Small heat shock protein Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin Anastasia V Pivovarova1,2, Natalia A Chebotareva1, Ivan S Chernik3, Nikolai B Gusev3 and Dmitrii I Levitsky1,4 A N Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia School of Bioengineering and Bioinformatics, Moscow State University, Russia Department of Biochemistry, School of Biology, Moscow State University, Russia A N Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia Keywords actin; analytical ultracentrifugation; dynamic light scattering; size exclusion chromatography; small heat shock proteins Correspondence D I Levitsky, A N Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky prosp 33, 119071 Moscow, Russia Fax: +7 495 954 2732 Tel: +7 495 952 1384 E-mail: levitsky@inbi.ras.ru (Received 24 July 2007, revised 10 September 2007, accepted 24 September 2007) doi:10.1111/j.1742-4658.2007.06117.x 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 Commun 331, 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  16 nm, a sedimentation coefficient of 17–20 S, and a molecular mass of about 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 Actin is one of the most ubiquitous and abundant proteins in nature It is one of the main constituents of the cell cytoskeleton, and its interaction with myosin motor coupled with ATP hydrolysis is the molecular basis of muscle contraction and a number of other events in cell motility Actin exists in monomeric (G) and polymeric (F) forms Monomeric G-actin is a globular protein with a molecular mass Abbreviations DLS, dynamic light scattering; DSC, differential scanning calorimetry; Hsp27, recombinant human heat shock protein with apparent molecular mass 27 kDa; Hsp27-3D, pseudophosphorylated Hsp27 with mutations S15D, S78D and S82D; Rh, hydrodynamic radius; sHSP, small heat shock protein; SEC, size exclusion chromatography FEBS Journal 274 (2007) 5937–5948 ª 2007 The Authors Journal compilation ª 2007 FEBS 5937 Stable complexes of small HSP with denatured actin A V Pivovarova et al of 42 kDa An important feature of actin is its ability to polymerize upon addition of neutral salts, with formation of long, polar F-actin filaments Different types of stress, e.g heat shock, can induce actin unfolding, leading to disruption of actin filaments and aggregation of fully or partially denatured actin [1,2] Accumulation of aggregated proteins is dangerous for the cell, and this is especially important in the case of abundant proteins, such as actin There are different mechanisms for preventing formation of insoluble aggregates, and the small heat shock proteins (sHSPs) play an important role in this process sHSPs comprise a large and diverse family of proteins with molecular masses from 12 to 43 kDa The members of this protein family share the so-called a-crystallin domain, consisting of 80–100 amino acids, which is located in the C-terminal part of the protein, whereas the N-terminal part differs in sequence and length [3–5] Almost all sHSPs assemble into large oligomeric complexes that vary in structure and number of monomers [3,6,7] In vitro, sHSPs act as molecular chaperones in preventing unfolded proteins from irreversible aggregation and insolubilization [5,8,9], and their chaperone activity is dependent on the quaternary structure [10,11] Different protein kinases phosphorylate sHSPs, and by this means might affect their oligomeric structure and chaperone activity [3,4,12] Expression of some sHSPs is increased in response to different kinds of injury, such as heat shock, and their content is especially high in heart, striated and smooth muscle [3,13,14], where the expression of actin is also very high It seems very likely that one of the main functions of sHSPs in muscles is their interaction with actin Many investigations [4,15] have been devoted to analyses of this interaction The most contradictory results were obtained in studies on the ability of sHSPs to affect actin polymerization and to interact with native actin filaments It was supposed that some sHSPs (Hsp25, Hsp27) may act as actincapping proteins, which inhibit actin polymerization depending on their oligomeric state and extent of phosphorylation [16–18] Recently published data indicate that Hsp27 interacts with monomeric actin and by this means might affect actin polymerization [19] However, direct involvement of Hsp27 in the regulation of actin polymerization still remains questionable, and has not been confirmed in other publications [20,21] Another sHSP, Hsp20, was also claimed to be a genuine actin-binding protein involved in the regulation of smooth muscle contraction [22] However, more recently, it was found that Hsp20 does not 5938 directly interact with actin filaments either in solution or in myofibrils obtained from smooth, cardiac or skeletal muscle [20] Thus, at present, it seems unlikely that the sHSPs can act as genuine actin-binding proteins under normal conditions It seems more likely that sHSPs interact with actin only under unfavorable