Methods for Identifying Factors That Control the Folding of Amyloid Proteins of Diverse Origin

Abstract

The present invention provides a yeast cell based system for determining factors that control the folding of amyloid proteins of diverse origins. Further the present invention provides methods of using such a system to screen for reagents that affect amyloid formation, a process that is integral to several devastating human disease including Creutzfeld-Jacob disease (CJD), fatal familial insomnia (FFI), Gertsmann-Straussler-Scheinker (GSS) syndrome, and kuru. The system of the present invention provides a rapid screening system to quickly and cheaply identify reagents that affect the folding and aggregation properties of the target protein.

1 . A method of identifying a candidate substance that inhibits the aggregation of an aggregate-prone amyloid protein, comprising:
 (a) contacting a yeast cell that expresses an aggregate-prone amyloid protein with said candidate substance under conditions effective to allow aggregated amyloid formation; and   (b) determining the ability of said candidate substance to inhibit the aggregation of the aggregate-prone amyloid protein.   
     
     
         2 . The method of  claim 1 , wherein the aggregate-prone amyloid protein comprises a Sup35 or URE3 polypeptide. 
     
     
         3 . The method of  claim 1 , wherein the aggregate-prone amyloid protein comprises a PrP or β-amyloid polypeptide. 
     
     
         4 . The method of  claim 1 , wherein the aggregate-prone amyloid protein is a chimeric protein. 
     
     
         5 . The method of  claim 4 , wherein the chimeric protein comprises at least the N-terminal domain of Sup35. 
     
     
         6 . The method of  claim 4 , wherein the chimeric protein comprises at least an aggregate forming domain of a mammalian amyloid polypeptide. 
     
     
         7 . The method of  claim 4 , wherein the chimeric protein comprises at least an aggregate forming domain of an aggregate-prone amyloid protein operably attached to a detectable marker protein. 
     
     
         8 . The method of  claim 7 , wherein said marker protein is green fluorescent protein or luciferase. 
     
     
         9 . The method of  claim 7 , wherein said marker protein is a drug-resistance marker protein. 
     
     
         10 . The method of  claim 7 , wherein said marker protein is a hormone receptor. 
     
     
         11 . The method of  claim 10 , wherein said hormone receptor is a glucocorticoid receptor. 
     
     
         12 . The method of  claim 6 , wherein the mammalian amyloid polypeptide is PrP or β-amyloid. 
     
     
         13 . The method of  claim 12 , wherein the chimeric protein comprises as least about amino acids 1-42 of β-amyloid protein. 
     
     
         14 . The method of  claim 4 , wherein the chimeric protein comprises Sup35 in which the N-terminal domain has been replaced by amino acids 1-42 of β-amyloid protein. 
     
     
         15 . The method of  claim 1 , wherein any aggregation of the aggregate-prone amyloid protein is detected by the ability of the aggregated protein to bind Congo Red. 
     
     
         16 . The method of  claim 1 , wherein any aggregation of the aggregate-prone amyloid protein is detected by increased protease resistance of the aggregated protein. 
     
     
         17 . The method of  claim 1 , wherein the aggregate-prone amyloid protein is labeled. 
     
     
         18 . The method of  claim 17 , wherein the label is a radioactive isotope, a fluorophore, or a chromophore. 
     
     
         19 . The method of  claim 18 , wherein the label is  35 S. 
     
     
         20 . The method of  claim 18 , wherein the fluorophore comprises a green fluorescent protein polypeptide. 
     
     
         21 . The method of  claim 1 , wherein any aggregation is determined by the presence of a [PSI+] phenotype. 
     
     
         22 . The method of  claim 1 , wherein said yeast cell overexpresses Hsp104. 
     
     
         23 . A method of identifying a candidate substance for therapeutic activity against an amyloidogenic disease in an animal, said method comprising:
 (a) contacting a yeast cell that expresses an aggregate-prone amyloid protein with said candidate substance under conditions effective to allow amyloid formation; and   (b) determining the ability of said candidate substance to inhibit aggregation of the aggregate-prone amyloid protein,   wherein the ability to inhibit aggregation is indicative of therapeutic activity.   
     
     
         24 . The method of  claim 23 , wherein the aggregate-prone amyloid protein comprises a PrP, β-amyloid, Sup35, or URE3 polypeptide. 
     
     
         25 . The method of  claim 23 , wherein the protein is a chimeric protein. 
     
     
         26 . The method of  claim 25 , wherein the chimeric protein comprises a Sup35 polypeptide. 
     
     
         27 . The method of  claim 25 , wherein the chimeric protein comprises a mammalian amyloid polypeptide. 
     
     
         28 . The method of  claim 27 , wherein the mammalian amyloid polypeptide is PrP or β-amyloid. 
     
     
         29 . The method of  claim 23 , wherein any aggregation of the aggregate-prone amyloid protein is detected by the ability of the aggregation to bind Congo Red. 
     
     
         30 . The method of  claim 23 , wherein the aggregate-prone amyloid protein is labeled. 
     
     
         31 . The method of  claim 30 , wherein the label is a radioactive isotope, a fluorophore, or a chromophore. 
     
     
         32 . The method of  claim 31 , wherein the label is  35 S. 
     
     
         33 . The method of  claim 31 , wherein the fluorophore comprises a green fluorescent protein polypeptide. 
     
     
         34 . The method of  claim 23 , wherein the aggregation of the aggregate-prone amyloid protein is determined by the presence of a [PSI+] phenotype. 
     
     
         35 . The method of  claim 23 , wherein the disease is selected from the group consisting of Alzheimer's disease, scrapie, spongiform encephalopathy in a mammal, kuru, Creutzfeldt-Jakob disease, Gestmann-Strausser-Scheinker disease, or fatal familial insomnia. 
     
     
         36 . The method of  claim 35 , wherein the mammal is bovine, feline, a mink, deer, elk, a mouse, a hamster, an ape, a monkey, or human.