Molecular mechanisms of heat shock factor 1 regulation Kmiecik, Szymon W.; Mayer, Matthias P.
Trends in biochemical sciences (Amsterdam. Regular ed.),
March 2022, 2022-03-00, 20220301, Volume:
47, Issue:
3
Journal Article
Peer reviewed
To thrive and to fulfill their functions, cells need to maintain proteome homeostasis even in the face of adverse environmental conditions or radical restructuring of the proteome during ...differentiation. At the center of the regulation of proteome homeostasis is an ancient transcriptional mechanism, the so-called heat shock response (HSR), orchestrated in all eukaryotic cells by heat shock transcription factor 1 (Hsf1). As Hsf1 is implicated in aging and several pathologies like cancer and neurodegenerative disorders, understanding the regulation of Hsf1 could open novel therapeutic opportunities. In this review, we discuss the regulation of Hsf1’s transcriptional activity by multiple layers of control circuits involving Hsf1 synthesis and degradation, conformational rearrangements and post-translational modifications (PTMs), and molecular chaperones in negative feedback loops.
Hsf1 is a thermosensor itself and directly senses elevated temperatures with conformational changes in the leucin zipper domains HR-A/B and HR-C.Hsp70, not Hsp90 as proposed in many studies, is the main chaperone regulating Hsf1 activity by monomerizing Hsf1 trimers and thereby dissociating Hsf1 from DNA.A complex network of post-translational modifications (PTMs) accompanies Hsf1 through its lifetime, providing fine-tuning of its activity to integrate many input signals and to meet the respective needs of the cell at any time.Hsf1 activity downregulation could be beneficial to treat cancer, whereas Hsf1 upregulation may help to counteract neurodegenerative diseases like Parkinson or Alzheimer, or could be a key to healthy aging by prolonging the optimal proteostasis capacity.
Intracellular amyloid fibrils linked to neurodegenerative disease typically accumulate in an age-related manner, suggesting inherent cellular capacity for counteracting amyloid formation in early ...life. Metazoan molecular chaperones assist native folding and block polymerization of amyloidogenic proteins, preempting amyloid fibril formation. Chaperone capacity for amyloid disassembly, however, is unclear. Here, we show that a specific combination of human Hsp70 disaggregase-associated chaperone components efficiently disassembles α-synuclein amyloid fibrils characteristic of Parkinson’s disease in vitro. Specifically, the Hsc70 chaperone, the class B J-protein DNAJB1, and an Hsp110 family nucleotide exchange factor (NEF) provide ATP-dependent activity that disassembles amyloids within minutes via combined fibril fragmentation and depolymerization. This ultimately generates non-toxic α-synuclein monomers. Concerted, rapid interaction cycles of all three chaperone components with fibrils generate the power stroke required for disassembly. This identifies a powerful human Hsp70 disaggregase activity that efficiently disassembles amyloid fibrils and points to crucial yet undefined biology underlying amyloid-based diseases.
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•A specific human Hsp70 chaperone system efficiently disassembles α-synuclein fibrils•Fragmentation and depolymerization generate short fibrils and non-toxic monomers•Coordinated chaperone binding cycles generate power stroke for disassembly•Fibril disassembly requires N- and C-terminal α-synuclein extensions
Gao et al. used a well-controlled in vitro system to identify a powerful human Hsp70 disaggregase that efficiently disassembles α-synuclein amyloid fibrils into less toxic monomers and small oligomers. They dissect the amyloid disassembly mechanism, a combination of fibril fragmentation and depolymerization.
The molecular chaperones of the Hsp70 family have been recognized as targets for anti-cancer therapy. Since several paralogs of Hsp70 proteins exist in cytosol, endoplasmic reticulum and ...mitochondria, we investigated which isoform needs to be down-regulated for reducing viability of cancer cells. For two recently identified small molecule inhibitors, VER-155008 and 2-phenylethynesulfonamide (PES), which are proposed to target different sites in Hsp70s, we analyzed the molecular mode of action in vitro. We found that for significant reduction of viability of cancer cells simultaneous knockdown of heat-inducible Hsp70 (HSPA1) and constitutive Hsc70 (HSPA8) is necessary. The compound VER-155008, which binds to the nucleotide binding site of Hsp70, arrests the nucleotide binding domain (NBD) in a half-open conformation and thereby acts as ATP-competitive inhibitor that prevents allosteric control between NBD and substrate binding domain (SBD). Compound PES interacts with the SBD of Hsp70 in an unspecific, detergent-like fashion, under the conditions tested. None of the two inhibitors investigated was isoform-specific.