conditions Disruption of actin filaments is among the most immediate early effects of various stresses Multiple publications indicate that different stress conditions, such as oxidative stress, acidosis, energy depletion, heat shock, or excessive contractile activity, might induce translocation of sHSPs from cytosol to cytoskeleton and that this translocation can result in stabilization of actin filaments [23–27] Very recently, it has been shown that, under heat shock conditions (upon incubation at 43 °C), aB-crystallin, a member of the sHSP family, directly interacts with actin in immunoprecipitation experiments, and associates with actin filaments in living cells, and that this in vivo interaction of aB-crystallin prevents heat-induced disorganization of actin filaments [28] However, no effects of aB-crystallin were observed in unstressed cells These facts agree with the data showing that in vitro sHSPs not interact with intact actin filaments [2,20], but prevent heatinduced aggregation of actin [2,20,21,29] Thus, it seems probable that sHSPs interact with actin filaments only under stress conditions, such as heat shock, but the exact molecular mechanism of this interaction is not clearly understood We have previously shown that, in solution, some recombinant sHSPs (chicken Hsp24, human Hsp27, and their 3D mutants mimicking phosphorylation) have no influence on the thermal unfolding of F-actin as measured by differential scanning calorimetry (DSC), but they effectively prevent aggregation of thermally denatured actin [2] Furthermore, we analyzed in cosedimentation experiments the interaction of denatured actin with the S15D ⁄ S78D ⁄ S82D mutant construct of Hsp27, hereafter referred to as Hsp27-3D, which has been proposed to mimic the properties of phosphorylated Hsp27 in vitro [12] It has been shown that, after heating of F-actin in the presence of Hsp273D, denatured actin does not precipitate upon highspeed centrifugation and is found in the supernatant together with Hsp27-3D, whereas both intact F-actin and F-actin heated in the absence of Hsp27-3D fully precipitate under the same conditions [2] From these data, we proposed that Hsp27-3D and other sHSPs can form relatively small, stable and highly soluble complexes with denatured actin, and this is the mechanism by which sHSPs prevent the aggregation of F-actin FEBS Journal 274 (2007) 5937–5948 ª 2007 The Authors Journal compilation ª 2007 FEBS A V Pivovarova et al In the present work, we performed further studies on the complexes of Hsp27-3D with denatured actin We applied dynamic light scattering (DLS), analytical ultracentrifugation and size exclusion chromatography (SEC) to examine some properties of these complexes, such as their size and stoichiometry Hsp27-3D is especially useful for such experiments, due to its very small size, it being much smaller than wild-type human Hsp27 [10,11,30,31] and many other sHSPs, which usually form large oligomers [3–7,9] This mutant imitates phosphorylation of Hsp27 by MAPKAP2 kinase [12], and this phosphorylation is induced by different stimuli and stress conditions [19,23,24] Phosphorylation (or mutations) induces dissociation of large oligomers of Hsp27 and formation of small dimers and tetramers that are much more useful for investigation of interactions with denatured actin than large, variably sized oligomers formed by nonphosphorylated Hsp27 Our results demonstrate that, upon heating, thermal unfolding of F-actin is accompanied by formation of stable, soluble complexes of Hsp27-3D with denatured actin that contain roughly equal quantities of denatured actin and Hsp27-3D Results DLS studies of actin–Hsp27-3D complexes formed upon thermal denaturation of F-actin Previously, we have shown that Hsp27-D has no influence on the thermal unfolding of F-actin as measured by DSC, but effectively prevents aggregation of thermally denatured actin [2] Here we applied DLS to investigate in more detail the Hsp27-3D effects on actin aggregation in the course of thermal denaturation of F-actin Previous studies have shown that the DLS method allows determination of the size of particles formed in the process of protein aggregation during heating [32–35] We performed the DLS experiments under similar conditions and at the same heating rate (1 C°Ỉmin)1) as used in the previously described DSC measurements [2], except that a lower actin concentration (0.5 mgỈmL)1 instead of 1.0 mgỈmL)1) was used Under these conditions, F-actin denatures within a temperature range of 55–70 °C, with a maximum at 61 °C [1,2] Before thermal denaturation (i.e at temperatures up to 55 °C), F-actin demonstrates, as expected, a very random distribution of hydrodynamic radius (Rh) values, from 10 nm to 1000 nm and even to a few micrometers (Figs 1A and 2A) Obviously, real Rh values cannot be obtained by DLS for long actin Stable complexes of small HSP with denatured actin Fig Formation of the complexes of denatured actin with Hsp273D as studied by DLS (A) F-actin (0.5 mgỈmL)1) was