Chaperone System Research Articles

Mitochondrial ClpX activates an essential biosynthetic enzyme through partial unfolding.

Mitochondria control the activity, quality, and lifetime of their proteins with an autonomous system of chaperones, but the signals that direct substrate-chaperone interactions and outcomes are poorly understood. We previously discovered that the mitochondrial AAA+ protein unfoldase ClpX (mtClpX) activates the initiating enzyme for heme biosynthesis, 5-aminolevulinic acid synthase (ALAS), by promoting cofactor incorporation. Here, we ask how mtClpX accomplishes this activation. Using S. cerevisiae proteins, we identified sequence and structural features within ALAS that position mtClpX and provide it with a grip for acting on ALAS. Observation of ALAS undergoing remodeling by mtClpX revealed that unfolding is limited to a region extending from the mtClpX-binding site to the active site. Unfolding along this path is required for mtClpX to gate cofactor binding to ALAS. This targeted unfolding contrasts with the global unfolding canonically executed by ClpX homologs and provides insight into how substrate-chaperone interactions direct the outcome of remodeling.

Investigation of Natural and Synthetic Aggregation Inhibitors using Microfluidic Applications

Microfluidic approaches offer an extension of conventional biophysical techniques for the study of the intricate nature of biomolecule recognition. Recent microfluidic applications show the possibility of performing binding studies in the condensed phase without the need for extensive dilution, sample surface immobilization or changes to the molecular environment from the liquid to the gas phase. Capitalizing on such approaches, we used a microfluidic diffusion device to demonstrate a detailed characterisation of molecular chaperone activity in suppressing protein aggregation and the underlying binding mechanisms leading to specific inhibitory phenomena. In particular, we have studied in detail the entropic binding dynamics of the sHsp αB-crystallin to α-synuclein fibrils and have shown that changes in heat capacity are indicative of chaperone structural changes prior to the binding. For clusterin and Brichos, chaperone systems previously described in the context of Aβ(42) aggregation, binding studies and aggregation kinetics have been conducted and revealed a complex suppression system in which each chaperone specifically inhibits distinct microscopic aggregations steps without overlap. These results together with further binding kinetics of Brichos form a distinct structural picture of elongation sites on fibrillar ends and secondary nucleation sites along the fibrillar surface. As secondary nucleation seems to be the major inhibitory target desired in order to prevent the formation of toxic oligomeric species, mimicking nature's specific inhibition of aggregation for therapeutic applications seems like an ideal solution. However, we show that potential therapeutic antibodies for Alzheimer's, are not, on their own, sufficient for inhibiting secondary nucleation. Each antibody shows individual binding properties either to Aβ(42) monomers, fibrils or to both, resulting in a specific inhibition pattern. A full biophysical characterisation of potential therapeutics and all intermolecular interactions involved could thus increase the positive outcome in drug development.

Augmentation of Bri2 molecular chaperone activity against amyloid-\u03b2 reduces neurotoxicity in mouse hippocampus in vitro

Molecular chaperones play important roles in preventing protein misfolding and its potentially harmful consequences. Deterioration of molecular chaperone systems upon ageing are thought to underlie age-related neurodegenerative diseases, and augmenting their activities could have therapeutic potential. The dementia relevant domain BRICHOS from the Bri2 protein shows qualitatively different chaperone activities depending on quaternary structure, and assembly of monomers into high-molecular weight oligomers reduces the ability to prevent neurotoxicity induced by the Alzheimer-associated amyloid-β peptide 1-42 (Aβ42). Here we design a Bri2 BRICHOS mutant (R221E) that forms stable monomers and selectively blocks a main source of toxic species during Aβ42 aggregation. Wild type Bri2 BRICHOS oligomers are partly disassembled into monomers in the presence of the R221E mutant, which leads to potentiated ability to prevent Aβ42 toxicity to neuronal network activity. These results suggest that the activity of endogenous molecular chaperones may be modulated to enhance anti-Aβ42 neurotoxic effects.

Structural and Biochemical Properties of Hsp40/Hsp70 Chaperone System.

