Commentary on paper: Small heat shock proteins and the cytoskeleton: an essential interplay for cell integrity? (Wettstein et al.).

The recently published paper by Wettstein et al. (2012) reviews the data of literature dealing with participation of small heat shock proteins (sHsp) in cytoskeleton regulation. Analyzing the effect of sHsp on microfilaments, the authors come to conclusion that depending on phosphorylation HspB1 can function as barbed-end-capping protein and can prevent aggregation of F-actin under stress conditions. The modern data do not confirm all these suggestions. We propose that stabilization effect of HspB1 on microfilaments is due to HspB1 interaction with partially unfolded actin or with genuine actin-binding proteins. In addition, HspB1 can exert its stabilizing effect on F-actin by modulating other elements of the cytoskeleton (intermediate filaments and microtubules) or by controlling homeostasis (for instance, redox state). Without being genuine actin-binding proteins, HspB1 and HspB6 predominantly protect microfilaments via an indirect mechanism that is yet to be characterized.

Cofilin weakly interacts with 14-3-3 and therefore can only indirectly participate in regulation of cell motility by small heat shock protein HspB6 (Hsp20).

It has been previously reported that phosphorylated cofilin interacted with 14-3-3ζ protein to generate a sub-micromolar K(d) binary complex. Here we challenge this hypothesis by analyzing the direct association of recombinant cofilin with 14-3-3ζ using different in vitro biochemical methods. Phosphorylated cofilin at high concentration binds to 14-3-3 immobilized on nitrocellulose, however no complex formation was detected by means of native gel electrophoresis or chemical crosslinking. Intact dimeric or mutant monomeric 14-3-3 was unable to form stable complexes with phosphorylated or unphosphorylated cofilin detected by size-exclusion chromatography. In co-sedimentation assay 14-3-3 did not affect interaction of cofilin with F-actin. The data of native gel electrophoresis indicate that 14-3-3 did not affect interaction of cofilin with G-actin. Thus, cofilin only weakly interacts with 14-3-3 and therefore cannot directly compete with phosphorylated small heat shock protein HspB6 for its binding to 14-3-3. It is hypothesized that phosphorylated HspB6 might affect interaction of 14-3-3 with protein phosphatases (and/or protein kinases) involved in dephosphorylation (or phosphorylation) of cofilin and by this means regulate cofilin-dependent reorganization of cytoskeleton.

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.

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 ß-strands forming two ß-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.

Large potentials of small heat shock proteins.

Modern classification of the family of human small heat shock proteins (the so-called HSPB) is presented, and the structure and properties of three members of this family are analyzed in detail. Ubiquitously expressed HSPB1 (HSP27) is involved in the control of protein folding and, when mutated, plays a significant role in the development of certain neurodegenerative disorders. HSPB1 directly or indirectly participates in the regulation of apoptosis, protects the cell against oxidative stress, and is involved in the regulation of the cytoskeleton. HSPB6 (HSP20) also possesses chaperone-like activity, is involved in regulation of smooth muscle contraction, has pronounced cardioprotective activity, and seems to participate in insulin-dependent regulation of muscle metabolism. HSPB8 (HSP22) prevents accumulation of aggregated proteins in the cell and participates in the regulation of proteolysis of unfolded proteins. HSPB8 also seems to be directly or indirectly involved in regulation of apoptosis and carcinogenesis, contributes to cardiac cell hypertrophy and survival and, when mutated, might be involved in development of neurodegenerative diseases. All small heat shock proteins play important "housekeeping" roles and regulate many vital processes; therefore, they are considered as attractive therapeutic targets.

Properties of the monomeric form of human 14-3-3ζ protein and its interaction with tau and HspB6.

Dimers formed by seven isoforms of the human 14-3-3 protein participate in multiple cellular processes. The dimeric form has been extensively characterized; however, little is known about the structure and properties of the monomeric form of 14-3-3. The monomeric form is involved in the assembly of homo- and heterodimers, which could partially dissociate back into monomers in response to phosphorylation at Ser58. To obtain monomeric forms of human 14-3-3ζ, we produced four protein constructs with different combinations of mutated (M) or wild-type (W) segments E(5), (12)LAE(14), and (82)YREKIE(87). Under a wide range of expression conditions in Escherichia coli, the MMM and WMM mutants were insoluble, whereas WMW and MMW mutants were soluble, highly expressed, and purified to homogeneity. WMW and MMW mutants remained monomeric over a wide range of concentrations while retaining the α-helical structure characteristic of wild-type 14-3-3. However, WMW and MMW mutants were highly susceptible to proteolysis and had much lower thermal stabilities than the wild-type protein. Using WMW and MMW mutants, we show that the monomeric form interacts with the tau protein and with the HspB6 protein, in both cases forming complexes with a 1:1 stoichiometry, in contrast to the 2:1 and/or 2:2 complexes formed by wild-type 14-3-3. Significantly, this interaction requires phosphorylation of tau protein and HspB6. Because of minimal changes in structure, MMW and especially WMW mutant proteins are promising candidates for analyzing the effect of monomerization on the physiologically important properties of 14-3-3ζ.

