EP1144603A3

EP1144603A3 - Protease

Info

Publication number: EP1144603A3
Application number: EP00903587A
Authority: EP
Inventors: Kay Hofmann
Original assignee: Memorec Medical Molecular Research Cologne Stoffel GmbH
Current assignee: MEMOREC STOFFEL GMBH-MEDIZINISCH-MOLEKULARE ENTWIC
Priority date: 1999-01-22
Filing date: 2000-01-19
Publication date: 2002-02-06
Also published as: AU2542100A; CA2360585A1; WO2000043505A2; WO2000043505A3; EP1144603A2

Abstract

The invention relates to protease with two aspartate radicals in a catalytically active structure, a first aspartate radical being in a sequence X1GX2GD and a second aspartate radical being in a sequence X3X4DX5, X1, X2, X3 and X5 being selected independently of each other from the following: Ala, Val, Leu, Met and Ile; and X4 being an aromatic amino acid. The sequences X1GX2GD and X3X4DX5 lie in a transmembrane region.

Description

Protease

The present invention relates to a protease encoding nucleic acids for the protease and the inhibitors, antibodies, medicaments and diagnostic agents associated therewith.

The present invention provides a protease with two aspartate residues in a catalytically active structure, wherein a first aspartate residue is in a motif X1GX2GD and a second aspartate residue is in a motif X ₃ X ₄ DX ₅ , wherein Xi, X ₂ , X3 and X _{5 are} independent are selected from each other from Ala, Val, Leu, Met and Ile and X _{4 is} an aromatic amino acid, and the motifs XιGX ₂ GD and X ₃ X ₄ DX _{5 are located} in a transmembrane region. The single letter code of the amino acids was used for the motifs, ie D = Asp, G = Gly etc.

Such proteases are very likely to be involved in the cleavage of the amyloid precursor protein (APP). In one embodiment of the invention, the protease according to the invention represents the hitherto unidentified γ-secretase which is involved in processing the APP to form the amyloid peptides referred to as Aß.

An overview of the role of γ-secretases in the development of Alzheimer's disease is given by S.L Ross et al. in J. of Biol. Chem. 273 (1998), 15309-15312.

Preferred proteases of the present invention additionally have a sequence PALX6YX7V, where X ₆ and X ₇ independently of one another have the same meaning as Xi. However, it is preferred that X ₆ and X _{7 are} leucine or isoleucine. In particular, the proteases are proteases from mammals, in particular from humans.

The proteases according to the invention preferably have catalytically active aspartate residues in a region which lies within a transmembrane region. Transmembrane regions can be predicted on the basis of different models if the sequence of a protein is known. They are characterized in that in one area there are predominantly hydrophobic amino acids which are flanked by areas in which there are more hydrophilic amino acids.

An aspartate in a transmembrane region can be detected, for example, by using the "GREASE" program, which is part of the FASTA 2.0 program package. With a window width of 17, this program must be used to calculate a hydrophobicity value of at least 80 for the aspartate. The FASTA program package is described in WR Pearson and DJ Lipman, Proc. Natl. Acad. Sci. USA 85 (1988) 2444-2448. The GREASE program uses the Kyte / Doolittle algorithm, described in J. Kyte and RF Doolittle, J. Mol. Biol 157: 105-132 (1982).

Particularly preferred proteases of the present invention are referred to as psl 1-5 (human psl 1-5: SEQ ID No. 1 to 4 + 19, murine psl 2-4: SEQ ID No. 5-7, sacc. Cerevisiae psl 3: SEQ ID No. 8, human psl2L SEQ ID No. 18).

The invention furthermore relates to variants of the proteases according to the invention. Variants are proteins which are derived from the proteases according to the invention by one or more mutations, insertions and deletions, in particular by conservative exchanges. special N- or C-terminal shortened or elongated forms.

