Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Structure and properties of avian small heat shock protein with molecular weight 25 kDa
Introduction
Small heat shock proteins (sHsp) are a diverse group of low molecular weight (15–42 kDa) chaperones widely presented in Archaea, bacteria, plants and different phyla of animals [1], [2]. As a rule, even under normal conditions these proteins are constitutively expressed and their expression is significantly increased after heat shock or under other stress conditions [3], [4], [5]. sHsp act as molecular chaperones preventing aggregation of many different partially unfolded proteins and keeping them in folding competent state [6], [7], [8]. In co-operation with other factors, e.g. Hsp70, Hsp110 and ATP, sHsp may facilitate productive refolding of unfolded proteins [9], [10].
Many mammalian tissues (and especially muscles) simultaneously express five or six types of sHsp. Among them are αB-crystallin, heat shock proteins with molecular weight 17, 20, 22, 25/27 kDa and myotonic distrophy protein kinase binding protein (MKBP) [11], [12]. All these proteins contain in the C-terminal part of their structure conservative α-crystallin domain consisting of 80–100 amino acid residues. In the case of sHsp with molecular weight 25/27 kDa, this domain provides for formation of small oligomers of sHsp containing two or four monomers [7], [13]. These small oligomers tend to aggregate, forming large oligomers containing 16–24 (or even more) monomers [13], [14]. Formation of these oligomers seems to be dependent on the structure and properties of the N-terminal part of sHsp [13]. However, detailed mechanism of formation of large oligomers is not completely understood and different oligomers of sHsp are described for bacteria [2], [15], nematodes [16] and mammals [6], [7], [13], [14].
Under stress conditions, sHsp with molecular weight 25/27 kDa is phosphorylated by the so-called mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP-2) that is activated by p38 mitogen-activated protein kinase (MAP-kinase) [1], [2], [7], [17]. Phosphorylation of two (or three sites) by MAPKAP-2 kinase results in partial dissociation of large oligomers of sHsp with molecular weight 25/27 kDa and αB-crystallin and decrease of their chaperone activity [6], [7], [13]. Therefore, it is supposed that phosphorylation may modulate quaternary structure of sHsp [7], [13], their distribution in the cell [17], [18] and chaperone activity [6], [7].
In spite of many common features, sHsp with molecular weight 25/27 kDa isolated from different species differ in their primary structure, oligomeric state, the number of the sites of phosphorylation, chaperone activity and their ability to interact with different unfolded proteins. At present the largest progress was achieved in the analysis of the structure and properties of mammalian sHsp [1], [2], [3], [6], [7], [8], [9], [13], [14], whereas sHsp from other phyla of animals were not analysed in details. However, comparison of the properties of sHsp isolated from different species may provide important information on the organisation of this type of chaperone and on the mechanism of their functioning. Avian Hsp25 was described in the end of 1980s–beginning of 1990s [19], [20]. This protein is abundant in avian muscles and effectively inhibits actin polymerisation [5], [19], [20]. However, little is known about physicochemical properties of this protein, its quaternary structure and chaperone activity. Therefore, this investigation deals with isolation of tissue and recombinant chicken sHsp with apparent molecular weight 25 kDa, preparation of point mutants mimicking phosphorylation of Hsp25 and analysis of the structure and properties of avian Hsp25.
Section snippets
Protein preparation
Purification of chicken gizzard Hsp25 was performed by slightly modified procedure of Miron et al. [19], [20] as described earlier [21].
For isolation of recombinant protein the full-length cDNA encoding Hsp25 was cloned into pET11c(+) vector (Novagen) under control of bacteriophage T7 RNA polymerase promoter. The coding region of the cDNA from pGC25-7 plasmid (kindly provided by Professor A. Nakai Department of Molecular and Cell Biology, Institute for Frontier Medical Sciences, Kyoto
Primary structure of chicken sHsp with apparent molecular weight 25 kDa
In the first publication dealing with the primary structure of chicken Hsp25 Miron et al. [20] claimed that this protein is highly homologous (67% identity, 80% similarity) to human Hsp with molecular weight 27 kDa. Although the overall primary structures of these two proteins were very similar, the structures in the vicinity of two potential sites of phosphorylation (Ser78 and Ser82 of the human sequence) were different. According to Miron et al. [20] the primary structure of chicken Hsp25
Discussion
Avian sHsp Hsp25 was described by Miron et al. [19], [20] in the end of 1980s–beginning of 1990s. This protein was highly homologous to human sHsp with molecular weight 27 kDa except for the linker between the N-terminal part of the molecule and its α-crystallin domain. As a rule, this part of molecule contains two potential sites of phosphorylation (marked by asterisks) and has a very conservative structure RAL(S*/N)RQ(L/M)S*SG for the heat shock proteins isolated from dog (SwissProt P42929),
Acknowledgements
The authors are grateful to Prof. A. Nakai (Department of Molecular and Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Japan) for providing cDNA of chicken Hsp25 and to Dr. Alexei H. Katrukha for preparation of monoclonal antibodies against chicken Hsp25. This investigation was supported by Russian Foundation for Basic Science, joint grant of Russian Foundation for Basic Science and INTAS and by the Wellcome Trust.
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