Phosphorylation of human small heat shock protein HspB8 (Hsp22) by ERK1 protein kinase.

A number of phosphomimicking mutants (replacement of Ser/Thr residues by Asp) of human small heat shock protein HspB8 were obtained and phosphorylation of the wild type HspB8 and its mutants by ERK1 kinase was analyzed in vitro. Mutation S159D does not affect phosphorylation, whereas mutations S24D and S27D equally moderately inhibited and mutation T87D strongly inhibited phosphorylation of HspB8. The double mutations S24D/T87D and S27D/T87D induced very strong inhibitory effect and the triple mutations S24D/S27D/T87D completely prevented phosphorylation catalyzed by ERK1. Thus, Ser24 and Thr87, found to be phosphorylated in vivo, are among the sites phosphorylated by ERK1 in HspB8 in vitro. Mutations S24D and T87D affect intrinsic tryptophan fluorescence and susceptibility to chymotrypsinolysis of HspB8. Phosphomimicking mutations and phosphorylation promote concentration-dependent association of HspB8 subunits. Mutations S24D and S27D decrease, whereas mutation T87D increases the chaperone-like activity of HspB8. It is concluded that phosphorylation catalyzed by ERK1 might affect the structure and chaperone-like activity of HspB8 and therefore can be important for regulation of interaction of HspB8 with different target proteins.

Phosphomimicking mutations of human 14-3-3ζ affect its interaction with tau protein and small heat shock protein HspB6.

Effect of phosphomimicking mutations of 14-3-3ζ on its interaction with phosphorylated shortest isoform of human tau protein and phosphorylated human small heat shock protein HspB6 (Hsp20) was analyzed. Chemical crosslinking and native gel electrophoresis indicate that mutations S184E and T232E weakly affect interaction of 14-3-3 with phosphorylated tau protein, whereas mutations S58E and S58E/S184E/T232E significantly impair interaction of 14-3-3 and tau. Size-exclusion chromatography, chemical crosslinking and immunoprecipitation revealed that phosphomimicking mutations S58E and S58E/S184E/T232E strongly decrease, mutation T232E weakly affects and mutation S184E improves interaction of 14-3-3 with phosphorylated HspB6. Thus, mutation mimicking phosphorylation of Ser58 dramatically decreases interaction of 14-3-3 with two target proteins and this effect might be due to destabilization of the dimeric structure of 14-3-3 and/or conformational changes of the target-binding site. The mutation mimicking phosphorylation of Thr232 weakly affects interaction of 14-3-3 with both proteins. The mutation mimicking phosphorylation of Ser184 does not markedly affect interaction with tau protein and improves the interaction of 14-3-3 with HspB6. Thus, effect of 14-3-3 phosphorylation depends on the nature of the target protein and therefore, phosphorylation of 14-3-3 might affect its target specificity.

Versatility of the small heat shock protein HSPB6 (Hsp20).

The recently published review by Dreiza et al. (Cell Stress and Chaperones DOI 10.1007/s12192-0090127-8 ) dealing with the functional role of HSPB6 in muscle regulation is critically analyzed. Published data indicate that the chaperone-like activity of HSPB6 is comparable with that of HSPB5 and that phosphorylation of HSPB6 does not affect its oligomeric structure. Different hypotheses concerning the molecular mechanisms of HSPB6 action on smooth muscle contraction and on the reorganization of the cytoskeleton are compared, and it is concluded that although HSPB6 is not a genuine actin-binding protein, it can affect the actin cytoskeleton indirectly. Phosphorylated HSPB6 interacts with 14-3-3 and thereby displaces other binding partners of 14-3-3; among them, certain phosphatases, protein kinases, and various actin-binding proteins, which can participate in the reorganization of the actin cytoskeleton. In addition, HSPB6 seems to regulate the activity of certain protein kinases. All of these processes are dependent on HSPB6 phosphorylation which in turn might be regulated by the formation of heterooligomeric complexes of HSPB6 with other small heat shock proteins.