heated at a constant rate of C°Ỉmin)1 in the presence of Hsp27-3D (0.125 mgỈmL)1), and Rh was plotted as a function of temperature (B) After being heated to 85 °C, the sample was cooled and incubated at 25 °C, and Rh was plotted as a function of incubation time Other conditions: 30 mM Hepes (pH 7.3), 100 mM NaCl, and mM MgCl2 DLS measurements were carried out at a scattering angle of 90° filaments of different length The Rh distribution was essentially the same for F-actin in the presence and absence of Hsp27-3D (Fig 2A) This agrees with our previous results [2] showing that, in vitro, Hsp27-3D does not interact with native actin filaments In the absence of Hsp-3D, thermal denaturation of F-actin led to the formation of very large aggregates with Rh up to 10 lm (Fig 2B) In contrast, in the presence of Hsp27-3D, the F-actin thermal denaturation was accompanied by complete disappearance of large particles with high Rh, and only small particles with Rh of  17 nm were detected (Fig 1A) When F-actin thermal denaturation was completed, at 70 °C we observed a very narrow Rh distribution, with an average Rh of 17 nm (Fig 2B) These small particles retained their size on following heating up to 80 °C, and some slight increase in Rh was only observed at temperatures above 80 °C (Fig 1A) The Rh reached 40–50 nm at 84 °C (Fig 1A), and this Rh value remained unchanged after cooling the sample to 25 °C (Fig 1B) Similar DLS experiments were performed under conditions when F-actin at a constant concentration of 0.5 mgỈmL)1 was heated in the presence of Hsp27-3D at various concentrations, from 0.015 to 0.5 mgỈmL)1 The results show that the Rh value for the complexes of Hsp27-3D with denatured actin strongly depends on the Hsp27-3D concentration in the sample (Fig 3) The Rh of the complexes decreased from 53 to 16– 17 nm with an increase in the concentration of added FEBS Journal 274 (2007) 5937–5948 ª 2007 The Authors Journal compilation ª 2007 FEBS 5939 Stable complexes of small HSP with denatured actin A V Pivovarova et al Fig Dependence of Rh for the complexes of denatured actin with Hsp27-3D on the Hsp27-3D concentration in the initial incubation mixture of Hsp27-3D with F-actin The Hsp27-3D concentration varied from 0.015 to 0.5 mgỈmL)1, and the F-actin concentration was constant and equal to 0.5 mgỈmL)1 Other conditions were the same as in Fig 1A The Rh values were determined from the Rh distributions obtained after F-actin thermal denaturation at 70 °C The Hsp27-3D ⁄ actin weight ratios in the initial mixture are indicated for each point Rh of 16 nm under saturation conditions) is much smaller than the corresponding values for native actin filaments or actin aggregates formed upon thermal denaturation of F-actin in the absence of Hsp27-3D Analytical ultracentrifugation of the Hsp27-3D complexes with denatured actin Fig Distribution of the particles by their size (Rh) for F-actin (0.5 mgỈmL)1) in the absence or in the presence of Hsp27-3D (0.125 mgỈmL)1) registered before F-actin thermal denaturation (at 30–35 °C) (A) and after F-actin denaturation (at 70 °C) (B) Conditions were the same as in Fig 1A Each plot is an average of 10 distributions obtained within the temperature range 30–35 °C in Fig 1A (A), or five distributions obtained within the range 69–71 °C (B) Hsp27-3D from 0.015 to 0.125 mgỈmL)1, and this Rh value ( 16 nm) remained almost unchanged upon a further increase in Hsp27-3D concentration up to 0.5 mgỈmL)1 These results suggest that the smallest complexes of Hsp27-3D with denatured actin are formed at Hsp27-3D concentrations above 0.125 mgỈmL)1, i.e at an Hsp27-3D ⁄ actin weight ratio higher than : Thus, the results of DLS experiments clearly demonstrate formation of stable complexes of Hsp27-3D with denatured actin The size of these complexes (average 5940 As already mentioned, the soluble complexes of Hsp27-3D with denatured actin, which are formed in the course of thermal denaturation of F-actin, retained their size after cooling to room temperature (Fig 1) This property of the complexes allows their investigation in sedimentation velocity experiments F-actin (0.5 mgỈmL)1) was heated at a constant rate of C°Ỉmin)1 up to 75 °C, i.e to complete actin denaturation, in the presence of Hsp27-3D at different concentrations, from 0.1 to 0.4 mgỈmL)1 In all cases, we did not observe any significant increase in light scattering, which normally accompanies thermal denaturation of F-actin in the absence of sHSPs, and this indicated that Hsp27-3D formed soluble and relatively small complexes with denatured actin After cooling, the samples were used for analytical ultracentrifugation to study the sedimentation behavior of these complexes The differential distributions c(s, f ⁄ f0) of sedimentation coefficients (s) and for the actin–Hsp27-3D FEBS Journal 274 (2007) 5937–5948 ª 2007 The Authors Journal compilation ª 2007 FEBS A V Pivovarova et al Fig Sedimentation velocity analysis of the complexes of Hsp273D with thermally denatured actin The complexes