Nikolai B. Gusev

Structure and properties of small heat shock proteins (sHsp) and their interaction with cytoskeleton proteins.

The modern classification of small heat shock proteins (sHsp) is presented and peculiarities of their primary structure and the mechanism of formation of oligomeric complexes are described. Data on phosphorylation of sHsp by different protein kinases are presented and the effect of phosphorylation on oligomeric state and chaperone activity of sHsp is discussed. Intracellular location of sHsp under normal and stress conditions is described and it is emphasized that under certain condition sHsp interact with different elements of cytoskeleton. The literature concerning the effect of sHsp on polymerization of actin in vitro is analyzed. An attempt is made to compare effects of sHsp on polymerization of actin in vitro with the results obtained on living cells under normal conditions and after heat shock or hormone action. The literature concerning possible effects of sHsp on cell motility is also analyzed.

Three-dimensional structure of α-crystallin domain dimers of human small heat shock proteins HSPB1 and HSPB6

Small heat shock proteins (sHSPs) are a family of evolutionary conserved ATP-independent chaperones. These proteins share a common architecture defined by a signature α-crystallin domain (ACD) flanked by highly variable N- and C-terminal extensions. The ACD, which has an immunoglobulin-like fold, plays an important role in sHSP assembly. This domain mediates dimer formation of individual protomers, which then may assemble into larger oligomers. In vertebrate sHSPs, the dimer interface is formed by the symmetrical antiparallel pairing of two β-strands (β7), generating an extended β-sheet on one face of the ACD dimer. Recent structural studies of isolated ACDs from a number of vertebrate sHSPs suggest a variability in the register of the β7/β7 strand interface, which may, in part, give rise to the polydispersity often associated with the full-length proteins. To further analyze the structure of ACD dimers, we have employed a combination of X-ray crystallography and solution small-angle X-ray scattering (SAXS) to study the ACD-containing fragments of human HSPB1 (HSP27) and HSPB6 (HSP20). Unexpectedly, the obtained crystal structure of the HSPB1 fragment does not reveal the typical β7/β7 dimers but, rather, hexamers formed by an asymmetric contact between the β4 and the β7 strands from adjacent ACDs. Nevertheless, in solution, both ACDs form stable dimers via the symmetric antiparallel interaction of β7 strands. Using SAXS, we show that it is possible to discriminate between different putative registers of the β7/β7 interface, with the results indicating that, under physiological conditions, there is only a single register of the strands for both proteins.

Small heat shock protein Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin

Previously, we have shown that the small heat shock protein with apparent molecular mass 27 kDa (Hsp27) does not affect the thermal unfolding of F‐actin, but effectively prevents aggregation of thermally denatured F‐actin [Pivovarova AV, Mikhailova VV, Chernik IS, Chebotareva NA, Levitsky DI & Gusev NB (2005) Biochem Biophys Res Commun331, 1548–1553], and supposed that Hsp27 prevents heat‐induced aggregation of F‐actin by forming soluble complexes with denatured actin. In the present work, we applied dynamic light scattering, analytical ultracentrifugation and size exclusion chromatography to examine the properties of complexes formed by denatured actin with a recombinant human Hsp27 mutant (Hsp27–3D) mimicking the naturally occurring phosphorylation of this protein at Ser15, Ser78, and Ser82. Our results show that formation of these complexes occurs upon heating and accompanies the F‐actin thermal denaturation. All the methods show that the size of actin–Hsp27‐3D complexes decreases with increasing Hsp27‐3D concentration in the incubation mixture and that saturation occurs at approximately equimolar concentrations of Hsp27‐3D and actin. Under these conditions, the complexes exhibit a hydrodynamic radius of ∼ 16 nm, a sedimentation coefficient of 17–20 S, and a molecular mass of about 2 MDa. It is supposed that Hsp27‐3D binds to denatured actin monomers or short oligomers dissociated from actin filaments upon heating and protects them from aggregation by forming relatively small and highly soluble complexes. This mechanism might explain how small heat shock proteins prevent aggregation of denatured actin and by this means protect the cytoskeleton and the whole cell from damage caused by accumulation of large insoluble aggregates under heat shock conditions.

