Human HspB1 Research Articles

Human HspB1, HspB3, HspB5 and HspB8: Shaping these disease factors during vertebrate evolution

Small heat shock proteins (sHSPs) emerged early in evolution and occur in all domains of life and nearly in all species, including humans. Mutations in four sHSPs (HspB1, HspB3, HspB5, HspB8) are associated with neuromuscular disorders. The aim of this study is to investigate the evolutionary forces shaping these sHSPs during vertebrate evolution. We performed comparative evolutionary analyses on a set of orthologous sHSP sequences, based on the ratio of non-synonymous: synonymous substitution rates for each codon. We found that these sHSPs had been historically exposed to different degrees of purifying selection, decreasing in this order: HspB8 > HspB1, HspB5 > HspB3. Within each sHSP, regions with different degrees of purifying selection can be discerned, resulting in characteristic selective pressure profiles. The conserved α-crystallin domains were exposed to the most stringent purifying selection compared to the flanking regions, supporting a 'dimorphic pattern' of evolution. Thus, during vertebrate evolution the different sequence partitions were exposed to different and measurable degrees of selective pressures. Among the disease-associated mutations, most are missense mutations primarily in HspB1 and to a lesser extent in the other sHSPs. Our data provide an explanation for this disparate incidence. Contrary to the expectation, most missense mutations cause dominant disease phenotypes. Theoretical considerations support a connection between the historic exposure of these sHSP genes to a high degree of purifying selection and the unusual prevalence of genetic dominance of the associated disease phenotypes. Our study puts the genetics of inheritable sHSP-borne diseases into the context of vertebrate evolution.

Disorder, quasi-order, order: a holistic view of small heat shock proteins

Small heat shock proteins (sHSPs) are cellular first responders to stress, with the ability to delay the onset of protein aggregation. Both the structures and mechanisms of sHSPs are enigmatic due in large part to the large polydisperse and dynamic oligomeric states they adopt. Previous studies using conventional “divide and conquer” approaches have provided important insights but have not cracked the code, as the whole is not the sum of the parts for sHSPs. We have taken a holistic approach to decipher the human sHSPs HSPB1 (Hsp27) and HSPB5 (alphaB-crystallin), using NMR, HDX/MS, and UV-crosslinking/MS, among other techniques.

Oligomeric Structural Transition of HspB1 from Chinese Hamster.

HspB1 is a mammalian sHsp that is ubiquitously expressed in almost all tissues and involved in regulating many vital functions. Although the recent crystal structure of human HspB1 showed that 24 monomers form the oligomeric complex of human HspB1 in a spherical configuration, the molecular architecture of HspB1 is still controversial. In this study, we examined the oligomeric structural change of CgHspB1 by sedimentation velocity analytical ultracentrifugation. At the low temperature of 4 °C, CgHspB1 exists as an 18-mer, probably a trimeric complex of hexamers. It is relatively unstable and partially dissociates into small oligomers, hexamers, and dodecamers. At elevated temperatures, the 24-mer was more stable than the 18-mer. The 24-mer is also in dynamic equilibrium with the dissociated oligomers in the hexameric unit. The hexamer further dissociates to dimers. The disulfide bond between conserved cysteine residues seems to be partly responsible for the stabilization of hexamers. The N-terminal domain is involved in the assembly of dimers and the interaction between hexamers. It is plausible that CgHspB1 expresses a chaperone function in the 24-mer structure.

The chaperone HSPB1 prepares protein aggregates for resolubilization by HSP70

In human cells under stress conditions, misfolded polypeptides can form potentially cytotoxic insoluble aggregates. To eliminate aggregates, the HSP70 chaperone machinery extracts and resolubilizes polypeptides for triage to refolding or degradation. Yeast and bacterial chaperones of the small heat-shock protein (sHSP) family can bind substrates at early stages of misfolding, during the aggregation process. The co-aggregated sHSPs then facilitate downstream disaggregation by HSP70. Because it is unknown whether a human sHSP has this activity, we investigated the disaggregation role of human HSPB1. HSPB1 co-aggregated with unfolded protein substrates, firefly luciferase and mammalian lactate dehydrogenase. The co-aggregates formed with HSPB1 were smaller and more regularly shaped than those formed in its absence. Importantly, co-aggregation promoted the efficient disaggregation and refolding of the substrates, led by HSP70. HSPB1 itself was also extracted during disaggregation, and its homo-oligomerization ability was not required. Therefore, we propose that a human sHSP is an integral part of the chaperone network for protein disaggregation.