were obtained by heating of F-actin (0.5 mgỈmL)1) to 75 °C at a constant rate of C°Ỉmin)1 in the presence of 0.1 mgỈmL)1 (A), 0.2 mgỈmL)1 (B) and 0.4 mgỈmL)1 (C) Hsp27-3D Differential sedimentation coefficient distributions [c(s, f ⁄ fo) versus s] were obtained at 20 °C (after cooling the species) and saved as the one-dimensional c(s,*) distributions The rotor speed was 30 000 r.p.m Other conditions were: 30 mM Hepes (pH 7.3), 100 mM NaCl, and mM MgCl2 complexes obtained at different Hsp27-3D concentrations are shown in Fig 4A–C In all cases, the c(s, f ⁄ f0) distributions of the complexes exhibit several peaks whose sedimentation coefficients depend on the Hsp27-3D concentration At the lowest Hsp27-3D concentration (0.1 mgỈmL)1), the sample exhibits a sedimentation coefficient in the range 10–45 S, and the c(s, f ⁄ f0) distribution is represented by four main peaks with maxima at 14–17 S, 22.5 S, 28 S, and 35 S (Fig 4A) Increasing the Hsp27-3D concentration in Stable complexes of small HSP with denatured actin the sample up to 0.2 mgỈmL)1 resulted in almost complete disappearance of the fractions with s > 30 S (Fig 4B) In this case, the c(s, f ⁄ f0) distribution of the complex is represented by the main, large-amplitude peak with a maximum at 21.6 S, a small peak at 29.8 S, and several badly resolved peaks at 8–18 S A further increase in the Hsp27-3D concentration (up to 0.4 mgỈmL)1) resulted in full disappearance of all peaks with s > 30 S, narrowing of the distribution curve, and shifting of the distribution curve to the lower s-values (Fig 4C) Under these conditions, the c(s, f ⁄ f0) distribution curve of the actin–Hsp27-3D complex shows three peaks of similar amplitude, with maxima at 14 S, 17 S, and 19.4 S, and several small peaks at S, 11.4 S, and 28.2 S Besides the above mentioned peaks, all c(s, f ⁄ f0) distribution curves also contain the peak at 3.0–3.2 S (Fig 4A–C) This peak is assigned to Hsp27-3D unbound to actin, in good agreement with previous reports that unheated Hsp27-3D has a sedimentation coefficient of  S [11,30,31], which is believed to correspond to Hsp27-3D dimers Previous studies have shown that isolated Hsp27-3D denatures at 70 °C, and its thermal denaturation is completely reversible [2] Thus, under the conditions used here, Hsp27-3D fully denatured when the samples were heated to 75 °C, and then fully renatured upon cooling prior to sedimentation experiments The results presented in Fig show that the denaturation–renaturation procedure had no significant influence on the sedimentation behavior of Hsp27-3D Increasing Hsp27-3D concentration in the sample increases the amplitude of the peak at 3.2 S (Fig 4A–C), and this indicates that the amount of actin-free Hsp27-3D, increases with increasing concentration of added Hsp27-3D Thus, the results of these experiments show that, under the conditions used, a proportion of Hsp273D is involved in the formation of stable complexes with denatured actin, which exhibit a sedimentation coefficient in the range 8–40 S, depending on the Hsp27-3D concentration, with average s20,w of about 17–20 S The remaining Hsp27-3D remains free and sediments with s20,w ¼ 3–3.2 S Knowing the total concentration of Hsp27-3D and determining the quantity of free Hsp27-3D, we can calculate the amount of Hsp27-3D involved in the complexes with denatured actin, and by this means estimate the stoichiometry Hsp27-3D ⁄ actin in these complexes Unfortunately, analytical ultracentrifugation cannot provide exact data on the concentration of free Hsp27-3D in the probes For this purpose, we applied SEC FEBS Journal 274 (2007) 5937–5948 ª 2007 The Authors Journal compilation ª 2007 FEBS 5941 Stable complexes of small HSP with denatured actin A V Pivovarova et al Stoichiometry of the Hsp27-3D–actin complexes analyzed by SEC In general, the probes for SEC experiments were prepared by the same means as for analytical ultracentrifugation, except that higher protein concentrations were used F-actin (1.0 mgỈmL)1) was heated at a constant rate of C°Ỉmin)1 up to 68 °C in the absence or in the presence of Hsp27-3D (0.25–1.0 mgỈmL)1) Under these conditions, F-actin was fully denatured both in the absence and in the presence of Hsp27-3D [2] In the absence of Hsp27-3D, thermal denaturation of F-actin was accompanied by a strong increase in light scattering, whereas in the presence of Hsp27-3D, only a small increase in light scattering was observed (Fig 5A) The amplitude of light scattering was dependent on the concentration of Hsp27-3D (Fig 5A) After cooling, the samples were subjected to highspeed centrifugation (20 at 140 000 g) to sediment protein aggregates, and the supernatants thus obtained (Fig 5B) were subjected to SEC to separate soluble complexes formed by Hsp27-3D with denatured actin from actin-free Hsp27-3D F-actin heated up to 68 °C in the absence of Hsp273D was fully precipitated upon ultracentrifugation, and therefore no peaks were detected on the elution profile (data not shown) When F-actin was heated in