Báo cáo khoa học: Small heat shock protein Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin docx

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 sm

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 Gusev3and

Dmitrii I Levitsky1,4

1 A N Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia

2 School of Bioengineering and Bioinformatics, Moscow State University, Russia

3 Department of Biochemistry, School of Biology, Moscow State University, Russia

4 A N Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia

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 mono-meric (G) and polymono-meric (F) forms Monomono-meric G-actin is a globular protein with a molecular mass

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

Septem-ber 2007, accepted 24 SeptemSeptem-ber 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 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

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; R h , hydrodynamic radius; sHSP, small heat shock protein; SEC, size exclusion chromatography.

of 42 kDa An important feature of actin is its

abil-ity 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

dena-tured actin [1,2] Accumulation of aggregated proteins

is dangerous for the cell, and this is especially

impor-tant in the case of abundant proteins, such as actin

There are different mechanisms for preventing

forma-tion of insoluble aggregates, and the small heat shock

proteins (sHSPs) play an important role in this

pro-cess

sHSPs comprise a large and diverse family of

pro-teins 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

olig-omeric 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

con-tradictory results were obtained in studies on the

abil-ity of sHSPs to affect actin polymerization and to

interact with native actin filaments It was supposed

that some sHSPs (Hsp25, Hsp27) may act as

actin-capping proteins, which inhibit actin polymerization

depending on their oligomeric state and extent of

phosphorylation [16–18] Recently published data

indi-cate that Hsp27 interacts with monomeric actin and by

this means might affect actin polymerization [19]

However, direct involvement of Hsp27 in the

regula-tion of actin polymerizaregula-tion still remains quesregula-tionable,

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

regula-tion of smooth muscle contracregula-tion [22] However,

more recently, it was found that Hsp20 does not

directly interact with actin filaments either in solution

or in myofibrils obtained from smooth, cardiac or skel-etal 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 fila-ments [23–27] Very recently, it has been shown that, under heat shock conditions (upon incubation at

43C), aB-crystallin, a member of the sHSP family, directly interacts with actin in immunoprecipitation experiments, and associates with actin filaments in liv-ing cells, and that this in vivo interaction of aB-crystal-lin 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 do not inter-act with intinter-act inter-actin filaments [2,20], but prevent heat-induced aggregation of actin [2,20,21,29] Thus, it seems probable that sHSPs interact with actin fila-ments 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 ana-lyzed 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 Hsp27-3D, denatured actin does not precipitate upon high-speed 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 mecha-nism by which sHSPs prevent the aggregation of F-actin

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

MAP-KAP2 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

forma-tion 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,

solu-ble 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

influ-ence on the thermal unfolding of F-actin as measured

by DSC, but effectively prevents aggregation of

ther-mally 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 CÆmin)1) as used in the previously described DSC

measurements [2], except that a lower actin

concentra-tion (0.5 mgÆmL)1 instead of 1.0 mgÆmL)1) was used

Under these conditions, F-actin denatures within a

temperature range of 55–70C, with a maximum at

61C [1,2]

Before thermal denaturation (i.e at temperatures up

to 55C), F-actin demonstrates, as expected, a very

random distribution of hydrodynamic radius (Rh)

val-ues, 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

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 denatur-ation 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 70C

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 80C, and some slight increase in Rh was only observed at temperatures above 80C (Fig 1A) The Rh reached 40–50 nm at 84C (Fig 1A), and this Rh value remained unchanged after cooling the sample to 25C (Fig 1B)

Similar DLS experiments were performed under con-ditions when F-actin at a constant concentration of 0.5 mgÆmL)1 was heated in the presence of Hsp27-3D

at various concentrations, from 0.015 to 0.5 mgÆmL)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

Fig 1 Formation of the complexes of denatured actin with Hsp27-3D as studied by DLS (A) F-actin (0.5 mgÆmL)1) was heated at

a constant rate of 1 CÆmin)1 in the presence of Hsp27-3D (0.125 mgÆmL)1), and Rhwas plotted as a function of temperature (B) After being heated to 85 C, the sample was cooled and incu-bated at 25 C, and R h was plotted as a function of incubation time Other conditions: 30 m M Hepes (pH 7.3), 100 m M NaCl, and 1 m M

MgCl2 DLS measurements were carried out at a scattering angle

of 90.

