Tài liệu Báo cáo Y học: Functional analysis of a small heat shock/a-crystallin protein from Artemia

Functional analysis of a small heat shock/a-crystallin protein

from Artemia franciscana

Oligomerization and thermotolerance

Julie A. Crack, Marc Mansour, Yu Sun and Thomas H. MacRae

Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada

Oviparously developing embryos of the brine shrimp,

Artemia franciscana, synthesize abundant quantities of a

small heat shock/a-crystallin protein, termed p26. Wild-type

p26 functions as a molecular chaperone in vitro and is

thought to help encysted Artemia embryos survive severe

physiological stress encountered during diapause and

anoxia. Full-length and truncated p26 cDNA derivatives

were generated by PCR amplification of p26-3-6-3, then

cloned in either pET21(+) or pRSETC and expressed in

Escherichia coli BL21(DE3). All constructs gave a polypep￾tide detectable on Western blots with either p26 specific

antibody, or with antibody to the His6 epitope tag encoded

by pRSETC. Full-length p26 in cell-free extracts of E. coli

was about equal in mass to that found in Artemia embryos,

but p26 lacking N- and C-terminal residues remained either

as monomers or small multimers. All p26 constructs

conferred thermotolerance on transformed E. coli, although

not all formed oligomers, and cells expressing N-terminal

truncated derivatives of p26 were more heat resistant than

bacteria expressing p26 with C-terminal deletions. The

C-terminal extension of p26 is seemingly more important for

thermotolerance than is the N-terminus, and p26 protects

E. coli against heat shock when oligomer size and protein

concentration are low. The findings have important impli￾cations for understanding the functional mechanisms of

small heat shock/a-crystallin proteins.

Keywords: small heat shock/a-crystallin protein; oligomeri￾zation; thermotolerance; diapause; Artemia franciscana.

Cells respond to stress by the enhanced synthesis of heat

shock or stress proteins, which are also developmentally

regulated under normal physiological conditions. Stress

proteins are divided into several families on the basis of size

and amino-acid sequence [1–5]. Moreover, they function as

molecular chaperones, facilitating proper folding, transport

and multimerization of nascent proteins, as well as

preventing the irreversible aggregation of denaturing

proteins. The small heat shock/a-crystallin proteins consti￾tute a structurally divergent, ubiquitous group within the

chaperone superfamily, ranging in molecular mass from 12

to 43 kDa [6]. A conserved region, termed the a-crystallin

domain, distinguishes all small heat shock/a-crystallin

proteins, and a two or three domain structure is proposed

for these proteins [7,8]. The a-crystallin domain, located

toward the C-terminus of the protein monomer, consists of

80–100 amino-acid residues and is important for oligomer

formation and chaperoning [9–13]. Flexible C-terminal

extensions of small heat shock/a-crystallin proteins,

enriched in polar and charged amino-acid residues, vary

in length and sequence [8,14,15]. Loss or modification of the

C-terminal extension has the potential to perturb function

and reduce solubility of these proteins and their complexes

with target proteins [15–19]. The N-terminus, which may be

partly buried within the mature protein, promotes oligomer

formation, subunit exchange, and capture of unfolding

proteins [12,18,20–26].

Small heat shock/a-crystallin proteins confer thermotol￾erance upon cells [27–33], protect against apoptotic death

[34,35] and have chaperone activity in vitro, wherein the

aggregation of client proteins is prevented [36–38]. Chap￾eroning is thought to depend upon formation of oligomers

that reach 800 kDa in mass and possess quaternary

structure modifiable by environmental parameters

[8,18,20,22,39,40]. Oligomers exhibit dynamic equilibrium

with constituent subunits, which can affect chaperoning but

is not in itself sufficient to ensure chaperone activity

[25,41,42]. A small heat shock/a-crystallin protein from

Methanococcus jannaschii, termed Mj hspl6.5, has been

crystallized, revealing highly ordered oligomers of 24

subunits with a hollow center [9]. Cryoelectron microscopy

of small heat shock/a-crystallin proteins from several

sources has shown, however, that oligomer structure ranges

from well defined to variable, leading to the idea that

structural plasticity elicits low specificity and permits

binding of different target proteins [10,24,42]. Several

molecules of denaturing proteins, present in an unstable

molten globule state, interact with a single oligomer when

chaperoning occurs. The proteins are protected from

irreversible aggregation under stress, their activity may be

preserved, and they either refold spontaneously or with the

assistance of other chaperones upon release [38,43–46].

Embryos of the brine shrimp, Artemia franciscana,

develop ovoviviparously, leading to release of swimming

Correspondence to T. H. MacRae, Department of Biology, Dalhousie

University, Halifax, Nova Scotia, B3H 4J1, Canada.

Fax: + 902 494 3736, Tel.: + 902 494 6525,

E-mail: [email protected]

Abbreviations: Gp4G, guanosine 5¢-tetraphospho-5¢-guanosine;

IPTG, isopropyl thio-b-D-galactoside; HRP, horseradish peroxidase.

(Received 12 October 2001, revised 3 December 2001, accepted 5

December 2001)

Eur. J. Biochem. 269, 933–942 (2002) Ó FEBS 2002