Neuroinflammatory processes are augmented in mice overexpressing human heat-shock protein B1 following ethanol-induced brain injury

BackgroundHeat-shock protein B1 (HSPB1) is among the most well-known and versatile member of the evolutionarily conserved family of small heat-shock proteins. It has been implicated to serve a neuroprotective role against various neurological disorders via its modulatory activity on inflammation, yet its exact role in neuroinflammation is poorly understood. In order to shed light on the exact mechanism of inflammation modulation by HSPB1, we investigated the effect of HSPB1 on neuroinflammatory processes in an in vivo and in vitro model of acute brain injury.MethodsIn this study, we used a transgenic mouse strain overexpressing the human HSPB1 protein. In the in vivo experiments, 7-day-old transgenic and wild-type mice were treated with ethanol. Apoptotic cells were detected using TUNEL assay. The mRNA and protein levels of cytokines and glial cell markers were examined using RT-PCR and immunohistochemistry in the brain. We also established primary neuronal, astrocyte, and microglial cultures which were subjected to cytokine and ethanol treatments. TNFα and hHSPB1 levels were measured from the supernates by ELISA, and intracellular hHSPB1 expression was analyzed using fluorescent immunohistochemistry.ResultsFollowing ethanol treatment, the brains of hHSPB1-overexpressing mice showed a significantly higher mRNA level of pro-inflammatory cytokines (Tnf, Il1b), microglia (Cd68, Arg1), and astrocyte (Gfap) markers compared to wild-type brains. Microglial activation, and 1 week later, reactive astrogliosis was higher in certain brain areas of ethanol-treated transgenic mice compared to those of wild-types. Despite the remarkably high expression of pro-apoptotic Tnf, hHSPB1-overexpressing mice did not exhibit higher level of apoptosis. Our data suggest that intracellular hHSPB1, showing the highest level in primary astrocytes, was responsible for the inflammation-regulating effects. Microglia cells were the main source of TNFα in our model. Microglia isolated from hHSPB1-overexpressing mice showed a significantly higher release of TNFα compared to wild-type cells under inflammatory conditions.ConclusionsOur work provides novel in vivo evidence that hHSPB1 overexpression has a regulating effect on acute neuroinflammation by intensifying the expression of pro-inflammatory cytokines and enhancing glial cell activation, but not increasing neuronal apoptosis. These results suggest that hHSPB1 may play a complex role in the modulation of the ethanol-induced neuroinflammatory response.

Release of a disordered domain enhances HspB1 chaperone activity toward tau

Small heat shock proteins (sHSPs) are a class of ATP-independent molecular chaperones that play vital roles in maintaining protein solubility and preventing aberrant protein aggregation. They form highly dynamic, polydisperse oligomeric ensembles and contain long intrinsically disordered regions. Experimental challenges posed by these properties have greatly impeded our understanding of sHSP structure and mechanism of action. Here we characterize interactions between the human sHSP HspB1 (Hsp27) and microtubule-associated protein tau, which is implicated in multiple dementias, including Alzheimer's disease. We show that tau binds both to a well-known binding groove within the structured alpha-crystallin domain (ACD) and to sites within the enigmatic, disordered N-terminal region (NTR) of HspB1. However, only interactions involving the NTR lead to productive chaperone activity, whereas ACD binding is uncorrelated with chaperone function. The tau-binding groove in the ACD also binds short hydrophobic regions within HspB1 itself, and HspB1 mutations that disrupt these intrinsic ACD-NTR interactions greatly enhance chaperone activity toward tau. This leads to a mechanism in which the release of the disordered NTR from a binding groove on the ACD enhances chaperone activity toward tau. The study advances understanding of the mechanisms by which sHSPs achieve their chaperone activity against amyloid-forming clients and how cells defend against pathological tau aggregation. Furthermore, the resulting mechanistic model points to ways in which sHSP chaperone activity may be increased, either by native factors within the cell or by therapeutic intervention.