The Hsp70–Hsp90 Chaperone Cascade in Protein Folding Morán Luengo, Tania; Mayer, Matthias P.; Rüdiger, Stefan G.D.
Trends in cell biology,
February 2019, 2019-02-00, 20190201, Volume:
29, Issue:
2
Journal Article
Peer reviewed
Open access
Conserved families of molecular chaperones assist protein folding in the cell. Here we review the conceptual advances on three major folding routes: (i) spontaneous, chaperone-independent folding; ...(ii) folding assisted by repetitive Hsp70 cycles; and (iii) folding by the Hsp70–Hsp90 cascades. These chaperones prepare their protein clients for folding on their own, without altering their folding path. A particularly interesting role is reserved for Hsp90. The function of Hsp90 in folding is its ancient function downstream of Hsp70, free of cochaperone regulation and present in all kingdoms of life. Eukaryotic signalling networks, however, embrace Hsp90 by a plethora of cochaperones, transforming the profolding machinery to a folding-on-demand factor. We discuss implications for biology and molecular medicine.
We discuss three productive pathways for protein folding in the cell: (i) spontaneous, chaperone-independent folding; (ii) Hsp70 cycling; and (iii) Hsp70–Hsp90 chaperone cascade.
Chaperone-assisted protein folding counts on the ATP-dependent action of Hsp70 and Hsp90 in the early stages in the folding reaction; protein folding proceeds subsequently in a chaperone-free fashion so that the global kinetics of the reaction remain unaltered.
Hsp90 has two defined functions: (i) the ancient, evolutionarily conserved function in protein folding downstream from Hsp70, independent of cochaperones; and (ii) the regulation of sophisticated signalling networks in the eukaryotic cytosol, finely tuned by a plethora of cochaperones.
Progress in understanding the Hsp70–Hsp90-assisted protein folding mechanism may inspire new therapeutic strategies for the treatment of proteinopathies.
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.
Synopsis
Heat shock transcription factor 1 (Hsf1) is a central regulator of the heat shock response (HSR) in eukaryotic cells. This study demonstrates that the Hsp70 system attenuates the metazoa HSR and restores the resting state, by monomerizing trimeric Hsf1 and thus dissociating it from DNA.
Hsp70 chaperones together with J‐domain co‐chaperones dissociate Hsf1 from DNA in vitro.
Hsf1 is bound by Hsp70s at distinct sites with different affinities, allowing Hsf1 to closely monitor cellular Hsp70 levels.
Starting at the binding site adjacent to the trimerization domain, Hsp70s monomerize Hsf1 trimers by successive cycles of entropic pulling.
Mutation or deletion of the identified binding sites potentiate Hsf1 activity in cell culture models.
Hsp70 and J‐domain chaperones attenuate the heat shock response in metazoa by monomerizing heat shock transcription factor 1 (Hsf1) and thus removing it from DNA.
Central to the protein folding activity of Hsp70 chaperones is their ability to interact with protein substrates in an ATP-controlled manner, which relies on allosteric regulation between their ...nucleotide-binding (NBD) and substrate-binding domains (SBD). Here we dissect this mechanism by analysing mutant variants of the Escherichia coli Hsp70 DnaK blocked at distinct steps of allosteric communication. We show that the SBD inhibits ATPase activity by interacting with the NBD through a highly conserved hydrogen bond network, and define the signal transduction pathway that allows bound substrates to trigger ATP hydrolysis. We identify variants deficient in only one direction of allosteric control and demonstrate that ATP-induced substrate release is more important for chaperone activity than substrate-stimulated ATP hydrolysis. These findings provide evidence of an unexpected dichotomic allostery mechanism in Hsp70 chaperones and provide the basis for a comprehensive mechanical model of allostery in Hsp70s.
