yeast expression vectors such as pYESHIS (Invitrogen, San Diego, USA)
  • fungi fungi.
  • reporter genes include, but are not limited to E. coli ⁇ -galactosidase ( ⁇ -gal, Luban and Goff, 1995; Curr Opin Biotechnol 6, 59-64), xanthine-guanine phosphoribosyl transferase (Chu and Berg, 1985; Nucleic Acids Res 13, 2921-2930), galactokinase (Schumperli et al, 1982; Proc Natl Acad Sci USA 79, 257-261), interleukin-2 (Cullen, 1986; Cell 46, 973- 982), thymidine kinase (Searle et al., 1985; Mol Cell Biol 5, 1480-1489), alkaline phosphatase (Toh et al., 1989; Eur J Biochem 182, 231-237; Henthorn et al., 1988; Proc Natl Acad Sci USA 85, 6342-6346), secretory alkaline phosphatase (
  • reporter genes are available from commercial sources.
  • Expression products of the reporter genes such as reporter enzymes can be measured using standard methods.
  • bioassays can be carried out for biologically active proteins such as interleukin-2.
  • Enzyme assays can be performed when the reporter gene product is a reporter enzyme such as alkaline phosphatase or ⁇ -galactosidase.
  • various types of immunoassays such as competitive immunoassays, direct immunoassays and indirect immunoassays may be used.
  • Such immunoassays involve the formation of immune complexes containing the reporter gene product and a measurable "reporter" or a "label".
  • reporter includes moieties that can be detected directly, such as fluorochromes and radiolabels, and moieties such as enzymes that must be reacted or derivatized to be detected.
  • a method according to the invention characterized in that cells are cultured expressing a fusion protein comprising the cleavage site of the ⁇ -secretase.
  • the fusion protein according to the invention is membrane- associated or membrane-integrated.
  • Yet another important embodiment of the present invention is a method according to the invention, characterized in that cells are cultured expressing a fusion protein comprising the
  • Yet another important embodiment of the present invention is a method according to the invention, characterized in that the fusion protein comprises the amyloid precursor protein or a s fragment thereof.
  • Presenilin cleavage can occur either autoproteolytically or by the presenilinase.
  • the fusion protein comprises presenilin 1 or a fragment thereof.
  • the fragments may be N-terminal fragments (NTF) of approx. 21-28 kDa or C-terminal o fragments (CTF) of 16-24 kDa (Haas et al., 1998; J Neural Transm Suppl 53, 159-67; Okochi et al., 1997; FEBS Lett 418, 162-6; Thinakaran et al, 1996 (Ref.
  • Presenilin 1 or fragments thereof are the substrates of the presenilinase and must at least contain one specific presenilinase cleavage site.
  • Yet another important embodiment of the present invention is a method according to the 5 invention, characterized in that the fusion protein comprises presenilin 2 or a fragment thereof.
  • the fragments may be N-terminal fragments (NTF) of approx.
  • Presenilin 2 or fragments thereof are the substrates of the o presenilinase and must at least contain one specific presenilinase cleavage site.
  • Yet another important embodiment of the present invention is a method according to the invention, characterized in that method is a high throughput screening (HTS).
  • HTS relates to an experimental setup wherein a large number of compounds is tested simultaneously.
  • said HTS setup may be carried out in microplates, may be partially or fully automated and may be linked to electronic devices such as computers for data storage, analysis, and interpretation using bioinformatics.
  • said automation may involve robots capable of handling large numbers of microplates and capable of carrying out several thousand tests per day.
  • a test compound which shows a desired inhibitory function in a cell-free system will also be tested in a cell-based system using a cell line according to the present invention.
  • the term HTS also comprises ultra high throughput screening formats (UHTS).
  • UHTS ultra high throughput screening formats
  • said UHTS formats may be carried out using 384- or 1536-well microplates, sub-microliter or sub-nanoliter pipettors, improved plate readers and procedures to deal with evaporation.
  • HTS methods are described e.g. in US 5876946 A or US 5902732 A.
  • the expert in the field can adapt the method described below to a HTS or UHTS format without the need of carrying out an inventive step.
  • Another preferred embodiment of the present invention is the use of a protein or peptide according to the invention for the identification of an inhibitor of the presenilinase or the autoproteolytic cleavage of presenilin.
  • Another preferred embodiment of the present invention is the use of a protein or peptide according to the invention for the identification of an inhibitor of the ⁇ -secretase.
  • Yet another preferred embodiment of the present invention is a substance identifiable with a method according to the invention (see above), characterized in that it is capable of specifically inhibiting the proteolytic cleavage of a ⁇ -secretase-substrate.
  • Yet another preferred embodiment of the present invention is a substance identifiable with a method according to the invention (see above), characterized in that it is capable of specifically inhibiting the proteolytic cleavage of a presenilinase-substrate.
  • said substrate is presenilin.
  • Said substance may be a protein or a chemical compound.
  • An example for such a substance is disclosed below:
  • Another important aspect of the present invention is the use of a substance according to the invention (see above) for the manufacture of a medicament in the treatment of neurodegenerative diseases, preferably Alzheimer's disease.
  • a pharmaceutically acceptable carrier can contain physiologically acceptable compounds that act, for example, to stabilize or to increase the absorption of an substance capable of inhibiting the proteolytic cleavage of a presenilinase or ⁇ -secretase-substrate.
  • physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients (see also e.g. Remington's Pharmaceutical Sciences (1990), 18th ed. Mack Publ., Easton).
  • a pharmaceutically acceptable carrier including a physiologically acceptable compound, depends, for example, on the route of administration of the composition.
