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.     

Chaperone activity of human small heat shock protein-GST fusion proteins

Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST fusion proteins with MDH and CS, is modulated by both sHsp oligomeric conformation and by variations of sHsp sequences.     

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.     

Sequestration of toxic oligomers by HspB1 as a cytoprotective mechanism

Small heat shock proteins (sHsps) are molecular chaperones that protect cells from cytotoxic effects of protein misfolding and aggregation. HspB1, an sHsp commonly associated with senile plaques in Alzheimer's disease (AD), prevents the toxic effects of Aβ aggregates in vitro. However, the mechanism of this chaperone activity is poorly understood. Here, we observed that in two distinct transgenic mouse models of AD, mouse HspB1 (Hsp25) localized to the penumbral areas of plaques. We have demonstrated that substoichiometric amounts of human HspB1 (Hsp27) abolish the toxicity of Aβ oligomers on N2a (mouse neuroblastoma) cells. Using biochemical methods, spectroscopy, light scattering, and microscopy methods, we found that HspB1 sequesters toxic Aβ oligomers and converts them into large nontoxic aggregates. HspB1 was overexpressed in N2a cells in response to treatment with Aβ oligomers. Cultured neurons from HspB1-deficient mice were more sensitive to oligomer-mediated toxicity than were those from wild-type mice. Our results suggest that sequestration of oligomers by HspB1 constitutes a novel cytoprotective mechanism of proteostasis. Whether chaperone-mediated cytoprotective sequestration of toxic aggregates may bear clues to plaque deposition and may have potential therapeutic implications must be investigated in the future.     

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 1000 kDa. 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.     

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.     

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.     

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.     

Mutations of small heat shock proteins and human congenital diseases

The structure and properties of different members of a large family of small heat shock proteins (sHsp) playing an important role in cell homeostasis are described. Participation of the N-terminal domain in formation of large oligomers and chaperone activity of sHsp is analyzed. The structure of the α-crystallin domain of sHsp is characterized and the role of this domain in sHsp dimerization and chaperone activity is discussed. The properties of the C-terminal region of sHsp are described, and its participation in formation of large oligomers and chaperone activity are analyzed. The data from the literature on HspB1 and HspB3 mutations are presented, and involvement of these mutations in development of certain neurodegenerative diseases is discussed. Mutations of HspB4 are described and data on involvement of these mutations in development of cataract are presented. Multiple effects of HspB5 mutations are analyzed, and data are presented indicating that mutations of this protein are accompanied by development of different congenital diseases, such as cataract and different types of myopathies. The data on HspB6 and HspB8 mutations are presented, and feasible effects of these mutations on proteins structure are analyzed. Probable mechanisms underlying sHsp mutation-induced development of different congenital diseases are discussed.     

Dynamic processes that reflect anti-apoptotic strategies set up by HspB1 (Hsp27)

Human HspB1 (also denoted Hsp27) is an oligomeric anti-apoptotic protein that has tumorigenic and metastatic roles. To approach the structural organizations of HspB1 that are active in response to apoptosis inducers acting through different pathways, we have analyzed the relative protective efficiency induced by this protein as well its localization, oligomerization and phosphorylation. HeLa cells, that constitutively express high levels of HspB1 were treated with either etoposide, Fas agonist antibody, staurosporine or cytochalasin D. Variability in HspB1 efficiency to interfere with the different apoptotic transduction pathways induced by these agents were detected. Moreover, inducer-specific dynamic changes in HspB1 localization, native size and phosphorylation were observed, that differed from those observed after heat shock. Etoposide and Fas treatments gradually shifted HspB1 towards large but differently phosphorylated oligomeric structures. In contrast, staurosporine and cytochalasin D induced the rapid but transient formation of small oligomers before large structures were formed. These events correlated with inducer-specific phosphorylations of HspB1. Of interest, the formation of small oligomers in response to staurosporine and cytochalasin D was time correlated with the rapid disruption of F-actin. The subsequent, or gradual in the case of etoposide and Fas, formation of large oligomeric structures was a later event concomitant with the early phase of caspase activation. These observations support the hypothesis that HspB1 has the ability, through specific changes in its structural organization, to adapt and interfere at several levels with challenges triggered by different signal transduction pathways upstream of the execution phase of apoptosis.     