Hsp70s are ubiquitous molecular chaperones that act in a myriad of cellular functions, affecting virtually all aspects in the life of proteins from synthesis to degradation. Hsp70 proteins act in the cell in cooperation with a large set of dedicated co-chaperones consisting of J-domain proteins and nucleotide exchange factors that regulate the Hsp70 chaperone cycle. Recent studies have made significant progress towards obtaining a better understanding of the mechanisms through which Hsp70s and their co-chaperones operate, providing insights into structural, kinetic, and functional features of the various members of this network. In this chapter we describe the emerging working principles of the Hsp70 machine and its co-chaperones, and highlight how mechanistic aspects of this network are tied to distinct protein folding functions.

Structure of the TELO2-TTI1-TTI2 Complex and its Function in TOR Recruitment to the R2TP Chaperone

The R2TP (RUVBL1-RUVBL2-RPAP3-PIH1D1) complex, in collaboration with HSP90, functions as a chaperone for the assembly and stability of protein complexes, including RNA polymerases, snRNPs and PI3 kinase-like kinases (PIKK) such as TOR and SMG1. PIKK stabilisation depends on an additional complex of TELO2, TTI1 and TTI2 (TTT), whose structure and function are poorly understood. We have now determined the cryo-EM structure of the human R2TP-TTT complex that together with biochemical experiments reveals the mechanism of TOR recruitment to the R2TP-TTT chaperone. The HEAT-repeat TTT complex binds the kinase domain of TOR, without blocking its activity, and delivers TOR to the R2TP chaperone. In addition, TTT regulates the R2TP chaperone by inhibiting RUVBL1-RUVBL2 ATPase activity and by modulating the conformation and interactions of the PIH1D1 and RPAP3 components of R2TP. Together, our results show how TTT couples the recruitment of TOR to R2TP with the regulation of this chaperone system.

Proteome-wide observation of the phenomenon of life on the edge of solubility

To function effectively proteins must avoid aberrant aggregation, and hence they are expected to be expressed at concentrations safely below their solubility limits. By analyzing proteome-wide mass spectrometry data of Caenorhabditis elegans, however, we show that the levels of about three-quarters of the nearly 4,000 proteins analyzed in adult animals are close to their intrinsic solubility limits, indeed exceeding them by about 10% on average. We next asked how aging and functional self-assembly influence these solubility limits. We found that despite the fact that the total quantity of proteins within the cellular environment remains approximately constant during aging, protein aggregation sharply increases between days 6 and 12 of adulthood, after the worms have reproduced, as individual proteins lose their stoichiometric balances and the cellular machinery that maintains solubility undergoes functional decline. These findings reveal that these proteins are highly prone to undergoing concentration-dependent phase separation, which on aging is rationalized in a decrease of their effective solubilities, in particular for proteins associated with translation, growth, reproduction, and the chaperone system.

E3 ubiquitin ligase CHIP attenuates cellular proliferation and invasion abilities in triple-negative breast cancer cells.

Carboxyl terminus of Hsc-70-interacting protein (CHIP), as U-box-type ubiquitin ligase, connects the chaperone and proteasome systems and plays a pivotal role in maintaining protein homeostasis in the cytoplasm. CHIP induces the ubiquitination and degradation of diverse oncogenic substrate proteins and therefore involves in the progression of tumorigenesis. In this study, the CHIP expression was examined in different human breast cancer cell lines and a group of breast cancer tissues. We found, for the first time, that CHIP expression was correlated with the molecular subtyping of breast cancer. CHIP was least expressed in the base-like subtype of breast cancer, which are triple-negative breast cancer (TNBC) breast cancer predominantly. Accordingly, CHIP expression was evidently decreased in the TNBC MDA-MB-231 breast cancer cell line. Enforced induction of CHIP in the MDA-MB-231 cells exerted no obvious influences on cellular growth and cell cycle. The apoptotic and proliferation cells in hCHIP cells were both reduced compared to the ctrl cells. The mRNA and protein expressions of the anti-apoptotic Bcl-2 and Bcl-xL were markedly increased in the hCHIP cells compared to that of the ctrl cells. The expression of RelA was significantly reduced in the nuclear extract in hCHIP cells compared to that in the ctrl cells. The protein expressions of IKKβ were markedly decreased in the hCHIP cells compared to the ctrl cells. The reduced cellular proliferation was largely due to the attenuated IKKβ-p65/NF-κB activity. Meanwhile, the invasion ability but not the migration ability was diminished when CHIP was overexpressed in the MDA-MB-231 cells. The activity of MMP2 but not MMP9 was significantly decreased in the hCHIP cells compared to the ctrl cells. Taken together, these observations here provide functional evidence for CHIP behaved as a tumor suppressor in the TNBC breast cancer cells. CHIP influenced diverse biological aspects of the MDA-MB-231 breast cancer cells. Importantly, CHIP expression is a useful indicator of the molecular subtyping of breast cancer.

