Structure and properties of avian small heat shock protein with molecular weight 25 kDa

The primary structure of chicken small heat shock protein (sHsp) with apparent molecular weight 25 kDa was refined and it was shown that this protein has conservative primary structure 74RALSRQLSSG(83) at Ser77 and Ser81, which are potential sites of phosphorylation. Recombinant wild-type chicken Hsp25, its three mutants, 1D (S15D), 2D (S77D+S81D) and 3D (S15D+S77D+S81D), as well as delR mutant with the primary structure 74RALS-ELSSG(82) at potential sites of phosphorylation were expressed and purified. It has been shown that the avian tissues contain three forms of Hsp25 having pI values similar to that of the wild-type protein, 1D and 2D mutants that presumably correspond to nonphosphorylated, mono- and di-phosphorylated forms of Hsp25. Recombinant wild-type protein, its 1D mutant and Hsp25, isolated from chicken gizzard, form stable high molecular weight oligomeric complexes. The delR, 2D and 3D mutants tend to dissociate and exist in the form of a mixture of high and low molecular weight oligomers. Point mutations mimicking phoshorylation decrease chaperone activity of Hsp25 measured by reduction of dithiothreitol induced aggregation of alpha-lactalbumin, but increase the chaperone activity of Hsp25 measured by heat induced aggregation of alcohol dehydrogenase. It is concluded that avian Hsp25 has a more stable quaternary structure than its mammalian counterparts and mutations mimicking phosphorylation differently affect chaperone activity of avian Hsp25, depending on the nature of target protein and the way of denaturing.     

Yeast actin with a mutation in the "hydrophobic plug" between subdomains 3 and 4 (L266D) displays a cold-sensitive polymerization defect

Holmes et al. (Holmes, K. C., D. Popp, W. Gebhard, and W. Kabsch. 1990. Nature [Lond.] 347: 44-49) hypothesized that between subdomains 3 and 4 of actin is a loop of 10 amino acids including a four residue hydrophobic plug that inserts into a hydrophobic pocket formed by two adjacent monomers on the opposing strand thereby stabilizing the F- actin helix. To test this hypothesis we created a mutant yeast actin (L266D) by substituting Asp for Leu266 in the plug to disrupt this postulated hydrophobic interaction. Haploid cells expressing only this mutant actin were viable with no obvious altered phenotype at temperatures above 20 degrees C but were moderately cold-sensitive for growth compared with wild-type cells. The critical concentration for polymerization increased 10-fold at 4 degrees C compared with wild-type actin. The length of the nucleation phase of polymerization increased as the temperature decreased. At 4 degrees C nucleation was barely detectable. Addition of phalloidin-stabilized F-actin nuclei and phalloidin restored L266D actin''s ability to polymerize at 4 degrees C. This mutation also affects the overall rate of elongation during polymerization. Small effects of the mutation were observed on the exchange rate of ATP from G-actin, the G-actin intrinsic ATPase activity, and the activation of myosin S1 ATPase activity. Circular dichroism measurements showed a 15 degrees C decrease in melting temperature for the mutant actin from 57 degrees C to 42 degrees C. Our results are consistent with the model of Holmes et al. (Holmes, K. C., D. Popp, W. Gebhard, and W. Kabsch. 1990. Nature [Lond.]. 347:44-49) involving the role of the hydrophobic plug in actin filament stabilization.     

Phosphorylation of Amoeba G-actin and its effect on actin polymerization

Mass culture of Amoeba proteus enabled us to do biochemical studies on this organism. Actin and profilin were purified from Amoeba to examine actin phosphorylation and polymerization. The apparent molecular weight of Amoeba actin was 44,000, and its isoelectric point was 5.8. The apparent molecular weight of Amoeba profilin was 12,000, and its isoelectric point was 4.9. It reduced the rate of actin polymerization as reported in the cases of profilins from other organisms. A protein of Mr = 44,000 (44 K protein) was phosphorylated in a Ca2+-dependent manner in cell homogenate of Amoeba without being inhibited by calmodulin antagonists. Using the homogenate as a kinase, purified Amoeba G-actin could be phosphorylated in proportion to the amount of actin. However, neither Amoeba F-actin nor rabbit skeletal muscle G-actin was phosphorylated. The phosphorylation of Amoeba actin with a kinase partially purified from A. proteus increased with dilution of the actin concentration. When Amoeba profilin was added, more than 80% of the actin was phosphorylated. By viscometry, electron microscopy, and ultracentrifugation analysis it was demonstrated that Amoeba G-actin phosphorylated in the presence of profilin and kinase did not polymerize in this solution. High-performance liquid chromatography analysis showed that phosphorylated Amoeba actin remained in a monomeric state even under conditions favorable for actin polymerization.     