the presence of Hsp27-3D, subjected to ultracentrifugation, and loaded on the SEC column, we detected two peaks on the elution profile (Fig 6A) According to the data of SDS ⁄ PAGE, the first asymmetric peak, eluted close to the void volume (8–10 mL), contained both actin and Hsp27-3D (data not shown), thus indicating that this peak contains soluble complexes formed by denatured actin and Hsp27-3D Surprisingly, the size of this peak on the elution profile was constant and independent of the initial concentration of Hsp27-3D (Fig 6A) We suppose that the majority of soluble complexes formed by denatured actin and Hps27-3D were retarded on the column filter, and only a small, nearly equal proportion of these complexes entered the column and was detected in the first peak on the elution profile The second peak, eluted at about 14.2 mL (apparent molecular mass about 100 kDa), contained isolated Hsp27-3D The size of this peak was clearly increased with increasing initial concentrations of Hsp27-3D in the incubation mixture Thus, knowing the initial total concentration of Hsp27-3D and the concentration of Hps27-3D remaining free, we were able to indirectly estimate the concentration of Hps27-3D bound to denatured actin Plotting the concentration of Hsp273D bound to denatured actin against the total concen5942 Fig Concentration-dependent effect of Hsp27-3D on the heatinduced aggregation of F-actin (A) F-actin (1.0 mgỈmL)1) was heated at a constant rate of C°Ỉmin)1 in the absence (curve 1) or in the presence (curves 2–4) of Hsp27-3D, and aggregation was followed by light scattering at 350 nm The Hsp27-3D concentration was equal to 0.25, 0.5 and 1.0 mgỈmL)1 for curves 2, 3, and 4, respectively Other conditions were the same as in Fig 1A After being heated to 68 °C, the samples were cooled and subjected to ultracentrifugation, and protein composition of supernatants was analyzed by SDS ⁄ PAGE (B) Lanes and represent control unheated F-actin (0.5 mgỈmL)1) and Hsp27-3D (0.5 mgỈmL)1), respectively Lanes 3–7: supernatants obtained from the samples subjected to heating up to 68 °C and ultracentrifugation Lanes 3–5: F-actin in the presence of 1.0, 0.5 and 0.25 mgỈmL)1 Hsp27-3D, respectively Lanes and 7: F-actin alone and Hsp27-3D alone (0.5 mgỈmL)1), respectively Positions of actin and Hsp27-3D are marked on the left tration of Hsp27-3D, we tried to determine the stoichiometry of the complexes formed Unfortunately, at fixed F-actin concentration (24 lm) and Hsp27-3D concentrations varying in the range 0–44 lm, we were unable to achieve saturation (Fig 6B) Probably, saturation can be reached at higher Hsp27-3D concentrations that were unattainable under the conditions used Therefore, we performed similar experiments under different conditions, i.e at a constant Hsp27-3D concentration of mgỈmL)1 ( 44 lm) and various F-actin concentrations (0.25–3.0 mgỈmL)1 or 6–70 lm) The probes containing different concentrations of actin and fixed concentration of Hsp27-3D were heated FEBS Journal 274 (2007) 5937–5948 ª 2007 The Authors Journal compilation ª 2007 FEBS A V Pivovarova et al Fig Analysis of actin–Hsp27-3D complexes by SEC The complexes were obtained at a constant F-actin concentration of 1.0 mgỈmL)1 and different Hsp27-3D concentrations as shown in Fig (A) Equal volumes (500 lL) of each sample were subjected to SEC on a Superdex 200 HR 10 ⁄ 30 column Curve-1 corresponds to Hsp27-3D alone (0.5 mgỈmL)1) Curves 2–4 correspond to the actin–Hsp29-3D complexes obtained at Hsp27-3D concentrations of 1.0, 0.5 and 0.25 mgỈmL)1, respectively (lanes 3–5 in Fig 5B) (B) Dependence of molar concentration of Hsp27-3D bound to denatured actin in their complexes obtained at a constant F-actin concentration on the concentration of added Hsp27-3D The concentration of bound Hsp27-3D was calculated as the difference between the concentration of added Hsp27-3D and that of actin-free Hsp27-3D in the samples as determined from (A) Stable complexes of small HSP with denatured actin detected on the elution profile (Fig 7A) The first peak contained soluble complexes containing denatured F-actin and Hsp27-3D (Fig 7B), whereas the second peak contained isolated Hps27-3D (Fig 7C) The first peak was eluted close to the void volume (8–10 mL), and its size was only slightly increased upon increase of the initial F-actin concentration (Fig 7, insert) For instance, a 12-fold increase of initial F-actin concentration was accompanied by a less than two-fold increase of the first peak This is probably because a large proportion of the complexes formed by denatured actin and Hsp27-3D that remains in the supernatant after ultracentrifugation was retarded on the column filter, and only a small proportion of these complexes entered the column and was detected in the first peak This means that the size of the first peak cannot be directly used for correct determination of the quantity of complexes formed by denatured actin and Hsp273D In contrast, the size of the second peak corresponding to isolated Hsp27-3D