Oligomerization, Chaperone Activity, and Nuclear Localization of p26, a Small Heat Shock Protein from Artemia franciscana

Abstract

Artemia franciscana embryos undergo encystment, developmental arrest and diapause, the last characterized by profound metabolic dormancy and extreme stress resistance. Encysted embryos contain an abundant small heat shock protein termed p26, a molecular chaperone that undoubtedly has an important role in development. To understand better the role of p26 in Artemia embryos, the structural and functional characteristics of full-length and truncated p26 expressed in Escherichia coli and COS-1 cells were determined. p26 chaperone activity declined with increasing truncation of the protein, and those deletions with the greatest adverse effect on protection of citrate synthase during thermal stress had the most influence on oligomerization. When produced in either prokaryotic or eukaryotic cells the p26 ␣-crystallin domain consisting of amino acid residues 61-152 existed predominantly as monomers, and p26 variants lacking the amino-terminal domain but with intact carboxyl-terminal extensions were mainly monomers and dimers. The amino terminus was, therefore, required for efficient dimer formation. Assembly of higher order oligomers was enhanced by the carboxyl-terminal extension, although removing the 10 carboxyl-terminal residues had relatively little effect on oligomerization and chaperoning. Full-length and carboxyl-terminal truncated p26 resided in the cytoplasm of transfected COS-1 cells; however, variants missing the complete amino-terminal domain and existing predominantly as monomers/dimers entered the nuclei. A mechanism whereby oligomer disassembly assisted entry of p26 into nuclei was suggested, this of importance because p26 translocates into Artemia embryo nuclei during development and stress. However, when examined in Artemia, the p26 oligomer size was unchanged under conditions that allowed movement into nuclei, suggesting a process more complex than just oligomer dissociation.The small heat shock proteins (sHsps), 1 characterized by a conserved ␣-crystallin domain exhibiting an immunoglobulinlike fold bordered by variable amino-and carboxyl-terminal extensions (1-7), function as molecular chaperones. The molecular mass of sHsps ranges from 12 to 43 kDa, and most oligomerize by a multistep process often with dimers as stable suboligomeric units (8, 9), although for bovine ␣B-crystallin monomers may be basic building blocks (10). For sHsps such as Hsp20 (11) and Hsp22 (12), dimers are the predominant complex, and they exhibit chaperone activity. Of medical significance, ␣A-crystallin truncations occur in mammalian lens, suggesting a relationship between carboxyl-terminal modification and cataract (13,14). Also, dropping either 13 or 25 carboxylterminal residues from human ␣B-crystallin causes myofibrillar myopathy, with modified proteins exerting dominant negative effects (15). The truncated and normal ␣B-crystallins appear to interact, yielding oligomers slightly smaller than those generated by wild type protein.Assembly mechanisms and the resulting oligomers vary for sHsps from different sources (2, 4,...

Stress tolerance during diapause and quiescence of the brine shrimp, Artemia

“…p26, with a calculated monomeric molecular mass of 20.7 kDa, has an α-crystallin domain flanked by variable amino-and carboxyl-terminal regions, and it assembles into oligomers (Liang et al 1997a;Crack et al 2002;Sun et al 2004Sun et al , 2006Sun and MacRae 2005). Only diapause-destined Artemia embryos synthesize p26, and expression of the p26 gene is indifferent to heat in spite of possessing up-stream heat shock elements (Liang and MacRae 1999;Qiu et al 2006;Qiu and MacRae 2008a).…”

“…Reversible translocation of p26 into nuclei occurs in cysts exposed to anoxia, heat, and acidic pH, and during entry into diapause (Clegg et al 1994(Clegg et al , 1995(Clegg et al , 2000Liang et al 1997b;Liang and MacRae 1999). p26, which locates to nuclei in transfected mammalian cells (Sun et al 2004, is thought to interact with Hsp70 and nuclear lamins in cyst nuclei, but has little effect on transcription Clegg 2001, 2002). Based on these findings, p26 was thought to act as a molecular chaperone, a conclusion bolstered by the observation that p26 purified from either A. franciscana (Liang et al 1997a;Day et al 2003) or transformed bacteria (Sun et al 2004Sun and MacRae 2005) prevents the denaturation of heated tubulin and citrate synthase, and insulin when exposed to dithiothreitol.…”

“…p26, which locates to nuclei in transfected mammalian cells (Sun et al 2004, is thought to interact with Hsp70 and nuclear lamins in cyst nuclei, but has little effect on transcription Clegg 2001, 2002). Based on these findings, p26 was thought to act as a molecular chaperone, a conclusion bolstered by the observation that p26 purified from either A. franciscana (Liang et al 1997a;Day et al 2003) or transformed bacteria (Sun et al 2004Sun and MacRae 2005) prevents the denaturation of heated tubulin and citrate synthase, and insulin when exposed to dithiothreitol. Sitedirected mutagenesis established the structural stability of p26, a characteristic that prolongs the life of the protein through diapause (Sun and MacRae 2005;Sun et al 2006;Wu and MacRae 2010).…”

Heat-shock dependent oligomeric status alters the function of a plant-specific thioredoxin-like protein, AtTDX

“…The substrate was incubated in a 50 mM Hepes-KOH (pH 8.0) buffer at 45°C with various concentrations of AtTDX. Thermal aggregation of the substrate was determined by monitoring the turbidity increase at A340 as described (14,32).…”

Deubiquitinating enzyme BAP1 is involved in the formation and maintenance of the diapause embryos of Artemia

“…After termination of diapause by certain environmental cues, the embryos resume development, given suitable environmental conditions (Abatzopoulos et al 2002). Although several molecules associated with diapause have been described, the identification of factors involved in diapause initiation is still in its infancy (Sun et al 2004;Viner and Clegg 2001;Sharon et al 2009). Recently, flow cytometry analysis revealed that the cells of encysted embryos are arrested at the G1/S stage (data not shown), which indicated a relationship between the induction of diapause and control of the cell cycle.…”