  • PSl G384A FAD associated mutation
  • the glycine at residue 384 was exchanged to amino acids containing aliphatic-, ⁇ -helix breaking-, aromatic-, as well as charged (positive and negative) side chains (35) (Fig. la).
  • PSl derivatives were stably transfected into K293 cells overexpressing Swedish mutant ⁇ APP
  • PSl holoprotein (Fig. lb). Efficient endoproteolysis of PSl holoproteins was observed in cell lines expressing the G384A, G384I, G384P and G384W mutants Fig. lb). In contrast, expression of G384K and G384D resulted in reduced amounts of the PSl CTF indicating diminished PSl endoproteolysis by the introduction of charged amino acids at this position. As reported before (23), the PSl D385A mutant also accumulated as an uncleaved holoprotein (Fig. lb).
  • PSl and PS2 Stable expression of PS derivatives results in the displacement of endogenous presenilins (PSl and PS2) (39) and is a prerequisite for functional expression of exogenous presenilins.
  • endogenous presenilins PSl and PS2
  • PS2 the expression of PS2 (38).
  • overexpression of all PSl G384 mutants as well as PSl D385A and wt PSl led to an almost complete displacement of endogenous PS2 CTFs.
  • G384 mutations can affect ⁇ APP CTF generation we analyzed the functional consequences of these variants on A ⁇ generation. For further analysis the mutations were s divided into three groups according to their effects on PS endoproteolysis and ⁇ APP CTF accumulation: (I) G384A: a natural occuring FAD associated mutation (31, 32) which undergoes PS endoproteolysis and accumulates low levels of ⁇ APP CTFs; (II) G384P, G384I and G384W: artificial mutants which do not affect PS endoproteolysis and cause the accumulation of high levels of ⁇ APP CTFs; (III) G384K and G384D: artificial mutants that undergo reduced PS o endoproteolysis and accumulate high levels of ⁇ APP CTFs.
  • G384A from group I, G384P from group II and G384K from group III.
  • Cell lines were labeled with 35 S-methionine and conditioned media were investigated for A ⁇ levels.
  • the FAD associated G384A mutant produced substantial amounts of total A ⁇ , including very high amounts of A ⁇ 42 [Fig. 2a; see (D) for quantitation].
  • the proline and lysine s substitution significantly reduced total A ⁇ production (Fig. 2a).
  • the PSl D385A mutation strongly inhibited total A ⁇ production (Fig. 2a). All mutations analyzed did not affect ⁇ -secretory processing or expression of the ⁇ APP holoprotein (Fig. 2a).
  • a ⁇ levels were quantitated from conditioned media of 35 S-methionine labeled cell lines by o phosphorimaging (Fig. 2b). Independent quantitations were obtained by analyzing A ⁇ concentrations in conditioned media of unlabeled cells using a previously described ELISA (Fig. 2c). Both assays confirmed that the proline and lysine mutation decreased A ⁇ production.
  • mutagenesis of the neighboring glycine residue can therefore have two fundamentally different effects. Depending on the amino acid inserted it can promote very high levels of pathological AJ342 generation or reduce total A ⁇ production.
  • TFPPs do not only contain a very similar active site but also mediate an endoproteolytic cleavage, which is reminiscent to the cleavage reactions supported/mediated by PSs.
  • TFPPs are known to remove leader peptides of selected substrates by cleaving between hydrophobic and hydrophilic domains close to the cytoplasmic site of the membrane (48), a cleavage reaction, which is strikingly similar to the ⁇ -secretase cleavage of Notch and ⁇ APP (10).
  • protease activity occurs as a high molecular weight complex (4, 5, 6, 49), which may contain additional proteins required for its function.
  • cD ⁇ As encoding PSl G384A, PSl G286I, PSl G286P, PSl G384W, PSl G384K and PSl G384D were constructed by oligomicleotide-directed mutagenesis using the polymerase s chain reaction as described previously (37). The respective PCR products were cloned into EcoRI/XhoI restriction sites of the expression vector pcD ⁇ A3.1 containing a zeocin resistance gene (Invitrogen) and sequenced to verify successful mutagenesis.
  • HEK 293 cells stably expressing PSl G384A, PSl G286I, PSl G286P, PSl G384W, PSl G384K, PSl G384D were generated by transfection of HEK 293 cells stably expressing ⁇ APP 0 containing the Swedish mutation (50).
  • Transfected HEK 293 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycine, 200 ⁇ g ml "1 G418 (to select for ⁇ APP expression) and 200 ⁇ g ml "1 zeocin (to select for presenilin expression).
  • PS proteins were detected by a combined immunoprecipitation/western blotting 5 procedure (51). Extracts from HEK 293 cells were prepared and subjected to immunoprecipitation using the polyclonal antibody 3027 to PSl or 3711 to PS2 (51). Following gel electrophoresis, immunoprecipitated PS proteins were identified by immunoblotting using the monoclonal antibody BI.3D7 [to PSl; (51)] or BI.HF5c [to PS2; (51)].
  • a ⁇ species were immunoprecipitated from conditioned media with antibody 3926 [to A ⁇ 1-42 (52)] and separated 5 on a previously described Tris-Bicine gel system (53), which allows the specific identification of A ⁇ 40 and AJ342 based on their characteristic running behavior. Quantitation of A ⁇ species was performed both by phosphoimager analysis and by a previously described ELISA (7). Antibody 5313 (22) was used to immunopreciptate the ⁇ APP holoprotein from cell lysates or APPs from conditioned media. 0 41. W. Song, et al., Proc Natl Acad Sci USA 96, 6959-63 (1999).
  • HEK293 cells stably expressing Swedish mutant ⁇ APP and PSl derivatives were stably transfected with the previously described expression plasmid containing myc-tagged Notch ⁇ E (N ⁇ E) cDNA (22, 43).
  • Transfected cells were cultured as described (36) except that 100 ⁇ g ml "1 hygromycin was added to the culture medium to select for Notch expression.
  • To analyze 5 cleavage of N ⁇ E cells were starved for 1 h in methionine- and serum-free MEM, metabolically labeled with 300 ⁇ Ci 35 S-methionine (Promix, Amersham) for 15 min, and chased for 1 h in medium containing excess amounts of unlabeled methionine.
  • Cell extracts were prepared and Notch derivatives were immunoprecipitated using the anti-myc antibody 9E10 as described (22).

