Alzheimer's Disease Secretase, APP Substrates Therefor and Uses Therefor

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.

18 . 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) an amino acid sequence set forth in SEQ ID NO: 4,   (b) the amino acid sequence at least 90% identical to a fragment of the aspartyl protease amino acid set forth as SEQ ID NO: 4 and the polypeptide exhibits aspartyl protease activity involved in processing APP into amyloid beta;   (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.   
     
     
         19 . A polypeptide encoded by the polynucleotide sequence of  claim 18 . 
     
     
         20 . The polynucleotide of  claim 18 , wherein the encoded polypeptide lacks a transmembrane domain. 
     
     
         21 . The polynucleotide of  claim 18 , wherein the encoded polypeptide exhibits aspartyl protease activity involved in processing APP into amyloid beta. 
     
     
         22 . A purified or isolated polynucleotide according to  claim 18 , wherein the polypeptide encoded by the polynucleotide further comprises a signal peptide. 
     
     
         23 . A purified or isolated polynucleotide according to  claim 18 , wherein the polypeptide encoded by the polynucleotide further comprises a heterologous tag. 
     
     
         24 . A host cell transformed or transfected with a polynucleotide according to  claim 18 . 
     
     
         25 . A vector comprising a polynucleotide according to  claim 18 . 
     
     
         26 . An expression vector comprising a polynucleotide according to any one of  claim 18  operably linked to a heterologous expression control sequence. 
     
     
         27 . A host cell transformed or transfected with an expression vector according to  claim 26 . 
     
     
         28 . A host cell according to  claim 27  that is a mammalian cell. 
     
     
         29 . A method of making an aspartyl protease polypeptide, comprising steps of growing a host cell according to  claim 28  in a growth media under conditions where the cell expresses the polypeptide encoded by the polynucleotide. 
     
     
         30 . A method according to  claim 29 , further comprising isolating or purifying the polypeptide from the cell or from the growth media. 
     
     
         31 . A method for identifying an agent that decreases the protease activity of an aspartyl protease polypeptide, comprising steps of:
 (a) expressing an aspartyl protease polypeptide by growing a host cell of  claim 27  under conditions wherein the cell expresses the polypeptide encoded by the polynucleotide,   (b) measuring proteolytic activity of said polypeptide in the presence and absence of a test agent; and   (c) comparing proteolytic activity of the polypeptide 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.   
     
     
         32 . 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 according to  claim 27  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.   
     
     
         33 . A method according to  claim 31  or  32  wherein step (b) comprises measuring the proteolytic activity of the polypeptide toward an amyloid precursor protein (APP) substrate. 
     
     
         34 . A method according to  claim 33 , wherein the APP substrate comprises an amyloid beta (A-beta) processing site. 
     
     
         35 . A method according to  claim 33 , wherein the APP substrate comprises the Swedish mutation (K→N, M→L). 
     
     
         36 . A method according to  claim 33 , wherein the APP substrate is a peptide comprising a β-secretase cleavage site that comprises the formula P2-P1-P1′-P2′ (SEQ ID NO: 74), wherein
 P2 is an amino acid selected from K and N;   P1 is an amino acid selected from M and L;   P1′ is the amino acid D; and   P2′ is the amino acid A.