Human Small Heat Shock Protein Research Articles

Interaction of the C-terminal immunoglobulin-like domains (Ig 22–24) of filamin C with human small heat shock proteins

Small heat shock proteins are the well-known regulators of the cytoskeleton integrity, yet their complexes with actin-binding proteins are underexplored. Filamin C, a dimeric 560 kDa protein, abundant in cardiac and skeletal muscles, crosslinks actin filaments and contributes to Z-disc formation and membrane-cytoskeleton attachment. Here, we analyzed the interaction of a human filamin C fragment containing immunoglobulin-like domains 22–24 (FLNC22-24) with five small heat shock proteins (HspB1, HspB5, HspB6, HspB7, HspB8) and their α-crystallin domains. On size-exclusion chromatography, only HspB7 or its α-crystallin domain formed complexes with FLNC22-24. Despite similar isoelectric points of the small heat shock proteins analyzed, only HspB7 and its α-crystallin domain interacted with FLNC22-24 on native gel electrophoresis. Crosslinking with glutaraldehyde confirmed the formation of complexes between HspB7 (or its α-crystallin domain) and the filamin С fragment, inhibiting intersubunit FLNC crosslinking. These data are consistent with the structure modeling using Alphafold. Thus, the C-terminal fragment (immunoglobulin-like domains 22–24) of filamin C contains the site for HspB7 (or its α-crystallin domain) interaction, which competes with FLNC22-24 dimerization and its probable interaction with different target proteins.

Site-Specific Glycation of Human Heat Shock Protein (Hsp27) Enhances Its Chaperone Activity.

Non-enzymatic posttranslational modifications are believed to affect at least 30% of human proteins, commonly termed glycation. Many of these modifications are implicated in various pathological conditions, e.g., cataract, diabetes, neurodegenerative diseases, and cancer. Chemical protein synthesis enables access to full-length proteins carrying site-specific modifications. One such modification, argpyrimidine (Apy), has been detected in human small heat shock protein Hsp27 and closely related proteins in patient-derived tissues. Thus far, studies have looked into only artificial mixtures of Apy modifications, and only one has analyzed Apy188. We were interested in understanding the impact of such individual Apy modifications on five different arginine sites within the crucial N-terminal domain of Hsp27. By combining protein semisynthesis with biochemical assays on semisynthetic Hsp27 analogues with single-point Apy modification at those sites, we have shown how a seemingly minimal modification within this region results in dramatically altered functional attributes.

Human Small Heat Shock Protein B8 Inhibits Protein Aggregation without Affecting the Native Folding Process.

Small Heat Shock Proteins (sHSPs) are key components of our Protein Quality Control system and are thought to act as reservoirs that neutralize irreversible protein aggregation. Yet, sHSPs can also act as sequestrases, promoting protein sequestration into aggregates, thus challenging our understanding of their exact mechanisms of action. Here, we employ optical tweezers to explore the mechanisms of action of the human small heat shock protein HSPB8 and its pathogenic mutant K141E, which is associated with neuromuscular disease. Through single-molecule manipulation experiments, we studied how HSPB8 and its K141E mutant affect the refolding and aggregation processes of the maltose binding protein. Our data show that HSPB8 selectively suppresses protein aggregation without affecting the native folding process. This anti-aggregation mechanism is distinct from previous models that rely on the stabilization of unfolded polypeptide chains or partially folded structures, as has been reported for other chaperones. Rather, it appears that HSPB8 selectively recognizes and binds to aggregated species formed at the early stages of aggregation, preventing them from growing into larger aggregated structures. Consistently, the K141E mutation specifically targets the affinity for aggregated structures without impacting native folding, and hence impairs its anti-aggregation activity.