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Abstract

L'invention concerne des protéines, des peptides ou des aspartyl-protéases comportant des motifs de consensus spécifiés et des acides nucléiques codant lesdites protéines, peptides ou aspartyl-protéases. L'invention concerne également le dépistage de substances capables d'inhiber lesdites aspartyl-protéases, des substances identifiables grâce audit procédé et enfin, des compositions pharmaceutiques comprenant lesdites substances.
PCT/EP2001/007512 2000-07-07 2001-06-30 Aspartyl-protease Ceased WO2002004644A2 (fr)

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Publication number Priority date Publication date Assignee Title
WO2004026331A1 (fr) * 2002-09-17 2004-04-01 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Peptides inhibant les activites de clivage specifiques des presenilines

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US5986054A (en) * 1995-04-28 1999-11-16 The Hospital For Sick Children, Hsc Research And Development Limited Partnership Genetic sequences and proteins related to alzheimer's disease
CA2191924A1 (fr) * 1995-12-05 1997-06-06 Kevin Felsenstein Derives de 5-amino-6-cyclohexyl-4-hydroxyhexanamide, inhibiteurs de la production de proteine .beta.-amyloide
GB9608657D0 (en) * 1996-04-26 1996-07-03 Smithkline Beecham Plc Novel treatment
AU4589297A (en) * 1996-10-07 1998-05-05 Scios Inc. Method to identify direct inhibitors of the beta-amyloid forming enzyme gamma-secretase
EP1200567A4 (fr) * 1999-07-12 2003-02-26 Dartmouth College Composes et procedes d'identification de composes qui inhibent une nouvelle classe d'aspartyl proteases

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004026331A1 (fr) * 2002-09-17 2004-04-01 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Peptides inhibant les activites de clivage specifiques des presenilines

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