Dynamic viscoelastic properties of cellulose carbamate dissolved in NaOH aqueous solution

Dynamic viscoelastic properties of cellulose carbamate (CC) dissolved in NaOH aqueous solution were systematically studied for the first time. CC was microwave-assisted synthesized from the mixture of cellulose and urea and then dissolved in 7 wt % NaOH aqueous solution precooled to -7 °C. The obtained CC solution is transparent and has good liquidity. To clarify the rheological behavior of the solution, the CC solutions were investigated by dynamic viscoelastic measurements. The shear storage modulus (G') and loss modulus (G') as a function of the angular frequency (ω), concentration (c), nitrogen content (N %), viscosity-average molecular weight (M(η)), temperature (T), and time (t) were analyzed and discussed in detail. The sol-gel transition temperature of CC (M(η) = 7.78 × 10(4)) solution decreased from 36.5 to 31.3 °C with an increase of the concentration from 3.0 to 4.3 wt % and decreased from 35.7 to 27.5 °C with an increase of the nitrogen content from 1.718 to 5.878%. The gelation temperature of a 3.8 wt % CC solution dropped from 38.2 to 34.4 °C with the M(η) of CC increased from 6.35 × 10(4) to 9.56 × 10(4). The gelation time of the CC solution was relatively short at 30 °C, but the solution was stable for a long time at about 15 °C. Moreover, the gels already formed at elevated temperature were irreversible; that is, after cooling to a lower temperature including the dissolution temperature (-7 °C), they could not be dissolved to become liquid.     

Reaction of small heat-shock proteins to different kinds of cellular stress in cultured rat hippocampal neurons

Upregulation of small heat-shock proteins (sHsps) in response to cellular stress is one mechanism to increase cell viability. We previously described that cultured rat hippocampal neurons express five of the 11 family members but only upregulate two of them (HspB1 and HspB5) at the protein level after heat stress. Since neurons have to cope with many other pathological conditions, we investigated in this study the expression of all five expressed sHsps on mRNA and protein level after sublethal sodium arsenite and oxidative and hyperosmotic stress. Under all three conditions, HspB1, HspB5, HspB6, and HspB8 but not HspB11 were consistently upregulated but showed differences in the time course of upregulation. The increase of sHsps always occurred earlier on mRNA level compared with protein levels. We conclude from our data that these four upregulated sHsps (HspB1, HspB5, HspB6, HspB8) act together in different proportions in the protection of neurons from various stress conditions.     

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.     

Structural and functional specificity of small heat shock protein HspB1 and HspB4, two cellular partners of HspB5: role of the in vitro hetero-complex formation in chaperone activity

The ubiquitous small heat shock proteins are essential elements in cellular protection, through a molecular chaperone activity. Among them, human small heat shock protein HspB1, HspB4 and HspB5 are involved in oncogenesis, anti-apoptotic activity and lens transparency. Therefore, these proteins are potential therapeutic targets in many diseases. Their general chaperone activity is related to their dynamic and multiple oligomeric structures, which are still poorly understood. The tissue selective distribution of HspB1 and HspB4, two cellular partners of HspB5, suggests that these two proteins might have evolved to play distinct physiological functions. Moreover, hetero-complex formation seems to be favoured in?vivo, yet the functional specificity of the HspB1-HspB5 and HspB4-HspB5 hetero-complexes compared to the homo-oligomers remains unclear in the stress response pathway. A powerful approach combining biochemistry, biophysics and bioinformatics, allowed us to compare the different assemblies, with a special emphasis on the structural data, subunit exchange properties, activity and sequence evolution. We showed that they all exhibit different properties, from structural organization in physiological versus stress conditions, to chaperone-like activity, whatever the level of sequence conservation. Subunit exchange kinetics leading to HspB1-HspB5 or HspB4-HspB5 hetero-complex formation is also different between these two complexes: HspB5 exchanges more rapidly subunits with HspB1 than with HspB4. The relative sequence conservation in the sHSP superfamily does hide important structural heterogeneity and flexibility, which confer an enlarged range of different surface necessary to efficiently form complexes with various stress-induced cellular targets. Our data suggest that the formation of hetero-complexes could be an original evolutionary strategy to gain new cellular functions.     

Abnormal interaction of motor neuropathy-associated mutant HspB8 (Hsp22) forms with the RNA helicase Ddx20 (gemin3)

A number of missense mutations in the two related small heat shock proteins HspB8 (Hsp22) and HspB1 (Hsp27) have been associated with the inherited motor neuron diseases (MND) distal hereditary motor neuropathy and Charcot-Marie-Tooth disease. HspB8 and HspB1 interact with each other, suggesting that these two etiologic factors may act through a common biochemical mechanism. However, their role in neuron biology and in MND is not understood. In a yeast two-hybrid screen, we identified the DEAD box protein Ddx20 (gemin3, DP103) as interacting partner of HspB8. Using co-immunoprecipitation, chemical cross-linking, and in vivo quantitative fluorescence resonance energy transfer, we confirmed this interaction. We also show that the two disease-associated mutant HspB8 forms have abnormally increased binding to Ddx20. Ddx20 itself binds to the survival-of-motor-neurons protein (SMN protein), and mutations in the SMN1 gene cause spinal muscular atrophy, another MND and one of the most prevalent genetic causes of infant mortality. Thus, these protein interaction data have linked the three etiologic factors HspB8, HspB1, and SMN protein, and mutations in any of their genes cause the various forms of MND. Ddx20 and SMN protein are involved in spliceosome assembly and pre-mRNA processing. RNase treatment affected the interaction of the mutant HspB8 with Ddx20 suggesting RNA involvement in this interaction and a potential role of HspB8 in ribonucleoprotein processing.     