The invention also relates to nudeic acids which code for the proteases according to the invention. Preferred nucleic acids according to the invention are those with SEQ ID No. 9 - 17 + 20 (human psl 1 - 5: SEQ ID No. 9 - 12 + 20, murine psl 2 - 4: SEQ ID No. 13 - 15, sacc. Cerevisiae psl 3: SEQ ID No. 16, human psl2L SEQ ID No. 17). Complementary nudeic acids are also part of the invention.

The proteases according to the invention are involved in the cleavage of the APP to Aß and are thus indirectly involved in the development of, for example, Alzheimer's disease. The invention therefore also relates to inhibitors which inhibit the expression or the activity of the proteases. Such inhibitors can be identified in simple procedures. Corresponding inhibitors can be identified, for example, by measuring the expression or the activity of the proteases in the presence of potential inhibitors. Antibodies directed against the aspartate protesases are particularly suitable for measuring expression and are therefore also part of the invention.

The aspartate proteases according to the invention are also involved in the cleavage of other transmembrane proteins, in particular the Notch receptor protein and related proteins which play a role in the development of the nervous system.

The proteolytic cleavage inside membranes is also involved in other important processes, e.g. :

• Proteolytic degradation of N-terminal signal peptides after their cleavage by the signal peptidase. • Proteolytic degradation of C-terminal propeptides, such as those caused by transamidase-catalyzed cleavage during the post-translational GPI anchoring of proteins.

• Generation of peptides for presentation by histocompatibility complex molecules of type I and II. In the case of soluble proteins, such peptides are primarily formed by the proteasome. However, peptides also arise from transmembrane regions of proteins, as can only be explained by cleavage inside the membrane.

• Proteolytic cleavage of the ER stress sensor protein Irel. The endoplasmic reticulum has a mechanism that detects the accumulation of unfolded or incorrectly folded proteins and sends a signal to the cell nucleus. This mechanism is called "unfolded protein response" or UPR, and leads to the increased formation of proteins that facilitate folding. In mammals there are two sensor proteins (Irelalpha and Irelbeta) that react to folding defects and are then proteolytically cleaved within the membrane.

The processes mentioned can be influenced therapeutically by using the protease or its inhibitors according to the invention.

Zeil lines which do not express any of the proteases or nucleic acids according to the invention and preferably also contain no homologous proteases or nucleic acids are particularly suitable for this. These can be used to test the activity of the proteases or to determine inhibitors according to Example 2, preferably in accordance with the method described in Example 1. Saccharomyces cerevisiae is particularly suitable for this. In Knowledge of the corresponding protease or the coding nucleic acid (SEQ ID No. 8 and 16) can be used to produce yeast strains by known methods which no longer contain this protein or the nucleic acid. They are therefore preferably suitable as an expression system for characterizing the aspartate proteases according to the invention or for identifying suitable inhibitors. Corresponding cell lines, preferably yeast cell lines and the use of the protein with SEQ ID No. 8 as aspartate protease and the nucleic acid with SEQ ID No. 16 for the expression of a protease are therefore also the subject of the invention.

The proteases, nudeic acids, inhibitors and antibodies according to the invention can be contained in medicinal and diagnostic agents. They are particularly suitable for the treatment or diagnosis of diseases which are causally linked to the cleavage of the amyloid precursor protein, in particular Alzheimer's disease.

Example 1:

