STRUCTURAL AND GENETIC ASPECTS OF TUBULIN INTERACTIONS

微管蛋白相互作用的结构和遗传方面

• 批准号: 3295613
• 负责人: LYNWOOD R YARBROUGH
• 金额: $ 14.7万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3295613
• 关键词: Saccharomyces; Trypanosoma; antimitotics; benzimidazole analog; binding proteins; brain; chemical binding; chemical structure function; chickens; circular dichroism; colchicine; fluorescence spectrometry; gene complementation; genetic manipulation; guanosine triphosphate; laboratory rat; microtubules; molecular cloning; plasmids; point mutation; protein engineering; protein reconstitution; protein sequence; species difference; structural genes; tubulin

Microtubules are multisubunit protein structures found in all eukaryotic cells which are involved in a variety of cell functions. They are composed primarily of two genetically related proteins of molecular weight 50,000 termed alpha and beta tubulin. Tubulins isolated from lower eukaryotes such as yeast, fungi, and protozoa (which may differ by as much as 30% in amino acid sequence from mammalian tubulins) exhibit greatly different assembly, stability, and ligand binding properties than tubulins isolated from mammalian brain. The long term goal of our research is to use genetic and biophysical techniques to determine how the structural and functional properties of microtubules relate to the amino acid sequence of the alpha and beta tubulins and to define functional domains of the tubulin monomers and protomers. The specific aims of this proposal are: 1. To prepare cloned tubulins of a protozoan (African trypanosome) and higher eukaryote brain (chicken), develop conditions for reconstitution of biologically active tubulin protomers, and compare the assembly, conformation, and ligand binding properties with authentic trypanosome and brain tubulin. 2. To purify and characterize authentic trypanosome tubulin in terms of its assembly, stability, and susceptibility to antimitotic agents such as colchicine and the benzimidazoles. 3. To define the molecular mechanisms of interactions of guanine nucleotides and antimitotic agents by assembly and characterization of interspecies dimers or genetically altered (chimeras and site-specific mutants) alpha and beta tubulins. 4. To determine whether the trypanosome or chick brain beta tubulins (or regions of them) are biologically functional in yeast. An understanding of the mechanisms by which guanine nucleotides interact with tubulin and modulate its properties could contribute significantly to our understanding of its role in other important biological systems such as the G proteins, ras oncogenes, and ribosome. Knowledge of the factors governing interaction of antimitotic agents with tubulin and their related effects on microtubule structure could lead to the development of new antimitotic agents useful for treatment of protozoan diseases such as trypanosomiasis or perhaps malignancies.

微管是多亚基蛋白质结构，存在于所有 涉及多种细胞功能的真核细胞。 它们主要由两种遗传相关的蛋白质组成 分子量为50,000的α和β微管蛋白。 微管蛋白分离自低等真核生物，如酵母，真菌， 原生动物（其氨基酸差异可高达30 来自哺乳动物微管蛋白的序列）表现出极大的不同 组装、稳定性和配体结合性能优于微管蛋白 从哺乳动物大脑中分离出来的 我们的长期目标 研究是使用遗传和生物物理技术来确定 微管的结构和功能特性 涉及α和β微管蛋白的氨基酸序列 并确定微管蛋白单体的功能结构域， 原聚体 这项建议的具体目标是： 1. 制备克隆的原生动物微管蛋白（非洲 锥虫）和高等真核生物脑（鸡），发展 生物活性微管蛋白重建的条件 原聚体，并比较组装，构象和配体 与真正的锥虫和脑微管蛋白的结合特性。 2. 为了纯化和鉴定真正的锥虫微管蛋白， 就其组装、稳定性和对抗有丝分裂药的敏感性而言， 药剂如秋水仙碱和苯并咪唑。 3. 确定鸟嘌呤相互作用的分子机制 核苷酸和抗有丝分裂剂， 种间二聚体或遗传改变的表征 （嵌合体和位点特异性突变体）α和β微管蛋白。 4. 为了确定锥虫或鸡脑β 微管蛋白（或其区域）在酵母中具有生物学功能。 了解鸟嘌呤核苷酸 与微管蛋白相互作用并调节其性质可能有助于 对我们理解其在其他重要领域中的作用有重大意义 生物系统，如G蛋白，ras癌基因， 核糖体 对影响生物多样性和生物多样性之间相互作用的因素的认识 具有微管蛋白的抗有丝分裂剂及其相关作用 微管结构可能导致新的 用于治疗原生动物疾病的抗有丝分裂剂 例如锥虫病或恶性肿瘤。