An intermediate state of G-actin between native and denatured: polymerization rate decreases but extent of polymerization remains unchanged

The rate of actin polymerization gradually decreased without changing the final level of polymerization, when incubated in the presence of 0.2 mM ATP at pH 8.0 and 25 degrees C. This change was much faster in Mg2+-actin than Ca2+-actin, and Mg2+-actin became denatured and unpolymerizable on prolonged incubation. The drop in the polymerization rate was due both to weakened nucleation and a slowed elongation rate in the incubated actin. The change in the polymerization rate was partially reversible by storing the sample at 0 degrees C. When the rate of polymerization dropped markedly on prolonged incubation, a gel filtration profile showed that Ca2+-actin existed as monomer not as oligomer. On the other hand, Mg2+-actin formed dimers, and other oligomers, as revealed by crosslinking analysis. There were changes in fluorescence intensities due to tyrosine and/or tryptophan residues of the actin molecule, and in difference absorption spectra, suggesting that conformational changes intermediate between native and denatured states occurred during incubation.     

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

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

The first step in the polymerisation of actin

In the presence of certain cations (e.g. K+ or Mg2+) actin polymerizes. Below a certain concentration (the critical concentration) the monomer G-actin does not polymerize on the addition of K+ or Mg2+. However, the proteolysis experiments of Rich and Estes [J. Mol. Biol. 104, 777--792 (1976)] strongly suggest that cations induce a change in conformation of G-actin leading to a novel form of actin, G*-actin. This conformational change may be the first step in the polymerization of actin. We have studied G*-actin induced by K+, by difference spectroscopy. We show that G*-actin is a monomer and we confirm that the bound ATP is not cleaved. We also studied the G-actin in equilibrium with G*-actin equilibrium at 4 degrees C as a function of K+ or Mg2+ concentration. With KCl, the transformation can be accounted for as a screening effect. The effect of Mg2+ is more specific and the change in conformation of the G-actin could result from the binding of two or three Mg2+ ions/molecule. We suggest that the G-actin in equilibrium with G*-actin transformation results from the neutralization of a polyanionic region on the actin surface and that this region could be the highly negatively charged N terminus.     

Effect of temperature on the mechanism of actin polymerization

The rate of the Mg2+-induced polymerization of rabbit skeletal muscle G-actin has been measured as as function of temperature at pH 8 by using various concentrations of Mg2+, Ca2+, and G-actin. A polymerization mechanism similar to that proposed at this pH [Frieden, C. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6513-6517] was found to fit the data from 10 to 35 degrees C. From the kinetic data, no evidence for actin filament fragmentation was found at any temperature. Dimer formation is the most temperature-sensitive step, with the ratio of forward and reverse rate constants changing 4 orders of magnitude from 10 to 35 degrees C. Over this temperature change, all other ratios of forward and reverse rate constants change 7-fold or less, and the critical concentration remains nearly constant. The reversible Mg2+-induced isomerization of G-actin monomer occurs to a greater extent with increasing temperature, measured either by using N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled actin or by simulation of the full-time course of the polymerization reaction. This is partially due to Mg2+ binding becoming tighter, and Ca2+ binding becoming weaker, with increasing temperature. Elongation rates from the filament-pointed end, determined by using actin nucleated by plasma gelsolin, show a temperature dependence slightly larger than that expected for a diffusion-limited reaction.     

Mutants in the Dictyostelium Arp2/3 complex and chemoattractant-induced actin polymerization

We have investigated the role of the Arp2/3 complex in Dictyostelium cell chemotaxis towards cyclic-AMP and in the actin polymerization that is triggered by this chemoattractant. We confirm that the Arp2/3 complex is recruited to the cell perimeter, or into a pseudopod, after cyclic-AMP stimulation and that this is coincident with actin polymerization. This recruitment is inhibited when actin polymerization is blocked using latrunculin suggesting that the complex binds to pre-existing actin filaments, rather than to a membrane associated signaling complex. We show genetically that an intact Arp2/3 complex is essential in Dictyostelium and have produced partially active mutants in two of its subunits. In these mutants both phases of actin polymerization in response to cyclic-AMP are greatly reduced. One mutant projects pseudopodia more slowly than wild type and has impaired chemotaxis, together with slower movement. The second mutant chemotaxes poorly due to an adhesion defect, suggesting that the Arp2/3 complex plays a crucial part in adhering cells to the substratum as they move. We conclude that the Arp2/3 complex largely mediates the actin polymerization response to chemotactic stimulation and contributes to cell motility, pseudopod extension and adhesion in Dictyostelium chemotaxis.     