was strongly dependent on the initial F-actin concentration, and an increase of actin concentration was accompanied by significant decrease in the Hsp27-3D remaining free Thus, the size of this peak provides information on the quantity of actin-free Hsp27-3D Isolated Hsp27-3D (1 mgỈmL)1) was either kept on ice or heated up to 70 °C in the absence of F-actin, and after ultracentrifugation was subjected to SEC (curves and in Fig 7A) The size of the peaks was not dependent on prior heating, thus indicating high thermal stability of isolated Hsp27-3D Measuring the size of this peak and comparing it with the size of corresponding peaks obtained in the presence of variable concentrations of F-actin, we were able to determine the concentration of Hsp27-3D remaining free at different actin concentrations The concentration of Hsp27-3D bound to denatured actin was determined by subtracting the concentration of free Hps27-3D from the total concentration of Hsp27-3D Plotting the concentration of actin-bound Hps27-3D against the F-actin ⁄ Hsp27-3D molar ratio in the initial mixture (Fig 7D), we found that saturation was achieved at a molar ratio close to : This means that under conditions of saturation, denatured actin and Hsp27-3D form equimolar complexes Discussion to 68 °C under the above mentioned conditions The samples were cooled, and subjected to ultracentrifugation, and the supernatants obtained were loaded on the Superdex 200 column Again, two peaks were This article expands our knowledge of the mechanism by which Hsp27-3D and probably other mammalian sHSPs protect F-actin from heat-induced aggregation Previous work has clearly demonstrated that sHSPs FEBS Journal 274 (2007) 5937–5948 ª 2007 The Authors Journal compilation ª 2007 FEBS 5943 Stable complexes of small HSP with denatured actin A V Pivovarova et al Fig SEC analysis of the actin–Hsp27-3D complexes obtained at a constant Hsp27-3D concentration The complexes were obtained by heating an F-actin–Hsp27-3D mixture to 70 °C at a constant rate of C°Ỉmin)1 Experiments were performed with a constant Hsp27-3D concentration equal to mgỈmL)1 and different F-actin concentrations, varying from 0.25 to 3.0 mgỈmL)1 After being cooled, the samples were subjected to ultracentrifugation, and equal volumes of the supernatants (500 lL) were analyzed by SEC (A) SEC curves and correspond, respectively, to control unheated Hsp27-3D and Hsp27-3D heated to 70 °C, both at a concentration of 0.5 mgỈmL)1 Curves 3–7 correspond to Hsp27-3D (1 mgỈmL)1) heated in the presence of 0.25, 0.5, 1.0, 2.0 and 3.0 mgỈmL)1 F-actin, respectively The inset expands the region of 8–11.5 mL elution volume for clarity (B) SDS ⁄ PAGE for the actin– Hsp27-3D complexes obtained at a constant Hsp27-3D concentration (1.0 mgỈmL)1) and different F-actin concentrations: 0.25 (3), 0.5 (4), 1.0 (5), 2.0 (6) and 3.0 mgỈmL)1 (7) The lane numbers correspond to the numbers of SEC curves in (A) In all cases, fractions with elution volumes from 8.5 to 9.0 mL in (A) were collected, combined, and subjected to SDS ⁄ PAGE (C) SDS ⁄ PAGE of free Hsp27-3D (1, 2) and in the presence of denatured actin (3–7) [fractions with an elution volume of 14 mL in (A)] The lane numbers correspond to those for SEC curves in (A) (D) Dependence of molar concentration of Hsp27-3D bound to denatured actin in their complexes obtained at a constant Hsp27-3D concentration (44 lM) on the F-actin ⁄ Hsp27-3D molar ratio in the initial incubation mixture The concentration of actin-bound Hsp27-3D was calculated as in Fig 6A, using SEC data from (A) and a molecular mass of Hsp27-3D monomer equal to 22.8 kDa have no effect on the F-actin thermal unfolding measured by DSC, but they effectively prevent aggregation of thermally denatured actin [2] Based on previous results of cosedimentation experiments [2], we have proposed that Hsp27-3D and probably other sHSPs prevent heat-induced aggregation of F-actin by forming relatively small, stable and highly soluble complexes with denatured actin In the present work, we studied the properties of these complexes using DLS, SEC, and analytical ultracentrifugation For this 5944 purpose, we used Hsp27-3D, as this Hsp27 mutant mimicking naturally occurring phosphorylation is known to exist in vitro in small-size oligomers that are much smaller than the large oligomers of many other sHSPs [3–5,7,9–11,30,31] Comparison of the DLS results shown here (Fig 1A) with DSC data obtained earlier [2] clearly shows that actin–Hsp27-3D complexes are formed during the course of F-actin thermal denaturation All the methods used here show that the size of these complexes depends on the Hsp27-3D ⁄ actin ratio in the initial mixture of Hsp27-3D and F-actin (Figs 3, and 7A) Each method (DLS, SEC, sedimentation velocity analysis) has some advantages and drawbacks [36] However, all the methods clearly show that the size (and mass) of the actin– Hsp27-3D complexes decreases with increase in the Hsp27-3D content in the initial mixture Saturation of the complexes with