Protein Folding and Function at Cold Temperature: Co-Evolution of the Chaperonin CCT and Tubulins from Antarctic Fishes

自3800万~2500万年前南极大陆冰川的出现、南美洲与南极半岛之间德雷克航道的开通以及南大洋开始降温以来，南极海洋生物的演化一直受到极端寒冷温度的驱动。由于生活在非常低和稳定的温度下，南极鱼类作为理解生物分子冷适应机制的模型特别有吸引力，生物分子冷适应或生物化学和生理系统的补偿性重组以保护生物功能在寒冷的热状态下。两个相互关联且可能共同进化的系统，即形成微管的微管蛋白和协助微管蛋白折叠的含伴侣蛋白的TCP1(t-复合蛋白-1)复合体(CCT)，为阐明这些机制提供了一个无与伦比的机会。这项研究将在以下方面产生新的重要知识：1)微管组装和伴侣功能的冷适应；2)CCT对微管蛋白结合特异性的共同进化起源。这项建议的第一个目标是确定在南极鱼类β-微管蛋白中发现的五个新的氨基酸替代对低温下微管组装的贡献。第二个目标是利用从南极鱼类睾丸组织中提纯的CCT建立伴侣蛋白体外折叠系统，并对其热性质和作用机制进行评价。第三个目标是，通过系统发育控制的对比，评估来自南极鱼类的CCT及其微管蛋白底物已经共同进化到在低温下发挥作用的假设。这项建议的更广泛影响包括向东北大学的研究生和REU本科生介绍与南极海洋生态系统的生态和环境问题有关的最先进的生化、细胞和分子生物学研究。由于许多关于冷适应蛋白质的生物发生和功能的研究将在帕尔默空间站的实地进行，这些学生将在探险生物科学的实际考虑方面获得宝贵的经验。这项研究还将增加关于地球极端环境之一的分子冷适应的知识，因此与提出关于火星或木卫二上潜在的外星生命的精炼假设有关。这种具有冷功能的伴侣蛋白折叠系统将对生物制药和生物技术行业用于折叠不溶蛋白具有重要价值。

AbstractSince the advent of Antarctic continental glaciation, the opening of the Drake Passage between South America and the Antarctic Peninsula, and the onset of cooling of the Southern Ocean ~38-25 million years ago, evolution of the Antarctic marine biota has been driven by the development of extreme cold temperatures. Because they live at very low and stable temperatures, Antarctic fishes of the suborder Nototheniodei are particularly attractive as models for understanding the mechanisms of biomolecular cold adaptation, or the compensatory restructuring of biochemical and physiological systems to preserve biological function in cold thermal regimes. Two interrelated and potentially co-evolved systems, the tubulins that form microtubules and the chaperonin-containing TCP1 (t-complex protein-1) complex (CCT) that assists the folding of tubulins, provide an unparalleled opportunity to elucidate these mechanisms. This research will yield new and important knowledge regarding: 1) cold adaptation of microtubule assembly and of chaperonin function; and 2) the co-evolutionary origin of tubulin-binding specificity by CCT. The first objective of this proposal is to determine the contributions of five novel amino acid substitutions found in Antarctic fish beta-tubulins to microtubule assembly at cold temperature. The second objective is to establish a chaperonin folding system in vitro using CCT purified from testis tissue of Antarctic fishes and to evaluate its thermal properties and mechanism. The third objective is to evaluate, through phylogenetically controlled contrasts, the hypothesis that CCT and its tubulin substrates from Antarctic fishes have co-evolved to function at cold temperatures. The broader impacts of this proposal include introduction of graduate and REU undergraduate students of Northeastern University to state-of-the-art biochemical, cellular, and molecular-biological research relevant to ecological and environmental issues of the Antarctic marine ecosystem. Because much of the research on the biogenesis and function of cold-adapted proteins will be performed in the field at Palmer Station, these students will gain invaluable experience in the practical considerations of expeditionary biological science. The research also will increase knowledge about molecular cold adaptation in one of the Earth's extreme environments, and hence is relevant to the formulation of refined hypotheses regarding potential extraterrestrial life on Mars or Europa. The cold-functioning chaperonin protein folding system will be of great value to the biopharmaceutical and biotechnological industries for use in folding insoluble proteins.