Mechanistic Analysis of Microtubule Based Motors

基于微管的电机的机理分析

基本信息

批准号：
8267668
负责人：
SUSAN P. GILBERT
金额：
$ 35.53万
依托单位：
RENSSELAER POLYTECHNIC INSTITUTE
依托单位国家：
美国
项目类别：
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/8267668
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 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 Depolymerization 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

项目摘要

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纺锤体组装的机制，以及复杂的马达阵列和微管 (MT) 相互作用蛋白如何正确协调染色体分离。有丝分裂缺陷会导致出生缺陷和癌症，因此，对有丝分裂机制的全面分子理解对于人类发育和健康至关重要。有丝分裂驱动蛋白在纺锤体功能的各个方面都发挥着重要作用——影响染色体运动和分离、细胞分裂以及调节微管聚合和解聚。虽然驱动蛋白具有共同的结构基序，但关键氨基酸的变化赋予每个驱动蛋白独特的机械化学特性，从而指定其细胞功能。因此，如果我们在体外阐明单个有丝分裂驱动蛋白的酶学特性，我们将深入了解其在存在其他分子马达、蛋白质和调节因子的复杂细胞环境中对纺锤体功能的特定作用。该研究评估了三种代表性的驱动蛋白，以解决有关染色体在微管晶格上进行运动的机械要求、MT滑动和纺锤体稳定性的MT-MT交联功能、纺锤体组装和动力学的MT缩短和MT伸长等问题。我们将使用前稳态动力学方法与平衡方法和荧光显微镜相结合来实现以下三个具体目标：1）定义 Kar3Cik1 机械化学的反平行 MT 在后期的交联功能。 2) 定义 Kar3Vik1 机械化学，用于其在 MT 负端的积累，以交联主轴极处的平行 MT。 3）定义CENP-E过程步进的机制基础。公共卫生相关性：CENP-E、Eg5/KSP 和 Kinesin-14 对于细胞分裂以及人类健康和发育至关重要。它们的选择性抑制可能会导致更有效的抗有丝分裂疗法，用于治疗癌症、症状性冠状动脉疾病和增殖性糖尿病视网膜病变等疾病。拟议的研究应该会产生新的高通量筛选策略，选择化合物来增强癌细胞死亡而不是化疗后的非整倍体。目前，已有多种针对Eg5/KSP和CENP-E的特异性化疗药物处于I/II期临床试验中。