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Identification of Mammalian Protein Quality Control Factors by High-Throughput Cellular Imaging
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...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3282772/ https://www.ncbi.nlm.nih.gov/pubmed/22363705 http://dx.doi.org/10.1371/journal.pone.0031684 |
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author | Pegoraro, Gianluca Voss, Ty C. Martin, Scott E. Tuzmen, Pinar Guha, Rajarshi Misteli, Tom |
author_facet | Pegoraro, Gianluca Voss, Ty C. Martin, Scott E. Tuzmen, Pinar Guha, Rajarshi Misteli, Tom |
author_sort | Pegoraro, Gianluca |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-3282772 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-32827722012-02-23 Identification of Mammalian Protein Quality Control Factors by High-Throughput Cellular Imaging Pegoraro, Gianluca Voss, Ty C. Martin, Scott E. Tuzmen, Pinar Guha, Rajarshi Misteli, Tom PLoS One Research Article 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. Public Library of Science 2012-02-20 /pmc/articles/PMC3282772/ /pubmed/22363705 http://dx.doi.org/10.1371/journal.pone.0031684 Text en This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. https://creativecommons.org/publicdomain/zero/1.0/ This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. |
spellingShingle | Research Article Pegoraro, Gianluca Voss, Ty C. Martin, Scott E. Tuzmen, Pinar Guha, Rajarshi Misteli, Tom Identification of Mammalian Protein Quality Control Factors by High-Throughput Cellular Imaging |
title | Identification of Mammalian Protein Quality Control Factors by High-Throughput Cellular Imaging |
title_full | Identification of Mammalian Protein Quality Control Factors by High-Throughput Cellular Imaging |
title_fullStr | Identification of Mammalian Protein Quality Control Factors by High-Throughput Cellular Imaging |
title_full_unstemmed | Identification of Mammalian Protein Quality Control Factors by High-Throughput Cellular Imaging |
title_short | Identification of Mammalian Protein Quality Control Factors by High-Throughput Cellular Imaging |
title_sort | identification of mammalian protein quality control factors by high-throughput cellular imaging |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3282772/ https://www.ncbi.nlm.nih.gov/pubmed/22363705 http://dx.doi.org/10.1371/journal.pone.0031684 |
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