Chaperone biomarkers of lifespan and penetrance track the dosages of many other proteins

Many traits vary among isogenic individuals in homogeneous environments. In microbes, plants and animals, variation in the protein chaperone system affects many such traits. In the animal model C. elegans, the expression level of hsp-16.2 chaperone biomarkers correlates with or predicts the penetrance of mutations and lifespan after heat shock. But the physiological mechanisms causing cells to express different amounts of the biomarker were unknown. Here, we used an in vivo microscopy approach to dissect different contributions to cell-to-cell variation in hsp-16.2 expression in the intestines of young adult animals, which generate the most lifespan predicting signal. While we detected both cell autonomous intrinsic noise and signaling noise, we found both contributions were relatively unimportant. The major contributor to cell-to-cell variation in biomarker expression was general differences in protein dosage. The hsp-16.2 biomarker reveals states of high or low effective dosage for many genes.

Sec17 (α-SNAP) and Sec18 (NSF) restrict membrane fusion to R-SNAREs, Q-SNAREs, and SM proteins from identical compartments

Membrane fusion at each organelle requires conserved proteins: Rab-GTPases, effector tethering complexes, Sec1/Munc18 (SM)-family SNARE chaperones, SNAREs of the R, Qa, Qb, and Qc families, and the Sec17/α-SNAP and ATP-dependent Sec18/NSF SNARE chaperone system. The basis of organelle-specific fusion, which is essential for accurate protein compartmentation, has been elusive. Rab family GTPases, SM proteins, and R- and Q-SNAREs may contribute to this specificity. We now report that the fusion supported by SNAREs alone is both inefficient and promiscuous with respect to organelle identity and to stimulation by SM family proteins or complexes. SNARE-only fusion is abolished by the disassembly chaperones Sec17 and Sec18. Efficient fusion in the presence of Sec17 and Sec18 requires a tripartite match between the organellar identities of the R-SNARE, the Q-SNAREs, and the SM protein or complex. The functions of Sec17 and Sec18 are not simply negative regulation; they stimulate fusion with either vacuolar SNAREs and their SM protein complex HOPS or endoplasmic reticulum/cis-Golgi SNAREs and their SM protein Sly1. The fusion complex of each organelle is assembled from its own functionally matching pieces to engage Sec17/Sec18 for fusion stimulation rather than inhibition.

UPR proteins IRE1 and PERK switch BiP from chaperone to ER stress sensor.

BiP is a major ER chaperone and suggested to act as primary sensor in the activation of the unfolded protein response (UPR). How BiP operates as a molecular chaperone and as an ER stress sensor is unknown. Here, by reconstituting components of human UPR, ER stress and BiP chaperone systems, we discover that the interaction of BiP with the luminal domains (LD) of UPR proteins, IRE1 and PERK, switch BiP from its chaperone cycle into an ER stress sensor cycle by preventing the binding of its cochaperones, with loss of ATPase stimulation. Furthermore, misfolded protein-dependent dissociation of BiP from IRE1 is primed by ATP but not ADP. Our data elucidate a previously unidentified mechanistic cycle of BiP function that explains its ability to act as a Hsp70 chaperone and ER stress sensor.

Duplicate divergence of two bacterial small heat shock proteins reduces the demand for Hsp70 in refolding of substrates.

