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.

Is the small heat shock protein HspB1 (Hsp27) a real and predominant target of methylglyoxal modification?

This study analyzed the interaction of commercial monoclonal anti-methylglyoxal antibodies that predominantly recognize argpyrimidine with unmodified and modified model proteins and small heat shock proteins. These antibodies specifically recognize methylglyoxal (MG)-modified bovine serum albumin and lysozyme, but they react equally well with both unmodified and MG-modified HspB1. Mutation R188W decreased the interaction of these antibodies with unmodified HspB1, thus indicating that this residue participates in the formation of antigenic determinant. However, these antibodies did not recognize either short (ESRAQ) or long (IPVTFESRAQLGGP) peptides with primary structure identical to that at Arg188 of HspB1. Neither of the peptides obtained after the cleavage of HspB1 at Met or Cys residues were recognized by anti-argpyrimidine antibodies. This means that unmodified HspB1 contains a discontinuous epitope that includes the sequence around Arg188 and that this epitope is recognized by anti-argpyrimidine antibodies in unmodified HspB1. Incubation of HspB1 with MG is accompanied by the accumulation of hydroimidazolones, but not argpyrimidines. Therefore, conclusions based on utilization of anti-argpyrimidine antibodies and indicating that HspB1 is the predominant and preferential target of MG modification in the cell require revision.

Interaction of small heat shock proteins with light component of neurofilaments (NFL).

The interaction of human small heat shock protein HspB1, its point mutants associated with distal hereditary motor neuropathy, and three other small heat shock proteins (HspB5, HspB6, HspB8) with the light component of neurofilaments (NFL) was analyzed by differential centrifugation, analytical ultracentrifugation, and fluorescent spectroscopy. The wild-type HspB1 decreased the quantity of NFL in pellets obtained after low- and high-speed centrifugation and increased the quantity of NFL remaining in the supernatant after high-speed centrifugation. Part of HspB1 was detected in the pellet of NFL after high-speed centrifugation, and at saturation, 1 mol of HspB1 monomer was bound per 2 mol of NFL. Point mutants of HspB1 associated with distal hereditary motor neuropathy (G84R, L99M, R140G, K141Q, and P182S) were almost as effective as the wild-type HspB1 in modulation of NFL assembly. At low ionic strength, HspB1 weakly interacted with NFL tetramers, and this interaction was increased upon salt-induced polymerization of NFL. HspB1 and HspB5 (αB-crystallin) decreased the rate of NFL polymerization measured by fluorescent spectroscopy. HspB6 (Hsp20) and HspB8 (Hsp22) were less effective than HspB1 (or HspB5) in modulation of NFL assembly. The data presented indicate that the small heat shock proteins affect NFL transition from tetramers to filaments, hydrodynamic properties of filaments, and their bundling and therefore probably modulate the formation of intermediate filament networks in neurons.

Characterization of human small heat shock protein HspB1 that carries C-terminal domain mutations associated with hereditary motor neuron diseases.

Physico-chemical properties of four mutants (T164A, T180I, P182S and R188W) of human small heat shock protein HspB1 (Hsp27) associated with neurodegenerative diseases were analyzed by means of fluorescence spectroscopy, dynamic light scattering, size-exclusion chromatography and measurement of chaperone-like activity. Mutation T164A was accompanied by destabilization of the quaternary structure and decrease of thermal stability without any significant changes of chaperone-like activity. Mutations T180I and P182S are adjacent or within the conserved C-terminal motif IPI/V. Replacement T180⇒I leading to the formation of hydrophobic cluster consisting of three Ile produced small increase of thermal stability without changes of chaperone-like activity. Mutation P182S induced the formation of metastable large oligomers of HspB1 with apparent molecular weight of more than 1000kDa. Oligomers of P182S have very low thermal stability and undergo irreversible aggregation at low temperature. The P182S mutant forms mixed oligomers with the wild type HspB1 and the properties of these mixed oligomers are intermediate between those of the wild type HspB1 and its mutant. Mutation R188W did not significantly affect quaternary structure or thermal stability of HspB1, but was accompanied by a pronounced decrease of its chaperone-like activity. All mutations analyzed are associated with hereditary motor neuropathies or Charcot-Marie-Tooth disease type 2; however, molecular mechanisms underlying pathological effects are specific for each of these mutants.

