Tài liệu Báo cáo khoa học: Interaction of the small heat shock protein with molecular mass 25 kDa

Interaction of the small heat shock protein with molecular mass

25 kDa (hsp25) with actin

Olesya O. Panasenko1

, Maria V. Kim1

, Steven B. Marston2 and Nikolai B. Gusev1

1

Department of Biochemistry, School of Biology, Moscow State University, Moscow, Russia; 2

Imperial College School of

Medicine at National Heart and Lung Institute, Dovehose Street, London, UK

The interaction of heat shock protein with molecular mass

25 kDa (HSP25) and its point mutants S77D + S81D (2D

mutant) and S15D + S77D + S81D (3D mutant) with

intact and thermally denatured actin was analyzed by means

of fluorescence spectroscopy and ultracentrifugation. Wild

type HSP25 did not affect the polymerization of intact actin.

The HSP25 3D mutant decreased the initial rate without

affecting the maximal extent of intact actin polymerization.

G-actin heated at 40–45
C was partially denatured, but

retained its ability to polymerize. The wild type HSP25 did

not affect polymerization of this partially denatured actin.

The 3D mutant of HSP25 increased the initial rate of poly￾merization of partially denatured actin. Heating at more

than 55
C induced complete denaturation of G-actin.

Completely denatured G-actin cannot polymerize, but it

aggregates at increased ionic strength. HSP25 and especially

its 2D and 3D mutants effectively prevent salt-induced

aggregation of completely denatured actin. It is concluded

that the interaction of HSP25 with actin depends on the state

of both actin and HSP25. HSP25 predominantly acts as a

chaperone and preferentially interacts with thermally

unfolded actin, preventing the formation of insoluble

aggregates.

Keywords: small heat shock protein; actin; thermal

denaturation.

Actin is the major component of the thin filaments of

muscle cells and of the cytoskeleton system of nonmuscle

cells. It is therefore a very abundant protein, and its

concentration in smooth muscle is close to 800–900 lM [1].

Actin has a rather complex and labile tertiary structure [2,3].

Different types of stress can induce actin unfolding [4,5],

aggregation of partially folded actin [5,6] and redistribution

of actin inside the cell [7–9]. Accumulation of partially

folded or aggregated proteins can induce significant damage

to cells. This is especially important in the case of abundant

proteins, such as actin. Therefore the cell has evolved

different mechanisms to prevent the formation of insoluble

aggregates, and heat shock proteins (HSPs) play an

important role in this process.

The data in the literature indicate that the small heat

shock protein with molecular mass 25–27 kDa (HSP25)

plays an important role in actin remodeling, contractility of

different cell types and protection of the cytoskeleton under

different unfavorable conditions [7,8,10]. Miron et al.

[11,12] showed that avian HSP25 effectively inhibits actin

polymerization and prevents gelation of actin induced by

filamin and/or a-actinin. These observations were confirmed

by Benndorf et al. [13], who showed that nonphosphoryl￾ated monomers of HSP25 effectively inhibit actin

polymerization, whereas phosphorylated monomers and

nonphosphorylated multimers of HSP25 are ineffective in

the regulation of actin polymerization. The protein seg￾ments of monomeric HSP25 involved in the inhibition of

actin polymerization were determined recently [14].

Although these data are of great interest, their application

to cell physiology is questionable as under physiological

conditions HSP25 forms high molecular mass oligomers

that are in equilibrium with low molecular mass oligomers

[15,16], but practically do not dissociate to monomers. The

actin depolymerizing effect ascribed to HSP25 [11–14]

contrasts with the stabilizing of microfilaments induced by

HSP25 or its phosphorylated forms [7,17]. Moreover,

recently Butt et al. [18] have shown that under in vitro

conditions HSP25 either does not affect or even activates the

polymerization of actin.

To explain the contradictory results described in the

literature we assumed that the mode of interaction is

dependent both on the state of HSP25 and actin. In this

paper we analyze the effect of recombinant avian HSP25

and its mutants mimicking phosphorylation on the heat￾induced aggregation and polymerization of intact and

partially denatured actin.

Materials and methods

Proteins

HSP25 from chicken gizzard was purified by the procedure

described previously [19]. Chicken HSP25 was cloned,

Correspondence to N. B. Gusev, Department of Biochemistry,

School of Biology, Moscow State University,

Moscow 119992, Russia. Tel./Fax: + 7 095 939 2747,

E-mail: NBGusev@mail.ru

Abbreviations: ANS, 8-anilinonaphtalene-1-sulfonic acid; HSP, heat

shock proteins; 1D mutant, chicken HSP25 with mutation S15D;

2D mutant, chicken HSP25 with mutation S77D + S81D;

3D mutant, chicken HSP25 with mutation S15D + S77D + S81D;

MAPKAP-2, mitogen-activated protein kinase-activated protein

kinase-2.

(Received 15 October 2002, revised 25 December 2002,

accepted 7 January 2003)

Eur. J. Biochem. 270, 892–901 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03449.x