The human HSP70 family of chaperones: where do we stand?

The 70-kDa heat shock protein (HSP70) family of molecular chaperones represents one of the most ubiquitous classes of chaperones and is highly conserved in all organisms. Members of the HSP70 family control all aspects of cellular proteostasis such as nascent protein chain folding, protein import into organelles, recovering of proteins from aggregation, and assembly of multi-protein complexes. These chaperones augment organismal survival and longevity in the face of proteotoxic stress by enhancing cell viability and facilitating protein damage repair. Extracellular HSP70s have a number of cytoprotective and immunomodulatory functions, the latter either in the context of facilitating the cross-presentation of immunogenic peptides via major histocompatibility complex (MHC) antigens or in the context of acting as “chaperokines” or stimulators of innate immune responses. Studies have linked the expression of HSP70s to several types of carcinoma, with Hsp70 expression being associated with therapeutic resistance, metastasis, and poor clinical outcome. In malignantly transformed cells, HSP70s protect cells from the proteotoxic stress associated with abnormally rapid proliferation, suppress cellular senescence, and confer resistance to stress-induced apoptosis including protection against cytostatic drugs and radiation therapy. All of the cellular activities of HSP70s depend on their adenosine-5′-triphosphate (ATP)-regulated ability to interact with exposed hydrophobic surfaces of proteins. ATP hydrolysis and adenosine diphosphate (ADP)/ATP exchange are key events for substrate binding and Hsp70 release during folding of nascent polypeptides. Several proteins that bind to distinct subdomains of Hsp70 and consequently modulate the activity of the chaperone have been identified as HSP70 co-chaperones. This review focuses on the regulation, function, and relevance of the molecular Hsp70 chaperone machinery to disease and its potential as a therapeutic target.     

The hsp110 and Grp1 70 stress proteins: newly recognized relatives of the Hsp70s

Both the Grp170 and Hsp110 families represent relatively conserved and distinct sets of stress proteins, within a more diverse category that also includes the Hsp70s. All of these families are found in a wide variety of organisms from yeasts to humans. Although Hsp110s or Grp170s are not Hsp70s any more than Hsp70s are Hsp110s or Grp170s, it is still reasonable to refer to this combination of related families as the Hsp70 superfamily based on arguments discussed above and since no obvious prokaryotic Hsp110 or Grp170 has yet been identified. These proteins are related to their counterparts in the Hsp70/Grp78 family of eukaryotic stress proteins but are characterized by significantly larger molecular weights. The members of the Grp170 family are characterized by C-terminal ER retention sequences and are ER localized in yeasts and mammals. As a Grp, Grp170 is recognized to be coregulated with other major Grps by a well-known set of stress conditions, sometimes referred to as the unfolded protein response (Kozutsumi et al 1988; Nakaki et al 1989). The Hsp110 family members are localized in the nucleus and cytoplasm and, with other major Hsps, are also coregulated by a specific set of stress conditions, most notably including hyperthermic exposures. Hsp110 is sometimes called Hsp105, although it would be preferable to have a uniform term. The large Hsp70-like proteins are structurally similar to the Hsp70s but differ from them in important ways. In both the Grp170 and Hspl10 families, there is a long loop structure that is interposed between the peptide-binding ,-domain and the alpha-helical lid. In the Hsp110 family and Grp170, there are differing degrees of expansion in the alpha-helical domain and the addition of a C-terminal loop. This gives the appearance of much larger lid domains for Hsp110 and Grp170 compared with Hsp70. Both Hsp110 and Grp170 families have relatively conserved short sequences in the alpha-helical domain in the lid, which are conserved motifs in numerous proteins (we termed these motifs Magic and TedWylee as discussed earlier). The structural differences detailed in this review result in functional differences between the large (Grp170 and Hspl10) members of the Hsp70 superfamily, the most distinctive being an increased ability of these proteins to bind (hold) denatured polypeptides compared with Hsc70, perhaps related to the enlarged C-terminal helical domain. However, there is also a major difference between these large stress proteins; Hsp110 does not bind ATP in vitro, whereas Grp170 binds ATP avidly. The role of the Grp170 and Hsp110 stress proteins in cellular physiology is not well understood. Overexpression of Hsp110 in cultured mammalian cells increases thermal tolerance. Grp170 binds to secreted proteins in the ER and may be cooperatively involved in folding these proteins appropriately. These roles are similar to those of the Hsp70 family members, and, therefore, the question arises as to the differential roles played by the larger members of the superfamily. We have discussed evidence that the large members of the superfamily cooperate with members of the Hsp70 family, and these chaperones probably interact with a large number of chaperones and cochaperones in their functional activities. The fundamental point is that Hsp110 is found in conjunction with Hsp70 in the cytoplasm (and nucleus) and Grp170 is found in conjunction with78 in tha ER in every eucaryotic cell examined from yeast to humans. This would strongly argue that Hsp110 Grp170 exhibit functions in eucaryotes not effectively performed by Hsp70s or Grp78, respectively. Of interest in this respect is the observation that all Hsp110s loss of function or deletion mutants listed in the Drosophila deletion project database are lethal. The important task for the future is to determine the roles these conserved molecular chaperones play in normal and physiologically stressed cells.     

