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