A small heat shock/α-crystallin protein from encysted Artemia embryos suppresses tubulin denaturation

Abstract

Small heat shock/alpha-crystallin proteins function as molecular chaperones, protecting other proteins from irreversible denaturation by an energy-independent process. The brine shrimp, Artemia franciscana, produces a small heat shock/alpha-crystallin protein termed p26, found in embryos undergoing encystment, diapause, and metabolic arrest. These embryos withstand long-term anoxia and other stresses normally expected to cause death, a property likely dependent on molecular chaperone activity. The association of p26 with tubulin in unfractionated cell-free extracts of Artemia embryos was established by affinity chromatography, suggesting that p26 chaperones tubulin during encystment. To test this possibility, both proteins were purified by modifying published protocols, thereby simplifying the procedures, enhancing p26 yield about 2-fold, and recovering less tubulin than before. The denaturation of purified tubulin as it "aged" and exposed hydrophobic sites during incubation at 35 degrees C was greatly reduced when p26 was present; however, tubulin polymerization into microtubules was reduced. On incubation at 35 degrees C, centrifugation in sucrose density gradients demonstrated the association of purified p26 with tubulin. This is the first study where the relationship between a small heat shock/alpha-crystallin protein and tubulin from the same physiologically stressed organism was examined. The results support the proposal that p26 binds tubulin and prevents its denaturation, thereby increasing the resistance of encysted Artemia embryos to stress. Additional factors are apparently required for release of tubulin from p26 and restoration of efficient assembly, events that would occur as embryos resume development and the need for microtubules is established.

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

“…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 90-kDa Heat Shock Protein Hsp90 Protects Tubulin against Thermal Denaturation

“…The soluble form of tubulin is highly sensitive to denaturation, but it is protected when incorporated into microtubules (31,32). Thus, we asked whether tubulin aggregation would be reduced in the presence of microtubules.…”

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

“…The sHsps bind proteins in the molten globular state which are primed for aggregation and potential irreversible precipitation (26). These substrates, involved in functions from transcription to secondary metabolism, are subsequently released and refolded, activities reported to depend upon ATP-requiring chaperones such as Hsp70 (1,21,(27)(28)(29)(30).Embryos of the brine shrimp, Artemia franciscana, undergo ovoviviparous and oviparous development, the former yielding nauplii and the latter encysted gastrulae or cysts (31). Cysts enter diapause (32, 33), characterized by deep reduction in metabolic activity (34) and resistance to extreme environmental stress such as long term anoxia, desiccation, and heat shock (35,36).…”

“…The sHsps bind proteins in the molten globular state which are primed for aggregation and potential irreversible precipitation (26). These substrates, involved in functions from transcription to secondary metabolism, are subsequently released and refolded, activities reported to depend upon ATP-requiring chaperones such as Hsp70 (1,21,(27)(28)(29)(30).…”