Yeast screens for treatment of human disease

Abstract

Screening methods for identifying substances that provide therapeutic value for various diseases associated with protein misfolding are provided. Genetic and chemical screening methods are provided using a yeast system. The methods of the invention provide a rapid and cost-effective method to screen for compounds that prevent protein misfolding and/or protein fibril formation and/or protein aggregation which includes numerous neurodegenerative diseases including Parkinson's disease, Alzheimer's disease, Huntington's disease as well as non-neuronal diseases such as type 2 diabetes.

1 - 68 . (canceled) 
     
     
         69 . A yeast cell comprising an expression construct, the expression construct comprising a promoter operably linked to a nucleic acid encoding a polypeptide comprising an alpha synuclein protein. 
     
     
         70 . The yeast cell of  claim 69 , wherein the alpha synuclein protein is a wild-type alpha synuclein protein. 
     
     
         71 . The yeast cell of  claim 70 , wherein the wild-type alpha synuclein protein is a human alpha synuclein protein. 
     
     
         72 . The yeast cell of  claim 71 , wherein the human alpha synuclein protein comprises the amino acid sequence of SEQ ID NO:2. 
     
     
         73 . The yeast cell of  claim 69 , wherein the alpha synuclein protein is a mutant alpha synuclein polypeptide. 
     
     
         74 . The yeast cell of  claim 73 , wherein the mutant alpha synuclein protein comprises a deletion, an insertion, or a substitution relative to SEQ ID NO:2. 
     
     
         75 . The yeast cell of  claim 74 , wherein the mutant alpha synuclein protein comprises an A53T mutation or an A30P mutation. 
     
     
         76 . The yeast cell of  claim 75 , wherein the mutant alpha synuclein protein comprises an A53T mutation. 
     
     
         77 . The yeast cell of  claim 69 , wherein the polypeptide is a fusion protein. 
     
     
         78 . The yeast cell of  claim 77 , wherein the fusion protein comprises a detectable marker. 
     
     
         79 . The yeast cell of  claim 78 , wherein the detectable marker is selected from the group consisting of a fluorescent protein, an enzyme, and an epitope. 
     
     
         80 . The yeast cell of  claim 79 , wherein the reporter polypeptide is a fluorescent protein. 
     
     
         81 . The yeast cell of  claim 80 , wherein the fluorescent protein is a green fluorescent protein. 
     
     
         82 . The yeast cell of  claim 69 , wherein the yeast cell is selected from the group consisting of  Saccharomyces cerevisiae, Schizosaccharomyces pombe, Saccharomyces uvae, Saccharomyces kluyveri, Saccharomyces uvarum, Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris, Pichia methanolica, Pichia kluyveri, Yarrowia lipolytica, Candida  sp.,  Candida utilis, Candida cacaoi, Geotrichum  sp., and  Geotrichum fermantans.    
     
     
         83 . The yeast cell of  claim 82 , wherein the yeast cell is  Saccharomyces cerevisiae.    
     
     
         84 . The yeast cell of  claim 69 , wherein the promoter is selected from the group consisting of GAL1-10, GAL1, GALL, GALS, GPD, ADH, TEF, CYC1, MRP7, and MET25. 
     
     
         85 . The yeast cell of  claim 69 , wherein the expression construct is an integrative plasmid or an episomal plasmid. 
     
     
         86 . The yeast cell of  claim 69 , wherein at least one gene that plays a role in drug efflux or cell permeability is disrupted. 
     
     
         87 . The yeast cell of  claim 86 , wherein the yeast gene that is disrupted is selected from the group consisting of PRD1, PDR3, and ERG6.