α-Crystallin Domains of Five Human Small Heat Shock Proteins (sHsps) Differ in Dimer Stabilities and Ability to Incorporate Themselves into Oligomers of Full-Length sHsps

The α-crystallin domain (ACD) is the hallmark of a diverse family of small heat shock proteins (sHsps). We investigated some of the ACD properties of five human sHsps as well as their interactions with different full-length sHsps. According to size-exclusion chromatography, at high concentrations, the ACDs of HspB1 (B1ACD), HspB5 (B5ACD) and HspB6 (B6ACD) formed dimers of different stabilities, which, upon dilution, dissociated to monomers to different degrees. Upon dilution, the B1ACD dimers possessed the highest stabilities, and those of B6ACD had the lowest. In striking contrast, the ACDs of HspB7 (B7ACD) and HspB8 (B8ACD) formed monomers in the same concentration range, which indicated the compromised stabilities of their dimer interfaces. B1ACD, B5ACD and B6ACD transiently interacted with full-length HspB1 and HspB5, which are known to form large oligomers, and modulated their oligomerization behavior. The small oligomers formed by the 3D mutant of HspB1 (mimicking phosphorylation at Ser15, Ser78 and Ser82) effectively interacted with B1ACD, B5ACD and B6ACD, incorporating these α-crystallin domains into their structures. The inherently dimeric full-length HspB6 readily formed heterooligomeric complexes with B1ACD and B5ACD. In sharp contrast to the abovementioned ACDs, B7ACD and B8ACD were unable to interact with full-length HspB1, the 3D mutant of HspB1, HspB5 or HspB6. Thus, their high sequence homology notwithstanding, B7ACD and B8ACD differ from the other three ACDs in their inability to form dimers and interact with the full-length small heat shock proteins. Having conservative primary structures and being apparently similar, the ACDs of the different sHsps differ in terms of their dimer stabilities, which can influence the heterooligomerization preferences of sHsps.

Abstract 1719: Activation of human small heat shock protein HSPB5 by zinc: Taking advantage of a multitude of possibilities

Abstract 1719: Activation of human small heat shock protein HSPB5 by zinc: Taking advantage of a multitude of possibilities

Is the small heat shock protein HSPB7 (cvHsp) a genuine actin-binding protein?

It is postulated that the small heat shock proteins directly interact with actin, affect formation and stabilize actin filaments. To verify this suggestion, we have analyzed interaction of recombinant human small heat shock protein HspB7 with skeletal muscle actin. In blot overlay HspB7 binds both G- and F-actin. The sites of interaction are located in the C-terminal large core domain of actin. In the course of ultracentrifugation F-actin and F-actin/tropomyosin complexes were pelleted and trapped HspB7. However, HspB7 pelleting was nonspecific and saturation was not achieved even at very high HspB7 concentration. HspB7 was unable to retard or prevent heat-induced F-actin aggregation. Native gel electrophoresis and chemical crosslinking failed to detect interaction of G-actin with HspB7, although both these methods clearly demonstrated formation of complexes formed by G-actin with DNAse I and cofilin-2. It is concluded that HspB7 is not a genuine actin-binding protein and its effect on actin filaments seems to be determined by interaction of HspB7 with minor regulatory proteins of actin filaments.

Insights on Human Small Heat Shock Proteins and Their Alterations in Diseases.

The family of the human small Heat Shock Proteins (HSPBs) consists of ten members of chaperones (HSPB1-HSPB10), characterized by a low molecular weight and capable of dimerization and oligomerization forming large homo- or hetero-complexes. All HSPBs possess a highly conserved centrally located α-crystallin domain and poorly conserved N- and C-terminal domains. The main feature of HSPBs is to exert cytoprotective functions by preserving proteostasis, assuring the structural maintenance of the cytoskeleton and acting in response to cellular stresses and apoptosis. HSPBs take part in cell homeostasis by acting as holdases, which is the ability to interact with a substrate preventing its aggregation. In addition, HSPBs cooperate in substrates refolding driven by other chaperones or, alternatively, promote substrate routing to degradation. Notably, while some HSPBs are ubiquitously expressed, others show peculiar tissue-specific expression. Cardiac muscle, skeletal muscle and neurons show high expression levels for a wide variety of HSPBs. Indeed, most of the mutations identified in HSPBs are associated to cardiomyopathies, myopathies, and motor neuropathies. Instead, mutations in HSPB4 and HSPB5, which are also expressed in lens, have been associated with cataract. Mutations of HSPBs family members encompass base substitutions, insertions, and deletions, resulting in single amino acid substitutions or in the generation of truncated or elongated proteins. This review will provide an updated overview of disease-related mutations in HSPBs focusing on the structural and biochemical effects of mutations and their functional consequences.