Small heat shock protein speciation: novel non-canonical 44 kDa HspB5-related protein species in rat and human tissues

When analyzing small stress proteins of rat and human tissues by electrophoretic methods followed by western blotting, and using the anti-HspB1/anti-HspB5 antibody clone 8A7, we unexpectedly found a protein with a molecular mass of ~44 kDa. On two-dimensional gels, this protein resolved into four distinct species. Electrophoretic and immunological evidence suggests that this 44 kDa protein is a derivative of HspB5, most likely a covalently linked HspB5 dimer. This HspB5-like 44 kDa protein (HspB5L-P44) is particularly abundant in rat heart, brain, and renal cortex and glomeruli. HspB5L-P44 was also found in human brains, including those from patients with Alexander disease, a condition distinguished by cerebral accumulation of HspB5. Gray matter of such a patient contained an elevated amount of HspB5L-P44. A spatial model of structurally ordered dimeric HspB5 α-crystallin domains reveals the exposed and adjacent position of the two peptide segments homologous to the HspB1-derived 8A7 antigen determinant peptide (epitope). This explains the observed extraordinary high avidity of the 8A7 antibody towards HspB5L-P44, as opposed to commonly used HspB5-specific antibodies which recognize other epitopes. This scenario also explains the remarkable fact that no previous study reported the existence of HspB5L-P44 species. Exposure of rat endothelial cells to UV light, an oxidative stress condition, temporarily increased HspB5L-P44, suggesting physiological regulation of the dimerization. The existence of HspB5L-P44 supports the protein speciation discourse and fits to the concept of the protein code, according to which the expression of a given gene is reflected only by the complete set of the derived protein species.     

Structural instability caused by a mutation at a conserved arginine in the alpha-crystallin domain of Chinese hamster heat shock protein 27

Mutations in the alpha-crystallin domain of 4 of the small heat shock proteins (sHsp) (Hsp27/HspB1, alphaA-crystallin/ HspB4, alphaB-crystallin/HspB5, and HspB8) are responsible for dominant inherited diseases in humans. One such mutation at a highly conserved arginine residue was shown to cause major conformational defects and intracellular aggregation of alphaA- and alphaB-crystallins and HspB8. Here, we studied the effect of this Arg mutation on the structure and function of Hsp27. Chinese hamster Hsp27 with Arg148 replaced by Gly (Hsp27R148G) formed dimers in vitro and in vivo, which contrasted with the 12- or 24-subunit oligomers formed by the wild-type protein (Hsp27WT). Despite these alterations, Hsp27R148G had a chaperone activity almost as high as Hsp27WT. The dimers of Hsp27R148G did not further deoligomerize on phosphorylation and like the dimers formed by phosphorylated Hsp27WT were not affected by the deletion of the N-terminal WD/EPF (single letter amino acid code) motif, suggesting that mutation of Arg148, deletion of the N-terminal WD/EPF motif, and phosphorylation of Ser90 may produce similar structural perturbations. Nevertheless, the structure of Hsp27R148G appeared unstable, and the mutated protein accumulated as aggregates in many cells. Both a lower basal level of phosphorylation of Hsp27R148G and the coexpression of Hsp27WT could reduce the frequency of formation of these aggregates, suggesting possible mechanisms regulating the onset of the sHsp-mediated inherited diseases.     

Mammalian HspB1 (Hsp27) is a molecular sensor linked to the physiology and environment of the cell

Constitutively expressed small heat shock protein HspB1 regulates many fundamental cellular processes and plays major roles in many human pathological diseases. In that regard, this chaperone has a huge number of apparently unrelated functions that appear linked to its ability to recognize many client polypeptides that are subsequently modified in their activity and/or half-life. A major parameter to understand how HspB1 is dedicated to interact with particular clients in defined cellular conditions relates to its complex oligomerization and phosphorylation properties. Indeed, HspB1 structural organization displays dynamic and complex rearrangements in response to changes in the cellular environment or when the cell physiology is modified. These structural modifications probably reflect the formation of structural platforms aimed at recognizing specific client polypeptides. Here, I have reviewed data from the literature and re-analyzed my own studies to describe and discuss these fascinating changes in HspB1 structural organization.     

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).     

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.