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.     

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.     

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.     

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.     

Small Heat Shock Proteins and Distal Hereditary Neuropathies

Classification of small heat shock proteins (sHsp) is presented and processes regulated by sHsp are described. Symptoms of hereditary distal neuropathy are described and the genes whose mutations are associated with development of this congenital disease are listed. The literature data and our own results concerning physicochemical properties of HspB1 mutants associated with Charcot–Marie–Tooth disease are analyzed. Mutations of HspB1, associated with hereditary motor neuron disease, can be accompanied by change of the size of HspB1 oligomers, by decreased stability under unfavorable conditions, by changes in the interaction with protein partners, and as a rule by decrease of chaperone-like activity. The largest part of these mutations is accompanied by change of oligomer stability (that can be either increased or decreased) or by change of intermonomer interaction inside an oligomer. Data on point mutation of HspB3 associated with axonal neuropathy are presented. Data concerning point mutations of Lys141 of HspB8 and those associated with hereditary neuropathy and different forms of Charcot–Marie–Tooth disease are analyzed. It is supposed that point mutations of sHsp associated with distal neuropathies lead either to loss of function (for instance, decrease of chaperone-like activity) or to gain of harmful functions (for instance, increase of interaction with certain protein partners).     

Phosphorylation by cyclic AMP-dependent protein kinase inhibits chaperone-like activity of human HSP22 <Emphasis Type="Italic">in vitro</Emphasis>

Human small heat shock protein with molecular mass 22 kD (HSP22, HspB8) contains two Ser residues (Ser24 and Ser57) in consensus sequence RXS and is effectively phosphorylated by cAMP-dependent protein kinase in vitro. Mutation S24D did not affect, whereas mutations S57D or S24,57D prevented phosphorylation of HSP22 by cAMP-dependent protein kinase thus indicating that Ser57 is the primary site of phosphorylation. Phosphorylation (or mutation) of Ser57 (or Ser24 and Ser57) resulted in changes of the local environment of tryptophan residues and increased HSP22 susceptibility to chymotrypsinolysis. Mutations mimicking phosphorylation decreased dissociation of HSP22 oligomer at low concentration without affecting its quaternary structure at high protein concentration. Mutations S24D, S57D, and especially S24,57D were accompanied by decrease of chaperone-like activity of HSP22 if insulin and rhodanase were used as substrates. Thus, phosphorylation by cAMP-dependent protein kinase affects the structure and decreases chaperone-like activity of HSP22 in vitro.     

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 1 L 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.     

Structure and properties of chimeric small heat shock proteins containing yellow fluorescent protein attached to their C-terminal ends

Recombinant chimeras of small heat shock proteins (sHsp) HspB1, HspB5, and HspB6 containing enhanced yellow fluorescent protein (EYFP) attached to their C-terminal ends were constructed and purified. Some properties of these chimeras were compared with the corresponding properties of the same chimeras containing EYFP attached to the N-terminal end of sHsp. The C-terminal fluorescent chimeras of HspB1 and HspB5 tend to aggregate and form a heterogeneous mixture of oligomers. The apparent molecular weight of the largest C-terminal chimeric oligomers was higher than that of the corresponding N-terminal chimeras or of the wild-type proteins; however, both homooligomers of N-terminal chimeras and homooligomers of C-terminal chimeras contained fewer subunits than the wild-type HspB1 or HspB5. Both N-terminal and C-terminal chimeras of HspB6 form small oligomers with an apparent molecular weight of 73–84 kDa. The C-terminal chimeras exchange their subunits with homologous wild-type proteins. Heterooligomers formed by the wild-type HspB1 (or HspB5) and the C-terminal chimeras of HspB6 differ in size and composition from heterooligomers formed by the corresponding wild-type proteins. As a rule, the N-terminal chimeras possess similar or slightly higher chaperone-like activity than the corresponding wild-type proteins, whereas the C-terminal chimeras always have a lower chaperone-like activity than the wild-type proteins. It is concluded that attachment of EYFP to either N-terminal or C-terminal ends of sHsp affects their oligomeric structure, their ability to form heterooligomers, and their chaperone-like activity. Therefore, the data obtained with fluorescent chimeras of sHsp expressed in the cell should be interpreted with caution.     

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.     

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.     