Alpha B-Crystallin in Muscle Disease Prevention: The Role of Physical Activity.

HSPB5 or alpha B-crystallin (CRYAB), originally identified as lens protein, is one of the most widespread and represented of the human small heat shock proteins (sHSPs). It is greatly expressed in tissue with high rates of oxidative metabolism, such as skeletal and cardiac muscles, where HSPB5 dysfunction is associated with a plethora of human diseases. Since HSPB5 has a major role in protecting muscle tissues from the alterations of protein stability (i.e., microfilaments, microtubules, and intermediate filament components), it is not surprising that this sHSP is specifically modulated by exercise. Considering the robust content and the protective function of HSPB5 in striated muscle tissues, as well as its specific response to muscle contraction, it is then realistic to predict a specific role for exercise-induced modulation of HSPB5 in the prevention of muscle diseases caused by protein misfolding. After offering an overview of the current knowledge on HSPB5 structure and function in muscle, this review aims to introduce the reader to the capacity that different exercise modalities have to induce and/or activate HSPB5 to levels sufficient to confer protection, with the potential to prevent or delay skeletal and cardiac muscle disorders.

Quaternary Structure and Hetero-Oligomerization of Recombinant Human Small Heat Shock Protein HspB7 (cvHsp).

In this study, a reliable and simple method of untagged recombinant human HspB7 preparation was developed. Recombinant HspB7 is presented in two oligomeric forms with an apparent molecular weight of 36 kDa (probably dimers) and oligomers with an apparent molecular weight of more than 600 kDa. By using hydrophobic and size-exclusion chromatography, we succeeded in preparation of HspB7 dimers. Mild oxidation promoted the formation of large oligomers, whereas the modification of Cys 126 by iodoacetamide prevented it. The deletion of the first 13 residues or deletion of the polySer motif (residues 17–29) also prevented the formation of large oligomers of HspB7. Cys-mutants of HspB6 and HspB8 containing a single-Cys residue in the central part of the β7 strand in a position homologous to that of Cys137 in HspB1 can be crosslinked to the wild-type HspB7 through a disulfide bond. Immobilized on monoclonal antibodies, the wild-type HspB6 interacted with the wild-type HspB7. We suppose that formation of heterodimers of HspB7 with HspB6 and HspB8 may be important for the functional activity of these small heat shock proteins.

Trends in HSPB5 research: a 36-year bibliometric analysis.

HSPB5 (heat shock protein B5), also known as αB-crystallin, is one of the most widespread and populous of the ten human small heat shock proteins (sHsps). Over the past decades, extensive research has been conducted on HSPB5. However, few studies have statistically analyzed these publications. Herein, we conducted a bibliometric analysis to track the global research trend and current development status of HSPB5 research from the Web of Science Core Collection (WoSCC) database between 1985 and 2020. Our results demonstrate that 1220 original articles cited 54,778 times in 391 scholarly journals were published. Visualization analyses reveal that the Journal of Biological Chemistry was the most influential journal with 85 articles. The USA dominated this field with 520 publications (42.62%), followed by Japan with 149 publications (12.21%), and Kato contributed the largest number of publications. Most related publications were published in journals focusing on biochemistry molecular biology, cell biology, neurosciences neurology, and ophthalmology. In addition, keyword co-occurrence analyses identify three predominant research topics: expression of HSPB5, chaperone studies for HSPB5, and pathological studies of HSPB5. This study provides valuable guidance for researchers and leads to collaborative opportunities between diverse research interests to be integrated for HSPB5 research.

