STRUCTURE/FUNCTION OF A BASAL BODY-ASSOCIATED CALCIUM-BP

基础体相关钙-BP的结构/功能

• 批准号: 2459385
• 负责人: BESSIE P HUANG
• 金额: $ 29.63万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2459385
• 关键词: Chlamydomonas; HeLa cells; X ray crystallography; calcium binding protein; centrosome; chromosome movement; computer assisted sequence analysis; eukaryote; gene expression; genetic library; genetic mapping; genetic regulation; human genetic material tag; immunoelectron microscopy; immunoprecipitation; kinetosome; light microscopy; microtubules; nuclear magnetic resonance spectroscopy; nucleic acid hybridization; nucleic acid sequence; protein biosynthesis; protein structure function; site directed mutagenesis; ultraviolet spectrometry

The temporal and spatial distribution of microtubules in eukaryotic cells is controlled by discrete organelles, known as microtubule-organizing centers (MTOCs). MTOCs exhibit remarkable structural variation among different organisms but have similar functions of organizing interphase microtubule arrays and determining the bipolarity of the mitotic spindle; as such, they are crucial to the fidelity of cellular reproduction and cytoplasmic organization. The specific aims of this proposal are to study the structure and function of caltractin, a calcium-binding protein that is a conserved component of MTOCs in divergent species. Caltractin, also known as "centrin," has been cloned at the DNA level from a number of different organisms including yeast, algae, frog, mouse and human. Based on amino acid sequence analysis these proteins have been found to represent a new subfamily within the superfamily of EF-hand calcium- binding proteins that includes calmodulin and troponin C. Genetic analyses indicate that caltractin in the alga Chlamydomonas reinhardtii and its yeast homolog in Saccharomyces cerevisiae, Cdc31p are required for the normal duplication and segregation of the basal body complex and spindle pole body, the major MTOC in the respective cell types. In this proposal, the localization and expression of human caltractin in HeLa cells, and the consequences of disrupting caltractin function in these cells will be studied to determine if, as in Chlamydomonas and yeast cells, the protein plays a role in the duplication and segregation of the centrosome, the major MTOC in animal cells. Protein structure/function relationships will be examined by studying the consequences of expressing site-specific mutations in caltractin in stable transformants of Chlamydomonas. Genetic and biochemical approaches will be used to identify other genes and gene products that affect the basal body complex in Chlamydomonas and proteins that may interact with caltractin. And finally, the in vitro functional and structural features of wild-type and mutant forms of algal and human caltractin expressed and purified from bacteria will be characterized. The aim of these in vitro studies is to identify features that distinguish caltractin from other closely related calcium-binding proteins, and to define at high resolution by NMR spectroscopy and potentially, X- ray crystallography, the consequences of calcium-binding on protein conformation. The long term objectives of this project are to define the mechanisms at the molecular level that regulate the assembly and function of MTOCs. Protein components of MTOCs that are characterized to be of fundamental importance in their physiology and/or replication constitute potential novel targets for developing new therapeutic approaches to influence microtubule function.

真核细胞微管的时空分布 由离散的细胞器控制，称为微管组织 中心（MTOC）。 MTOC 之间表现出显着的结构差异 不同的生物体但具有相似的组织间期功能 微管阵列并确定有丝分裂纺锤体的双极性； 因此，它们对于细胞繁殖的保真度至关重要 细胞质组织。本提案的具体目标是研究 钙结合蛋白（一种钙结合蛋白）的结构和功能 是不同物种中 MTOC 的保守组成部分。钙拮抗剂，还有 被称为“centrin”，已在 DNA 水平上从许多 不同的生物体，包括酵母、藻类、青蛙、小鼠和人类。基于 氨基酸序列分析发现这些蛋白质 代表 EF-hand 钙超家族中的一个新亚家族- 结合蛋白，包括钙调蛋白和肌钙蛋白 C。 遗传分析 表明藻类莱茵衣藻及其 酿酒酵母中的酵母同源物 Cdc31p 是 基底体复合体和纺锤体的正常复制和分离 极体，各个电池类型中的主要 MTOC。在这个提案中， 人钙拮抗剂在 HeLa 细胞中的定位和表达，以及 破坏这些细胞中钙牵蛋白功能的后果将是 研究以确定蛋白质是否像衣藻和酵母细胞中那样 在中心体的复制和分离中发挥作用， 动物细胞中的主要MTOC。蛋白质结构/功能关系 通过研究表达位点特异性的后果来检查 衣藻稳定转化体中钙乳蛋白的突变。遗传 生化方法将用于鉴定其他基因和基因 影响衣藻和蛋白质基础体复合物的产品 可能与钙勒素相互作用。最后，体外功能 以及藻类和人类野生型和突变型的结构特征 从细菌中表达和纯化的钙牵蛋白将被表征。这 这些体外研究的目的是确定区分的特征 来自其他密切相关的钙结合蛋白的钙拮抗剂，以及 通过 NMR 光谱和潜在的 X 射线以高分辨率定义 晶体学，钙结合对蛋白质的影响 构象。该项目的长期目标是定义 调节组装和功能的分子水平机制 MTOC。 MTOC 的蛋白质成分的特征是 在其生理学和/或复制中具有根本重要性 开发新治疗方法的潜在新靶标 影响微管功能。