Hsp27-3D molecules occurs at approximately equimolar concentrations of Hsp27-3D and actin (Figs and 7D) This agrees with previous studies on the sHSP complexes with various denatured proteins, suggesting a maximum binding capacity of one protein subunit per one sHSP subunit [37] Under these conditions, the actin–Hsp27-3D FEBS Journal 274 (2007) 5937–5948 ª 2007 The Authors Journal compilation ª 2007 FEBS A V Pivovarova et al complexes exhibit Rh of  16 nm and s20,w of about 17–20 S (Figs and 4) It should be noted that Hsp27-3D (Fig 5) and other sHSPs [2] effectively prevent F-actin thermal aggregation, even at rather low sHSP concentrations, when the sHSP ⁄ actin molar ratio is much lower than : It is noteworthy that the complexes of a similar size, with Rh of about 16 nm and s20,w of about 17– 20 S, were observed previously in DLS and analytical ultracentrifugation experiments not only with Hsp273D, but also after heating of F-actin in the presence of a-crystallin [38] In that case, however, we could not clearly separate the complexes of a-crystallin with denatured actin from actin-free a-crystallin, which formed large oligomers with Rh  11 nm [33] and s20,w ¼ 18 ± S [39,40] (Incidentally, this was the reason why we used only Hsp27-3D in the present work) Nevertheless, the similarity between Hsp27-3D and a-crystallin in the size of their complexes with denatured actin suggests that this parameter of the complexes is mainly determined by the target protein (denatured actin), but independent of the sHSP used This agrees with previous studies showing that mouse Hsp25 and yeast Hsp26, the two members of the sHSP family that significantly differ in their quaternary structure, form similar complexes with various denatured proteins, and the size of these complexes is dependent only on the target protein [37] Taken together, all these results support a viewpoint that the formation of soluble complexes with non-native proteins is a conserved feature of the sHSP family of chaperones, and the morphology of these complexes is substrate-dependent, but independent of the sHSP used [37] Previous electron microscopy studies showed spherical, regularly shaped particles formed by Hsp25 or Hsp26 with various denatured target proteins [37] Assuming a spherical shape for the actin–Hsp27-3D complexes, we can estimate the apparent molecular mass of the complex using an empirical relationship between the relative molecular mass and the hydrodynamic radius: Mr ¼ (1.68Rh)2.3394 [41] According to this estimation, the particles with Rh of  16 nm (i.e the actin–Hsp27-3D complexes formed under saturation conditions) have a molecular mass of about MDa If we take into account equal amounts of actin (42 kDa) and Hsp27-3D (molecular mass of the monomer 22.8 kDa) in their complexes, then the complexes with Rh of  16 nm should contain about 30 denatured actin monomers and an equal quantity of Hsp27-3D monomers Thus, the number of denatured actin molecules in their complexes with Hsp27-3D is much lower than Stable complexes of small HSP with denatured actin in intact actin filaments, which contain hundreds and even thousands of actin subunits This is consistent with a recently proposed dissociative mechanism of F-actin thermal denaturation [1] One of the main features of this mechanism is that the actin filament denatures not as a whole, but as separate monomers or short oligomers that dissociate from the filament during heating In the absence of sHSP, denatured actin monomers (or short oligomers) easily aggregate, and during this process even undamaged actin filaments become trapped and are precipitated The results presented here, together with the data obtained earlier [2], suggest that sHSPs bind to denatured actin monomers or short oligomers and protect them from aggregation by forming relatively small and highly soluble complexes, whose size is much less than that of intact F-actin We suppose that this is the mechanism by which sHSPs prevent the aggregation of F-actin during its thermal denaturation In many respects, this mechanism is similar to that postulated earlier for different soluble enzymes [37,42,43] Generally, sHSPs cannot protect target proteins from denaturation and cannot refold denatured substrate However, sHSPs prevent the aggregation of denatured target proteins, forming a reservoir of folding intermediates that can either be refolded by the network of cell chaperones or passed to proteasomes for degradation Our results suggest that this reservoir, in the case of F-actin, is presented as soluble and relatively small complexes formed by sHSPs with denatured actin molecules obtained during the heating of F-actin In conclusion, the analysis of the properties of the complexes formed between sHSP and denatured actin, as performed by different methods, provides new insights into the mechanism by which sHSPs prevent the aggregation of F-actin induced by its thermal denaturation This mechanism may explain how sHSPs protect the cytoskeleton and the whole cell from