Mechanistic Analysis of Microtubule Based Motors

基于微管的电机的机理分析

• 批准号: 7912055
• 负责人: SUSAN P. GILBERT
• 金额: $ 11.4万
• 依托单位: RENSSELAER POLYTECHNIC INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7912055
• 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(申请人提供)：生物医学研究中的一个主要挑战是确定有丝分裂纺锤体组装的机制，以及复杂的马达和微管(MT)相互作用蛋白阵列如何正确协调染色体分离。有丝分裂的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人类的发育和健康至关重要。有丝分裂动蛋白在纺锤体功能的各个方面都发挥着重要的作用--影响染色体的移动和分离、细胞的分裂，以及调节微管的聚合和解聚。虽然动蛋白具有共同的结构基序，但关键氨基酸的变化赋予了每种动蛋白独特的机械力化学性质，从而指定了其细胞功能。因此，如果我们在体外阐明单个有丝分裂动蛋白的酶性质，我们就可以深入了解它在复杂的细胞环境中对纺锤体功能的特定作用，在这个复杂的环境中，还有其他分子马达、蛋白质和调节因子。这项研究提出了三种具有代表性的动蛋白，以解决染色体在微管晶格上进行运动的机制要求，MT-MT对MT滑动和纺锤体稳定性的交联作用，以及MT缩短和MT延长对纺锤体组装和动力学的影响。我们将使用稳态前动力学方法，结合平衡方法和荧光显微镜来解决以下三个具体目标：1)确定Kar3Cik1机械力化学在后期的反平行MTS的交联功能。2)定义了Kar3Vik1机械力化学，使其在MT负端积累，在纺锤体极交联平行的MT。3)明确了CENP-E过程步进的机理基础。 公共卫生相关性：CENP-E、EG5/KSP和Kinesin-14对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。拟议的研究应该会导致高通量筛选的新策略，即选择化合物来增强化疗后的癌细胞死亡，而不是非整倍体。目前，有许多针对EG5/KSP和CENP-E的特异性化疗药物正在进行I/II期临床试验。