Profiling Ssb-Nascent Chain Interactions Reveals Principles of Hsp70-Assisted Folding.

The yeast Hsp70 chaperone Ssb interacts with ribosomes and nascent polypeptides to assist protein folding. To reveal its working principle, we determined the nascent chain-binding pattern of Ssb at near-residue resolution by in vivo selective ribosome profiling. Ssb associates broadly with cytosolic, nuclear, and hitherto unknown substrate classes of mitochondrial and endoplasmic reticulum (ER) nascent proteins, supporting its general chaperone function. Ssb engages most substrates by multiple binding-release cycles to a degenerate sequence enriched in positively charged and aromatic amino acids. Timely association with this motif upon emergence at the ribosomal tunnel exit requires ribosome-associated complex (RAC) but not nascent polypeptide-associated complex (NAC). Ribosome footprint densities along orfs reveal faster translation at times of Ssb binding, mainly imposed by biases in mRNA secondary structure, codon usage, and Ssb action. Ssb thus employs substrate-tailored dynamic nascent chain associations to coordinate co-translational protein folding, facilitate accelerated translation, and support membrane targeting of organellar proteins.

The Hsp70 chaperone network.

The 70-kDa heat shock proteins (Hsp70s) are ubiquitous molecular chaperones that act in a large variety of cellular protein folding and remodelling processes. They function virtually at all stages of the life of proteins from synthesis to degradation and are thus crucial for maintaining protein homeostasis, with direct implications for human health. A large set of co-chaperones comprising J-domain proteins and nucleotide exchange factors regulate the ATPase cycle of Hsp70s, which is allosterically coupled to substrate binding and release. Moreover, Hsp70s cooperate with other cellular chaperone systems including Hsp90, Hsp60 chaperonins, small heat shock proteins and Hsp100 AAA+ disaggregases, together constituting a dynamic and functionally versatile network for protein folding, unfolding, regulation, targeting, aggregation and disaggregation, as well as degradation. In this Review we describe recent advances that have increased our understanding of the molecular mechanisms and working principles of the Hsp70 network. This knowledge showcases how the Hsp70 chaperone system controls diverse cellular functions, and offers new opportunities for the development of chemical compounds that modulate disease-related Hsp70 activities.

The unfolding story of a redox chaperone.

Oxidative stress, especially in combination with heat stress, poses a life-threatening challenge to many organisms by causing protein misfolding and aggregation. In this issue, Reichmann et al. demonstrate how a destabilized linker region of the bacterial chaperone Hsp33 prevents aggregation of a denatured protein by stabilizing structural elements.

Hsp70- and Hsp90-Mediated Regulation of the Conformation of p53 DNA Binding Domain and p53 Cancer Variants.

The activity of the tumor suppressor p53 has to be timed and balanced closely to prevent untimely induction of cell death. The stability of p53 depends on the ubiquitin ligase Mdm2 but also on Hsp70 and Hsp90 chaperones that interact with its DNA binding domain (DBD). Using hydrogen exchange mass spectrometry and biochemical methods, we analyzed conformational states of wild-type p53-DBD at physiological temperatures and conformational perturbations in three frequent p53 cancer mutants. We demonstrate that the Hsp70/Hdj1 system shifts the conformational equilibrium of p53 toward a flexible, more mutant-like, DNA binding inactive state by binding to the DNA binding loop. The analyzed cancer mutants are likewise destabilized by interaction with the Hsp70/Hdj1 system. In contrast, Hsp90 protects the DBD of p53 wild-type and mutant proteins from unfolding. We propose that the Hsp70 and Hsp90 chaperone systems assume complementary functions to optimally balance conformational plasticity with conformational stability.

Molecular mechanisms of heat shock factor 1 regulation.

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.

Molecular Mechanism of J-Domain-Triggered ATP Hydrolysis by Hsp70 Chaperones.