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.

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.

The Role of the Arginine in the Conserved N-Terminal Domain RLFDQxFG Motif of Human Small Heat Shock Proteins HspB1, HspB4, HspB5, HspB6, and HspB8.

Although the N-terminal domain of vertebrate small heat shock proteins (sHsp) is poorly conserved, it contains a core motif preserved in many members of the sHsp family. The role of this RLFDQxFG motif remains elusive. We analyzed the specific role of the first arginine residue of this conserved octet sequence in five human sHsps (HspB1, HspB4, HspB5, HspB6, and HspB8). Substitution of this arginine with an alanine induced changes in thermal stability and/or intrinsic fluorescence of the related HspB1 and HspB8, but yielded only modest changes in the same biophysical properties of HspB4, HspB5, and HspB6 which together belong to another clade of vertebrate sHsps. Removal of the positively charged Arg side chain resulted in destabilization of the large oligomers of HspB1 and formation of smaller size oligomers of HspB5. The mutation induced only minor changes in the structure of HspB4 and HspB6. In contrast, the mutation in HspB8 was accompanied by shifting the equilibrium from dimers towards the formation of larger oligomers. We conclude that the RLFDQxFG motif plays distinct roles in the structure of several sHsp orthologs. This role correlates with the evolutionary relationship of the respective sHsps, but ultimately, it reflects the sequence context of this motif.

Engineering of a Polydisperse Small Heat-Shock Protein Reveals Conserved Motifs of Oligomer Plasticity

Small heat-shock proteins (sHSPs) are molecular chaperones that bind partially and globally unfolded states of their client proteins. Previously, we discovered that the archaeal Hsp16.5, which forms ordered and symmetric 24-subunit oligomers, can be engineered to transition to an ordered and symmetric 48-subunit oligomer by insertion of a peptide from human HspB1 (Hsp27). Here, we uncovered the existence of an array of oligomeric states (30-38 subunits) that can be populated as a consequence of altering the sequence and length of the inserted peptide. Polydisperse Hsp16.5 oligomers displayed higher affinity to a model client protein consistent with a general mechanism for recognition and binding that involves increased access of the hydrophobic N-terminal region. Our findings, which integrate structural and functional analyses from evolutionarily distant sHSPs, support a model wherein the modular architecture of these proteins encodes motifs of oligomer polydispersity, dissociation, and expansion to achieve functional diversity and regulation.

The preferential heterodimerization of human small heat shock proteins HSPB1 and HSPB6 is dictated by the N-terminal domain

Small heat shock proteins are ATP-independent molecular chaperones. Their function is to bind partially unfolded proteins under stress conditions. In vivo, members of this chaperone family are known to preferentially assemble together forming large, polydisperse heterooligomers. The exact molecular mechanisms that drive specific heteroassociation are currently unknown. Here we study the oligomers formed between human HSPB1 and HSPB6. Using small-angle X-ray scattering we could characterize two distinct heterooligomeric species present in solution. By employing native mass spectrometry we show that such assemblies are formed purely from heterodimeric building blocks, in line with earlier cross-linking studies. Crucially, a detailed analysis of truncation variants reveals that the preferential association between these two sHSPs is solely mediated by their disordered N-terminal domains.

Characterization of New Transgenic Mouse Models for Two Charcot-Marie-Tooth-Causing HspB1 Mutations using the Rosa26 Locus.