Dominant Negative Mutant Actins Identified in Flightless Drosophila Can Be Classified into Three Classes

Strongly dominant negative mutant actins, identified by An and Mogami (An, H. S., and Mogami, K. (1996) J. Mol. Biol. 260, 492–505), in the indirect flight muscle of Drosophila impaired its flight, even when three copies of the wild-type gene were present. Understanding how these strongly dominant negative mutant actins disrupt the function of wild-type actin would provide useful information about the molecular mechanism by which actin functions in vivo. Here, we expressed and purified six of these strongly dominant negative mutant actins in Dictyostelium and classified them into three groups based on their biochemical phenotypes. The first group, G156D, G156S, and G268D actins, showed impaired polymerization and a tendency to aggregate under conditions favoring polymerization. G63D actin of the second group was also unable to polymerize but, unlike those in the first group, remained soluble under polymerizing conditions. Kinetic analyses using G63D actin or G63D actin·gelsolin complexes suggested that the pointed end surface is defective, which would alter the polymerization kinetics of wild-type actin when mixed and could affect formation of thin filament structures in indirect flight muscle. The third group, R95C and E226K actins, was normal in terms of polymerization, but their motility on heavy meromyosin surfaces in the presence of tropomyosin-troponin indicated altered sensitivity to Ca²⁺. Cofilaments in which R95C or E226K actins were copolymerized with a 3-fold excess of wild-type actin also showed altered Ca²⁺ sensitivity in the presence of tropomyosin-troponin.     

Mechanism of action of phalloidin on the polymerization of muscle actin

Under conditions where muscle actin only partially polymerizes, or where it does not polymerize at all, a significant enhancement of polymerization was observed if equimolar phalloidin was also present. The increased extent of polymerization in the the presence of phalloidin can be explained by the reduced critical actin concentration of partially polymerized populations at equilibrium. Under such conditions, the rate of polymerization, as judged by the length of time to reach half the viscosity plateau, was found to be essentially independent of the phalloidin concentration. Moreover, the initial rate of polymerization of actin was also found to be independent of phalloidin concentration. However, phalloidin apparently causes a reduction in the magnitude of the reverse rates in the polymerization reaction, as was demonstrated by the lack of depolymerization of phalloidin-treated actin polymers. This effect of phalloidin is also supported by the identification of actin nuclei and short polymers in populations of G-actin incubated with phalloidin in the absence of added KCl. Our conclusion, then, is that phalloidin influences the polymerization of actin by stabilizing nuclei and polymers as they are formed.     

The binding of heat-treated myosin subfragment 1 to actin and substructure considerations

Heat treatment of myosin subfragment 1 at 35 degrees C caused about 95% inactivation of the catalytic function but did not block its binding to actin. Heat-treated subfragment 1 showed specific, strong, and close to stoichiometric binding to actin. MgATP but not MgADP dissociated these complexes. However, in contrast to intact subfragment 1, the heat-treated protein did not polymerize G-actin and was not protected from trypsin by the binding to actin. Tryptic degradation of the 50K fragment abolished, or reduced greatly, the binding of heat-treated subfragment 1 to actin in solution but not on nitrocellulose overlays. These results are discussed in the context of subfragment 1 substructure.     

Detection of conformational changes in actin by proteolytic digestion: Evidence for a new monomeric species

