α-Crystallin as a molecular chaperone

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Abstract

The role of α-crystallin as a molecular chaperone may explain how the lens stays transparent for so long. α-Crystallin prevents the aggregation of other lens crystallins and proteins that have become unfolded by “trapping” the protein in a high molecular weight complex. It also protects enzyme activities. The substrate protein may interact while in a molten globule state. α-Crystallin predominantly binds to proteins very early in the denaturation pathways. The amphiphilic nature of α-crystallin, a polar C-terminal-region and a hydrophobic N-terminal-region are all essential for chaperone function. The flexible C-terminal extension maintains solubility and can bind to opposing charged residues of unfolding proteins. Hydrophobic regions in the N-terminal region then hold the unfolded protein. Specific areas important for chaperone binding and function have been identified throughout the N-terminal-region, connecting peptide and C-terminal extension. After a substantial amount of chemical data and models, cryo-EM images of α-crystallin have confirmed a variable 3D surface with a hollow interior. α-Crystallin taken from the lens nucleus shows an age-dependent decrease in chaperone function. High molecular weight aggregates and α-crystallin found within the nucleus from clear and cataract lenses have reduced chaperone function. Post-translational modifications, known to occur during ageing, such as glycation, carbamylation, oxidation, phosphorylation and truncation cause a decrease in chaperone function. α-Crystallin is expressed outside the lens. αB-Crystallin can be induced by heat shock in many tissues where it is translocated from cytoplasm to nucleus. Increased expression of αB-crystallin has been seen in many pathological states. Conformational disorders, including cataract may have a common aetiology and potentially a common therapy.

Introduction

Many biochemical processes involve proteins in a partially unfolded conformation making them liable to aggregation and thus preventing formation of a functional structure. Mechanisms for coping with such processes, either inductive or constitutive, have arisen through evolution to guarantee survival. For example, when living cells are exposed to elevated temperatures they respond by over-expressing specific sets of genes that synthesise heat shock proteins (HSPs). These stress proteins appear to maintain the correct conformations and prevent the incorrect intermolecular association of unfolded polypeptide chains, which could result in their aggregation. This protective response is present in virtually every cell, tissue and organ. HSPs can be induced by a wide variety of other stresses including pH extremes, nutrient limitation, osmotic variation, hypoxia, chemotherapeutic agents and noxious chemicals. These stresses activate heat shock transcription factors, which in turn bind to specific DNA sequences known as heat shock elements. Heat shock elements are located in the promoter regions of the HSP gene and upon activation increase transcription of the respective gene (Sorger, 1991). The earliest response to heat shock was observed in Drosophila melanogaster whose salivary gland chromosomes showed “puffs” after heat stress (Tissieres et al., 1974). These are sites of very efficient, very rapid gene transcription, which takes precedence over existing transcripts while under stress.

Many stress proteins have been discovered and characterised in an extraordinary variety of organisms. They are highly conserved and are classified into families according to their subunit molecular mass, which ranges from 8 to 200-kDa. Curiously, these functionally-related proteins are structurally unrelated (Section 2). Stress proteins are located in various compartments of the cell. In eukaryotes they occur in the nucleus, nucleolus, cytosol, mitochondria, endoplasmic reticulum and chloroplasts. In prokaryotes they occur in the cytoplasm, cytoplasmic membrane and periplasmic space. Stress proteins within the cytosol are known to migrate inside or around the nucleus during stress, thereby protecting the nuclear structure. Most stress proteins are synthesised under normal cellular conditions i.e. they are constitutively expressed and therefore involved in essential basic cellular roles (Fig. 1). Pelham, (1996)proposed that members of the HSP70 family, in animal and microbial cells, are involved in the assembly and disassembly of proteins in the nucleus, cytosol and endoplasmic reticulum. Pelham suggested that under normal cellular conditions these abundant proteins are involved in protein folding and breakdown. Under stress conditions they prevent damage by binding to exposed hydrophobic surfaces and help to refold damaged protein. These ideas are the basis of the concept of molecular chaperones and have resulted in a re-examination of protein folding.

