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.

1 . A method for identifying agents that inhibit the activity of human Asp2 aspartyl protease (Hu-Asp2), comprising the steps of: 
 (a) contacting amyloid precursor protein (APP) and purified and isolated Hu-Asp2 in the presence and absence of a test agent;    (b) determining the APP processing activity of the Hu-Asp2 in the presence and absence of the test agent; and    (c) comparing the APP processing activity of the Hu-Asp2 polypeptide in the presence of the test agent to the activity in the absence of the test agent to identify an agent that inhibits the APP processing activity of Hu-Asp2, wherein reduced activity in the presence of the test agent identifies an agent that inhibits Hu-Asp2 activity.    
     
     
         2 . A method according to  claim 1 , wherein the Hu-Asp2 comprises a polypeptide purified and isolated from a cell transformed or transfected with a polynucleotide comprising a nucleotide sequence that encodes the Hu-Asp2.  
     
     
         3 . A method according to  claim 2  wherein the nucleotide sequence is selected from the group consisting of: 
 (a) a nucleotide sequence encoding the Hu-Asp2(a) amino acid sequence set forth in SEQ ID NO:4;  
 (b) a nucleotide sequence encoding the Hu-Asp2(b) amino acid sequence set forth in SEQ ID NO:6;  
 (c) a nucleotide sequence encoding a fragment of Hu-Asp2(a) (SEQ ID NO:4) or Hu-Asp2(b) (SEQ ID NO:6), wherein said fragment exhibits aspartyl protease activity characteristic of Hu-Asp2(a) or Hu-Asp2(b); and  
 (d) a nucleotide sequence of a polynucleotide that hybridizes under stringent hybridization conditions to a Hu-Asp2-encoding polynucleotide selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:5.  
 
     
     
         4 . A method according to  claim 2  wherein the Hu-Asp2 comprises the Hu-Asp2(a) amino acid sequence set forth in SEQ ID NO:4.  
     
     
         5 . A method according to  claim 2  wherein the Hu-Asp2 comprises the Hu-Asp2(b) amino acid sequence set forth in SEQ ID NO:6.  
     
     
         6 . A method according to  claim 2  wherein the Hu-Asp2 comprises a fragment of Hu-Asp2(a) (SEQ ID NO:4) or Hu-Asp2(b) (SEQ ID NO:6), wherein said fragment exhibits aspartyl protease activity characteristic of Hu-Asp2(a) or Hu-Asp2(b).  
     
     
         7 . A method according to  claim 1 , wherein the APP comprises the Swedish mutation (K→N, M→L) adjacent to the β-secretase processing site.  
     
     
         8 . A method according to  claim 1 , further comprising a step of treating Alzheimer's Disease with an agent identified as an inhibitor of Hu-Asp2 according to steps (a)-(c).  
     
     
         9 . A method for identifying agents that inhibit the activity of human Asp2 aspartyl protease (Hu-Asp2), comprising the steps of: 
 (a) contacting Hu-Asp2 and amyloid precursor protein (APP) in the presence and absence of a test agent, wherein the APP comprises a carboxy-terminal di-lysine (KK) and wherein the contacting comprises growing a host cell that expresses the APP in the presence and absence of the test agent;    (b) determining the APP processing activity of the Hu-Asp2 in the presence and absence of the test agent; and    (c) comparing the APP processing activity of the Hu-Asp2 polypeptide in the presence of the test agent to the activity in the absence of the test agent to identify an agent that inhibits the activity of Hu-Asp2, wherein reduced activity in the presence of the test agent identifies an agent that inhibits Hu-Asp2 activity.    
     
     
         10 . A method according to  claim 9 , wherein the APP further comprises the Swedish mutation (K→N, M→L) adjacent to the β-secretase processing site.  
     
     
         11 . A method according to  claim 9 , wherein the host cell has been transformed or transfected with a polynucleotide comprising a nucleotide sequence that encodes a Hu-Asp2, wherein said nucleotide sequence is selected from the group consisting of: 
 (a) a nucleotide sequence encoding the Hu-Asp2(a) amino acid sequence set forth in SEQ ID NO:4;    (b) a nucleotide sequence encoding the Hu-Asp2(b) amino acid sequence set forth in SEQ ID NO:6;    (c) a nucleotide sequence encoding a fragment of Hu-Asp2(a) (SEQ ID NO:4) or Hu-Asp2(b) (SEQ ID NO:6), wherein said fragment exhibits aspartyl protease activity characteristic of Hu-Asp2(a) or Hu-Asp2(b); and    (d) a nucleotide sequence of a polynucleotide that hybridizes under stringent hybridization conditions to a Hu-Asp2-encoding polynucleotide selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:5.    
     
     
         12 . A method according to  claim 9 , further comprising a step of treating Alzheimer's Disease with an agent identified as an inhibitor of Hu-Asp2 according to steps (a)-(c).  
     