Charcot-Marie-Tooth (CMT) and associated neuropathies, the most common inherited diseases of the peripheral nervous system, remain so far incurable. Three existing murine models of Charcot-Marie-Tooth type 2F (CMT2F) and/or distal hereditary motor neuropathy type IIb (dHMNIIb), caused by mutations in the small heat shock protein B1 gene (HSPB1/HSP27), partially recapitulate the hallmarks of peripheral neuropathy. Because these models overexpress the HSPB1 mutant proteins they differ from the patients' situation. To overcome the possible bias induced by overexpression, we generated and characterized a transgenic model in which the wild type or mutant HSPB1 protein was expressed at a moderate, more physiologically relevant level. We generated a new transgenic mouse model in which a human wild type (hHSPB1WT) or mutant (hHSPB1R127W; hHSPB1P182L) HSPB1 transgene was integrated in the mouse ROSA26 locus. The motor and sensory functions of the mice was assessed at 3, 6, 9, 12 and 18 month. However, the mice expressing the mutant hHSPB1 do not develop motor or sensory deficits and do not show any sign of axonal degeneration, even at late age. Quantitative PCR analyses reveal contrasting tissue-specific expression pattern for the endogenous mouse and exogenous human HSPB1 and show that the ratio of human HSPB1 to the endogenous mouse HspB1 is lower in the sciatic nerve and spinal cord compared to the brain. These results suggest that expressing the transgene at a physiological level using the ROSA26 locus may not be sufficient to model inherited peripheral neuropathies caused by mutation in HSPB1.

Characterization of Mutants of Human Small Heat Shock Protein HspB1 Carrying Replacements in the N-Terminal Domain and Associated with Hereditary Motor Neuron Diseases.

Physico-chemical properties of the mutations G34R, P39L and E41K in the N-terminal domain of human heat shock protein B1 (HspB1), which have been associated with hereditary motor neuron neuropathy, were analyzed. Heat-induced aggregation of all mutants started at lower temperatures than for the wild type protein. All mutations decreased susceptibility of the N- and C-terminal parts of HspB1 to chymotrypsinolysis. All mutants formed stable homooligomers with a slightly larger apparent molecular weight compared to the wild type protein. All mutations analyzed decreased or completely prevented phosphorylation-induced dissociation of HspB1 oligomers. When mixed with HspB6 and heated, all mutants yielded heterooligomers with apparent molecular weights close to ~400 kDa. Finally, the three HspB1 mutants possessed lower chaperone-like activity towards model substrates (lysozyme, malate dehydrogenase and insulin) compared to the wild type protein, conversely the environmental probe bis-ANS yielded higher fluorescence with the mutants than with the wild type protein. Thus, in vitro the analyzed N-terminal mutations increase stability of large HspB1 homooligomers, prevent their phosphorylation-dependent dissociation, modulate their interaction with HspB6 and decrease their chaperoning capacity, preventing normal functioning of HspB1.

Regulation of Actin Interacting Protein Drebrin by Mutations in HSPB1

Heat shock protein B1 (HspB1) is an ATP‐independent molecular chaperone with a conserved α‐crystalline domain in the C‐termini region. A number of mutations in HspB1 have been identified in families with Charcot Marie Tooth type IIF (CMT‐IIF) disease or with distal hereditary motor neuropathy II (dHMN II). Mutations outside the crystalline domain (P39L, G84L, P128S, P182L) associate with dHMN. Mutations S135F, R136W and R127W associate with a CMT‐IIF phenotype.Wild type (wt) HspB1 reduces RhoA activation by translational inhibition of PDZ‐RhoGEF, a RhoA specific GEF. Decreased expression of PDZ‐RhoGEF by HspB1 was accomplished by enhancing expression of specific microRNAs (miR20a and miR128) that inhibit the translation of PDZ‐RhoGEF and resulted in an increased neurite growth response. The regulatory effect of HspB1 on RhoA activity is blocked by mutation in S135F of HspB1 but not by R136W and R127W. This suggested either that these HspB1 mutations alter independent cellular mechanisms or that their effects may converge downstream of RhoA GTPase signaling to cause similar disease phenotype.We constructed lentiviral vectors carrying full length human HspB1 cDNA of wt or mutant S135F, R136W or R127W. Cortical neurons derived from SD rat pups E15 were transfected to expressed wt or mutant HspB1transgene to determine their effect on actin cytoskeleton.The studies showed S135F, R16W and R127W mutations cause HspB1 to partition more readily with the cytoskeletal fraction and enhance the binding of HspB1 to F‐actin leading to the loss of Drebrin, an F‐actin side‐binding protein, from the F‐actin domain. These findings highlight the diverse mechanisms by which HspB1 functions as an important regulatory protein of the actin cytoskeleton.

