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.

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.

Role of Small Heat Shock Proteins in the Remodeling of Actin Microfilaments.

Small heat shock proteins (sHsps) play an important role in the maintenance of proteome stability and, particularly, in stabilization of the cytoskeleton and cell contractile apparatus. Cell exposure to different types of stress is accompanied by the translocation of sHsps onto actin filaments; therefore, it is commonly believed that the sHsps are true actin-binding proteins. Investigations of last years have shown that this assumption is incorrect. Stress-induced translocation of sHsp to actin filaments is not the result of direct interaction of these proteins with intact actin, but results from the chaperone-like activity of sHsps and their interaction with various actin-binding proteins. HspB1 and HspB5 interact with giant elastic proteins titin and filamin thus providing an integrity of the contractile apparatus and its proper localization in the cell. HspB6 binds to the universal adapter protein 14-3-3 and only indirectly affects the structure of actin filament. HspB7 interacts with filamin C and controls actin filament assembly. HspB8 forms tight complex with the universal regulatory and adapter protein Bag3 and participates in the chaperone-assisted selective autophagy (CASA) of actin-binding proteins (e.g., filamin), as well as in the actin-depending processes taking place in mitoses. Hence, the mechanisms of sHsp participation in the maintenance of the contractile apparatus and cytoskeleton are much more complicated and diverse than it has been postulated earlier and are not limited to direct interactions of sHsps with actin. The old hypothesis on the direct binding of sHsps to intact actin should be revised and further detailed investigation on the sHsp interaction with minor proteins participating in the formation and remodeling of actin filaments is required.

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.

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 ß7 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.

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.

Effect of cataract-associated mutations in the N-terminal domain of αB-crystallin (HspB5).

Physico-chemical properties of three cataract-associated missense mutants of αB-crystallin (HspB5) (R11H, P20S, R56W) were analyzed. The oligomers formed by the R11H mutant were smaller, whereas the oligomers of the P20S and R56W mutants were larger than those of the wild-type protein. The P20S mutant possessed lower thermal stability than the wild-type HspB5 or two other HspB5 mutants. All HspB5 mutants were able to form heterooligomeric complexes with αA-crystallin (HspB4), a genuine component of eye lens. However, the P20S and R56W mutants were less effective in the formation of these complexes and properties of heterooligomeric complexes formed by these mutants and HspB4 and analyzed by ion-exchange chromatography were different from those formed by the wild-type HspB5 and HspB4. All HspB5 variants also heterooligomerized with another partner protein, HspB6. Specifically for the P20S mutant forming two distinct sizes of homooligomers, only the smaller homooligomer population was able to interact with HspB6. P20S and R56W mutants possessed lower chaperone-like activity than the wild-type HspB5 when UV-irradiated ßL-crystallin was used as a model substrate. Importantly, all three mutations are localized in three earlier postulated short α-helical regions present in the N-terminal domain of αB-crystallin. These observations suggest an important structural and functional role of these regions. Correspondingly, therein localized mutations ultimately result in clinically relevant cataracts.

Mutations in HspB1 and hereditary neuropathies.

Charcot-Marie-Tooth (CMT) disease is major hereditary neuropathy. CMT has been linked to mutations in a range of proteins, including the small heat shock protein HspB1. Here we review the properties of several HspB1 mutants associated with CMT. In vitro, mutations in the N-terminal domain lead to a formation of larger HspB1 oligomers when compared with the wild-type (WT) protein. These mutants are resistant to phosphorylation-induced dissociation and reveal lower chaperone-like activity than the WT on a range of model substrates. Mutations in the α-crystallin domain lead to the formation of yet larger HspB1 oligomers tending to dissociate at low protein concentration and having variable chaperone-like activity. Mutations in the conservative IPV motif within the C-terminal domain induce the formation of very large oligomers with low chaperone-like activity. Most mutants interact with a partner small heat shock protein, HspB6, in a manner different from that of the WT protein. The link between the altered physico-chemical properties and the pathological CMT phenotype is a subject of discussion. Certain HspB1 mutations appear to have an effect on cytoskeletal elements such as intermediate filaments and/or microtubules, and by this means damage the axonal transport. In addition, mutations of HspB1 can affect the metabolism in astroglia and indirectly modulate the viability of motor neurons. While the mechanisms of pathological mutations in HspB1 are likely to vary greatly across different mutations, further in vitro and in vivo studies are required for a better understanding of the CMT disease at molecular level.

Characterization of human small heat shock protein HSPB1 α-crystallin domain localized mutants associated with hereditary motor neuron diseases.

Congenital mutations in human small heat shock protein HSPB1 (HSP27) have been linked to Charcot-Marie-Tooth disease, a commonly occurring peripheral neuropathy. Understanding the molecular mechanism of such mutations is indispensable towards developing future therapies for this currently incurable disorder. Here we describe the physico-chemical properties of the autosomal dominant HSPB1 mutants R127W, S135F and R136W. Despite having a nominal effect on thermal stability, the three mutations induce dramatic changes to quaternary structure. At high concentrations or under crowding conditions, the mutants form assemblies that are approximately two times larger than those formed by the wild-type protein. At low concentrations, the mutants have a higher propensity to dissociate into small oligomers, while the dissociation of R127W and R135F mutants is enhanced by MAPKAP kinase-2 mediated phosphorylation. Specific differences are observed in the ability to form hetero-oligomers with the homologue HSPB6 (HSP20). For wild-type HSPB1 this only occurs at or above physiological temperature, whereas the R127W and S135F mutants form hetero-oligomers with HSPB6 at 4 °C, and the R136W mutant fails to form hetero-oligomers. Combined, the results suggest that the disease-related mutations of HSPB1 modify its self-assembly and interaction with partner proteins thus affecting normal functioning of HSPB1 in the cell.

Effect of methylglyoxal modification on the structure and properties of human small heat shock protein HspB6 (Hsp20).

Human small heat shock protein HspB6 (Hsp20) was modified by metabolic α-dicarbonyl compound methylglyoxal (MGO). At low MGO/HspB6 molar ratio, Arg13, Arg14, Arg27, and Arg102 were the primary sites of MGO modification. At high MGO/HspB6 ratio, practically, all Arg and Lys residues of HspB6 were modified. Both mild and extensive MGO modification decreased susceptibility of HspB6 to trypsinolysis and prevented its heat-induced aggregation. Modification by MGO was accompanied by formation of small quantities of chemically crosslinked dimers and did not dramatically affect quaternary structure of HspB6. Mild modification by MGO did not affect whereas extensive modification decreased interaction of HspB6 with HspB1. Phosphorylation of HspB6 by cyclic adenosine monophosphate (cAMP)-dependent protein kinase was inhibited after mild modification and completely prevented after extensive modification by MGO. Chaperone-like activity of HspB6 measured with subfragment 1 of skeletal myosin was enhanced after MGO modifications. It is concluded that Arg residues located in the N-terminal domain of HspB6 are easily accessible to MGO modification and that even mild modification by MGO affects susceptibility to trypsinolysis, phosphorylation by cAMP-dependent protein kinase, and chaperone-like activity of HspB6.

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.