Efficient targeting of Hsp70 chaperones to substrate proteins depends on J-domain cochaperones, which in synergism with substrates trigger ATP hydrolysis in Hsp70s and concomitant substrate trapping. We present the crystal structure of the J-domain of Escherichia coli DnaJ in complex with the E. coli Hsp70 DnaK. The J-domain interacts not only with DnaK's nucleotide-binding domain (NBD) but also with its substrate-binding domain (SBD) and packs against the highly conserved interdomain linker. Mutational replacement of contacts between J-domain and SBD strongly reduces the ability of substrates to stimulate ATP hydrolysis in the presence of DnaJ and compromises viability at heat shock temperatures. Our data demonstrate that the J-domain and the substrate do not deliver completely independent signals for ATP hydrolysis, but the J-domain, in addition to its direct influence on Hsp70s catalytic center, makes Hsp70 more responsive for the hydrolysis-inducing signal of the substrate, resulting in efficient substrate trapping.

Hsp90 Breaks the Deadlock of the Hsp70 Chaperone System.

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.

Conformational dynamics of the Hsp70 chaperone throughout key steps of its ATPase cycle.

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.

Ubiquitin-specific protease 11 structure in complex with an engineered substrate mimetic reveals a molecular feature for deubiquitination selectivity.

Ubiquitin-specific proteases (USPs) are crucial for controlling cellular proteostasis and signaling pathways but how deubiquitination is selective remains poorly understood, in particular between paralogues. Here, we developed a fusion tag method by mining the Protein Data Bank and trapped USP11, a key regulator of DNA double-strand break repair, in complex with a novel engineered substrate mimetic. Together, this enabled structure determination of USP11 as a Michaelis-like complex that revealed key S1 and S1' binding site interactions with a substrate. Combined mutational, enzymatic, and binding experiments identified Met77 in linear diubiquitin as a significant residue that leads to substrate discrimination. We identified an aspartate "gatekeeper" residue in the S1' site of USP11 as a contributing feature for discriminating against linear diubiquitin. When mutated to a glycine, the corresponding residue in paralog USP15, USP11 acquired elevated activity toward linear diubiquitin in-gel shift assays, but not controls. The reverse mutation in USP15 confirmed that this position confers paralog-specific differences impacting diubiquitin cleavage rates. The results advance our understanding of the molecular basis for the higher selectivity of USP11 compared to USP15 and may aid targeted inhibitor development. Moreover, the reported carrier-based crystallization strategy may be applicable to other challenging targets.

Feedback regulation of heat shock factor 1 (Hsf1) activity by Hsp70-mediated trimer unzipping and dissociation from DNA.

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.

Targeted Proteomics Reveals Quantitative Differences in Low-Abundance Glycosyltransferases of Patients with Congenital Disorders of Glycosylation.

Protein glycosylation is an essential post-translational modification in all domains of life. Its impairment in humans can result in severe diseases named congenital disorders of glycosylation (CDGs). Most of the glycosyltransferases (GTs) responsible for proper glycosylation are polytopic membrane proteins that represent challenging targets in proteomics. We established a multiple reaction monitoring (MRM) assay to comprehensively quantify GTs involved in the processes of N-glycosylation and O- and C-mannosylation in the endoplasmic reticulum. High robustness was achieved by using an enriched membrane protein fraction of isotopically labeled HEK 293T cells as an internal protein standard. The analysis of primary skin fibroblasts from eight CDG type I patients with impaired ALG1, ALG2, and ALG11 genes, respectively, revealed a substantial reduction in the corresponding protein levels. The abundance of the other GTs, however, remained unchanged at the transcript and protein levels, indicating that there is no fail-safe mechanism for the early steps of glycosylation in the endoplasmic reticulum. The established MRM assay was shared with the scientific community via the commonly used open source Skyline software environment, including Skyline Batch for automated data analysis. We demonstrate that another research group could easily reproduce all analysis steps, even while using different LC-MS hardware.

Co-chaperone involvement in knob biogenesis implicates host-derived chaperones in malaria virulence.

The pathology associated with malaria infection is largely due to the ability of infected human RBCs to adhere to a number of receptors on endothelial cells within tissues and organs. This phenomenon is driven by the export of parasite-encoded proteins to the host cell, the exact function of many of which is still unknown. Here we inactivate the function of one of these exported proteins, PFA66, a member of the J-domain protein family. Although parasites lacking this protein were still able to grow in cell culture, we observed severe defects in normal host cell modification, including aberrant morphology of surface knobs, disrupted presentation of the cytoadherence molecule PfEMP1, and a total lack of cytoadherence, despite the presence of the knob associated protein KAHRP. Complementation assays demonstrate that an intact J-domain is required for recovery to a wild-type phenotype and suggest that PFA66 functions in concert with a HSP70 to carry out host cell modification. Strikingly, this HSP70 is likely to be of host origin. ATPase assays on recombinant protein verify a functional interaction between PFA66 and residual host cell HSP70. Taken together, our data reveal a role for PFA66 in host cell modification, strongly implicate human HSP70s as being essential in this process and uncover a new KAHRP-independent molecular factor required for correct knob biogenesis.