The evolutionary and ecological role of heat shock proteins

Most heat shock proteins (Hsp) function as molecular chaperones that help organisms to cope with stress of both an internal and external nature. Here, we review the recent evidence of the relationship between stress resistance and inducible Hsp expression, including a characterization of factors that induce the heat shock response and a discussion of the associated costs. We report on studies of stress resistance including mild stress, effects of high larval densities, inbreeding and age on Hsp expression, as well as on natural variation in the expression of Hsps. The relationship between Hsps and life history traits is discussed with special emphasis on the ecological and evolutionary relevance of Hsps. It is known that up‐regulation of the Hsps is a common cellular response to increased levels of non‐native proteins that facilitates correct protein folding/refolding or degradation of non‐functional proteins. However, we also suggest that the expression level of Hsp in each species and population is a balance between benefits and costs, i.e. a negative impact on growth, development rate and fertility as a result of overexpression of Hsps. To date, investigations have focused primarily on the Hsp70 family. There is evidence that representatives of this Hsp family and other molecular chaperones play significant roles in relation to stress resistance. Future studies including genomic and proteonomic analyses will increase our understanding of molecular chaperones in stress research.     

Autoantibodies to heat shock protein 60, 70, and 90 are not altered in the anti-SARS-CoV-2 IgG-seropositive humans without or with mild symptoms

Highly conserved heat shock proteins (Hsps) are localized in the cytoplasm and cellular organelles, and act as molecular chaperones or proteases. Members of Hsp families are released into the extracellular milieu under both normal and stress conditions. It is hypothesized that the severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) has the potential to elicit autoimmunity due to molecular mimicry between human extracellular Hsps and immunogenic proteins of the virus. To confirm the above hypothesis, levels of circulating autoantibodies directed to the key human chaperones i.e., Hsp60, Hsp70, and Hsp90 in the anti-SARS-CoV-2 IgG-seropositive participants have been evaluated. Twenty-six healthy volunteers who got two doses of the mRNA vaccine encoding the viral spike protein, anti-SARS-CoV-2 IgG-positive participants (n = 15), and healthy naïve (anti-SARS-CoV-2 IgG-negative) volunteers (n = 51) have been included in this study. We found that the serum levels of anti-Hsp60, anti-Hsp70, and anti-Hsp90 autoantibodies of the IgG, IgM, or IgA isotype remained unchanged in either the anti-COVID-19-immunized humans or the anti-SARS-CoV-2 IgG-positive participants when compared to healthy naïve volunteers, as measured by enzyme-linked immunosorbent assay. Our results showing that the humoral immune response to SARS-CoV-2 did not include the production of anti-SARS-CoV-2 antibodies that also recognized extracellular heat shock protein 60, 70, and 90 represent a partial evaluation of the autoimmunity hypothesis stated above. Further testing for cell-based immunity will be necessary to fully evaluate this hypothesis.     

Heat shock protein 70 is a potent activator of the human complement system

According to new hypotheses, extracellular heat shock proteins (Hsps) may represent an ancestral danger signal of cellular death or lysis-activating innate immunity. Recent studies demonstrating a dual role for Hsp70 as both a chaperone and cytokine, inducing potent proinflammatory response in human monocytes, provided support for the hypothesis that extracellular Hsp is a messenger of stress. Our previous work focused on the complement-activating ability of human Hsp60. We demonstrated that Hsp60 complexed with specific antibodies induces a strong classical pathway (CP) activation. Here, we show that another chaperone molecule also possesses complement-activating ability. Solid-phase enzyme-linked immunosorbent assay was applied for the experiments. Human Hsp70 activated the CP independently of antibodies. No complement activation was found in the case of human Hsp90. Our data further support the hypothesis that chaperones may messenger stress to other cells. Complement-like molecules and primitive immune cells appeared together early in evolution. A joint action of these arms of innate immunity in response to free chaperones, the most abundant cellular proteins displaying a stress signal, may further strengthen the effectiveness of immune reactions.     

