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

abstractopp - 0089451 - p。在过去的2500万年里，随着南大洋的冷却，南极沿海水域的鱼类进化出了生化和生理适应能力，维持了细胞骨架功能和基因转录等基本细胞过程。例如，它们的微管在体温（-1.8至+1摄氏度）下组装和运作，远低于恒温动物和温带变温动物。该研究的长期目标是在分子水平上确定增强微管组装、运动蛋白马达功能以及珠蛋白和微管蛋白基因表达的适应性。具体目标有三个：1)确定促进南极鱼类微管在低温下高效聚合的初级序列变化和翻译后修饰；2)评估低温条件下南极鱼类脑运动蛋白高效运作所需的生化适应；3)研究南极岩鳕鱼（Notothenia coriiceps）和温带岩鳕鱼（N. angustata）的珠蛋白和微管蛋白基因的结构、组织和启动子驱动表达。南极鱼类的脑微管与温带和温血脊椎动物的不同之处在于不同寻常的初级序列取代（主要位于微管单体的侧环和核心）和翻译后c端谷氨酰化。南极鱼类微管蛋白潜在的一级序列适应性将直接通过生产野生型和定点微管蛋白突变体进行体外功能分析来测试。突变和野生型鱼微管形成“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。在Chionodraco rastrospinosus的运动蛋白区域中，三个不寻常的取代可能通过改变ATP的结合和/或马达的速度来增强低温下的机械化学活性。为了测试这些变化的功能意义，鱼的残留物将被单独或一致地转化为哺乳动物脑运动蛋白中发现的那些。相互替换将被引入哺乳动物运动域的框架。在大肠杆菌中生产和纯化后，将通过测量其atp酶和运动活性的温度依赖性来评估突变体运动结构域的功能性能。我们将利用a-珠蛋白/b-珠蛋白基因对和a-微管蛋白基因簇来分析该基因表达的分子适应性。通过启动子/荧光素酶报告质粒构建物在可诱导红细胞和神经元模型细胞系统中的瞬时转染试验，以及荧光素酶报告活性的测定，来评估支持高效表达的山蕨球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。总之，这些研究应该揭示南极鱼类在低温下维持有效的细胞骨架组装、机械化学运动功能和基因表达的分子适应性。从最广泛的意义上说，这个研究项目应该促进对生命的准热模式的分子理解。

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.