Structure, Function, and Expression of Tubulins, Globins, and Microtubule-Dependent Motors from Cold-Adapted Antarctic Fishes

摘要OPP-0089451P.I.William Detrich在过去的2500万年里，随着南大洋的冷却，南极沿海的鱼类进化出了维持细胞骨架功能和基因转录等基本细胞过程的生化和生理适应。例如，它们的微管在远低于恒温动物和温带变温动物的体温(-1.8至1摄氏度)下组装和运行。这项拟议研究的长期目标是在分子水平上确定增强微管组装的适应性、运动蛋白马达的功能以及珠蛋白和微管蛋白基因的表达。具体目标有三：1)确定有助于南极鱼类微管蛋白在低温下高效聚合的初级序列变化和翻译后修饰；2)评估南极鱼类在低温下高效发挥脑动蛋白马达功能所需的生化适应；以及3)表征南极岩鳕鱼(Notothenia Coriicep)和温带同源物(N.angustata)珠蛋白和微管蛋白基因的结构、组织和启动子驱动的表达。南极鱼类的脑微管蛋白与温带和温血脊椎动物的不同之处在于不寻常的初级序列替换(主要位于微管蛋白单体的侧环和核心)和翻译后C末端谷氨酸化。南极鱼类微管蛋白潜在的初级序列适应性将直接通过生产野生型和定点突变体进行测试，用于体外功能分析。突变和野生型鱼类微管蛋白形成“冷稳定”微管的能力将通过测量其组装的临界浓度和通过视频增强显微镜分析其动力学来确定。通过改变三磷酸腺苷的结合和/或马达的速度，脊椎动物运动域中的三个不寻常的替换可能会增强低温下的机械力化学活性。为了测试这些变化的功能意义，鱼的残留物将单独和一致地转换为哺乳动物脑运动蛋白中的残留物。相互替代将被引入到哺乳动物运动领域的框架中。在大肠杆菌中表达和纯化后，将通过测量其ATPase和运动活性的温度依赖性来评估突变运动域的功能性能。将使用a-珠蛋白/b-珠蛋白基因对和a-微管蛋白基因簇来分析冠毛虫基因表达的分子适应性。支持高效表达的棒状杆菌珠蛋白和微管蛋白基因调控序列(启动子和增强子)的结构特征将通过在可诱导的红细胞和神经模型细胞系统中瞬时转染启动子/荧光素酶报告质粒构建并随后检测荧光素酶报告活性来评估。总之，这些研究应该揭示南极鱼类在低温下保持有效的细胞骨架组装、机械力化学运动功能和基因表达的分子适应。在最广泛的意义上，这项研究计划应该会促进分子对高热生命模式的理解。

AbstractOPP-0089451P.I. William Detrich As the Southern Ocean cooled during the past 25 million years, the fishes of Antarctic coastal waters evolved biochemical and physiological adaptations that maintain essential cellular processes such as cytoskeletal function and gene transcription. Their microtubules, for example, assemble and function at body temperatures (-1.8 to +1 oC) well below those of homeotherms and temperate poikilotherms. The long range goals of the proposed research are to determine, at the molecular level, the adaptations that enhance the assembly of microtubules, the function of kinesin motors, and the expression of globin and tubulin genes. The specific objectives are three: 1) to determine the primary sequence changes and posttranslational modifications that contribute to the efficient polymerization of Antarctic fish tubulins at low temperatures; 2) to evaluate the biochemical adaptations required for efficient function of the brain kinesin motor of Antarctic fishes at low temperatures; and 3) to characterize the structure, organization, and promoter-driven expression of globin and tubulin genes from an Antarctic rockcod (Notothenia coriiceps) and a temperate congener (N. angustata). Brain tubulins from Antarctic fishes differ from those of temperate and warm-blooded vertebrates both in unusual primary sequence substitutions (located primarily in lateral loops and the cores of tubulin monomers) and in posttranslational C-terminal glutamylation. Potential primary sequence adaptations of the Antarctic fish tubulins will be tested directly by production of wild-type and site directed tubulin mutants for functional analysis in vitro. The capacity of mutated and wild-type fish tubulins to form "cold-stable" microtubules will be determined by measurement of their critical concentrations for assembly and by analysis of their dynamics by video-enhanced microscopy. Three unusual substitutions in the kinesin motor domain of Chionodraco rastrospinosus may enhance mechanochemical activity at low temperature by modifying the binding of ATP and/or the velocity of the motor. To test the functional significance of these changes, the fish residues will be converted individually, and in concert, to those found in mammalian brain kinesin. Reciprocal substitutions will be introduced into the framework of the mammalian kinesin motor domain. After production in Escherichia coli and purification, the functional performance of the mutant motor domains will be evaluated by measurement of the temperature dependence of their ATPase and motility activities. Molecular adaptation of gene expression in N. coriiceps will be analyzed using an a-globin/b-globin gene pair and an a-tubulin gene cluster. Structural features of N. coriiceps globin and tubulin gene regulatory sequences (promoters and enhancers) that support efficient expression will be assessed by transient transfection assay of promoter/luciferase reporter plasmid constructs in inducible erythrocytic and neuronal model cell systems followed by assay of luciferase reporter activity. Together, these studies should reveal the molecular adaptations of Antarctic fishes that maintain efficient cytoskeletal assembly, mechanochemical motor function, and gene expression at low temperatures. In the broadest sense, this research program should advance the molecular understanding of the poikilothermic mode of life.