Purification, Structure and In vitro Molecular‐Chaperone Activity of Artemia P26, a Small Heat‐Shockh/α‐Crystallin Protein

Abstract

Encysted brine-shrimp gastrulae bring their metabolism to a reversible standstill during diapause and quiescence, demonstrating a remarkable resistance to unfavourable environmental conditions. For example, mortality of Artemia embryos under normal temperature and hydration is very low, even after two years of anoxia, and embryos commonly experience complete desiccation as part of their developmental program. Previous evidence from our laboratories indicated that p26, an abundant low-molecular-mass cyst-specific protein capable of translocation into the nucleus, may have a protective function in Artemia cysts. p26 was purified to apparent homogeneity and a continuous sequence of 141 of its amino acids was determined by peptide sequencing, revealing that it is a member of the small-heat-shock/u-crystallin family of proteins. As determined by molecular-sieve chromatography and sucrose-density-gradient centrifugation, native p26 is a multimer of about 27 monomers with a molecular mass of approximately 700 kDa. Inactivation of citrate synthase was less when the enzyme was heated in the presence rather than the absence of p26. Additionally, the renaturation of heat-inactivated citrate synthase was promoted by p26. These results indicated that p26 possesses molecular-chaperone activity, a property of other small heat-shock/u-crystallin proteins. Our findings demonstrate that p26 has the potential to protect the macromolecular components of Artemia embryos, either as they encyst or upon exposure to environmental extremes. Protection may depend upon the ability of p26 to function as a molecular chaperone.Keywords: chaperone ; heat-shocWu-crystallin protein; diapause ; Arternia The crustacean, Artemia franciscana reproduces via two alternative pathways. In the ovoviviparous mode, embryos develop in the ovisac and are released from the female as freeswimming nauplii. In contrast, for the oviparous route, development is arrested at the late gastrula stage, the embryos encyst and they are released from the female as encysted gastrulae, hence the term cysts [1]. The cysts, composed of about 4000 cells and enclosed in a shell that is impermeable to non-volatile molecules, are in a dormant state known as diapause [l-71. They are resistant to severe insults, including exposure to several organic solvents, y-irradiation, temperature extremes, desiccation and anoxia [1-3, 81. Moreover, the embryos withstand repeated cycles of hydration and desiccation, losing this ability at about the time they emerge from the cyst as larvae [X, 91. The period of post-gastrula development within the cyst occurs in the absence of DNA synthesis and mitosis [4, 101, implying extensive morphological rearrangement and differentiation of preexisting cells. Because cysts are readily available, their development has been investigated extensively at the molecularhiochemical level [11-141. Diapause in Artemia has been studied to a Iesser extent [2, 31, but it is known that its occurrence is 'anticipated', and embryos following ovoviviparous and oviparous r...

Stress response in tardigrades: differential gene expression of molecular chaperones

“…However, the importance of small heat-shock proteins is clearly demonstrated in Artemia franciscana. A massive accumulation of the sHsp p26 occurs in diapausing embryos of this brine shrimp (Liang et al 1997a;Liang et al 1997b). The protein p26 is able to move into the nucleus (Clegg et al 1995) and is thought to protect and/or chaperone, in cooperation with Hsp70, the nuclear matrix proteins (Willsie and Clegg 2002).…”

“…The investigation of transcripts and encoded stress proteins is based on their well-known function as molecular chaperones (Gething and Sambrook 1992;Georgopoulos and Welch 1993;Jakob et al 1993). Results derived from several other organisms that tolerate dehydration or suspended animation like nematodes (Chen et al 2006), crustaceans (Liang et al 1997b;MacRae 2003), insects (Tammariello et al 1999;Hayward et al 2004;Bahrndorff et al 2008;LopezMartinez et al 2009) and plants (Alamillo et al 1995;Ingram and Bartels 1996) suggest a versatile role for the stress response in dormant stages.…”

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).…”

“…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).…”

The induction of anhydrobiosis in the sleeping chironomid: Current status of our knowledge

“…Likewise, sHSPs act as desiccation protectants in animal cells. In Artemia cysts, massive amount of p26, a sHSP, occurs in the nucleus (28,29) to protect macromolecules from stress-induced denaturation (30). In tardigrades, HSP70 may be involved in physiological and biochemical system underlying desiccation tolerance to repair denatured proteins after rehydration (31).…”