Alzheimer's disease secretase, app substrates therefor, and uses therefor
AssigneePharmacia & Upjohn Company
InventorsMark E. Gurney, Michael J. Bienkowaki, Robert L. Heinrikson, Luis A. Parodi, Riqiang Yan
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Abstract
The present invention provides the enzyme and enzymatic procedures for cleaving the β secretase cleavage site of the APP protein and associated nucleic acids, peptides, vectors, cells and cell isolates and assays. The invention further provides a modified APP protein and associated nucleic acids, peptides, vectors, cells, and cell isolates, and assays that are particularly useful for identifying candidate therapeutics for treatment or prevention of Alzheimer's disease.
Claims17
A purified or isolated polynucleotide comprising a nucleotide sequence that hybridizes under the following stringent hybridization conditions to the complement of SEQ ID NO:
- 3 or SEQ ID NO:
- 5:
- (1) hybridization at 42° C. in a hybridization buffer comprising 6×SSC and 0.1% SDS, and
- (2) washing at 65° C. in a wash solution comprising 1×SSC and 0.1% SDS;
- wherein the polynucleotide encodes a polypeptide that comprises an amino acid sequence selected from the group consisting of:
- (a) the amino acid sequence set forth in SEQ ID NO:
- 4;
- (b) the amino acid sequence set forth in SEQ ID NO:
- 6;
- (c) fragments of (a) or (b) that include the aspartyl protease active site tripeptides DTG and DSG and exhibit aspartyl protease activity involved in processing APP into amyloid beta, and
- (d) sequence variants of (a), (b) or (c), wherein substitution differences between the amino acid sequence of the polypeptide and the amino acid sequence of (a) or (b) consist of conservative substitutions, and wherein the polypeptide exhibits aspartyl protease activity involved in processing APP into amyloid beta.
6 An expression vector comprising a polynucleotide according to any one of claims 1-3 operably linked to a heterologous expression control sequence.
A host cell transformed or transfected with an expression vector according to claim 6.
A method for identifying an agent that decreases the protease activity of an aspartyl protease polypeptide, comprising steps of:
- (a) growing a host cell transformed or transfected with a polynucleotide in the presence and absence of a test agent;
- under conditions wherein the cell expresses the polypeptide encoded by the polynucleotide,
- wherein the polynucleotide encodes an aspartyl protease polypeptide comprising the amino acid sequence set forth in SEQ ID NO:
- 4 or SEQ ID NO:
- 6 or a continuous fragment thereof that retains the aspartyl protease activity toward APP,
- wherein said fragment includes aspartyl protease active site tripeptides DTG and DSG, and
- (b) measuring proteolytic activity of the aspartyl protease polypeptide in the presence and absence of a test agent, and
- (c) comparing proteolytic activity of the aspartyl protease polypeptide expressed by the cell in the presence and absence of the test agent, wherein decreased proteolytic activity in the presence of the test agent identifies the test agent as an agent that decreases the protease activity of the aspartyl protease polypeptide.
Dependencies are inferred from the claim text.