title: Rapid profiling of disease alleles using a tunable reporter of protein misfolding.
abstract: Many human diseases are caused by genetic mutations that decrease protein stability. Such mutations may not specifically affect an active site, but can alter protein folding, abundance, or localization. Here we describe a high-throughput cell-based stability assay, IDESA (intra-DHFR enzyme stability assay), where stability is coupled to cell proliferation in the model yeast, Saccharomyces cerevisiae. The assay requires no prior knowledge of a protein's structure or activity, allowing the assessment of stability of proteins that have unknown or difficult to characterize activities, and we demonstrate use with a range of disease-relevant targets, including human alanine:glyoxylate aminotransferase (AGT), superoxide dismutase (SOD-1), DJ-1, p53, and SMN1. The assay can be carried out on hundreds of disease alleles in parallel or used to identify stabilizing small molecules (pharmacological chaperones) for unstable alleles. As demonstration of the general utility of this assay, we analyze stability of disease alleles of AGT, deficiency of which results in the kidney stone disease, primary hyperoxaluria type I, identifying mutations that specifically affect the protein-active site chemistry.
authors.affiliation: Department of Biology, Duke University, Durham, North Carolina 27708, USA.
authors.forename: Adrianne M C
authors.initials: AM
authors.lastname: Pittman
authors.forename: Melissa D
authors.initials: MD
authors.lastname: Lage
authors.forename: Vladimir
authors.initials: V
authors.lastname: Poltoratsky
authors.forename: Justin D
authors.initials: JD
authors.lastname: Vrana
authors.forename: Alessandro
authors.initials: A
authors.lastname: Paiardini
authors.forename: Alessandro
authors.initials: A
authors.lastname: Roncador
authors.forename: Barbara
authors.initials: B
authors.lastname: Cellini
authors.forename: Robert M
authors.initials: RM
authors.lastname: Hughes
authors.forename: Chandra L
authors.initials: CL
authors.lastname: Tucker
mesh_terms: D000483:Alleles; D004353:Drug Evaluation, Preclinical; D004795:Enzyme Stability / Q000187:drug effects / Q000235:genetics*; D017930:Genes, Reporter; D056726:Genetic Association Studies; D057166:High-Throughput Screening Assays; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding / Q000187:drug effects; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D012680:Sensitivity and Specificity; D000637:Transaminases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D000637:Transaminases