Small heat shock proteins (sHsps) are a conserved class of ATP-independent chaperones that bind to aggregation-prone polypeptides at stress conditions. sHsps encage these polypeptides in assemblies, shielding them from further aggregation. To facilitate their subsequent solubilization and refolding by Hsp70 (DnaK) and Hsp100 (ClpB) chaperones, first, sHsps need to dissociate from the assemblies. In most γ-proteobacteria, these functions are fulfilled by a single sHsp (IbpA), but in a subset of Enterobacterales, a two-protein sHsp (IbpA and IbpB) system has evolved. To gain insight into the emergence of complexity within this chaperone system, we reconstructed the phylogeny of γ-proteobacteria and their sHsps. We selected proteins representative of systems comprising either one or two sHsps and analysed their ability to form sHsps-substrate assemblies. All the tested IbpA proteins, but not IbpBs, stably interact with an aggregating substrate. Moreover, in Escherichia coli cells, ibpA but not ibpB suppress the growth defect associated with low DnaK level, which points to the major protective role of IbpA during the breakdown of protein quality control. We also examined how sHsps affect the association of Hsp70 with the assemblies at the initial phase of disaggregation and how they affect protein recovery after stress. Our results suggest that a single gene duplication event has given rise to the sHsp system consisting of a strong canonical binder, IbpA, and its non-canonical paralog IbpB that enhances sHsps dissociation from the assemblies. The cooperation between the sHsps reduces the demand for Hsp70 needed to outcompete them from the assemblies by promoting sHsps dissociation without compromising assembly formation at heat shock. This potentially increases the robustness and elasticity of sHsps protection against irreversible aggregation.

Structural and functional analysis of the Hsp70/Hsp40 chaperone system.

As one of the most abundant and highly conserved molecular chaperones, the 70-kDa heat shock proteins (Hsp70s) play a key role in maintaining cellular protein homeostasis (proteostasis), one of the most fundamental tasks for every living organism. In this role, Hsp70s are inextricably linked to many human diseases, most notably cancers and neurodegenerative diseases, and are increasingly recognized as important drug targets for developing novel therapeutics for these diseases. Hsp40s are a class of essential and universal partners for Hsp70s in almost all aspects of proteostasis. Thus, Hsp70s and Hsp40s together constitute one of the most important chaperone systems across all kingdoms of life. In recent years, we have witnessed significant progress in understanding the molecular mechanism of this chaperone system through structural and functional analysis. This review will focus on this recent progress, mainly from a structural perspective.

Small Heat Shock Proteins, Big Impact on Protein Aggregation in Neurodegenerative Disease.

Misfolding, aggregation, and aberrant accumulation of proteins are central components in the progression of neurodegenerative disease. Cellular molecular chaperone systems modulate proteostasis, and, therefore, are primed to influence aberrant protein-induced neurotoxicity and disease progression. Molecular chaperones have a wide range of functions from facilitating proper nascent folding and refolding to degradation or sequestration of misfolded substrates. In disease states, molecular chaperones can display protective or aberrant effects, including the promotion and stabilization of toxic protein aggregates. This seems to be dependent on the aggregating protein and discrete chaperone interaction. Small heat shock proteins (sHsps) are a class of molecular chaperones that typically associate early with misfolded proteins. These interactions hold proteins in a reversible state that helps facilitate refolding or degradation by other chaperones and co-factors. These sHsp interactions require dynamic oligomerization state changes in response to diverse cellular triggers and, unlike later steps in the chaperone cascade of events, are ATP-independent. Here, we review evidence for modulation of neurodegenerative disease-relevant protein aggregation by sHsps. This includes data supporting direct physical interactions and potential roles of sHsps in the stewardship of pathological protein aggregates in brain. A greater understanding of the mechanisms of sHsp chaperone activity may help in the development of novel therapeutic strategies to modulate the aggregation of pathological, amyloidogenic proteins. sHsps-targeting strategies including modulators of expression or post-translational modification of endogenous sHsps, small molecules targeted to sHsp domains, and delivery of engineered molecular chaperones, are also discussed.

Individualized management of genetic diversity in Niemann-Pick C1 through modulation of the Hsp70 chaperone system.

