Mechanistic Analysis of Microtubule Based Motors

基于微管的电机的机理分析

• 批准号: 7654581
• 负责人: SUSAN P. GILBERT
• 金额: $ 36.25万
• 依托单位: RENSSELAER POLYTECHNIC INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7654581
• 关键词: ATP phosphohydrolase; Address; Amino Acids; Anaphase; Aneuploidy; Behavior; Binding Proteins; Binding Sites; Biology; Biomedical Research; C-terminal; CENP-E protein; Cell Death; Cell Separation; Cell division; Cell physiology; Cells; Chromosome Segregation; Chromosomes; Clinical Trials; Collaborations; Communication; Complex; Congenital Abnormality; Coronary Arteriosclerosis; Cryoelectron Microscopy; Defect; Development; Disease; Environment; Equilibrium; Exhibits; Fluorescence Microscopy; Goals; Head; Health; Human; Human Development; In Vitro; Individual; Kinesin; Kinetics; Kinetochores; Lead; Malignant Neoplasms; Mediating; Metaphase Plate; Methodology; Microtubule Polymerization; Microtubules; Minus End of the Microtubule; Mitosis; Mitotic; Mitotic Checkpoint; Mitotic spindle; Molecular; Molecular Motors; Motor; Nucleotides; Output; Performance; Phase; Play; Plus End of the Microtubule; Property; Proteins; Public Health; Regulation; Regulatory Pathway; Reporting; Research; Resolution; Role; S-Phase Fraction; Saccharomyces cerevisiae; Slide; Specific qualifier value; Step Tests; Structure; Testing; Therapeutic; Therapeutic Intervention; Translating; Work; base; cancer cell; chemotherapy; chromosome movement; crosslink; depolymerization; drug discovery; genetic regulatory protein; high throughput screening; insight; proliferative diabetic retinopathy; public health relevance; research study; sensor

DESCRIPTION (provided by applicant): A major challenge in biomedical research is to define the mechanisms for mitotic spindle assembly and how a complex array of motors and microtubule (MT) interacting proteins correctly orchestrate chromosome segregation. Defects in mitosis result in birth defects and cancer, and therefore, a full molecular understanding of mitotic mechanisms is critical for human development and health. Mitotic kinesins play essential roles in all facets of spindle function- effecting chromosome movement and segregation, cell cleavage, and regulating microtubule polymerization and depolymerization. While kinesins share common structural motifs, key amino acid changes confer unique mechanochemical properties to each kinesin which specifies its cellular function. Therefore, if we elucidate the enzymatic properties of an individual mitotic kinesin in vitro, we will gain insight into its specific role for spindle function in the complex environment of the cell where there are other molecular motors, proteins, and regulatory factors. The research proposed evaluates three representative kinesins to address questions about the mechanistic requirements for processive movement of chromosomes on the microtubule lattice, MT-MT crosslinking function for MT sliding and spindle stability, and MT shortening and MT elongation for spindle assembly and dynamics. We will use presteady-state kinetic methodologies in combination with equilibrium approaches and fluorescence microscopy to address the following three specific aims: 1) Define the Kar3Cik1 mechanochemistry for its crosslinking function of anti-parallel MTs at anaphase. 2) Define the Kar3Vik1 mechanochemistry for its accumulation at MT minus-ends to crosslink parallel MTs at the spindle poles. 3) Define the mechanistic basis of CENP-E processive stepping. PUBLIC HEALTH RELEVANCE: CENP-E, Eg5/KSP, and Kinesin-14s are essential for cell division and therefore human health and development. Their selective inhibition may lead to more effective anti-mitotic therapeutics for treatment of diseases such as cancer, symptomatic coronary artery disease, and proliferative diabetic retinopathy. The proposed studies should lead to new strategies for high throughput screens that select compounds to enhance cancer cell death rather than aneuploidy after chemotherapy. Presently, there are a number of specific chemotherapeutics targeted to Eg5/KSP and CENP-E in Phase I/II Clinical trials.

描述（由申请人提供）：生物医学研究的一个主要挑战是定义有丝分裂主轴组件的机制，以及如何正确编排染色体分离的复杂电动机和微管（MT）相互作用的蛋白质。有丝分裂的缺陷导致先天缺陷和癌症，因此，对有丝分裂机制的全部分子理解对于人类的发展和健康至关重要。有丝分裂驱动蛋白在主轴功能的所有方面都起着重要作用 - 影响染色体运动和分离，细胞裂解以及调节微管聚合和去聚合。尽管驱动蛋白具有共同的结构基序，但关键氨基酸会改变为每个驱动蛋白指定其细胞功能的独特机械化学特性。因此，如果我们在体外阐明单个有丝分裂驱动蛋白的酶促性能，我们将深入了解其在具有其他分子电机，蛋白质和调节因子的细胞复杂环境中对纺锤体功能的特定作用。该研究提议评估三种代表性动力素，以解决有关染色体在微管晶格上的加工机制要求的问题，MT滑动和纺锤体稳定性的MT-MT交联功能以及用于纺锤体组装和动力学的MT缩短和MT缩短。我们将使用Presteady-STATE动力学方法与平衡方法和荧光显微镜结合使用以下三个特定目的：1）定义KAR3CIK1机械化学的抗平行MT的交联功能。 2）定义KAR3VIK1机械化学在MT减去末端的积累，以在纺锤极处交联MTS。 3）定义CENP-E处理阶梯的机理基础。 公共卫生相关性：CENP-E，EG5/KSP和运动蛋白14s对于细胞分裂以及人类健康和发展至关重要。它们的选择性抑制作用可能导致更有效的抗隔离疗法治疗癌症，有症状的冠状动脉疾病和增殖性糖尿病性视网膜病。拟议的研究应导致高通量筛选的新策略，以选择化合物以增强癌细胞死亡，而不是化学疗法后的非整倍性。目前，在I/II期临床试验中，有许多针对EG5/KSP和CENP-E的特定化学治疗剂。