Caught with their PAMPs down? The extracellular signalling actions of molecular chaperones are not due to microbial contaminants

In recent years, it has been hypothesised that a new signalling system may exist in vertebrates in which secreted molecular chaperones form a dynamic continuum between the cellular stress response and corresponding homeostatic physiological mechanisms. This hypothesis seems to be supported by the finding that many molecular chaperones are released from cells and act as extracellular signals for a range of cells. However, this nascent field of biological research seems to suffer from an excessive criticism that the biological activities of molecular chaperones are due to undefined components of the microbial expression hosts used to generate recombinant versions of these proteins. In this article, a number of the proponents of the cell signalling actions of molecular chaperones take this criticism head-on. They show that sufficient evidence exists to support fully the hypothesis that molecular chaperones have cell–cell signalling actions that are likely to be part of the homeostatic mechanism of the vertebrate.     

The Hsp27 gene is not required for Drosophila development but its activity is associated with starvation resistance

Heat shock proteins are induced under stress conditions and they act as molecular chaperones to refold denatured polypeptides. Stress resistances including thermotolerance generally are correlated with levels of the heat shock proteins. We investigated a fruit fly gene encoding a small heat shock protein, Hsp27, to determine if it functions in stress response of Drosophila melanogaster. A knockout Hsp27 allele was generated. Flies homozygous for this allele were viable, without obvious defects, and fertile, indicating Hsp27 is not essential for development. In stress-response tests, loss of the Hsp27 gene caused no defects in resistance to heat shock or oxidative treatments. However, a significant reduction in starvation resistance was associated with the genotype without a functional Hsp27 gene. The data suggest that the Drosophila HSP27 protein acts as a chaperone to provide cellular stress resistance, although its function may be limited to a subset of the stress response such as the starvation resistance.     

Morgana and melusin: Two fairies chaperoning signal transduction

Chaperones and scaffold proteins are key elements involved in controlling the assembly of molecular complexes required for coordinated signal transduction. Here we describe morgana and melusin, two phylogenetically conserved chaperones that cooperate with Hsp90 and regulate signal transduction in important physiopathological processes. While morgana is ubiquitously expressed, melusin expression is restricted to striated muscles. Despite high sequence homology, the two chaperones have distinct functions. Morgana controls genomic stability by regulating the centrosome cycle via ROCKII kinase. Melusin, however, organizes ERK signal transduction in cardiomyocytes and regulates cardiac compensatory hypertrophy in response to different stress stimuli.     

Multiple Hsp70 Isoforms in the Eukaryotic Cytosol: Mere Redundancy or Functional Specificity?

Hsp70 molecular chaperones play a variety of functions in every organism, cell type and organelle, and their activities have been implicated in a number of human pathologies, ranging from cancer to neurodegenerative diseases. The functions, regulations and structure of Hsp70s were intensively studied for about three decades, yet much still remains to be learned about these essential folding enzymes. Genome sequencing efforts revealed that most genomes contain multiple members of the Hsp70 family, some of which co-exist in the same cellular compartment. For example, the human cytosol and nucleus contain six highly homologous Hsp70 proteins while the yeast Saccharomyces cerevisiae contains four canonical Hsp70s and three fungal-specific ribosome-associated and specialized Hsp70s. The reasons and significance of the requirement for multiple Hsp70s is still a subject of debate. It has been postulated for a long time that these Hsp70 isoforms are functionally redundant and differ only by their spatio-temporal expression patterns. However, several studies in yeast and higher eukaryotic organisms challenged this widely accepted idea by demonstrating functional specificity among Hsp70 isoforms. Another element of complexity is brought about by specific cofactors, such as Hsp40s or nucleotide exchange factors that modulate the activity of Hsp70s and their binding to client proteins. Hence, a dynamic network of chaperone/co-chaperone interactions has evolved in each organism to efficiently take advantage of the multiple cellular roles Hsp70s can play. We summarize here our current knowledge of the functions and regulations of these molecular chaperones, and shed light on the known functional specificities among isoforms.     