Replacement of Arg in the conserved N-terminal RLFDQxFG motif affects physico-chemical properties and chaperone-like activity of human small heat shock protein HspB8 (Hsp22).

The small heat shock protein (sHsp) called HspB8 (formerly, Hsp22) is one of the least typical sHsp members, whose oligomerization status remains debatable. Here we analyze the effect of mutations in a highly conservative sequence located in the N-terminal domain of human HspB8 on its physico-chemical properties and chaperone-like activity. According to size-exclusion chromatography coupled to multi-angle light scattering, the wild type (WT) HspB8 is present as dominating monomeric species (~24 kDa) and a small fraction of oligomers (~60 kDa). The R29A amino acid substitution leads to the predominant formation of 60-kDa oligomers, leaving only a small fraction of monomers. Deletion of the 28–32 pentapeptide (Δ mutant) results in the formation of minor quantities of dimers (~49 kDa) and large quantities of the 24-kDa monomers. Both the WT protein and its Δ mutant efficiently bind a hydrophobic probe bis-ANS and are relatively rapidly hydrolyzed by chymotrypsin, whereas the R29A mutant weakly binds bis-ANS and resists chymotrypsinolysis. In contrast to HspB8 WT and its Δ mutant, which are well phosphorylated by cAMP-dependent and ERK1 protein kinases, the R29A mutant is poorly phosphorylated. R29A mutation affects the chaperone-like activity of HspB8 measured in vitro. It is concluded that the irreplaceable Arg residue located in the only highly conservative motif in the N-terminal domain of all sHsp proteins affects the oligomeric structure and key properties of HspB8.

Small Hsps as Therapeutic Targets of Cystic Fibrosis Transmembrane Conductance Regulator Protein.

Human small heat shock proteins are molecular chaperones that regulate fundamental cellular processes in normal and pathological cells. Here, we have reviewed the role played by HspB1, HspB4 and HspB5 in the context of Cystic Fibrosis (CF), a severe monogenic autosomal recessive disease linked to mutations in Cystic Fibrosis Transmembrane conductance Regulator protein (CFTR) some of which trigger its misfolding and rapid degradation, particularly the most frequent one, F508del-CFTR. While HspB1 and HspB4 favor the degradation of CFTR mutants, HspB5 and particularly one of its phosphorylated forms positively enhance the transport at the plasma membrane, stability and function of the CFTR mutant. Moreover, HspB5 molecules stimulate the cellular efficiency of currently used CF therapeutic molecules. Different strategies are suggested to modulate the level of expression or the activity of these small heat shock proteins in view of potential in vivo therapeutic approaches. We then conclude with other small heat shock proteins that should be tested or further studied to improve our knowledge of CFTR processing.

Cardio-Vascular Heat Shock Protein (cvHsp, HspB7), an Unusual Representative of Small Heat Shock Protein Family.

HspB7 is one of ten human small heat shock proteins. This protein is expressed only in insulin-dependent tissues (heart, skeletal muscle, and fat tissue), and expression of HspB7 is regulated by many different factors. Single nucleotide polymorphism is characteristic for the HspB7 gene and this polymorphism correlates with cardio-vascular diseases and obesity. HspB7 has an unusual N-terminal sequence, a conservative α-crystallin domain, and very short C-terminal domain lacking conservative IPV tripeptide involved in a small heat shock proteins oligomer formation. Nevertheless, in the isolated state HspB7 forms both small oligomers (probably dimers) and very large oligomers (aggregates). HspB7 is ineffective in suppression of amorphous aggregation of model proteins induced by heating or reduction of disulfide bonds, however it is very effective in prevention of aggregation of huntingtin fragments enriched with Gln residues. HspB7 can be an effective sensor of electrophilic agents. This protein interacts with the contractile and cytoskeleton proteins (filamin C, titin, and actin) and participates in protection of the contractile apparatus and cytoskeleton from different adverse conditions. HspB7 possesses tumor suppressive activity. Further investigations are required to understand molecular mechanisms of HspB7 participation in numerous biological processes.