Structural Aspects and Chaperone Activity of Human HspB3: Role of the “C-Terminal Extension”

HspB3, an as yet uncharacterized sHsp, is present in muscle, brain, heart, and in fetal tissues. A point mutation correlates with the development of axonal motor neuropathy. We purified recombinant human HspB3. Circular dichroism studies indicate that it exhibits β-sheet structure. Gel filtration and sedimentation velocity experiments show that HspB3 exhibits polydisperse populations with predominantly trimeric species. HspB3 exhibits molecular chaperone-like activity in preventing the heat-induced aggregation of alcohol dehydrogenase (ADH). It exhibits moderate chaperone-like activity towards heat-induced aggregation of citrate synthase. However, it does not prevent the DTT-induced aggregation of insulin, indicating that it exhibits target protein-dependent molecular chaperone-like activity. Unlike other sHsps, it has a very short C-terminal extension. Fusion of the C-terminal extension of αB-crystallin results in altered tertiary and quaternary structure, and increase in polydispersity of the chimeric protein, HspB3αB-CT. The chimeric protein shows comparable chaperone-like activity towards heat-induced aggregation of ADH and citrate synthase. However, it shows enhanced activity towards DTT-induced aggregation of insulin. Our study, for the first time, provides the structural and chaperone functional characterization of HspB3 and also sheds light on the role of the C-terminal extension of sHsps.     

The Function of Ile-X-Ile Motif in the Oligomerization and Chaperone-Like Activity of Small Heat Shock Protein AgsA at Room Temperature

Small heat shock proteins assemble as large oligomers in vitro and exhibit ATP-independent chaperone activities. Ile-X-Ile motif is essential in both the function and oligomer formation. AgsA of Salmonella enterica serovar Typhimurium has been demonstrated to adopt large oligomeric structure and possess strong chaperone activity. Size exclusion chromatography, non-denaturing pore gradient PAGE, and negatively stain electron microscopic analysis of the various C-terminal truncated mutants were performed to investigate the role of Ile-X-Ile motif in the oligomer assembly of AgsA. By measuring the ability to prevent insulin from aggregating induced by TCEP, the chaperone-like activity of AgsA and the C-terminal truncated mutants at room temperature were determined. We found that the truncated mutants with Ile-X-Ile motif partially or fully deleted lost the ability to form large oligomers. Contrast to wild type AgsA which displayed weak chaperone-like activity, those mutants shown significantly enhanced activities at room temperature. In summary, biochemical experiment, activity assay and electron microscopic analysis suggested that Ile-X-Ile motif is essential in oligomer assembly of AgsA and might take the role of an inhibitor for its chaperone-like activity at room temperature.     

The chaperone-like activity of rat HspB8/Hsp22 and dynamic molecular transition related to oligomeric architectures in vitro

HspB8/Hsp22 is a functionally distinct small heat shock proteins (sHsp) and is preferentially expressed in brain, heart, skeletal, and smooth muscle. HspB8 is also associated with neuromuscular function and protein quality control by proteasomes in cardiac hypertrophy. However, the molecular properties in vitro and molecular oligomerization remain uncertain. In this investigation, the rat HspB8 gene was expressed in E.coli cells, and mature HspB8 protein was efficiently prepared. The chaperone-like activity of HspB8 in vitro was quantitatively analyzed by model substrates. Size exclusion chromatography revealed that HspB8 had polydisperse oligomers and underwent dynamic molecular transition in solution, existing in a dynamic equilibrium between various oligomers. In a nonphysiological solution, HspB8 was predominantly octamers. In a physiological solution (pH 7.4), HspB8 mainly formed tetramers. The dynamic interactive transition maybe was helpful to maintain its molecular complxes in solution. In a FRET assay, subunit exchange occurred frequently between the various oligomers with a rate of 0.12, 0.089, and 0.064 min(-1) at 50°C, 43°C, and 37°C, respectively. It also demonstrated the dynamic molecular properties of HspB8 in solution.     