Genetic diversity provides a rich repository for understanding the role of proteostasis in the management of the protein fold in human biology. Failure in proteostasis can trigger multiple disease states, affecting both human health and lifespan. Niemann-Pick C1 (NPC1) disease is a rare genetic disorder triggered by mutations in NPC1, a multi-spanning transmembrane protein that is trafficked through the exocytic pathway to late endosomes (LE) and lysosomes (Ly) (LE/Ly) to globally manage cholesterol homeostasis. Defects triggered by >300 NPC1 variants found in the human population inhibit export of NPC1 protein from the endoplasmic reticulum (ER) and/or function in downstream LE/Ly, leading to cholesterol accumulation and onset of neurodegeneration in childhood. We now show that the allosteric inhibitor JG98, that targets the cytosolic Hsp70 chaperone/co-chaperone complex, can significantly improve the trafficking and post-ER protein level of diverse NPC1 variants. Using a new approach to model genetic diversity in human disease, referred to as variation spatial profiling, we show quantitatively how JG98 alters the Hsp70 chaperone/co-chaperone system to adjust the spatial covariance (SCV) tolerance and set-points on an amino acid residue-by-residue basis in NPC1 to differentially regulate variant trafficking, stability, and cholesterol homeostasis, results consistent with the role of BCL2-associated athanogene family co-chaperones in managing the folding status of NPC1 variants. We propose that targeting the cytosolic Hsp70 system by allosteric regulation of its chaperone/co-chaperone based client relationships can be used to adjust the SCV tolerance of proteostasis buffering capacity to provide an approach to mitigate systemic and neurological disease in the NPC1 population.

Partners in Mischief: Functional Networks of Heat Shock Proteins of Plasmodium falciparum and Their Influence on Parasite Virulence.

The survival of the human malaria parasite Plasmodium falciparum under the physiologically distinct environments associated with their development in the cold-blooded invertebrate mosquito vectors and the warm-blooded vertebrate human host requires a genome that caters to adaptability. To this end, a robust stress response system coupled to an efficient protein quality control system are essential features of the parasite. Heat shock proteins constitute the main molecular chaperone system of the cell, accounting for approximately two percent of the malaria genome. Some heat shock proteins of parasites constitute a large part (5%) of the ‘exportome’ (parasite proteins that are exported to the infected host erythrocyte) that modify the host cell, promoting its cyto-adherence. In light of their importance in protein folding and refolding, and thus the survival of the parasite, heat shock proteins of P. falciparum have been a major subject of study. Emerging evidence points to their role not only being cyto-protection of the parasite, as they are also implicated in regulating parasite virulence. In undertaking their roles, heat shock proteins operate in networks that involve not only partners of parasite origin, but also potentially functionally associate with human proteins to facilitate parasite survival and pathogenicity. This review seeks to highlight these interplays and their roles in parasite pathogenicity. We further discuss the prospects of targeting the parasite heat shock protein network towards the developments of alternative antimalarial chemotherapies.

Alpha-2-Macroglobulin, a Hypochlorite-Regulated Chaperone and Immune System Modulator.

Alpha-macroglobulins are ancient proteins that include monomeric, dimeric, and tetrameric family members. In humans, and many other mammals, the predominant alpha-macroglobulin is alpha-2-macroglobulin (α2M), a tetrameric protein that is constitutively abundant in biological fluids (e.g., blood plasma, cerebral spinal fluid, synovial fluid, ocular fluid, and interstitial fluid). α2M is best known for its remarkable ability to inhibit a broad spectrum of proteases, but the full gamut of its activities affects diverse biological processes. For example, α2M can stabilise and facilitate the clearance of the Alzheimer's disease-associated amyloid beta (Aβ) peptide. Additionally, α2M can influence the signalling of cytokines and growth factors including neurotrophins. The results of several studies support the idea that the functions of α2M are uniquely regulated by hypochlorite, an oxidant that is generated during inflammation, which induces the native α2M tetramer to dissociate into dimers. This review will discuss the evidence for hypochlorite-induced regulation of α2M and the possible implications of this in neuroinflammation and neurodegeneration.