The pivotal role of the beta 7 strand in the intersubunit contacts of different human small heat shock proteins.

Human alpha B-crystallin and small heat shock proteins HspB6 and HspB8 were mutated so that all endogenous Cys residues were replaced by Ser and the single Cys residue was inserted in a position homologous to that of Cys137 of human HspB1, i.e. in a position presumably located in the central part of beta 7 strand of the alpha-crystallin domain. The secondary, tertiary, and quaternary structures of thus obtained Cys-mutants as well as their chaperone-like activity were similar to those of their wild-type counterparts. Mild oxidation of Cys-mutants leads to formation of disulfide bond crosslinking neighboring monomers thus indicating participation of the beta 7 strand in intersubunit interaction. Oxidation weakly affects the secondary and tertiary structure, does not affect the quaternary structure of alpha B-crystallin and HspB6, and shifts equilibrium between monomer and dimer of HspB8 towards dimer formation. It is concluded that the beta 7 strand participates in the intersubunit interaction of four human small heat shock proteins (alpha B-crystallin, HspB1, HspB6, HspB8) having different structure of beta2 strand of alpha-crystallin domain and different length and composition of variable N- and C-terminal tails.

Phosphorylation of more than one site is required for tight interaction of human tau protein with 14-3-3zeta.

Serine residues phosphorylated by protein kinase A (PKA) in the shortest isoform of human tau protein (tau3) were sequentially replaced by alanine and interaction of phosphorylated tau3 and its mutants with 14-3-3 was investigated. Mutation S156A slightly decreased interaction of phosphorylated tau3 with 14-3-3. Double mutations S156A/S267A and especially S156A/S235A, strongly inhibited interaction of phosphorylated tau3 with 14-3-3. Thus, two sites located in the Pro-rich region and in the pseudo repeats of tau3 are involved in phosphorylation-dependent interaction of tau3 with 14-3-3. The state of tau3 phosphorylation affects the mode of 14-3-3 binding and by this means might modify tau filament formation.

Effect of phosphorylation on interaction of human tau protein with 14-3-3zeta.

Interaction of the shortest isoform of tau protein (tau3) with human 14-3-3zeta was analyzed by means of native gel electrophoresis, chemical crosslinking and size-exclusion chromatography. Phosphorylation by cAMP-dependent protein kinase (up to 2 mole of phosphate per mole of tau3) strongly enhanced interaction of tau3 with 14-3-3. Apparent K(D) of the complexes formed by phosphorylated tau3 and 14-3-3 was close to 2 microM, whereas the corresponding constant for unphosphorylated tau3 was at least 10 times higher. The stoichiometry of the complexes formed by phosphorylated tau3 and 14-3-3 was variable and was different from 1:1. 14-3-3 decreased the probability of formation of chemically crosslinked large homooligomers of phosphorylated tau3 and at the same time induced formation of crosslinked heterooligomeric complexes of tau3 and 14-3-3 with an apparent molecular mass of 120-140 kDa.

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.

Effect of mutations mimicking phosphorylation on the structure and properties of human 14-3-3zeta.

Effect of mutations mimicking phosphorylation on the structure of human 14-3-3zeta protein was analyzed by different methods. Mutation S58E increased intrinsic Trp fluorescence and binding of bis-ANS to 14-3-3. At low protein concentration mutation S58E increased the probability of dissociation of dimeric 14-3-3 and its susceptibility to proteolysis. Mutation S184E slightly increased Stokes radius and thermal stability of 14-3-3. Mutation T232E induced only small increase of Stokes radius and sedimentation coefficient that probably reflect the changes in the size or shape of 14-3-3. At low protein concentration the triple mutant S58E/S184E/T232E tended to dissociate, whereas at high concentration its properties were comparable with those of the wild type protein. The triple mutant was highly susceptible to proteolysis. Thus, mutation mimicking phosphorylation of Ser58 destabilized, whereas mutation of Ser184 induced stabilization of 14-3-3zeta structure.

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 alpha-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-beta 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.

Effect of mutations in the beta5-beta7 loop on the structure and properties of human small heat shock protein HSP22 (HspB8, H11).