Heterooligomeric complexes of human small heat shock proteins

Oligomeric association of human small heat shock proteins HspB1, HspB5, HspB6 and HspB8 was analyzed by means of size-exclusion chromatography, analytical ultracentrifugation and chemical cross-linking. Wild-type HspB1 and Cys mutants of HspB5, HspB6 and HspB8 containing a single Cys residue in position homologous to that of Cys137 of human HspB1 were able to generate heterodimers cross-linked by disulfide bond. Cross-linked heterodimers between HspB1/HspB5, HspB1/HspB6 and HspB5/HspB6 were easily produced upon mixing, whereas formation of any heterodimers with participation of HspB8 was significantly less efficient. The size of heterooligomers formed by HspB1/HspB6 and HspB5/HspB6 was different from the size of the corresponding homooligomers. Disulfide cross-linked homodimers of small heat shock proteins were unable to participate in heterooligomer formation. Thus, monomers can be involved in subunit exchange leading to heterooligomer formation and restriction of flexibility induced by disulfide cross-linking prevents subunit exchange.

Heterooligomeric complexes formed by human small heat shock proteins HspB1 (Hsp27) and HspB6 (Hsp20).

Formation of heterooligomeric complexes of human small heat shock proteins (sHsp) HspB6 (Hsp20) and HspB1 (Hsp27) was analyzed by means of native gel electrophoresis, analytical ultracentrifugation, chemical cross-linking and size-exclusion chromatography. HspB6 and HspB1 form at least two different complexes with apparent molecular masses 100-150 and 250-300 kDa, and formation of heterooligomeric complexes is temperature dependent. These complexes are highly mobile, easily exchange their subunits and are interconvertible. The stoichiometry of HspB1 and HspB6 in both complexes is close to 1/1 and smaller complexes are predominantly formed at low, whereas larger complexes are predominantly formed at high protein concentration. Formation of heterooligomeric complexes does not affect the chaperone-like activity of HspB1 and HspB6 if insulin or skeletal muscle F-actin was used as model protein substrates. After formation of heterooligomeric complexes the wild type HspB1 inhibits the rate of phosphorylation of HspB6 by cAMP-dependent protein kinase. The 3D mutant mimicking phosphorylation of HspB1 also forms heterooligomeric complexes with HspB6, but is ineffective in inhibition of HspB6 phosphorylation. Inside of heterooligomeric complexes HspB6 inhibits phosphorylation of HspB1 by MAPKAP2 kinase. Thus, in heterooligomeric complexes HspB6 and HspB1 mutually affect the structure of each other and formation of heterooligomeric complexes might influence diverse processes depending on small heat shock proteins.

Small heat shock protein Hsp20 (HspB6) as a partner of 14-3-3γ

Interaction of human 14-3-3γ with the small heat shock protein Hsp20 was analyzed by means of size-exclusion chromatography and chemical crosslinking. Unphosphorylated Hsp20 and its mutant S16D mimicking phosphorylation by cAMP-dependent protein kinase did not interact with 14-3-3. Phosphorylated Hsp20 formed a tight complex with 14-3-3 in which dimer of 14-3-3 was bound to dimer of Hsp20. 14-3-3 did not affect the chaperone activity of unphosphorylated Hsp20 but increased the chaperone activity of phosphorylated Hsp20 if insulin was used as a model substrate. Estimation of the effect of 14-3-3 on the chaperone activity of Hsp20 with other model substrates was complicated by the fact that under in vitro conditions isolated 14-3-3 possessed its own high chaperone activity. Taken into account high content of Hsp20 in different muscles it is supposed that upon phosphorylation Hsp20 might effectively compete with multiple protein targets of 14-3-3 and by this means indirectly affect many intracellular processes.

14-3-3 Proteins and regulation of cytoskeleton

The proteins of the 14-3-3 family are universal adapters participating in multiple processes running in the cell. We describe the structure, isoform composition, and distribution of 14-3-3 proteins in different tissues. Different elements of 14-3-3 structure important for dimer formation and recognition of protein targets are analyzed in detail. Special attention is paid to analysis of posttranslational modifications playing important roles in regulation of 14-3-3 function. The data of the literature concerning participation of 14-3-3 in regulation of intercellular contacts and different elements of cytoskeleton formed by microfilaments are analyzed. We also describe participation of 14-3-3 in regulation of small G-proteins and protein kinases important for proper functioning of cytoskeleton. The data on the interaction of 14-3-3 with different components of microtubules are presented, and the probable role of 14-3-3 in developing of certain neurodegenerative diseases is discussed. The data of the literature concerning the role of 14-3-3 in formation and normal functioning of intermediate filaments are also reviewed. It is concluded that due to its adapter properties 14-3-3 plays an important role in cytoskeleton regulation. The cytoskeletal proteins that are abundant in the cell might compete with the other protein targets of 14-3-3 and therefore can indirectly regulate many intracellular processes that are dependent on 14-3-3.