Attempt to optimize some properties of fluorescent chimeras of human small heat shock protein HspB1 by modifying linker length and nature.

Chimerical proteins consisting of enhanced yellow fluorescent protein (EYFP) connected by linkers of different length and nature to the N-terminal end of small heat shock protein HspB1 were obtained and characterized. To obtain fluorescent chimeras with properties similar to those of unmodified small heat shock protein, we used either 12-residue-long linkers of different nature (highly flexible Gly-Ser linker (L1), rigid α-helical linker (L2), or rigid Pro-Ala linker (L3)) or highly flexible Gly-Ser linker consisting of 12, 18, or 21 residues. The wild-type HspB1 formed large stable oligomers consisting of more than 20 subunits. Independent of the length or the nature of the linker, all the fluorescent chimeras formed small (5-9 subunits) oligomers tending to dissociate at low protein concentration. Chaperone-like activity of the wild-type HspB1 and its fluorescent chimeras were compared using lysozyme as a model protein substrate. Under the conditions used, all the fluorescent chimeras possessed higher chaperone-like activity than the wild-type HspB1. Chaperone-like activity of fluorescent chimeras with L1 and L3 linkers was less different from that of the wild-type HspB1 compare to the chaperone-like activity of chimeras with rigid L2 linker. Increase in the length of L1 linker from 12 up to 21 residues leads to decrease in the difference in the chaperone-like activity between the wild-type protein and its fluorescent chimeras. Since the N-terminal domain of small heat shock proteins participates in formation of large oligomers, any way of attachment of fluorescent protein to the N-terminal end of HspB1 leads to dramatic changes in its oligomeric structure. Long flexible linkers should be used to obtain fluorescent chimeras with chaperone-like properties similar to those of the wild-type HspB1.

Effect of disulfide crosslinking on thermal transitions and chaperone-like activity of human small heat shock protein HspB1.

Temperature-induced conformational changes of reduced and oxidized HspB1 crosslinked by disulfide bond between single Cys137 of neighboring monomers were analyzed by means of different techniques. Heating of reduced HspB1 was accompanied by irreversible changes of Trp fluorescence, whereas oxidized HspB1 underwent completely reversible changes of fluorescence. Increase of the temperature in the range of 20-70 °C was accompanied by self-association of both reduced and oxidized protein. Further increase of the temperature led to formation of heterogeneous mixture of large self-associated complexes of reduced HspB1 and to formation of smaller and less heterogeneous complexes of oxidized HspB1. Heat-induced changes of oligomeric state of reduced HspB1 were only partially reversible, whereas the corresponding changes of oligomeric state of oxidized HspB1 were almost completely reversible. Oxidation resulted in decrease of chaperone-like activity of HspB1. It is concluded that oxidative stress, inducing formation of disulfide bond, can affect stability and conformational mobility of human HspB1.

Human heat shock protein-specific cytotoxic T lymphocytes display potent antitumour immunity in multiple myeloma.

Tumour cell-derived heat shock proteins (HSPs) are used as vaccines for immunotherapy of cancer patients. However, it is proposed that the peptide chaperoned on HSPs, not HSPs themselves, elicited a potent immune response. Given that HSPs are highly expressed by most myeloma cells and vital to myeloma cell survival, we reasoned that HSPs themselves might be an ideal myeloma antigen. In the present study, we explored the feasibility of targeting HSPs themselves for treating multiple myeloma. We identified and chose HLA-A*0201-binding peptides from human HSPB1 (HSP27) and HSP90AA1 (HSP90), and confirmed their immunogenicity in HLA-A*0201 transgenic mice. Dendritic cells pulsed with HSPB1 and HSP90AA1 peptides were used to stimulate peripheral blood mononuclear cells from healthy volunteers and myeloma patients to generate HSP peptide-specific cytotoxic T lymphocytes (CTLs). HSP peptide-specific CTLs efficiently lysed HLA-A*0201(+) myeloma cells (established cell lines and primary plasma cells) but not HLA-A*0201(-) myeloma cells in vitro, indicating that myeloma cells naturally express HSP peptides in the context of major histocompatibility complex class I molecules. More importantly, HSP peptide-specific CTLs effectively reduced tumour burden in the xenograft mouse model of myeloma. Our study clearly demonstrated that HSPs might be novel tumour antigens for immunotherapy of myeloma.