Small heat shock proteins (sHsp) constitute an evolutionary conserved yet diverse family of chaperones acting as first line of defence against proteotoxic stress. sHsps coaggregate with misfolded ...proteins but the molecular basis and functional implications of these interactions, as well as potential sHsp specific differences, are poorly explored. In a comparative analysis of the two yeast sHsps, Hsp26 and Hsp42, we show in vitro that model substrates retain near-native state and are kept physically separated when complexed with either sHsp, while being completely unfolded when aggregated without sHsps. Hsp42 acts as aggregase to promote protein aggregation and specifically ensures cellular fitness during heat stress. Hsp26 in contrast lacks aggregase function but is superior in facilitating Hsp70/Hsp100-dependent post-stress refolding. Our findings indicate the sHsps of a cell functionally diversify in stress defence, but share the working principle to promote sequestration of misfolding proteins for storage in native-like conformation.
Protein folding in the cell requires ATP-driven chaperone machines such as the conserved Hsp70 and Hsp90. It is enigmatic how these machines fold proteins. Here, we show that Hsp90 takes a key role ...in protein folding by breaking an Hsp70-inflicted folding block, empowering protein clients to fold on their own. At physiological concentrations, Hsp70 stalls productive folding by binding hydrophobic, core-forming segments. Hsp90 breaks this deadlock and restarts folding. Remarkably, neither Hsp70 nor Hsp90 alters the folding rate despite ensuring high folding yields. In fact, ATP-dependent chaperoning is restricted to the early folding phase. Thus, the Hsp70-Hsp90 cascade does not fold proteins, but instead prepares them for spontaneous, productive folding. This stop-start mechanism is conserved from bacteria to man, assigning also a general function to bacterial Hsp90, HtpG. We speculate that the decreasing hydrophobicity along the Hsp70-Hsp90 cascade may be crucial for enabling spontaneous folding.
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•At physiological concentration, Hsp70 blocks effective protein folding•Hsp90 restarts folding, overcoming the Hsp70-inflicted folding block•The Hsp70-Hsp90 cascade increases folding yields, but does not alter folding kinetics•Short ATP depending chaperone phase is followed by slow Anfinsen folding
Chaperones help proteins to fold into their native state. Here, Morán Luengo et al. show that, contrary to expectations, the chaperone Hsp70 can block protein folding and that Hsp90 relieves the block to restart the folding reaction.
The 70 kDa heat shock proteins (Hsp70s) are highly versatile molecular chaperones that assist in a wide variety of protein-folding processes. They exert their functions by continuously cycling ...between states of low and high affinity for client polypeptides, driven by ATP-binding and hydrolysis. This cycling is tuned by cochaperones and clients. Although structures for the high and low client affinity conformations of Hsp70 and Hsp70 domains in complex with various cochaperones and peptide clients are available, it is unclear how structural rearrangements in the presence of cochaperones and clients are orchestrated in space and time. Here, we report insights into the conformational dynamics of the prokaryotic model Hsp70 DnaK throughout its adenosine-5'-triphosphate hydrolysis (ATPase) cycle using proximity-induced fluorescence quenching. Our data suggest that ATP and cochaperone-induced structural rearrangements in DnaK occur in a sequential manner and resolve hitherto unpredicted cochaperone and client-induced structural rearrangements. Peptides induce large conformational changes in DnaK·ATP prior to ATP hydrolysis, whereas a protein client induces significantly smaller changes but is much more effective in stimulating ATP hydrolysis. Analysis of the enthalpies of activation for the ATP-induced opening of the DnaK lid in the presence of clients indicates that the lid does not exert an enthalpic pulling force onto bound clients, suggesting entropic pulling as a major mechanism for client unfolding. Our data reveal important insights into the mechanics, allostery, and dynamics of Hsp70 chaperones. We established a methodology for understanding the link between dynamics and function, Hsp70 diversity, and activity modulation.
Hsp70 chaperones interact with a wide spectrum of substrates ranging from unfolded to natively folded and aggregated proteins. Structural evidence suggests that bound substrates are entirely enclosed ...in a β-sheet cavity covered by a helical lid, which requires structural rearrangements including lid opening to allow substrate access. We analyzed the mechanics of the lid movement of bacterial DnaK by disulfide fixation of lid elements to the β-sheet and by electron paramagnetic resonance spectroscopy using spin labels in the lid and β-sheet. Our results indicate that the lid-forming helix B adopts at least three conformational states and, notably, does not close over bound proteins, implying that DnaK does not only bind to extended peptide stretches of protein substrates but can also accommodate regions with substantial tertiary structure. This flexible binding mechanism provides a basis for the broad spectrum of substrate conformers of Hsp70s.
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