title: Coupled assays for monitoring protein refolding in Saccharomyces cerevisiae.
abstract: Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences (1). External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases (10). It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme (12). Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 (13), known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system (5), to protein refolding to validate this approach.
authors.affiliation: Department of Microbiology and Molecular Genetics, Graduate School of Biomedical Sciences, University of Texas Medical School.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Abrams
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Morano
mesh_terms: D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism*; D049409:Luciferases, Firefly / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C070972:HsP104 protein, S cerevisiae; D049452:Green Fluorescent Proteins; D049409:Luciferases, Firefly
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