Structure, properties, and functions of the human small heat-shock protein HSP22 (HspB8, H11, E2IG1): a critical review.

The recently described human HSP22 belongs to the superfamily of small heat‐shock proteins containing a conservative α‐crystallin domain. HSP22 seems to be involved in regulation of cell proliferation, cardiac hypertrophy, apoptosis, and carcinogenesis, and expression of point mutants of HSP22 correlates with development of different neuromuscular diseases. Therefore, an investigation of the structure and properties of HSP22 is desirable for understanding its multiple functions. HSP22 seems to belong to the group of so‐called intrinsically disordered proteins and possesses a highly flexible structure. HSP22 tends to form small‐molecular‐mass oligomers and interacts with biological membranes and many different proteins, among them glycolytic enzymes and different protein kinases. HSP22 possesses chaperonelike activity and prevents aggregation of denatured proteins both in vitro and in vivo. Depending on the cell type and its expression, HSP22 might have either pro‐ or anti‐apoptotic effects. Chaperonelike activity seems to be important for antiapoptotic effects, whereas interaction with and regulation of certain protein kinases might be important for the proapoptotic effects of HSP22. Expression of K141N or K141E mutants of HSP22 correlates with development of distal hereditary motor neuropathy and/or Charcot‐Marie‐Tooth disease. These mutations destabilize the structure of HSP22, affect its interaction with other small heat‐shock proteins, and decrease its chaperonelike activity. HSP22 decreases or prevents aggregation of Huntingtin fragments and amyloid‐β peptide 1–40 of the Dutch type. Thus, HSP22 seems to play an important role in the nervous system, and further investigations are needed to understand the molecular mechanisms of its functioning.

The role of intrinsically disordered regions in the structure and functioning of small heat shock proteins

Small heat shock proteins (sHsp) form a large ubiquitous family of proteins expressed in all phyla of living organisms. The members of this family have low molecular masses (13-43 kDa) and contain a conservative α-crystallin domain. This domain (about 90 residues) consists of several β-strands forming two β-sheets packed in immunoglobulinlike manner. The α-crystallin domain plays an important role in formation of stable sHsp dimers, which are the building blocks of the large sHsp oligomers. A large N-terminal domain and a short C-terminal extension flank the α-crystallin domain. Both the N-terminal domain and the C-terminal extension are flexible, susceptible to proteolysis, prone to posttranslational modifications, and are predominantly intrinsically disordered. Differently oriented N-terminal domains interact with each other, with the core α-crystallin domain of the same or neighboring dimers and play important role in formation of large sHsp oligomers. Phosphorylation of certain sites in the N-terminal domain affects the sHsp quaternary structure, the sHsp interaction with target proteins and the sHsp chaperone-like activity. The C-terminal extension often carrying the conservative tripeptide (I/V/L)-X-(I/V/L) is capable of binding to a hydrophobic groove on the surface of the core α-crystallin domain of neighboring dimer, thus affecting the plasticity and the overall structure of sHsp oligomers. The Cterminal extension interacts with target proteins and affects their interaction with the α-crystallin domain increasing solubility of the complexes formed by sHsp and their targets. Thus, disordered N- and C-terminal sequences play important role in the structure, regulation and functioning of sHsp.

Interaction of smooth muscle caldesmon with S‐100 protein

The interaction of caldesmon with certain Ca‐binding proteins was investigated by means of electrophoresis under non‐denaturating conditions. In the presence of Ca2+ calmodulin, troponin C and S‐100 protein form a complex with caldesmon. No complex formation takes place in the absence of Ca2+. Lactalbumin and pike parvalbumin (pI4.2) do not interact with caldesmon independently of Ca‐concentration. Both S‐100 protein and calmodulin effectively inhibit phosphorylation of caldesmon by Ca‐phospholipid‐dependent protein kinase. At low ionic strength S‐100 protein reverses the inhibitory action of caldesmon on the skeletal muscle acto‐heavy meromyosin ATPase more effectively than calmodulin. It is supposed that in certain tissues and cell compartments the proteins belonging to the S‐100 family are able to substitute for calmodulin in the caldesmondependent regulation of actin and myosin interaction.