Hsp90: breaking the symmetry.

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.

Human Hsp70 Disaggregase Reverses Parkinson's-Linked α-Synuclein Amyloid Fibrils.

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.

Heat shock transcription factor 1 is SUMOylated in the activated trimeric state.

The heat shock response is a transcriptional program of organisms to counteract an imbalance in protein homeostasis. It is orchestrated in all eukaryotic cells by heat shock transcription factor 1 (Hsf1). Despite very intensive research, the intricacies of the Hsf1 activation-attenuation cycle remain elusive at a molecular level. Post-translational modifications belong to one of the key mechanisms proposed to adapt the Hsf1 activity to the needs of individual cells, and phosphorylation of Hsf1 at multiple sites has attracted much attention. According to cell biological and proteomics data, Hsf1 is also modified by small ubiquitin-like modifier (SUMO) at several sites. How SUMOylation affects Hsf1 activity at a molecular level is still unclear. Here, we analyzed Hsf1 SUMOylation in vitro with purified components to address questions that could not be answered in cell culture models. In vitro Hsf1 is primarily conjugated at lysine 298 with a single SUMO, though we did detect low-level SUMOylation at other sites. Different SUMO E3 ligases such as protein inhibitor of activated STAT 4 enhanced the efficiency of in vitro modification but did not alter SUMO site preferences. We provide evidence that Hsf1 trimerization and phosphorylation at serines 303 and 307 increases SUMOylation efficiency, suggesting that Hsf1 is SUMOylated in its activated state. Hsf1 can be SUMOylated when DNA bound, and SUMOylation of Hsf1 does neither alter DNA-binding affinity nor affects heat shock cognate 71kDa protein (HSPA8)+DnaJ homolog subfamily B member 1-mediated monomerization of Hsf1 trimers and concomitant dislocation from DNA. We propose that SUMOylation acts at the transcription level of the heat shock response.

Alternative modes of client binding enable functional plasticity of Hsp70.

The Hsp70 system is a central hub of chaperone activity in all domains of life. Hsp70 performs a plethora of tasks, including folding assistance, protection against aggregation, protein trafficking, and enzyme activity regulation, and interacts with non-folded chains, as well as near-native, misfolded, and aggregated proteins. Hsp70 is thought to achieve its many physiological roles by binding peptide segments that extend from these different protein conformers within a groove that can be covered by an ATP-driven helical lid. However, it has been difficult to test directly how Hsp70 interacts with protein substrates in different stages of folding and how it affects their structure. Moreover, recent indications of diverse lid conformations in Hsp70-substrate complexes raise the possibility of additional interaction mechanisms. Addressing these issues is technically challenging, given the conformational dynamics of both chaperone and client, the transient nature of their interaction, and the involvement of co-chaperones and the ATP hydrolysis cycle. Here, using optical tweezers, we show that the bacterial Hsp70 homologue (DnaK) binds and stabilizes not only extended peptide segments, but also partially folded and near-native protein structures. The Hsp70 lid and groove act synergistically when stabilizing folded structures: stabilization is abolished when the lid is truncated and less efficient when the groove is mutated. The diversity of binding modes has important consequences: Hsp70 can both stabilize and destabilize folded structures, in a nucleotide-regulated manner; like Hsp90 and GroEL, Hsp70 can affect the late stages of protein folding; and Hsp70 can suppress aggregation by protecting partially folded structures as well as unfolded protein chains. Overall, these findings in the DnaK system indicate an extension of the Hsp70 canonical model that potentially affects a wide range of physiological roles of the Hsp70 system.

Functional diversity between HSP70 paralogs caused by variable interactions with specific co-chaperones.