Plant Hsp70 molecular chaperones: Protein structure, gene family, expression and function

The Hsp70 molecular chaperones of plants are encoded by a multi-gene family whose members are developmentally regulated and differentially expressed in response to temperature stress and other conditions that interrupt normal protein folding or favor protein denaturation. Under non-stressful conditions, Hsp70 cognates function in concert with a variety of co-chaperones to facilitate folding of de novo synthesized proteins, assist in transport of precursor proteins into organelles and to help target damaged proteins for degradation. Stress-induced Hsp70s function to mitigate aggregation of stress-denatured proteins and to refold non-native proteins restoring their biological function through iterative cycles of adenine nucleotide hydrolysis-dependent peptide binding and release. Much of what is known about how plant Hsp70s function comes from the study of Hsp70s from other types of organisms. Owing to their unique biology, much remains to be learned about the many functions Hsp70s play in plants.     

Heat shock proteins 90 kDa: diversity, structure, functions

The review presents data on diversity, structure, functions and gene expression of the high conserved family of heat shock proteins 90 kDa (Hsp90). They are specialized molecular chaperones that fulfill the folding, maintenance of structural integrity and conformational regulation of a subset of proteins involved in important cellular processes, such as transduction of signals, cell cycle control etc. A composition and functioning of the Hsp90 chaperone machine are considered. Hsp90s play a significant role in growth and development of organisms carrying out conformational regulation of many regulatory proteins and protecting cells under stress. The review summarizes the results of investigations of different organisms, mainly animals and yeasts, with emphasis on the facts on Hsp90s in plants.     

Chaperones as integrators of cellular networks: changes of cellular integrity in stress and diseases

The complex integrity of the cells and its sudden, but often predictable changes can be described and understood by the topology and dynamism of cellular networks. All these networks undergo both local and global rearrangements during stress and development of diseases. Here, we illustrate this by showing the stress-induced structural rearrangement of the yeast protein-protein interaction network (interactome). In an unstressed state, the yeast interactome is highly compact, and the centrally organized modules have a large overlap. During stress, several original modules became more separated, and a number of novel modules also appear. A few basic functions such as theproteasome preserve their central position; however, several functions with high energy demand, such the cell-cycle regulation loose their original centrality during stress. A number of key stress-dependent protein complexes, such as the disaggregation-specific chaperone, Hsp104 gain centrality in the stressed yeast interactome. Molecular chaperones, heat shock, or stress proteins became established as key elements in our molecular understanding of the cellular stress response. Chaperones form complex interaction networks (the chaperome) with each other and their partners. Here, we show that the human chaperome recovers the segregation of protein synthesis-coupled and stress-related chaperones observed in yeast recently. Examination of yeast and human interactomes shows that chaperones 1) are intermodular integrators of protein-protein interaction networks, which 2) often bridge hubs and 3) are favorite candidates for extensive phosphorylation. Moreover, chaperones 4) become more central in the organization of the isolated modules of the stressed yeast protein-protein interaction network, which highlights their importance in the decoupling and recoupling of network modules during and after stress. Chaperone-mediated evolvability of cellular networks may play a key role in cellular adaptation during stress and various polygenic and chronic diseases, such as cancer, diabetes or neurodegeneration.     

Partner proteins determine multiple functions of Hsp70

The 70 kDa heat shock proteins (Hsp70s) are ubiquitous molecular chaperones that are best known for their participation in protein folding. However, evidence is accumulating that Hsp70s perform several other cellular functions in cooperation with specific soluble or membrane-bound partner proteins. While the basic function of Hsp70s is explained by their ability to bind unfolded polypeptide segments, the partner proteins appear to customize them for specific roles such as involvement in protein traffic and folding, translocation of preproteins across membranes, and gene regulation.     