γ-secretase assay

The putative γ-secretases are transfected stably or transiently in cos-7 cells, which additionally express SpA4CT (signal peptide fused to ßA4 followed by the APP C-terminus). γ-Secretase activity is recognizable in this system by the generation of a 4.6 KDa peptide or by the disappearance of the 11 KDa band of the complete SpA4CT. If the pathologically relevant γ-secretase is present, both fragments should be detectable inside the cell. In the supernatant of the cells there is always ßA4, which is generated by an endogenous plasma membrane-constant γ-secretase activity, which, however, plays no role in the pathogenesis of Alzheimer's. The transfected cells are washed three times with cold DMEM and then harvested on ice with a cell scraper. The cells (approx. 5X10 ⁶ cells) are collected by centrifugation and in 1 ml Lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 1% NP-40, 1% Triton-x-100, 2 mM EDTA) lysed. The kernels are centrifuged off at 11,000 g. The supernatant is subjected to immunoprecipitation. For this purpose, 1 μg of the cell lysate is mixed with 2 μg / ml WO2 immunoglobulin (anti-ßA4 antibody) and shaken overhead at 4 ° C. for 0.5 h. Subsequently, 20 ul protein-G Sepharose suspension (1: 1) are added and shaken overhead at 4 ° C for 5 h. The Protein-G Sepharose is consecutively two times with the buffers A, B and C (A: 150 mM NaCI, 10 mM Tris-HCl pH 7.5, 0.2% NP-40, 2 mM EDTA; B: 500 mM NaCI, 10 mM Tris-HCI pH 7.5, 0.2% NP-40, 2 mM EDTA; C: 10 mM Tris-HCI pH 7.5), mixed with 20 μl 3X sample buffer, heated to 95 ° C and the supernatant to a 12% Tris Tricine gel applied. After the gel electrophoretic size fractionation, the proteins are transferred to a PVDF membrane and subsequently detected with an anti-ßA4 antibody.

Example 2:

Identification of a γ-secretase inhibitor

To identify an inhibitor of the pathologically relevant γ-secretase, the enzyme is coexpressed according to the above instructions in Cos-7 cells with SpA4CT. The cells are brought into contact with the substance to be investigated in a suitable manner (in the presence or absence of membrane-permeabilizing agents). The intracellularly formed βA4 is then detected as described above. A reduction in the amount of βA4 formed suggests that the substance is effective as a γ-secretase inhibitor.

Claims

claims

1. Protease with two aspartate residues in a catalytically active structure, with a first aspartate residue in a XiGX ₂ GD motif and a second aspartate residue in a XX ₄ DX ₅ motif, with Xi, X ₂ , X ₃ and X5 being selected independently of one another from Ala, Val, Leu, Met and Ile and X _{4 is} an aromatic amino acid, and the motifs X ₁ GX ₂ GD and X3X4DX5 lie in a transmembrane region.

2. Protease according to claim 1, characterized in that the protease has the sequence PALX ₆ YX? V, where X ₆ and X7 independently of one another have the same meaning as Xi and are preferably Leu or Ile.

3. Protease according to one of claims 1 to 2, characterized in that the protease is one of the sequences SEQ ID No. 1-8 and 18, 19.

4. Nudeic acids coding for a protease according to at least one of claims 1 to 3, preferably with SEQ ID No. 9 - 17 or 20.

5. Inhibitors, characterized in that they inhibit the expression or activity of the protease according to any one of claims 1 to 3.

6. Antibody directed against proteases according to one of claims 1 to 3.

7. A method for identifying inhibitors, characterized in that the activity of the proteases according to one of claims 1 to 3 is measured in the presence of potential inhibitors.

8. Medicament or diagnostic agent containing a protease according to one of claims 1 to 3, a nucleic acid according to claim 4, an inhibitor according to claim 5 and / or an antibody according to claim 6.

9. Use of the medicament or diagnostic agent according to claim 8 for the diagnosis or treatment of diseases which are causally connected with the cleavage of the amyloid precursor protein, in particular Alzheimer's disease.

10. Use of the medicament or diagnostic agent according to claim 8 for the diagnosis or treatment of diseases which are causally associated with an impaired degradation of hydrophobic signal peptides.

11. Use of the medicament or diagnostic agent according to claim 8 for the diagnosis or treatment of diseases which are causally associated with the accumulation of unfolded proteins in the endoplasmic reticulum.

12. Use of the medicament according to claim 8 for influencing the presentation of hydrophobic peptides by histocompatibility complex molecules in conditions such as viral infection, cancer or a rejection reaction after transplantation.

13. Zeil line, characterized in that the Zeil line expresses no protease according to at least one of claims 1 to 3 and / or contains no nucleic acid according to claim 4.