Hsp27-3D from 0.015 to 0.125 mgÆmL)1, and this Rh

value ( 16 nm) remained almost unchanged upon a

further increase in Hsp27-3D concentration up to

0.5 mgÆmL)1 These results suggest that the smallest

complexes of Hsp27-3D with denatured actin are

formed at Hsp27-3D concentrations above

0.125 mgÆmL)1, i.e at an Hsp27-3D⁄ actin weight ratio

higher than 1 : 4

Thus, the results of DLS experiments clearly

demon-strate formation of stable complexes of Hsp27-3D with

denatured actin The size of these complexes (average

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

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 investiga-tion in sedimentainvestiga-tion velocity experiments

F-actin (0.5 mgÆmL)1) was heated at a constant rate

of 1 CÆmin)1 up to 75C, i.e to complete actin dena-turation, in the presence of Hsp27-3D at different con-centrations, from 0.1 to 0.4 mgÆmL)1 In all cases, we did not observe any significant increase in light scat-tering, which normally accompanies thermal denatur-ation 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 ultracentrifuga-tion to study the sedimentaultracentrifuga-tion behavior of these complexes

The differential distributions c(s, f⁄ f0) of sedimenta-tion coefficients (s) and for the actin–Hsp27-3D

Fig 2 Distribution of the particles by their size (R h ) for F-actin

(0.5 mgÆmL)1) in the absence or in the presence of Hsp27-3D

(0.125 mgÆmL)1) registered before F-actin thermal denaturation (at

30–35 C) (A) and after F-actin denaturation (at 70 C) (B)

Condi-tions 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).

Fig 3 Dependence of Rh for the complexes of denatured actin with Hsp27-3D on the Hsp27-3D concentration in the initial incuba-tion mixture of Hsp27-3D with F-actin The Hsp27-3D concentraincuba-tion varied from 0.015 to 0.5 mgÆmL)1, and the F-actin concentration was constant and equal to 0.5 mgÆmL)1 Other conditions were the same as in Fig 1A The R h values were determined from the R h

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.

complexes obtained at different Hsp27-3D

concentra-tions 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

con-centration (0.1 mgÆmL)1), the sample exhibits a

sedi-mentation 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

the sample up to 0.2 mgÆmL)1resulted in almost com-plete 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 mgÆmL)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 9 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  3 S [11,30,31], which is believed to correspond to Hsp27-3D dimers Previous studies have shown that isolated Hsp27-3D denatures at

70C, and its thermal denaturation is completely reversible [2] Thus, under the conditions used here, Hsp27-3D fully denatured when the samples were heated to 75C, and then fully renatured upon cool-ing prior to sedimentation experiments The results presented in Fig 4 show that the denaturation–rena-turation 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 Hsp27-3D 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,w¼ 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

Fig 4 Sedimentation velocity analysis of the complexes of

Hsp27-3D with thermally denatured actin The complexes were obtained

by heating of F-actin (0.5 mgÆmL)1) to 75 C at a constant rate of

1 CÆmin)1 in the presence of 0.1 mgÆmL)1 (A), 0.2 mgÆmL)1 (B)

and 0.4 mgÆmL)1 (C) Hsp27-3D Differential sedimentation

coeffi-cient distributions [c(s, f ⁄ f o ) versus s] were obtained at 20 C (after

cooling the species) and saved as the one-dimensional c(s,*)

distri-butions The rotor speed was 30 000 r.p.m Other conditions were:

30 m M Hepes (pH 7.3), 100 m M NaCl, and 1 m M MgCl2.

