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Updated: Jan 26, 2026

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells
Published on: May 24, 2014
Reversible ubiquitination regulates HSF1 phase separation in response to proteotoxic stress
Yue Zhu1, Jianhua Wang2, Shuhong Tang2
1The Fifth People's Hospital of Dalian, Dalian Medical University, Dalian, China; Institute of Cancer Stem Cell, Dalian Medical University, Dalian, China.
Aims:
Heat shock factor 1 (HSF1) undergoes phase separation to form nuclear condensates in response to proteotoxic stress, which governs cell fate. Protein ubiquitination is a widely existed post-translational modification and closely participated in liquid-liquid phase separation. However, the exact roles of ubiquitination in HSF1 condensation remain obscure. Here, we aimed to investigate the regulation of HSF1 phase separation by ubiquitination in cells under proteotoxic conditions.
Materials And Methods:
Endogenous and exogenous HSF1 condensate formation was detected by immunofluorescence assays. HSF1 phase separation properties were analyzed by fluorescence recovery after photobleaching (FRAP) analysis. Western blotting, immunofluorescence, immunoprecipitation and co-transfection assays were performed to identify E3 ubiquitin ligases and deubiquitinating enzymes (DUBs) regulating HSF1 phase separation. The influence of ubiquitination sites on HSF1 phase separation was investigated by site-directed mutagenesis.
Key Findings:
Proteasome inhibitor PS341 induced HSF1 to form nuclear phase-separated condensates in a time-dependent manner. E3 ubiquitin ligase STUB1 promoted the phase separation of HSF1 by enhancing HSF1 ubiquitination, while DUBs USP13 and ATXN3 negatively regulated HSF1 condensation. Mutations of all nine ubiquitination sites within HSF1 dramatically inhibited condensate formation, with single-site mutations also showing significantly attenuated phase separation.
Significance:
Ubiquitination at multiple lysine residues jointly drives HSF1 phase separation in cells exposed to proteotoxic stress, which is coordinately regulated by E3 ubiquitin ligase STUB1 and DUBs including USP13 and ATXN3.
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