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.

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.

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.

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.

Physico-chemical properties of two point mutants of small heat shock protein HspB6 (Hsp20) with abrogated cardioprotection.

Physico-chemical properties of HspB6 S10F and P20L mutants with abrogated cardioprotective activity and associated with different forms of cardiomyopathy were analyzed. Under normal conditions both the wild-type HspB6 and its mutants formed small size oligomers (dimers) with apparent molecular weight of 50-60 kDa. Under crowding conditions (0.5 M trimethylamine N-oxide, TMAO) the wild-type HspB6 remained predominantly dimeric or formed small molecular weight complexes, whereas both mutants tended to form high molecular weight complexes. Catalytic subunit of cAMP-dependent protein kinase phosphorylated the wild-type HspB6 and its S10F mutant with comparable rate. The rate of P20L mutant phosphorylation was higher than that of the wild-type HspB6. S10F and P20L mutations did not affect interaction of phosphorylated HspB6 with universal adapter proteins 14-3-3. The wild-type HspB6 was resistant to heat-induced denaturation and aggregation, whereas both its mutants were denatured and started to aggregate at temperature much lower than its wild-type counterpart. Titration with fluorescent probe bis-ANS was accompanied by larger increase of fluorescence in the case of both mutants than in the case of the wild-type HspB6. Both mutants possessed higher chaperone-like activity than the wild-type protein. It is concluded that both S10F and P20L mutations are accompanied by increase of hydrophobicity of the very N-terminal region of HspB6 leading to increased aggregation at elevated temperature, formation of large complexes under crowding conditions and increased chaperone-like activity measured in vitro. Increased hydrophobicity and self-association can affect substrate specificity and interaction with certain target proteins thus leading to decrease or complete abrogation of cardioprotective activity.

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.