Single-molecule fluorescence-based approach reveals novel mechanistic insights into human small heat shock protein chaperone function

Small heat shock proteins (sHsps) are a family of ubiquitous intracellular molecular chaperones; some sHsp family members are upregulated under stress conditions and play a vital role in protein homeostasis (proteostasis). It is commonly accepted that these chaperones work by trapping misfolded proteins to prevent their aggregation; however, fundamental questions regarding the molecular mechanism by which sHsps interact with misfolded proteins remain unanswered. The dynamic and polydisperse nature of sHsp oligomers has made studying them challenging using traditional biochemical approaches. Therefore, we have utilized a single-molecule fluorescence-based approach to observe the chaperone action of human alphaB-crystallin (αBc, HSPB5). Using this approach we have, for the first time, determined the stoichiometries of complexes formed between αBc and a model client protein, chloride intracellular channel 1. By examining the dispersity and stoichiometries of these complexes over time, and in response to different concentrations of αBc, we have uncovered unique and important insights into a two-step mechanism by which αBc interacts with misfolded client proteins to prevent their aggregation.

The Heterooligomerization of Human Small Heat Shock Proteins Is Controlled by Conserved Motif Located in the N-Terminal Domain.

Ubiquitously expressed human small heat shock proteins (sHsps) HspB1, HspB5, HspB6 and HspB8 contain a conserved motif (S/G)RLFD in their N-terminal domain. For each of them, we prepared mutants with a replacement of the conserved R by A (R/A mutants) and a complete deletion of the pentapeptide (Δ mutants) and analyzed their heterooligomerization with other wild-type (WT) human sHsps. We found that WT HspB1 and HspB5 formed heterooligomers with HspB6 only upon heating. In contrast, both HspB1 mutants interacted with WT HspB6 even at low temperature. HspB1/HspB6 heterooligomers revealed a broad size distribution with equimolar ratio suggestive of heterodimers as building blocks, while HspB5/HspB6 heterooligomers had an approximate 2:1 ratio. In contrast, R/A or Δ mutants of HspB6, when mixed with either HspB1 or HspB5, resulted in heterooligomers with a highly variable molar ratio and a decreased HspB6 incorporation. No heterooligomerization of HspB8 or its mutants with either HspB1 or HspB5 could be detected. Finally, R/A or Δ mutations had no effect on heterooligomerization of HspB1 and HspB5 as analyzed by ion exchange chromatography. We conclude that the conserved N-terminal motif plays an important role in heterooligomer formation, as especially pronounced in HspB6 lacking the C-terminal IXI motif.

Structural aspects of the human small heat shock proteins related to their functional activities

Small heat shock proteins function as chaperones by binding unfolding substrate proteins in an ATP-independent manner to keep them in a folding-competent state and to prevent irreversible aggregation. They play crucial roles in diseases that are characterized by protein aggregation, such as neurodegenerative and neuromuscular diseases, but are also involved in cataract, cancer, and congenital disorders. For this reason, these proteins are interesting therapeutic targets for finding molecules that could affect the chaperone activity or compensate specific mutations. This review will give an overview of the available knowledge on the structural complexity of human small heat shock proteins, which may aid in the search for such therapeutic molecules.

Hsp27 chaperones FUS phase separation under the modulation of stress-induced phosphorylation.

Protein phase separation drives the assembly of membraneless organelles, but little is known about how these membraneless organelles are maintained in a metastable liquid- or gel-like phase rather than proceeding to solid aggregation. Here, we find that human small heat-shock protein 27 (Hsp27), a canonical chaperone that localizes to stress granules (SGs), prevents FUS from undergoing liquid-liquid phase separation (LLPS) via weak interactions with the FUS low complexity (LC) domain. Remarkably, stress-induced phosphorylation of Hsp27 alters its activity, leading Hsp27 to partition with FUS LC to preserve the liquid phase against amyloid fibril formation. NMR spectroscopy demonstrates that Hsp27 uses distinct structural mechanisms for both functions. Our work reveals a fine-tuned regulation of Hsp27 for chaperoning FUS into either a polydispersed state or a LLPS state and suggests an essential role for Hsp27 in stabilizing the dynamic phase of stress granules.