The human genome encodes ten different small heat shock proteins, each of which contains the so-called alpha-crystallin domain consisting of 80-100 residues and located in the C-terminal part of the molecule. The alpha-crystallin domain consists of six or seven beta-strands connected by different size loops and combined in two beta-sheets. Mutations in the loop connecting the beta5 and beta7 strands and conservative residues of beta7 in alphaA-, alphaB-crystallin and HSP27 correlate with the development of different congenital diseases. To understand the role of this part of molecule in the structure and function of small heat shock proteins, we mutated two highly conservative residues (K137 and K141) of human HSP22 and investigated the properties of the K137E and K137,141E mutants. These mutations lead to a decrease in intrinsic Trp fluorescence and the double mutation decreased fluorescence resonance energy transfer from Trp to bis-ANS bound to HSP22. Mutations K137E and especially K137,141E lead to an increase in unordered structure in HSP22 and increased susceptibility to trypsinolysis. Both mutations decreased the probability of dissociation of small oligomers of HSP22, and mutation K137E increased the probability of HSP22 crosslinking. The wild-type HSP22 possessed higher chaperone-like activity than their mutants when insulin or rhodanase were used as the model substrates. Because conservative Lys residues located in the beta5-beta7 loop and in the beta7 strand appear to play an important role in the structure and properties of HSP22, mutations in this part of the small heat shock protein molecule might have a deleterious effect and often correlate with the development of different congenital diseases.

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

Interaction of human 14-3-3gamma 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.

Structure and properties of K141E mutant of small heat shock protein HSP22 (HspB8, H11) that is expressed in human neuromuscular disorders.

Some properties of the K141E mutant of human HSP22 that is expressed in distal hereditary motor neuropathy were investigated. This mutation slightly decreased intrinsic fluorescence of HSP22 and induced changes in the far UV CD spectra that correlate with increase of disordered structure. Destabilized K141E mutant was more susceptible to trypsinolysis than the wild type protein. Mutation K141E did not significantly affect the hydrophobic properties measured by bis-ANS binding and did not affect the quaternary structure of HSP22. With insulin as a substrate the chaperone-like activity of K141E mutant and the wild type protein were similar. However with alcohol dehydrogenase and rhodanese the chaperone-like activity of K141E mutant was remarkably lower than the corresponding activity of the wild type protein. It is concluded that K141E mutation induces destabilization of HSP22 structure and probably by this means diminish the chaperone-like activity of HSP22 with certain protein substrates.

The problem of protein kinase activity of small heat shock protein Hsp22 (H11 or HspB8).

The recently described protein denoted H11, Hsp22 or HspB8 seems to participate in regulation of proliferation, apoptosis, and cardiac hypertrophy. Mutation of Hsp22 causes distal motor neuropathy. Multitude action of Hsp22 is supposed to be due to its protein kinase and/or chaperone-like activities. There are many indirect evidences indicating that Hsp22 possesses intrinsic protein kinase activity. However, low homology to protein kinases, low extent of autophosphorylation, lack of significant protein kinase activity with commonly used substrates, and lack of information on stoichiometry, kinetics, and substrate specificity make the existence of intrinsic protein kinase activity of Hsp22 questionable. It is supposed that protein kinase activity ascribed to Hsp22 is due to contaminating protein kinases. Hsp22 is highly homologous to small heat shock proteins and effectively prevents aggregation of denatured protein both in vitro and in vivo. Therefore, it is supposed that chaperone-like activity is of great importance for Hsp22 functioning.

Some properties of human small heat shock protein Hsp22 (H11 or HspB8).

Untagged recombinant human small heat shock protein with apparent molecular mass 22 kDa (Hsp22) was obtained in homogeneous state. Size exclusion chromatography and chemical crosslinking with dimethylsuberimidate indicate that Hsp22 forms stable dimers. Being highly susceptible to oxidation Hsp22 forms disulfide crosslinked dimers and poorly soluble high molecular mass oligomers. According to CD spectroscopy oxidation of Hsp22 results in disturbing of both secondary and tertiary structure. Hsp22 possesses a negligibly low autophosphorylation activity and under the conditions used is unable to phosphorylate casein or histone. Hsp22 effectively prevents heat-induced aggregation of yeast alcohol dehydrogenase and bovine liver rhodanese with chaperone activity comparable to that of recombinant human small heat shock protein with apparent molecular mass 20 kDa (Hsp20).