Microtubule Function, Protein Folding, and Embryogenesis in Antarctic Fishes: An Integrative Approach

自4000万~ 2500万年前南极大陆冰川出现、南美洲与南极半岛之间的德雷克海峡开通、南大洋开始降温以来，南极海洋生物群的进化一直受到极冷温度发展的驱动。生活在南大洋的变温动物面临的生化和生理挑战包括反应速率和代谢通量的降低以及大分子相互作用的普遍减弱。然而，南大洋变温动物现在正受到几个世纪或更短时间内变暖的威胁。本研究旨在了解南极海洋鱼类冷适应的分子机制，并评估这些生物抵抗或补偿海洋快速变暖的生理能力。P.I.将描述两个重要的相互作用的蛋白质系统，形成微管的微管蛋白和伴侣蛋白CCT（细胞质中含有伴侣蛋白的TCP-1，一个帮助小管折叠的蛋白质家族）。通过研究南极鱼类胚胎裂解（一个微管依赖过程）的热依赖性，将分析对全球温度变化的更高水平的综合响应。目的是：(1)确定在南极鱼类β -微管蛋白中发现的五种新型氨基酸取代对低温下微管组装的贡献。(2)比较南极鱼类和哺乳动物睾丸组织CCT的功能特性。(3)评价温度升高对南极鱼类胚胎发生的影响。该研究将向研究生和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物化学，细胞和分子生物学研究。这项工作还将通过开发一种具有冷功能的伴侣蛋白折叠系统来造福社会，这对生物制药和生物技术行业用于折叠不溶性蛋白质具有重要价值。

Since 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 ~40-25 million years ago, evolution of the Antarctic marine biota has been driven by the development of extreme cold temperatures. The biochemical and physiological challenges facing ectotherms living in the Southern Ocean include the reduction of reaction rates and metabolic fluxes and a pervasive weakening of macromolecular interactions. Yet, Southern Ocean ectotherms are now threatened by warming over periods measured in centuries or less. The proposed research seeks to understand the molecular mechanisms of cold adaptation in Antarctic marine fishes and to assess the physiological capacity of these organisms to resist or compensate for rapid oceanic warming. The P.I. will characterize two important and interacting protein systems, the tubulins that form microtubules and the chaperonin CCT (cytoplasmic chaperonin-containing TCP-1, a family of proteins that assists the folding of the tubulins). Higher-level, integrative responses to global temperature change will be analyzed by studying the thermal dependence of cleavage in Antarctic fish embryos (a microtubule-dependent process). The objectives are (1) to determine the contributions of five novel amino acid substitutions found in Antarctic fish beta-tubulins to microtubule assembly at cold temperature. (2) to compare the functional properties of CCT from testis tissues of Antarctic fishes and mammals. (3) to evaluate the effects of increased temperature on embryogenesis in Antarctic fishes. The research will introduce graduate and REU undergraduate students to state-of-the-art biochemical, cellular, and molecular-biological research relevant to ecological and environmental issues of the Antarctic marine ecosystem. The proposed work also will benefit society by developing a cold-functioning chaperonin protein folding system, of great value to the biopharmaceutical and biotechnological industries for use in folding insoluble proteins.