Structural and functional analysis of p26, a small heat shock protein from Artemia franciscana.

摘要

The brine shrimp, Artemia franciscana, undergoes encystment and diapause, exhibiting a remarkable resistance to environmental stresses during this time. Encysted Artemia embryos contain an abundant small heat shock protein termed p26, a molecular chaperone contributing to development and survival. To investigate the role of p26 during Artemia development and upon exposure to stress, the structural and functional characteristics of wild type and modified p26 were determined. Wild type p26 resided predominantly in the cytoplasm of transiently transfected COS-1 cells, however mutated p26 occurred in both cytoplasm and nuclei. Translocation into nuclei was especially evident for variants missing the complete amino-terminal region, a single site mutant R114A, and a multiple-arginine-deleted mutant "R". When produced in either prokaryotic or eukaryotic cells, modified p26 lacking the amino-terminal region occurred mainly as monomers and dimers, indicating the relevance of the amino-terminus for p26 oligomer formation. Assembly of higher order oligomers was strengthened by the carboxy-terminal extension, although removing the ten carboxy-terminal residues had only a marginal effect on oligomerization. A mechanism whereby oligomer disassembly assists translocation of p26 from cytoplasm into nuclei was suggested, this of importance because p26 translocates into Artemia embryo nuclei upon exposure to anoxia and thermal shock. However, when examined in Artemia, the results suggested a process more complex than just oligomer dissociation for regulation of p26 nuclear translocation. Bacterial thermotolerance and protein chaperoning assays demonstrated that modified p26 had decreased chaperoning capability, with truncated variants, mutant R114A and mutant "R" most dramatically affected. Oligomerization is necessary but not sufficient for full chaperone activity, since mutant R114A formed large oligomers while exhibiting limited chaperone capability. Circular dichroism revealed that secondary structures of most mutants, in comparison with wild type p26, possessed increased alpha-helices and decreased beta-conformations. Intrinsic fluorescence and ANS-binding studies demonstrated altered aromatic residue microenvironments and reduced global hydrophobicity for p26 variants. Comparative modeling indicated the p26 alpha-crystallin domain is arranged in an immunoglobulin-like fold as a beta-sandwich consisting of eight beta-strands forming two antiparallel beta-sheets. This work, which has important implications for understanding the relationship between p26 structural organization and functional mechanisms during Artemia development and stress, provides a model for study of other small heat shock proteins. As such, it may lead to a better appreciation of diseases, including ischemia/reperfusion injury, cataract, desmin-related myopathy and neurodegenerative disorders.