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Löw, Christian Frank (2014): Regulation of the cytosolic stress response: identification of positive and negative modulators by a Genome-Wide RNA interference screen. Dissertation, LMU München: Faculty of Chemistry and Pharmacy



The cytosolic stress response, also known as the heat-shock response (HSR), is one of the major defense mechanisms activated by cells to maintain the integrity of the cellular proteome under proteotoxic environmental conditions. It is characterized by the increased synthesis of heat-shock proteins (Hsps), mainly molecular chaperones and proteases which prevent the aggregation of misfolded proteins and mediate their refolding or degradation. It is generally accepted that the induction of the HSR is coordinated by the heat-shock transcription factor 1 (HSF1). However, many mechanistic aspects of the HSF1 regulation remain unclear. In the present study, a genome-wide RNA interference screen was combined with an extensive biochemical analysis and quantitative proteomics to better understand the regulation of the HSR upon thermal stress. In the screening experiments novel positive and negative modulators of the stress response were identified, including proteins involved in chromatin remodeling, transcription, mRNA splicing, DNA damage repair, and proteolytic degradation. The diversity of the identified regulators suggests that induction and attenuation of the HSR integrate signals from different cellular pathways and are rather multi-factorial processes than single gene/protein events. The modulator proteins are localized in multiple cellular compartments with the majority having their primary location in the nucleus. A protein-protein interaction analysis revealed a HSR regulatory network, with chromatin modifiers and nuclear protein quality control components occupying hub positions. These observations are supported by quantitative proteomics experiments, which showed specific stress-induced reorganizations of the nuclear proteome, including the transient accumulation of chaperones and proteasomal subunits. The histone acetyltransferase EP300 was shown to specifically control the cellular level of HSF1 by stabilizing it against proteasomal turnover under normal conditions. Moreover, the ubiquitin-proteasome system (UPS) was found to participate in the attenuation of the HSR by degrading stress-activated, hyperphosphorylated HSF1. Since HSF1 competes with stress-denatured proteins for access to the proteasome, the extent of cellular protein damage modulates the rate of HSR attenuation. In addition to thermal stress, various other proteotoxic stresses are known to induce the HSR such as the proteasome inhibitor MG132 and the triterpenoid celastrol, which activates HSF1 by an unknown mechanism. Therefore, the networks regulating HSF1 activation upon thermal stress, proteasome inhibition and celastrol treatment were compared in this study. Whereas there is a large overlap between the sets of regulatory factors activated after heat stress and proteasomal impairment, HSF1 activation after celastrol treatment seems to bypass the HSR regulatory network to a large extent. Nevertheless, comparison of the regulatory networks under different proteotoxic conditions revealed a set of HSR core components, including factors involved in chromatin remodeling, DNA damage repair, RNA transport, transcription, and ion transport. The various cellular functions and localizations of these core components reinforce the multifaceted nature of the HSR regulation. The results obtained in this study can help to identify potential targets for the pharmacologic manipulation of the HSR in the treatment of aggregate deposition diseases and cancer.