damage caused by accumulation of large, insoluble aggregates under heat shock conditions Experimental procedures Proteins Rabbit skeletal actin was prepared by the method of Spudich & Watt [44] Its concentration was determined by its absorbance at 290 nm, using an E1% of 6.3 cm)1 Monomeric G-actin in G buffer (2 mm Tris ⁄ HCl, pH 8.0, 0.2 mm ATP, 0.2 mm CaCl2, 0.5 mm b-mercaptoethanol, mm NaN3) was polymerized into F-actin filaments by the addition of MgCl2 to a final concentration of mm FEBS Journal 274 (2007) 5937–5948 ª 2007 The Authors Journal compilation ª 2007 FEBS 5945 Stable complexes of small HSP with denatured actin A V Pivovarova et al Prior to experiments, F-actin was diluted to a final concentration (from 0.25 to 3.0 mgỈmL)1) with 30 mm Hepes (pH 7.3), containing 100 mm NaCl and mm MgCl2 Recombinant human Hsp27-3D was cloned, expressed and purified as described previously [21,45] All proteins were homogeneous according to SDS ⁄ PAGE [46] Soluble complexes of Hsp27-3D with denatured actin were formed by heating the mixture of F-actin and Hsp27-3D at a constant rate of C°Ỉmin)1 up to a temperature at which full, irreversible denaturation of F-actin occurred (above 68 °C) [2] Insoluble aggregates were removed, if necessary, by high-speed centrifugation of the samples (20 at 140 000 g) Analytical ultracentrifugation Sedimentation velocity experiments were carried out in a model E analytical ultracentrifuge (Beckman) equipped with absorbance optics, a photoelectric scanner, a monochromator, and a computer on-line A four-hole rotor An-F Ti and 12 mm double sector cells were used The sedimentation profiles of the actin–Hsp27-3D complexes were recorded by measuring the absorbance at 280 nm All cells were scanned simultaneously The time interval between scans was The sedimentation coefficients were estimated from the differential sedimentation coefficient distribution [c(s, f ⁄ f0) versus s], which was analyzed using the sedfit program [47,48] F-actin aggregation Thermally induced aggregation of F-actin was detected by changes in light scattering at 90° as described previously [1,2] The measurements were performed on a Cary Eclipse fluorescence spectrophotometer (Varian Australia Pty Ltd, Mulgrave, Victoria, Australia) equipped with a temperature controller and thermoprobes F-actin in the absence or in the presence of Hsp27-3D was heated at a constant rate of C°Ỉmin)1 from 30 °C up to 68–75 °C The light scattering at 350 nm was measured with excitation and emission slits of 2.5 and 1.5 nm, respectively When the heating was completed, the samples were cooled, and the aliquots were withdrawn and subjected to ultracentrifugation at 140 000 g for 20–30 on a Beckman airfuge (Beckman Instruments Inc., Palo Alto, CA, USA) The protein composition of the supernatants and pellets was determined by SDS ⁄ PAGE [46] DLS DLS measurements were performed on a Photocor Complex apparatus (Photocor Instruments Inc., College Park, MD, USA) equipped with a temperature controller [33,34] The sample protein solution was illuminated by a 633 nm laser light, and the scattering signal was observed at an angle of 90° During the course of measurements, the temperature fluctuations were approximately ± 0.1 °C DLS data were accumulated and analyzed with the multifunctional real-time correlator Photocor-FC dynals software (Alango, Tirat Carmel, Israel) was used for polydisperse analysis of DLS data The mean hydrodynamic radius of the particles, Rh, was calculated from the Stokes–Einstein equation: D ¼ kBT ⁄ 6pgRh, where D is the diffusion coefficient obtained from the DLS measurements, kB is Boltzmann’s constant, T is the absolute temperature, and g is the shear viscosity of the solvent The viscosity of the solutions was measured on an AMVn Automated Micro Viscosimiter (Anton Paar, Graz, Austria) The data were further analyzed and plotted using origin 7.0 software (OriginLab Corp., Northampton, MA, USA) 5946 SEC Analytical SEC was carried out on a Superdex 200 HR 10 ⁄ 30 column using the ACTA-FPLC system (Amersham Pharmacia, Biotech Europe GmbH, Helsinki, Finland) The column was equilibrated with 30 mm Hepes ⁄ KOH (pH 7.3) containing 100 mm NaCl and mm MgCl2 The samples (500 lL) were loaded on the column and eluted at a rate of 0.5 mLỈmin)1 The column was calibrated with the following molecular mass markers: thyroglobulin (669 kDa), catalase (240 kDa), glyceraldehyde-3-phosphate dehydrogenase (122 kDa), BSA (68 kDa), and ovalbumin (43 kDa) Acknowledgements This work was supported by the Russian Foundation for Basic Research (grants 06-04-48343 to D I Levitsky and 07-04-00115 to N B Gusev), the Program ‘Molecular and Cell Biology’ of the Russian Academy of Sciences, and by INTAS (grant 03-51-4813) References Mikhailova VV, Kurganov BI, Pivovarova AV & Levitsky DI (2006) Dissociative mechanism of F-actin thermal denaturation Biochemistry (Moscow) 71, 1261– 1269 Pivovarova AV, Mikhailova VV, Chernik IS, Chebotareva NA, 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