Heat Shock Protein B1-Deficient Mice Display Impaired Wound Healing

There is large literature describing in vitro experiments on heat shock protein (hsp)B1 but understanding of its function in vivo is limited to studies in mice overexpressing human hspB1 protein. Experiments in cells have shown that hspB1 has chaperone activity, a cytoprotective role, regulates inflammatory gene expression, and drives cell proliferation. To investigate the function of the protein in vivo we generated hspB1-deficient mice. HspB1-deficient fibroblasts display increased expression of the pro-inflammatory cytokine, interleukin-6, compared to wild-type cells, but reduced proliferation. HspB1-deficient fibroblasts exhibit reduced entry into S phase and increased expression of cyclin-dependent kinase inhibitors p27kip1 and p21waf1. The expression of hspB1 protein and mRNA is also controlled by the cell cycle. To investigate the physiological function of hspB1 in regulating inflammation and cell proliferation we used an excisional cutaneous wound healing model. There was a significant impairment in the rate of healing of wounds in hspB1-deficient mice, characterised by reduced re-epithelialisation and collagen deposition but also increased inflammation. HspB1 deficiency augments neutrophil infiltration in wounds, driven by increased chemokine (C-X-C motif) ligand 1 expression. This appears to be a general mechanism as similar results were obtained in the air-pouch and peritonitis models of acute inflammation.

Structure and properties of G84R and L99M mutants of human small heat shock protein HspB1 correlating with motor neuropathy

Some properties of G84R and L99M mutants of HspB1 associated with peripheral distal neuropathies were investigated. Homooligomers formed by these mutants are larger than those of the wild type HspB1. Large oligomers of G84R and L99M mutants have compromised stability and tend to dissociate at low protein concentration. G84R and L99M mutations promote phosphorylation-dependent dissociation of HspB1 oligomers without affecting kinetics of HspB1 phosphorylation by MAPKAP2 kinase. Both mutants weakly interact with HspB6 forming small heterooligomers and being unable to form large heterooligomers characteristic for the wild type HspB1. G84R and L99M mutants possess lower chaperone-like activity than the wild type HspB1 with several model substrates. We suggest that G84R mutation affects mobility and accessibility of the N-terminal domain thus modifying interdimer contacts in HspB1 oligomers. The L99M mutation is located within the hydrophobic core of the α-crystallin domain close to the key R140 residue, and could affect the dimer stability.

Physico-chemical properties of R140G and K141Q mutants of human small heat shock protein HspB1 associated with hereditary peripheral neuropathies

Some physico-chemical properties of R140G and K141Q mutants of human small heat shock protein HspB1 associated with hereditary peripheral neuropathy were analyzed. Mutation K141Q did not affect intrinsic Trp fluorescence and interaction with hydrophobic probe bis-ANS, whereas mutation R140G decreased both intrinsic fluorescence and fluorescence of bis-ANS bound to HspB1. Both mutations decreased thermal stability of HspB1. Mutation R140G increased, whereas mutation K141Q decreased the rate of trypsinolysis of the central part (residues 5-188) of HspB1. Both the wild type HspB1 and its K141Q mutant formed large oligomers with apparent molecular weight ∼560kDa. The R140G mutant formed two types of oligomers, i.e. large oligomers tending to aggregate and small oligomers with apparent molecular weight ∼70kDa. The wild type HspB1 formed mixed homooligomers with R140G mutant with apparent molecular weight ∼610kDa. The R140G mutant was unable to form high molecular weight heterooligomers with HspB6, whereas the K141Q mutant formed two types of heterooligomers with HspB6. Invitro measured chaperone-like activity of the wild type HspB1 was comparable with that of K141Q mutant and was much higher than that of R140G mutant. Mutations of homologous hot-spot Arg (R140G of HspB1 and R120G of αB-crystallin) induced similar changes in the properties of two small heat shock proteins, whereas mutations of two neighboring residues (R140 and K141) induced different changes in the properties of HspB1.