Anfinsen proposed that all the information required for a polypeptide chain to fold into its biologically active three-dimensional structure is in its amino acid sequence. This was based upon work with ribonuclease A, which when unfolded in vitro regains its biological activity on removal of the denaturing agent, suggesting that proteins are self-organising (Anfinsen, 1973). However various large, oligomeric and multidomain proteins including subtilisin E (Ikemura et al., 1987) and α-lytic protease (Silen et al., 1989) did not spontaneously refold which suggests that additional factors may be required to mediate folding. Recombinant protein expression tended to produce large insoluble protein aggregates termed inclusion bodies (Rudolph and Lilie, 1996). These bodies were observed in an Escherichia coli mutant defective in heat shock proteins thereby implicating HSPs (Gragerov et al., 1991). Anfinsen's proposal still holds true although the mechanism has been amended to account for the discovery of proteins that assist in the attainment of the native polypeptide structure, including enzymes (protein disulphide isomerase and peptidyl prolyl cis-trans isomerase) and molecular chaperones (Gething and Sambrook, 1992).

Section snippets

Role of chaperones

Molecular chaperones are a functional class of unrelated families of protein that assist the correct non-covalent assembly of other polypeptide-containing structures in vivo, but are not components of these assembled structures when they are performing their normal biological function (Ellis, 1993). The predominant function of molecular chaperones is the prevention of incorrect associations and aggregation of unfolded polypeptide chains. The ability to recognise unfolded or partially denatured

The small heat shock protein family

The small heat shock proteins (sHSPs) have subunit weights less than 35-kDa and are the lowest molecular weight family. All organisms produce one or more sHSP, but they are most numerous in plants (Waters et al., 1996). The sHSP families have characteristic features including a) significant sequence homology; b) similarities in predicted protein structure; and c) similar intracellular distribution.

Chaperone activity of α-crystallin

The role of α-crystallin as a sHSP was emphasised by its accumulation in NIH3T3 cell lines in response to cellular stress (Klemenz et al., 1991a). The αB-crystallin gene promoter is subject to stress-mediated transcriptional control. Horwitz (1992)first characterised α-crystallin as a molecular chaperone in vitro, based on its ability to prevent heat-induced aggregation of lens proteins and enzymes.

These protective capabilities have been repeated with other in vitro assays including prevention

Regions important for chaperone activity

A variety of methods have been employed in an attempt to identify those regions playing a role in chaperone activity, perhaps forming the binding region.

Ageing

The manner of lens development is unique. Ectodermal tissue, under direction from the optic cup, invaginates, then seals forming a vesicle of epithelial cells. Posterior epithelial cells elongate and eventually fill the lumen of the vesicle. They then differentiate to form primary fibre cells under the influence of growth factors and start crystallin synthesis. The anterior epithelial cells in the pre-equatorial region migrate to the equator and then elongate both along the posterior capsule

Post-translational modifications

Many proteins are modified after translation by reactions catalysed and controlled by enzymes, e.g., phosphorylation, glycation, acetylation, and hydroxylation. However, longer-lived proteins such as lens proteins and collagen are candidates for the accumulation of non-enzymatic modifications (Harding et al., 1989a). Modifications of short-lived proteins may result in removal and replacement, essentially keeping modifications of reactive proteins to a minimum. There is no removal of lens

Role outside the lens

Although originally thought of as only contributing to the structural and refractive properties of the lens, α-crystallin is now known to be a member of the sHSP family. Another breakthrough was the discovery that expression of α-crystallin is not restricted to the lens and may have wider functional significance.

The extra-lenticular expression of αB-crystallin has been reported in many tissues (see below) but αA-crystallin was found only at very low levels in the spleen and thymus (Kato et al.,

Conclusion and future directions

α-Crystallin has many of the properties of a molecular chaperone, in particular protecting various proteins against aggregation induced by heating, by chaotropic agents, by reduction, and by chemical modification. It protects several enzymes against modification-induced loss of activity. In some studies protective complexes have been identified.

The discovery that α-crystallin behaves as a molecular chaperone has resulted in a tremendous surge in experimentation and reassessment of the role of α

Acknowledgements

The authors are indebted to Research into Ageing and the Trustees of the Oxford Eye Hospital for their financial support. We also wish to thank Prof. M. James C. Crabbe for his critical reading of the manuscript.

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