ABSTRACT
The heat shock response is a universal transcriptional response to proteotoxic stress orchestrated by heat shock transcription factor Hsf1 in all eukaryotic cells. Despite over 40 years of intense research, the mechanism of Hsf1 activity regulation remains poorly understood at the molecular level. In metazoa, Hsf1 trimerizes upon heat shock through a leucine-zipper domain and binds to DNA. How Hsf1 is dislodged from DNA and monomerized remained enigmatic. Here, using purified proteins, we demonstrate that unmodified trimeric Hsf1 is dissociated from DNA in vitro by Hsc70 and DnaJB1. Hsc70 binds to multiple sites in Hsf1 with different affinities. Hsf1 trimers are monomerized by successive cycles of entropic pulling, unzipping the triple leucine-zipper. Starting this unzipping at several protomers of the Hsf1 trimer results in faster monomerization. This process directly monitors the concentration of Hsc70 and DnaJB1. During heat shock adaptation, Hsc70 first binds to a high-affinity site in the transactivation domain, leading to partial attenuation of the response, and subsequently, at higher concentrations, Hsc70 removes Hsf1 from DNA to restore the resting state.
Subject(s)
DNA , HSC70 Heat-Shock Proteins , Heat Shock Transcription Factors , Protein Multimerization , Animals , Cell Line , DNA/chemistry , DNA/genetics , DNA/metabolism , HSC70 Heat-Shock Proteins/chemistry , HSC70 Heat-Shock Proteins/genetics , HSC70 Heat-Shock Proteins/metabolism , HSP40 Heat-Shock Proteins/chemistry , HSP40 Heat-Shock Proteins/genetics , HSP40 Heat-Shock Proteins/metabolism , Heat Shock Transcription Factors/chemistry , Heat Shock Transcription Factors/genetics , Heat Shock Transcription Factors/metabolism , Humans , Mice , Mice, Knockout , Protein DomainsABSTRACT
ATP-driven bacterial AAA+ proteases have been recognized as drug targets. They possess an AAA+ protein (e.g., ClpC), which threads substrate proteins into an associated peptidase (e.g., ClpP). ATPase activity and substrate selection of AAA+ proteins are regulated by adapter proteins that bind to regulatory domains, such as the N-terminal domain (NTD). The antibacterial peptide Cyclomarin A (CymA) kills Mycobacterium tuberculosis cells by binding to the NTD of ClpC. How CymA affects ClpC function is unknown. Here, we reveal the mechanism of CymA-induced toxicity. We engineered a CymA-sensitized ClpC chimera and show that CymA activates ATPase and proteolytic activities. CymA mimics adapter binding and enables autonomous protein degradation by ClpC/ClpP with relaxed substrate selectivity. We reconstitute CymA toxicity in E. coli cells expressing engineered ClpC and ClpP, demonstrating that gain of uncontrolled proteolytic activity causes cell death. This validates drug-induced overriding of AAA+ protease activity control as effective antibacterial strategy.
Subject(s)
ATPases Associated with Diverse Cellular Activities/antagonists & inhibitors , Anti-Bacterial Agents/pharmacology , Escherichia coli/chemistry , Oligopeptides/pharmacology , ATPases Associated with Diverse Cellular Activities/genetics , ATPases Associated with Diverse Cellular Activities/metabolism , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/isolation & purification , Escherichia coli/cytology , Models, Molecular , Molecular Conformation , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/enzymology , Oligopeptides/chemistry , Oligopeptides/isolation & purificationABSTRACT
The 90-kDa heat shock proteins (Hsp90s) assist the maturation of many key regulators of signal transduction pathways and cellular control circuits like protein kinases and transcription factors and chaperone their stability and activity. In this function, Hsp90s cooperate with some 30 cochaperones and they are themselves subject to regulation by numerous post-translational modifications. In vertebrates, two major isoforms exist in the cytosol, Hsp90α and Hsp90ß, which share a high degree of sequence identity and are expressed in tissue- and environmental condition-dependent manner. We identified an isoform-specific phosphorylation site in human Hsp90ß. This phosphorylation site seems to be linked to vertebrate evolution since it is not found in invertebrata but in all tetrapoda and many but not all fish species. We provide data suggesting that this phosphorylation is important for the activation of Hsp90 clients like glucocorticoid receptor and a protein kinase. Replacement of the phosphorylation site by glutamate affects the conformational dynamics of Hsp90 and interaction with the kinase-specific cochaperone Cdc37.
Subject(s)
Cell Cycle Proteins/metabolism , Chaperonins/metabolism , HSP90 Heat-Shock Proteins/metabolism , Amino Acid Sequence , Cell Cycle Proteins/genetics , Chaperonins/genetics , HEK293 Cells , HSP90 Heat-Shock Proteins/genetics , Humans , Phosphorylation , Protein Conformation , Protein Isoforms , Protein Kinases/metabolism , Protein Processing, Post-Translational , Receptors, Glucocorticoid/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Signal Transduction , Transcription Factors/metabolismABSTRACT
The heat shock response in yeast is regulated by the interaction between a chaperone protein and a heat shock transcription factor, and fine-tuned by phosphorylation.
Subject(s)
DNA-Binding Proteins , Heat-Shock Proteins , Heat Shock Transcription Factors , Heat-Shock Response , Phosphorylation , Transcription FactorsABSTRACT
The heat shock response is a universal homeostatic cell autonomous reaction of organisms to cope with adverse environmental conditions. In mammalian cells, this response is mediated by the heat shock transcription factor Hsf1, which is monomeric in unstressed cells and upon activation trimerizes, and binds to promoters of heat shock genes. To understand the basic principle of Hsf1 activation we analyzed temperature-induced alterations in the conformational dynamics of Hsf1 by hydrogen exchange mass spectrometry. We found a temperature-dependent unfolding of Hsf1 in the regulatory region happening concomitant to tighter packing in the trimerization region. The transition to the active DNA binding-competent state occurred highly cooperative and was concentration dependent. Surprisingly, Hsp90, known to inhibit Hsf1 activation, lowered the midpoint temperature of trimerization and reduced cooperativity of the process thus widening the response window. Based on our data we propose a kinetic model of Hsf1 trimerization.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Protein Multimerization/radiation effects , Temperature , Transcription Factors/chemistry , Transcription Factors/metabolism , Heat Shock Transcription Factors , Humans , Kinetics , Mass Spectrometry , Protein Conformation/radiation effects , Protein Folding/radiation effectsABSTRACT
Hsp90 chaperones receive much attention due to their role in cancer and other pathological conditions, and a tremendous effort of many laboratories has contributed in the past decades to considerable progress in the understanding of their functions. Hsp90 chaperones exist as dimers and, with the help of cochaperones, promote the folding of numerous client proteins. Although the original view of these interactions suggested that these dimeric complexes were symmetrical, it is now clear that many features are asymmetrical. In this review we discuss several recent advances that highlight how asymmetric interactions with cochaperones as well as asymmetric posttranslational modifications provide mechanisms to regulate client interactions and the progression through Hsp90's chaperone cycle.