Stoichiometry of the Hsp27-3D–actin complexes

analyzed by SEC

In general, the probes for SEC experiments were

pre-pared by the same means as for analytical

ultracentrif-ugation, except that higher protein concentrations were

used F-actin (1.0 mgÆmL)1) was heated at a constant

rate of 1 CÆmin)1up to 68C in the absence or in the

presence of Hsp27-3D (0.25–1.0 mgÆmL)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

depen-dent on the concentration of Hsp27-3D (Fig 5A)

After cooling, the samples were subjected to

high-speed centrifugation (20 min 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 68C in the absence of

Hsp27-3D 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

ultracentrifuga-tion, 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

indi-cating that this peak contains soluble complexes

formed by denatured actin and Hsp27-3D

Surpris-ingly, 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

Hsp27-3D bound to denatured actin against the total

concen-tration of Hsp27-3D, we tried to determine the stoichi-ometry 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, satu-ration can be reached at higher Hsp27-3D concentra-tions that were unattainable under the condiconcentra-tions used Therefore, we performed similar experiments under different conditions, i.e at a constant Hsp27-3D con-centration of 1 mgÆmL)1 ( 44 lm) and various F-actin concentrations (0.25–3.0 mgÆmL)1or 6–70 lm) The probes containing different concentrations of actin and fixed concentration of Hsp27-3D were heated

Fig 5 Concentration-dependent effect of Hsp27-3D on the heat-induced aggregation of F-actin (A) F-actin (1.0 mgÆmL)1) was heated at a constant rate of 1 CÆmin)1in the absence (curve 1) or

in the presence (curves 2–4) of Hsp27-3D, and aggregation was fol-lowed by light scattering at 350 nm The Hsp27-3D concentration was equal to 0.25, 0.5 and 1.0 mgÆmL)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 1 and 2 represent control unheated F-actin (0.5 mgÆmL)1) and Hsp27-3D (0.5 mgÆmL)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 mgÆmL)1Hsp27-3D, respectively Lanes 6 and 7: F-actin alone and Hsp27-3D alone (0.5 mgÆmL)1), respectively Positions of actin and Hsp27-3D are marked on the left.

to 68C under the above mentioned conditions The

samples were cooled, and subjected to

ultracentrifuga-tion, and the supernatants obtained were loaded on

the Superdex 200 column Again, two peaks were

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 concentra-tion was accompanied by a less than two-fold increase

of the first peak This is probably because a large pro-portion 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 Hsp27-3D

In contrast, the size of the second peak correspond-ing 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 mgÆmL)1) was either kept on ice or heated up to

70C in the absence of F-actin, and after ultracentri-fugation was subjected to SEC (curves 1 and 2 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 concen-trations The concentration of Hsp27-3D bound to denatured actin was determined by subtracting the concentration of free Hps27-3D from the total concen-tration of Hsp27-3D Plotting the concenconcen-tration 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

1 : 1 This means that under conditions of saturation, denatured actin and Hsp27-3D form equimolar com-plexes

Discussion

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

Fig 6 Analysis of actin–Hsp27-3D complexes by SEC The

com-plexes were obtained at a constant F-actin concentration of

1.0 mgÆmL)1and different Hsp27-3D concentrations as shown in

Fig 5 (A) Equal volumes (500 lL) of each sample were

sub-jected to SEC on a Superdex 200 HR 10 ⁄ 30 column Curve-1

corresponds to Hsp27-3D alone (0.5 mgÆmL)1) Curves 2–4

corre-spond to the actin–Hsp29-3D complexes obtained at Hsp27-3D

concentrations of 1.0, 0.5 and 0.25 mgÆmL)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).

