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.
Highlights
Cellular proteins must assume and maintain their native 3D conformation in order to be functionally active
By using the same criteria (EGFP/DsRedExpress2 Z-score.2), we identified 13 primary hits from this library (Table S4), among them UBE2D3, one of the 4 human genes encoding for a protein orthologous to the Ubc4/5 E2 ubiquitin ligase, which was previously shown to participate in the ubiquitination of misfolded proteins in S. cerevisiae [25]
We describe and use a high-content cell-based assay for the discovery of human Protein Quality Control (PQC) factors involved in the degradation of misfolded proteins
Summary
Cellular proteins must assume and maintain their native 3D conformation in order to be functionally active. Partial folding or misfolding renders proteins non-functional, and improperly folded proteins may become toxic to the cell [1,2]. Accurate folding of proteins is critical to prevent the formation of cellular aggregates and is implicated in human disease. Misfolded proteins tend to expose highly hydrophobic surfaces that are normally buried in their interior. Given the hydrophilic nature of the cellular medium, hydrophobic surfaces from different misfolded proteins tend to interact with each other and to form cellular aggregates [1]. Protein misfolding can lead to the disruption of protein homeostasis in a dominant negative fashion and may cause cell death as seen in Parkinson, Alzheimer and Huntington disease [3,4]
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