Heat shock protein 70 (HSP70) chaperones play a central role in protein quality control and are crucial for many cellular processes, including protein folding, degradation, and disaggregation. Human HSP70s compose a family of 13 members that carry out their functions with the aid of even larger families of co-chaperones. A delicate interplay between HSP70s and co-chaperone recruitment is thought to determine substrate fate, yet it has been generally assumed that all Hsp70 paralogs have similar activities and are largely functionally redundant. However, here we found that when expressed in human cells, two highly homologous HSP70s, HSPA1A and HSPA1L, have opposing effects on cellular handling of various substrates. For example, HSPA1A reduced aggregation of the amyotrophic lateral sclerosis-associated protein variant superoxide dismutase 1 (SOD1)-A4V, whereas HSPA1L enhanced its aggregation. Intriguingly, variations in the substrate-binding domain of these HSP70s did not play a role in this difference. Instead, we observed that substrate fate is determined by differential interactions of the HSP70s with co-chaperones. Whereas most co-chaperones bound equally well to these two HSP70s, Hsp70/Hsp90-organizing protein (HOP) preferentially bound to HSPA1L, and the Hsp110 nucleotide-exchange factor HSPH2 preferred HSPA1A. The role of HSPH2 was especially crucial for the HSPA1A-mediated reduction in SOD1-A4V aggregation. These findings reveal a remarkable functional diversity at the level of the cellular HSP70s and indicate that this diversity is defined by their affinities for specific co-chaperones such as HSPH2.

Crucial HSP70 co-chaperone complex unlocks metazoan protein disaggregation.

Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states. Healthy metazoan cells effectively eliminate intracellular protein aggregates, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control.

Recent advances in the structural and mechanistic aspects of Hsp70 molecular chaperones.

Hsp70 chaperones are central hubs of the protein quality control network and collaborate with co-chaperones having a J-domain (an ∼70-residue-long helical hairpin with a flexible loop and a conserved His-Pro-Asp motif required for ATP hydrolysis by Hsp70s) and also with nucleotide exchange factors to facilitate many protein-folding processes that (re)establish protein homeostasis. The Hsp70s are highly dynamic nanomachines that modulate the conformation of their substrate polypeptides by transiently binding to short, mostly hydrophobic stretches. This interaction is regulated by an intricate allosteric mechanism. The J-domain co-chaperones target Hsp70 to their polypeptide substrates, and the nucleotide exchange factors regulate the lifetime of the Hsp70-substrate complexes. Significant advances in recent years are beginning to unravel the molecular mechanism of this chaperone machine and how they treat their substrate proteins.

Heat Shock Protein 90α-Dependent B-Cell-2-Associated Transcription Factor 1 Promotes Hepatocellular Carcinoma Proliferation by Regulating MYC Proto-Oncogene c-MYC mRNA Stability.

B-cell lymphoma 2 (Bcl-2)-associated transcription factor 1 (Bclaf1) is known to be involved in diverse biological processes, but, to date, there has been no evidence for any functional role of Bclaf1 in hepatocellular carcinoma (HCC) progression. Here, we demonstrate that Bclaf1 is frequently up-regulated in HCC and that Bclaf1 up-regulation is associated with Edmondson grade, lower overall survival rates, and poor prognosis. Overexpression of Bclaf1 in HCC cell lines HepG2 and Huh7 promoted proliferation considerably, whereas Bclaf1 knockdown had the opposite effect. Xenograft tumors grown from Bclaf1 knockdown Huh7 cells had smaller tumor volumes than tumors grown from control cells. Furthermore, our study describes MYC proto-oncogene (c-Myc) as a downstream target of Bclaf1, given that Bclaf1 regulates c-MYC expression posttranscriptionally by its RS domain. To exert this function, Bclaf1 must interact with the molecular chaperone, heat shock protein 90 alpha (Hsp90α). In HCC tissue samples, Hsp90α levels were also increased significantly and Hsp90α-Bclaf1 interaction was enhanced. Bclaf1 interacts with the C-terminal domain of Hsp90α, and this interaction is disrupted by the C-terminal domain inhibitor, novobiocin (NB), resulting in proteasome-dependent degradation of Bclaf1. Moreover, NB-induced disruption of Hsp90α-Bclaf1 interaction dampened the production of mature c-MYC mRNA and attenuated tumor cell growth in vitro and in vivo. Conclusion: Our findings suggest that Bclaf1 affects HCC progression by manipulating c-MYC mRNA stability and that the Hsp90α/Bclaf1/c-Myc axis might be a potential target for therapeutic intervention in HCC.