     
         13 . A method for identifying agents that inhibit the activity of human Asp2 aspartyl protease (Hu-Asp2), comprising the steps of: 
 (a) contacting Hu-Asp2 and amyloid precursor protein (APP) in the presence and absence of a test agent, wherein the contacting comprises growing a host cell transformed or transfected with a polynucleotide comprising a nucleotide sequence encoding the Hu-Asp2 in the presence and absence of the test agent;    (b) determining the APP processing activity of the Hu-Asp2 in the presence and absence of the test agent; and    (c) comparing the APP processing activity of the Hu-Asp2 polypeptide in the presence of the test agent to the activity in the absence of the test agent to identify an agent that inhibits the activity of Hu-Asp2, wherein reduced activity in the presence of the test agent identifies an agent that inhibits Hu-Asp2 activity.    
     
     
         14 . A method according to  claim 13 , wherein the host cell expresses APP.  
     
     
         15 . A method according to  claim 14  wherein the determining step comprises measuring the production of amyloid beta peptide by the cell in the presence and absence of the test agent.  
     
     
         16 . A method according to  claim 14 , wherein the host cell expresses an APP having an amino acid sequence that includes a carboxy-terminal di-lysine.  
     
     
         17 . A method according to  claim 14 , wherein the host cell expresses an APP comprising the Swedish mutation (K→N, M→L) adjacent to the β-secretase processing site.  
     
     
         18 . A method according to  claim 13 , wherein the host cell is a human embryonic kidney cell line 293 (HEK293) cell.  
     
     
         19 . A method according to  claim 13  wherein the nucleotide sequence is selected from the group consisting of: 
 (a) a nucleotide sequence encoding the Hu-Asp2(a) amino acid sequence set forth in SEQ ID NO:4;  
 (b) a nucleotide sequence encoding the Hu-Asp2(b) amino acid sequence set forth in SEQ ID NO:6;  
 (c) a nucleotide sequence encoding a fragment of Hu-Asp2(a) (SEQ ID NO:4) or Hu-Asp2(b) (SEQ ID NO:6), wherein said fragment exhibits aspartyl protease activity characteristic of Hu-Asp2(a) or Hu-Asp2(b); and  
 (d) a nucleotide sequence of a polynucleotide that hybridizes under stringent hybridization conditions to a Hu-Asp2-encoding polynucleotide selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:5.  
 
     
     
         20 . A method according to  claim 13 , wherein the host cell comprises a vector that comprises the polynucleotide.  
     
     
         21 . A method according to  claim 13  wherein the polynucleotide comprises a nucleotide sequence encoding the Hu-Asp2(a) amino acid sequence set forth in SEQ ID NO:4.  
     
     
         22 . A method according to  claim 13  wherein the polynucleotide comprises a nucleotide sequence encoding the Hu-Asp2(b) amino acid sequence set forth in SEQ ID NO:6.  
     
     
         23 . A method according to  claim 13  wherein the polynucleotide comprises a nucleotide sequence encoding a polypeptide comprising a fragment of Hu-Asp2(a) (SEQ ID NO:4) or Hu-Asp2(b) (SEQ ID NO:6), wherein said fragment exhibits aspartyl protease activity characteristic of Hu-Asp2(a) or Hu-Asp2 (b).  
     
     
         24 . A method according to  claim 13  wherein the Hu-Asp2 is encoded by a nucleotide sequence of a polynucleotide that hybridizes under stringent hybridization conditions to a Hu-Asp2-encoding polynucleotide selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:6.  
     
     
         25 . A method according to  claim 13 , further comprising a step of treating Alzheimer's Disease with an agent identified as an inhibitor of Hu-Asp2 according to steps (a)-(c).  
     
     
         26 . A method for identifying agents that modulate the activity of Asp2 aspartyl protease, comprising the steps of: 
 (a) contacting an Asp2 aspartyl protease and amyloid precursor protein (APP) in the presence and absence of a test agent, wherein the Asp2 aspartyl protease is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions to a Hu-Asp2-encoding polynucleotide selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:6;    (b) determining the APP processing activity of the Asp2 aspartyl protease in the presence and absence of the test agent; and    (c) comparing the APP processing activity of the Asp2 aspartyl protease in the presence of the test agent to the activity in the absence of the agent to identify agents that modulate the activity of the Asp2 aspartyl protease, wherein a modulator that is an Asp2 inhibitor reduces APP processing and a modulator that is an Asp2 agonist increases such processing.    
     
     
         27 . A method according to  claim 26 , wherein the Asp2 aspartyl protease is purified and isolated.  
     
     
         28 . A method according to  claim 26 , further comprising a step of treating Alzheimer's Disease with an agent identified as an inhibitor of Hu-Asp2 according to steps (a)-(c).