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.

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 ∼560 kDa. The R140G mutant formed two types of oligomers, i.e. large oligomers tending to aggregate and small oligomers with apparent molecular weight ∼70 kDa. The wild type HspB1 formed mixed homooligomers with R140G mutant with apparent molecular weight ∼610 kDa. The R140G mutant was unable to form high molecular weight heterooligomers with HspB6, whereas the K141Q mutant formed two types of heterooligomers with HspB6. In vitro 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.

Monomeric 14-3-3ζ has a chaperone-like activity and is stabilized by phosphorylated HspB6.

Members of the 14-3-3 eukaryotic protein family predominantly function as dimers. The dimeric form can be converted into monomers upon phosphorylation of Ser(58) located at the subunit interface. Monomers are less stable than dimers and have been considered to be either less active or even inactive during binding and regulation of phosphorylated client proteins. However, like dimers, monomers contain the phosphoserine-binding site and therefore can retain some functions of the dimeric 14-3-3. Furthermore, 14-3-3 monomers may possess additional functional roles owing to their exposed intersubunit surfaces. Previously we have found that the monomeric mutant of 14-3-3ζ (14-3-3ζ(m)), like the wild type protein, is able to bind phosphorylated small heat shock protein HspB6 (pHspB6), which is involved in the regulation of smooth muscle contraction and cardioprotection. Here we report characterization of the 14-3-3ζ(m)/pHspB6 complex by biophysical and biochemical techniques. We find that formation of the complex retards proteolytic degradation and increases thermal stability of the monomeric 14-3-3, indicating that interaction with phosphorylated targets could be a general mechanism of 14-3-3 monomers stabilization. Furthermore, by using myosin subfragment 1 (S1) as a model substrate we find that the monomer has significantly higher chaperone-like activity than either the dimeric 14-3-3ζ protein or even HspB6 itself. These observations indicate that 14-3-3ζ and possibly other 14-3-3 isoforms may have additional functional roles conducted by the monomeric state.

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.

Expression, purification and some properties of fluorescent chimeras of human small heat shock proteins.

Small heat shock proteins (sHsp) are ubiquitously expressed in all human tissues and have an important housekeeping role in preventing the accumulation of aggregates of improperly folded or denatured proteins. They also participate in the regulation of the cytoskeleton, proliferation, apoptosis and many other vital processes. Fluorescent chimeras composed of sHsp and enhanced fluorescent proteins have been used to determine the intracellular locations of small heat shock proteins and to analyse the hetero-oligomeric complexes formed by different sHsp. However, the biochemical properties and chaperone-like activities of these chimeras have not been investigated. To determine the properties of these chimeras, we fused enhanced yellow and cyan fluorescent proteins (EYFP and ECFP) to the N-termini of four ubiquitously expressed human small heat shock proteins: HspB1, HspB5, HspB6, and HspB8. The eight fluorescent chimeras of small heat shock proteins and isolated fluorescent proteins were expressed in Escherichia coli. The chimeric proteins were isolated and purified via ammonium sulphate fractionation, ion exchange and size-exclusion chromatography. This method provided 20-100 mg of fluorescent chimeras from 1L of bacterial culture. The spectral properties of the chimeras were similar to those of the isolated fluorescent proteins. The fusion of fluorescent proteins to HspB6 and HspB8, which typically form dimers, did not affect their quaternary structures. Oligomers of the fluorescent chimeras of HspB1 and HspB5 were less stable and contained fewer subunits than oligomers formed by the wild-type proteins. Fusion with EYFP decreased the chaperone-like activity of HspB5 and HspB6 whereas fusion with ECFP increased chaperone-like activity. All fluorescent chimeras of HspB1 and HspB8 had higher chaperone-like activity than the wild-type proteins. Thus, although fluorescent chimeras are useful for many purposes, the fluorescent proteins used to form these chimeras may affect certain important properties of sHsp.

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.

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.

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.