Monocyte cytokine synthesis in response to extracellular cell stress proteins suggests these proteins exhibit network behaviour

Human peripheral blood monocytes were exposed to single or pairs of cell stress proteins (CSPs), specifically Hsp10, Hsp27, Hsp60 and Hsp70—the former two having anti-inflammatory actions while the latter pair being assumed to be pro-inflammatory in activity. This study was to test if these proteins exhibited any network behaviour. To control for possible lipopolysaccharide contamination, polymyxin B was used. Surprisingly, at concentrations higher than 1 μg/ml, polymyxin B itself could induce cytokine synthesis. A number of commercial sources of the molecular chaperones were tested, and marked variations in monocyte cytokine synthesis were found. All four CSPs stimulated the same profile of IL-1/IL-6 synthesis and IL-10/TNF-α synthesis although the kinetics of production of these two pairs of cytokines were very different. A key question was whether extracellular molecular chaperones exhibited network behaviour. To test this, monocytes were cultured with suboptimal concentrations of single CSP and pairs of CSP to look for additive, synergistic or antagonistic cell responses. The major finding was that pairs of molecular chaperones, including chaperones thought to stimulate monocyte cytokine synthesis, could produce significant antagonistic cellular responses. This demonstrates that extracellular CSPs constitute an additional potent layer within the complex cytokine network and furthermore suggests that monocytes have evolved to dampen their immune responses upon exposure to extracellular networks of CSPs—perhaps as a mechanism for protecting cells against detrimental cellular stress responses.     

Proteotoxicity is not the reason for the dependence of cancer cells on the major chaperone Hsp70

Several years ago a hypothesis was proposed that the survival of cancer cells depend on elevated expression of molecular chaperones because these cells are prone to proteotoxic stress. A critical prediction of this hypothesis is that depletion of chaperones in cancer cells should lead to proteotoxicity. Here, using the major chaperone Hsp70 as example, we demonstrate that its depletion does not trigger proteotoxic stress, thus refuting the model. Accordingly, other functions of chaperones, e.g., their role in cell signaling, might define the requirements for chaperones in cancer cells, which is critical for rational targeting Hsp70 in cancer treatment.     

The heat shock response: life on the verge of death

Organisms must survive a variety of stressful conditions, including sudden temperature increases that damage important cellular structures and interfere with essential functions. In response to heat stress, cells activate an ancient signaling pathway leading to the transient expression of heat shock or heat stress proteins (Hsps). Hsps exhibit sophisticated protection mechanisms, and the most conserved Hsps are molecular chaperones that prevent the formation of nonspecific protein aggregates and assist proteins in the acquisition of their native structures. In this Review, we summarize the concepts of the protective Hsp network.     

Mapping the road to recovery: the ClpB/Hsp104 molecular chaperone

The AAA(+)-ATPases are a family of molecular motors which have been seconded into a plethora of cellular tasks. One subset, the Hsp100 molecular chaperones, are general protein remodellers that help to maintain the integrity of the cellular proteome by means of protein destruction or resurrection. In this review we focus on one family of Hsp100s, the homologous ClpB and Hsp104 molecular chaperones that convey thermotolerance by resolubilising and rescuing proteins from aggregates. We explore how the nucleotide binding and hydrolysis properties at the twelve nucleotide-binding domains of these hexameric rings are coupled to protein disaggregation, highlighting similarities and differences between ClpB and Hsp104.     

Meta-analysis of heat- and chemically upregulated chaperone genes in plant and human cells

Molecular chaperones are central to cellular protein homeostasis. In mammals, protein misfolding diseases and aging cause inflammation and progressive tissue loss, in correlation with the accumulation of toxic protein aggregates and the defective expression of chaperone genes. Bacteria and non-diseased, non-aged eukaryotic cells effectively respond to heat shock by inducing the accumulation of heat-shock proteins (HSPs), many of which molecular chaperones involved in protein homeostasis, in reducing stress damages and promoting cellular recovery and thermotolerance. We performed a meta-analysis of published microarray data and compared expression profiles of HSP genes from mammalian and plant cells in response to heat or isothermal treatments with drugs. The differences and overlaps between HSP and chaperone genes were analyzed, and expression patterns were clustered and organized in a network. HSPs and chaperones only partly overlapped. Heat-shock induced a subset of chaperones primarily targeted to the cytoplasm and organelles but not to the endoplasmic reticulum, which organized into a network with a central core of Hsp90s, Hsp70s, and sHSPs. Heat was best mimicked by isothermal treatments with Hsp90 inhibitors, whereas less toxic drugs, some of which non-steroidal anti-inflammatory drugs, weakly expressed different subsets of Hsp chaperones. This type of analysis may uncover new HSP-inducing drugs to improve protein homeostasis in misfolding and aging diseases.