When KCl is added to a solution of G-actin to induce full polymerization, a decrease in the rate at which actin undergoes enzymatic proteolysis occurs. This decrease cannot be accounted for by factors affecting the enzymes employed, but rather appears to be due to a change in the conformation of G-actin. Partially polymerized actin solutions also show a reduction in digestibility which is dependent on the F-actin content, suggesting that F-actin is essentially indigestible. Moreover, low rates of digestion were also observed at sub-critical actin concentrations, where actin in the presence of 0.1 m-KCl does not polymerize. This indicates that a confomational change occurs in G-actin before the polymerization step.At sub-critical concentrations in 0.1 m-KCl, actin is in a truly monomeric state as judged by its viscosity characteristics, its inability to enhance the rate of polymerization of G-actin and its possession of ATP as the actin-bound nucleotide. These data support the existence of a new species of actin, called F-ATP-actin monomer, which has the same physical properties and the same bound nucleotide as G-actin, but digestion characteristics like F-actin. Since F-ATP-actin monomers have the same low susceptibility to proteolysis as F-ADP-actin polymers, and because both G-ATP-actin and G-ADP-actin have similar high rates of digestion, the observed change in the conformation of actin cannot be due to the phosphorylated state of the actin-bound nucleotide. Instead, the conformational change appears to be caused by the addition of KCl to G-actin.The newly-detected monomeric species is considered to be an intermediate in the polymerization process where F-ATP-actin monomers form a population of polymerizable molecules which must reach a critical concentration before nucleation and F-actin polymer formation begin.     

Maleimidobenzoyl-G-actin: structural properties and interaction with skeletal myosin subfragment-1

We have investigated various structural and interaction properties of maleimidobenzoyl-G-actin (MBS-actin), a new, internally cross-linked G-actin derivative that does not exhibit, at moderate protein concentration, the salt--and myosin subfragment 1 (S-1)-induced polymerizations of G-actin and reacts reversibly and covalently in solution with S-1 at or near the F-actin binding region of the heavy chain (Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6028-6032). The far-ultraviolet CD spectrum and alpha-helix content of the MBS-actin were identical with those displayed by native G-actin. 45Ca2+ measurements showed the same content of tightly bound Ca2+ in MBS-actin as in G-actin and the EDTA treatment of the modified protein promoted the same red shift of the intrinsic fluorescence spectrum as observed with native G-actin. Incubation of concentrated MBS-actin solutions with 100 mM KCl + 5 mM MgCl2 led to the polymerization of the actin derivative when the critical monomer concentration reached 1.6 mg/mL, at 25 degrees C, pH 8.0. The MBS-F-actin formed activated the Mg2(+)-ATPase of S-1 to the same extent as native F-actin. The MBS-G-actin exhibited a DNase I inhibitor activity very close to that found with native G-actin and was not to be at all affected by its specific covalent conjugation to S-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermostability of bacteriophage T4 fibritin and its deletion mutants]

The thermal stability of a series of recently obtained mutants of fibritin from bacteriophage T4 (a superhelical fibrous homotrimer with parallel-packed subunits each containing 486 amino acid residues) progressively truncated from the subunit N-end was studied during incubation at 40-90 degrees C in the presence of a surfactant (2% SDS). The mutant fibritins, G, B, C, and E, contained 443, 276, 231, and 120 amino acid residues, respectively. One more truncated mutant (fibritin S1, 108 amino acid residues) was obtained. The 2% SDS-PAGE showed that the migration mobilities of all these proteins corresponded to apparent molecular masses substantially greater than those of the preliminarily heated samples (3 min at 100 degrees C). The heating of the intact fibritin and the mutant G at 50-70 degrees C for 10 min resulted in the formation of a form with an apparent molecular mass higher than 200 kDa. This form probably represented a trimeric protein with a partly denatured N-terminal part. Fibritins B and C were more stable and were only partly denatured into monomers even at 70-90 degrees C. The short mutants E and S1 dissociated into monomers at temperatures from 45 to 50 degrees C. The denaturation of mutants B, C, E, and S1 proceeded in one stage without formation of any intermediate form. The stability of the trimeric molecules of native fibritin under PAGE denaturing conditions and the behavior of the intact protein during heating in the temperature range of 50-70 degrees C might be used for the identification of fibritin intermediate forms upon folding in vivo. The refolding capability was found for fibritin and its mutants denatured by heating at low temperatures in the presence of 2% SDS.     

Characterization of carbethoxylated actin

The carbethoxylation of histidine residues in G-actin impairs actin polymerization. The histidine residue essential for polymerization was identified as histidine-40 [Hegyi, G., Premecz, G., Sain, B., & Mühlrad, A. (1974) Eur. J. Biochem. 44, 7-12]. Non-polymerizable actin was separated from the polymerizable fraction after partial carbethoxylation. The non-polymerizable actin recovered the ability to polymerize following addition of phalloidin. Taking into account the evidence that phalloidin does not bind to G-actin in the absence of salt, the results indicate that the actin monomer undergoes a conformational change and subsequently binds phalloidin before polymerization. The resulting polymers activated S1 ATPase activity as effectively as control F-actin. In the presence of tropomyosin and troponin, a strong inhibition of actin-activated ATPase activity was observed in the absence of Ca2+, although no inhibition was observed in the presence of Ca2+. These results indicate that His-40 is not directly involved in a myosin binding site nor in a tropomyosin-troponin binding site.     