have no effect on the F-actin thermal unfolding

mea-sured 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

form-ing relatively small, stable and highly soluble

com-plexes with denatured actin In the present work, we

studied the properties of these complexes using DLS,

SEC, and analytical ultracentrifugation For this

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, 4 and 7A) Each method (DLS, SEC, sedi-mentation 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 3 and 7D) This agrees with previous studies on the sHSP complexes with various dena-tured proteins, suggesting a maximum binding capac-ity of one protein subunit per one sHSP subunit [37] Under these conditions, the actin–Hsp27-3D

Fig 7 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 1 CÆmin)1 Experiments were performed with a constant Hsp27-3D concentration equal to 1 mgÆmL)1 and different F-actin concentrations, varying from 0.25 to 3.0 mgÆmL)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 1 and 2 correspond, respectively, to control unheated Hsp27-3D and Hsp27-3D heated to 70 C, both at a con-centration of 0.5 mgÆmL)1 Curves 3–7 correspond to Hsp27-3D (1 mgÆmL)1) heated in the presence of 0.25, 0.5, 1.0, 2.0 and 3.0 mgÆmL)1F-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 concentra-tion (1.0 mgÆmL)1) and different F-actin concentrations: 0.25 (3), 0.5 (4), 1.0 (5), 2.0 (6) and 3.0 mgÆmL)1(7) The lane numbers cor-respond 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) [frac-tions 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 l M )

on the F-actin ⁄ Hsp27-3D molar ratio in the initial incubation mix-ture 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.

complexes exhibit Rh of  16 nm and s20,w of about

17–20 S (Figs 3 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 1 : 1

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

Hsp27-3D, 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,w¼ 18 ± 2 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

quater-nary 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

pro-teins 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

spheri-cal, 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

hydro-dynamic radius: Mr¼ (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

satura-tion condisatura-tions) have a molecular mass of about

2 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

com-plexes 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

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 fila-ments become trapped and are precipitated The results presented here, together with the data obtained earlier [2], suggest that sHSPs bind to dena-tured 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 aggrega-tion of F-actin during its thermal denaturaaggrega-tion In many respects, this mechanism is similar to that pos-tulated 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 inter-mediates that can either be refolded by the network

of cell chaperones or passed to proteasomes for de-gradation Our results suggest that this reservoir, in the case of F-actin, is presented as soluble and rela-tively small complexes formed by sHSPs with dena-tured 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 dena-turation This mechanism may explain how sHSPs pro-tect 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,

1 mm NaN3) was polymerized into F-actin filaments by the addition of MgCl2 to a final concentration of 2 mm

Prior to experiments, F-actin was diluted to a final

con-centration (from 0.25 to 3.0 mgÆmL)1) with 30 mm Hepes

(pH 7.3), containing 100 mm NaCl and 1 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

com-plexes of Hsp27-3D with denatured actin were formed by

heating the mixture of F-actin and Hsp27-3D at a

con-stant rate of 1 CÆmin)1 up to a temperature at which full,

irreversible denaturation of F-actin occurred (above 68C)

[2] Insoluble aggregates were removed, if necessary, by

high-speed centrifugation of the samples (20 min at

140 000 g)

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

1 CÆmin)1from 30C 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

com-pleted, the samples were cooled, and the aliquots were

with-drawn and subjected to ultracentrifugation at 140 000 g for

20–30 min 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

Com-plex 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

tem-perature fluctuations were approximately ± 0.1C DLS

data were accumulated and analyzed with the

multifunc-tional 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: D¼ 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)

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 sed-imentation 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 3 min The sedimentation coefficients were esti-mated from the differential sedimentation coefficient distri-bution [c(s, f⁄ f0) versus s], which was analyzed using the sedfitprogram [47,48]

SEC

Analytical SEC was carried out on a Super-dex 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 1 mm MgCl2 The samples (500 lL) were loaded on the column and eluted at a rate of 0.5 mLÆmin)1 The column was calibrated with the following molecular mass markers: thyroglobulin (669 kDa), catalase (240 kDa), glyceralde-hyde-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 Levit-sky 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

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