title: Identification of mammalian protein quality control factors by high-throughput cellular imaging.
abstract: Protein Quality Control (PQC) pathways are essential to maintain the equilibrium between protein folding and the clearance of misfolded proteins. In order to discover novel human PQC factors, we developed a high-content, high-throughput cell-based assay to assess PQC activity. The assay is based on a fluorescently tagged, temperature sensitive PQC substrate and measures its degradation relative to a temperature insensitive internal control. In a targeted screen of 1591 siRNA genes involved in the Ubiquitin-Proteasome System (UPS) we identified 25 of the 33 genes encoding for 26S proteasome subunits and discovered several novel PQC factors. An unbiased genome-wide siRNA screen revealed the protein translation machinery, and in particular the EIF3 translation initiation complex, as a novel key modulator of misfolded protein stability. These results represent a comprehensive unbiased survey of human PQC components and establish an experimental tool for the discovery of genes that are required for the degradation of misfolded proteins under conditions of proteotoxic stress.
authors.affiliation: Cell Biology of Genomes, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Pegoraro
authors.forename: Ty C
authors.initials: TC
authors.lastname: Voss
authors.forename: Scott E
authors.initials: SE
authors.lastname: Martin
authors.forename: Pinar
authors.initials: P
authors.lastname: Tuzmen
authors.forename: Rajarshi
authors.initials: R
authors.lastname: Guha
authors.forename: Tom
authors.initials: T
authors.lastname: Misteli
mesh_terms: D000483:Alleles; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism; D001681:Biological Assay; D039621:Eukaryotic Initiation Factor-3 / Q000378:metabolism; D020868:Gene Silencing; D015894:Genome, Human / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008322:Mammals / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D017510:Protein Folding; D055550:Protein Stability; D011506:Proteins / Q000378:metabolism*; D059748:Proteolysis; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D013312:Stress, Physiological; D013696:Temperature; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D000952:Antigens, Polyomavirus Transforming; D039621:Eukaryotic Initiation Factor-3; D018832:Molecular Chaperones; D011506:Proteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; C485184:enhanced green fluorescent protein; C110856:proteasome maturation protein; D049452:Green Fluorescent Proteins; D046988:Proteasome Endopeptidase Complex