Abstract 1736: Targeting Hsp90-Cdc37 complex overcomes drug resistance in glioma cells harboring FGFR3-TACC3 fusion gene

Abstract Glioblastoma is one of the most lethal malignancies with the poorest prognosis among tumors of the central nervous system. Fusion genes are common chromosomal aberrations in many cancers that may give rise to an in-frame fusion protein with oncogenic function. These fusion genes can be used as prognostic markers as well as drug targets in clinical practice. The FGFR3-TACC3 (F3T3) fusion gene was discovered in glioblastoma, with an occurrence rate of up to 8.3% and leading to uncontrolled proliferation and chromosomal instability. Our studies have shown that glioblastoma cells harboring the F3T3 are resistant to the frontline glioblastoma drug Temozolomide (TMZ). Using the Comet assay, we show that TMZ-mediated DNA damage is repaired more rapidly in cells harboring the F3T3 compared to control. Our studies on the mechanism of this drug resistance show that the F3T3 confers an aberrant activation of FGFR and ERK pathways when treated with TMZ. To further explore druggable target(s) to overcome resistance caused by F3T3, we immunoprecipitated (IP) the proteins that interact with F3T3 in U251 glioblastoma cells overexpressing the F3T3 or wildtype FGFR3, followed by LC-MS/MS analyses. Our quantitative proteomic analysis revealed interactions of the F3T3 fusion protein with Hsp90α and Cdc37 proteins. These interactions were further validated by reciprocal IPs followed by Western blotting. Activation of many kinases, such as FGFRs, depends on their interaction with the Hsp90 molecular chaperone system. Hsp90 recruitment is mediated by the co-chaperone adaptor protein Cdc37, which simultaneously binds to both the kinase and Hsp90. To test inhibition of F3T3 association with the Hsp90-Cdc37 complex, we treated U251 cells constitutively expressing F3T3 with the Hsp90 inhibitor Onalespib. We show that Onalespib treatment at 26 nM significantly suppresses the proliferation of F3T3 expressing U251 cells in comparison to micromolar levels of the pan-FGFR inhibitor BGJ398 and PD173074. Our results provide evidence that the F3T3 fusion gene contributes to drug resistance via multiple chemo-resistance pathways. Importantly, our findings establish that F3T3 is an Hsp90 client that shows strong addiction to the Hsp90-Cdc37 complex for cell growth and suggests a novel strategy for targeting F3T3 fusion gene in glioma. Citation Format: Tao Li, Farideh Mehraein-Ghomi, Sanjeev V. Namjoshi, Lynette M. Phillips, Elizabeth A. Ballard, Mary E. Forbes, ping-Chieh Chou, Xuejun Yang, Wei Zhang. Targeting Hsp90-Cdc37 complex overcomes drug resistance in glioma cells harboring FGFR3-TACC3 fusion gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1736.

HSP90 and co-chaperones: a multitaskers' view on plant hormone biology.

In order to survive under ever-changing conditions plants must be able to adaptively respond to their environment. Plant hormones and the signaling cross-talk among them play a key role in integrating external and internal cues, enabling the plants to acclimate accordingly. HSP90 and several of its co-chaperones are known as pleiotropic factors involved in the signaling pathways of multiple stress responses, including temperature, drought, and pathogen infection. Recently, hormone receptor components for auxin and jasmonic acid, respectively, have been identified as clients of the HSP90 chaperone system, suggesting a direct HSP90-dependent link to hormone signaling. In this review, we give an overview of the multiple roles of HSP90 and its co-chaperones in plant hormone biology and discuss the largely unexplored targets for signal integration that the activity of these apparent multitaskers may suggest.

The Hsp70 Chaperone System Stabilizes a Thermo-sensitive Subproteome in E. coli

Stress-inducible molecular chaperones have essential roles in maintaining protein homeostasis, but the extent to which they affect overall proteome stability remains unclear. Here, we analyze the effects of the DnaK (Hsp70) system on protein stability in Escherichia coli using pulse proteolysis combined with quantitative proteomics. We quantify ∼1,500 soluble proteins and find ∼500 of these to be protease sensitive under normal growth conditions, indicating a high prevalence of conformationally dynamic proteins, forming a metastable subproteome. Acute heat stress results in the unfolding of an additional ∼200 proteins, reflected in the exposure of otherwise buried hydrophobic regions. Overexpression of the DnaK chaperone system markedly stabilizes numerous thermo-sensitive proteins, including multiple ribosomal proteins and large, hetero-oligomeric proteins containing the evolutionarily ancient c.37 fold (P loop nucleoside triphosphate hydrolases). Thus, the Hsp70 system, in addition to its known chaperone functions, has a remarkable capacity to stabilize proteins in their folded states under denaturing stress conditions.

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 heatshock 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.