Analysis of the Dominant Effects Mediated by Wild Type or R120G Mutant of αB-crystallin (HspB5) towards Hsp27 (HspB1)

Several human small heat shock proteins (sHsps) are phosphorylated oligomeric chaperones that enhance stress resistance. They are characterized by their ability to interact and form polydispersed hetero-oligomeric complexes. We have analyzed the cellular consequences of the stable expression of either wild type HspB5 or its cataracts and myopathies inducing R120G mutant in growing and oxidative stress treated HeLa cells that originally express only HspB1. Here, we describe that wild type and mutant HspB5 induce drastic and opposite effects on cell morphology and oxidative stress resistance. The cellular distribution and phosphorylation of these polypeptides as well as the oligomerization profile of the resulting hetero-oligomeric complexes formed by HspB1 with the two types of exogenous polypeptides revealed the dominant effects induced by HspB5 polypeptides towards HspB1. The R120G mutation enhanced the native size and salt resistance of HspB1-HspB5 complex. However, in oxidative conditions the interaction between HspB1 and mutant HspB5 was drastically modified resulting in the aggregation of both partners. The mutation also induced the redistribution of HspB1 phosphorylated at serine 15, originally observed at the level of the small oligomers that do not interact with wild type HspB5, to the large oligomeric complex formed with mutant HspB5. This phosphorylation stabilized the interaction of HspB1 with mutant HspB5. A dominant negative effect towards HspB1 appears therefore as an important event in the cellular sensitivity to oxidative stress mediated by mutated HspB5 expression. These observations provide novel data that describe how a mutated sHsp can alter the protective activity of another member of this family of chaperones.     

Transmembrane segment 5 of the dipeptide transporter hPepT1 forms a part of the substrate translocation pathway

This study is the first systematic attempt to investigate the role of transmembrane segment 5 of hPepT1, the most conserved segment across different species, in forming a part of the aqueous substrate translocation pathway. We used cysteine-scanning mutagenesis in conjunction with the sulfhydryl-specific reagents, MTSEA and MTSET. Neither of these reagents reduced wild-type-hPepT1 transport activity in HEK293 cells and Xenopus oocytes. Twenty-one single cysteine mutations in hPepT1 were created by replacing each residue within TMS5 with a cysteine. HEK293 cells were then transfected with each mutated protein and the steady-state protein level, [3H]Gly-Sar uptake activity, and sensitivity to the MTS reagents were measured. S164C-, L168C-, G173C-, and I179C-hPepT1 were not expressed on the plasma membrane. Y167C-, N171C-, and S174C-hPepT1 showed 相似文献   

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.     

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.     

Utilization of fluorescent chimeras for investigation of heterooligomeric complexes formed by human small heat shock proteins

Fluorescent chimeras composed of enhanced cyan (or enhanced yellow) fluorescent proteins (ECFP or EYFP) and one of the four human small heat shock proteins (HspB1, HspB5, HspB6 or HspB8) were expressed in E. coli and purified. Fluorescent chimeras were used for investigation of heterooligomeric complexes formed by different small heat shock proteins (sHsp) and for analysis of their subunit exchange. EYFP-HspB1 and ECFP-HspB6 form heterooligomeric complex with apparent molecular weight of ∼280 kDa containing equimolar quantities of both sHsp. EYFP-HspB5 and ECFP-HspB6 formed heterogeneous oligomeric complexes. Fluorescent proteins inside heterooligomeric complexes formed by HspB1/HspB6 and HspB5/HspB6 chimeras are closely located, making possible effective fluorescence resonance energy transfer (FRET). Neither the wild type HspB8 nor its fluorescent chimeras were able to form stable heterooligomeric complexes with the wild type HspB1 and HspB5. Homo- and hetero-FRET was used for analysis of subunit exchange of small heat shock proteins. The apparent rate constant of subunit exchange was temperature-dependent and was higher for HspB6 forming small oligomers than for HspB1 forming large oligomers. Replacement induced by homologous subunits was more rapid than the replacement induced by heterologous subunits of small heat shock proteins. Fusion of fluorescent proteins might affect oligomeric structure of small heat shock proteins, however fluorescent chimeras can be useful for investigation of heterooligomeric complexes formed by sHsp and for analysis of kinetics of their subunit exchange.     

The pivotal role of the β7 strand in the intersubunit contacts of different human small heat shock proteins

Human αB-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 β7 strand of the α-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 β7 strand in intersubunit interaction. Oxidation weakly affects the secondary and tertiary structure, does not affect the quaternary structure of αB-crystallin and HspB6, and shifts equilibrium between monomer and dimer of HspB8 towards dimer formation. It is concluded that the β7 strand participates in the intersubunit interaction of four human small heat shock proteins (αB-crystallin, HspB1, HspB6, HspB8) having different structure of β2 strand of α-crystallin domain and different length and composition of variable N- and C-terminal tails.     

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.