Interplay of disordered and ordered regions of a human small heat shock protein yields an ensemble of 'quasi-ordered' states.

Small heat shock proteins (sHSPs) are nature's 'first responders' to cellular stress, interacting with affected proteins to prevent their aggregation. Little is known about sHSP structure beyond its structured α-crystallin domain (ACD), which is flanked by disordered regions. In the human sHSP HSPB1, the disordered N-terminal region (NTR) represents nearly 50% of the sequence. Here, we present a hybrid approach involving NMR, hydrogen-deuterium exchange mass spectrometry, and modeling to provide the first residue-level characterization of the NTR. The results support a model in which multiple grooves on the ACD interact with specific NTR regions, creating an ensemble of 'quasi-ordered' NTR states that can give rise to the known heterogeneity and plasticity of HSPB1. Phosphorylation-dependent interactions inform a mechanism by which HSPB1 is activated under stress conditions. Additionally, we examine the effects of disease-associated NTR mutations on HSPB1 structure and dynamics, leveraging our emerging structural insights.

The N terminus of the small heat shock protein HSPB7 drives its polyQ aggregation–suppressing activity

Heat shock protein family B (small) member 7 (HSPB7) is a unique, relatively unexplored member within the family of human small heat shock proteins (HSPBs). Unlike most HSPB family members, HSPB7 does not oligomerize and so far has not been shown to associate with any other member of the HSPB family. Intriguingly, it was found to be the most potent member within the HSPB family to prevent aggregation of proteins with expanded polyglutamine (polyQ) stretches. How HSPB7 suppresses polyQ aggregation has remained elusive so far. Here, using several experimental strategies, including in vitro aggregation assay, immunoblotting and fluorescence approaches, we show that the polyQ aggregation-inhibiting activity of HSPB7 is fully dependent on its flexible N-terminal domain (NTD). We observed that the NTD of HSPB7 is both required for association with and inhibition of polyQ aggregation. Remarkably, replacing the NTD of HSPB1, which itself cannot suppress polyQ aggregation, with the NTD of HSPB7 resulted in a hybrid protein that gained anti-polyQ aggregation activity. The hybrid NTDHSPB7-HSPB1 protein displayed a reduction in oligomer size and, unlike WT HSPB1, associated with polyQ. However, experiments with phospho-mimicking HSPB1 mutants revealed that de-oligomerization of HSPB1 alone does not suffice to gain polyQ aggregation-inhibiting activity. Together, our results reveal that the NTD of HSPB7 is both necessary and sufficient to bind to and suppress the aggregation of polyQ-containing proteins.

Chaperone-like activity of the N-terminal region of a human small heat shock protein and chaperone-functionalized nanoparticles.

Small heat shock proteins (sHsps) are molecular chaperones employed to interact with a diverse range of substrates as the first line of defense against cellular protein aggregation. The N-terminal region (NTR) is implicated in defining features of sHsps; notably in their ability to form dynamic and polydisperse oligomers, and chaperone activity. The physiological relevance of oligomerization and chemical-scale mode(s) of chaperone function remain undefined. We present novel chemical tools to investigate chaperone activity and substrate specificity of human HspB1 (B1NTR), through isolation of B1NTR and development of peptide-conjugated gold nanoparticles (AuNPs). We demonstrate that B1NTR exhibits chaperone capacity for some substrates, determined by anti-aggregation assays and size-exclusion chromatography. The importance of protein dynamics and multivalency on chaperone capacity was investigated using B1NTR-conjugated AuNPs, which exhibit concentration-dependent chaperone activity for some substrates. Our results implicate sHsp NTRs in chaperone activity, and demonstrate the therapeutic potential of sHsp-AuNPs in rescuing aberrant protein aggregation.