Overexpression of murine small heat shock protein HSP25 interferes with chondrocyte differentiation and decreases cell adhesion

Although multiple functions for the small heat shock protein HSP25 have been proposed, its specific role during developmental and differentiation processes is not known. Cartilage is one of the tissues in which HSP25 is specifically and highly expressed during development. C1 cells, able to form aggregates in vitro, can be induced to differentiate into chondrocytes. In this study, we generated two stable transfected clones overexpressing HSP25 at two different levels. Cell morphology and growth rate were modified in both clones, although the actin content and distribution did not seem to be altered. Overexpressing clones had more difficulties in coalescing, leading to smaller aggregates and they did not differentiate into chondrocytes. Subsequently, these aggregates tended to dissociate into loose masses of dying cells. The strength of all these effects was directly correlated to the level of HSP25 overexpression. These data suggest that overexpressing HSP25 decreases cellular adhesion and interferes with chondrocyte differentiation.     

The reactivity and function of thiol groups in trout actin

1. Considerable differences were found between the rates and degrees of modification of native trout actin with iodo[2-(14)C]acetate and iodo[1-(14)C]acetamide. 2. With iodoacetate, G- and F-actin were both labelled in the N-terminal peptide only. This modification had little effect on the ability of the actin to polymerize. 3. Iodoacetamide labelled three cysteine residues in both G- and F-actin. The modified cysteine residues were identified from the position of the corresponding tryptic peptides on peptide ;maps'. 4. The modification had little effect on the ability of G-actin to polymerize, to bind ATP or to bind Ca(2+), or on the ability of F-actin to depolymerize. 5. It is concluded that the three cysteine residues present on the ;surface' of the native trout actin molecule have no direct role in the polymerization processes, the binding of ATP, or the binding of Ca(2+).     

Some properties of complexes formed by small heat shock proteins with denaturated actin

The effects of a recombinant small heat shock protein with an apparent molecular weight of 27 kDa (Hsp27) and both wild type (Hsp27-wt) and S15D, S78D, S82D mutants (Hsp27-3D), which mimic the naturally occurring phosphorylation of this protein, on the thermal denaturation and aggregation of F-actin were studied. It has been shown that at a Hsp27/actin weight ration of 1/4, both Hsp27-wt and Hsp27-3D do not affect the thermal denaturation of F-actin, but efficiently prevent its further aggregation by forming soluble complexes with denatured actin. Formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. Hsp27-wt is known to exist as a high-molecular-weight oligomer, whereas Hsp27-3D forms only small dimers or tetramers. However, the complexes formed by Hsp27-wt and Hsp27-3D with denatured actin did not differ in their size, as measured by dynamic light scattering, and demonstrated the same hydrodynamic radius of 17–18 nm. On the other hand, the sedimentation coefficients of these complexes differed: they ranged 10–45 S in the case of Hsp27-3D and 18–60 S in case of Hsp27-wt. Thus, the initial oligomeric state of Hsp27 does not significantly affect its capacity to form small soluble complexes with denatured actin.     

Defined sequence segments of the small heat shock proteins HSP25 and alphaB-crystallin inhibit actin polymerization.

The interaction of small heat shock proteins (sHSPs) with the actin cytoskeleton has been described and some members of this family, e.g. chicken and murine HSP25 (HSP27), inhibit the polymerization of actin in vitro. To analyse the molecular basis of this interaction, we synthesized a set of overlapping peptides covering the complete sequence of murine HSP25 and tested the effect of these peptides on actin polymerization in vitro by fluorescence spectroscopy and electron microscopy. Two peptides comprising the sequences W43 to R57 (peptide 6) and I92 to N106 (peptide 11) of HSP25 were found to be potent inhibitors of actin polymerization. Phosphorylation of N-terminally extended peptide 11 at serine residues known to be phosphorylated in vivo resulted in decline of their inhibitory activity. Interestingly, peptides derived from the homologous peptide 11 sequence of murine alphaB-crystallin showed the same behaviour. The results suggest that both HSP25 and alphaB-crystallin have the potential to inhibit actin polymerization and that this activity is regulated by phosphorylation.