Mechanistic studies of a novel G-alpha nucleotide cycle

新型G-α核苷酸循环的机制研究

• 批准号: 7242518
• 负责人: David P. Siderovski
• 金额: $ 22.26万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7242518
• 关键词: Address; Affect; Antineoplastic Agents; Binding; Biochemical; Biochemical Genetics; Biochemistry; Biosensor; Cell Cycle; Cell Death; Cell Polarity; Cell Survival; Cell division; Cells; Cyclin-Dependent Kinases; DNA; Defect; Development; Dissociation; Functional disorder; Future; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Generations; Genetic; Goals; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Health; Heterotrimeric G Protein Subunit; Heterotrimeric GTP-Binding Proteins; Human; In Vitro; Lead; Life; Maps; Microtubules; Mitosis; Mitotic spindle; Molecular; Mutation; Nature; Neurodegenerative Disorders; Nucleotides; Organism; Outcome; Peptides; Phenotype; Process; Proteins; RGS Domain; Range; Regulation; Research; Series; Specificity; Stem cells; Testing; Tubulin; Tubulin Interaction; anti-cancer therapeutic; drug discovery; genetic regulatory protein; inhibitor/antagonist; insight; mutant; novel; programs; receptor; relating to nervous system; segregation; spatiotemporal; stem cell fate

DESCRIPTION (provided by applicant): Proper chromosomal segregation by microtubule (MT) dynamics at the mitotic spindle is an essential component of cell division in all organisms; aberration of this process can lead to severe developmental abnormalities. Recent disparate genetic and biochemical evidence suggests the existence of a nucleotide cycle for G-protein alpha subunits required for proper mitotic spindle function during cell division. This cycle employs G-alpha regulatory proteins conserved across metazoa, including GoLoco motif nucleotide dissociation inhibitors, RGS-domain-containing GTPase-accelerating proteins, and the guanine nucleotide exchange factor RIC-8. Our long-term objective is to define the molecular determinants and spatiotemporal dynamics that underlie this novel G-alpha regulatory cycle and its effects on MT dynamics. Aim 1 is to delineate the functional interplay between the novel G-alpha regulatory factors that impinge on G-alpha subunit activity in cell division via a series of protein biochemical studies. Aim 2 details development and use of novel peptide biosensors for G-alpha nucleotide state to determine the active species that modulates MT dynamics and the spatial and temporal dynamics of this process in live cells. A novel GoLoco-insensitivity mutation to G-alpha will be employed to address the necessity of the G-alpha/GoLoco interaction to these processes. Aim 3 is to map the functional determinants of RGS14 and G-alpha-i1 proteins that directly modulate MT dynamics using in vitro biochemistry and cell viability, MT network, and cell cycle transit studies, thereby testing our initial hypothesis that the coordinated action of G-alpha and G-alpha regulators directly on tubulin and MTs might represent the force generator in mitotic spindle function during mitosis. This research program will lead to a new understanding of the diversity of G-protein action and its impact on microtubule dynamics, and further the ultimate goal of defining the precise molecular mechanisms of microtubule force generation at mitosis. This should facilitate new drug discovery for anticancer therapeutics, as many current anti-proliferative agents target microtubule dynamics. These studies will also afford insight into the regulation of asymmetric cell division and, consequently, mechanisms of cell polarity and cell-fate determination that have specific relevance to neural specification, developmental defects, and the potential future uses of neuroprogenitor stem cells as treatment for neurodegenerative disorders.

描述（由申请人提供）：通过有丝分裂纺锤体的微管（MT）动力学进行适当的染色体分离是所有生物体细胞分裂的重要组成部分；这一过程的异常可能导致严重的发育异常。最近不同的遗传和生化证据表明，细胞分裂过程中适当的有丝分裂纺锤体功能所需的 G 蛋白 α 亚基的核苷酸循环的存在。该循环采用跨后生动物保守的 G-α 调节蛋白，包括 GoLoco 基序核苷酸解离抑制剂、含有 RGS 结构域的 GTP 酶加速蛋白和鸟嘌呤核苷酸交换因子 RIC-8。我们的长期目标是定义这种新型 G-α 调节循环及其对 MT 动力学影响的分子决定因素和时空动力学。目标 1 是通过一系列蛋白质生化研究来描述影响细胞分裂中 G-α 亚基活性的新型 G-α 调节因子之间的功能相互作用。目标 2 详细介绍了用于 G-α 核苷酸状态的新型肽生物传感器的开发和使用，以确定调节活细胞中 MT 动力学以及该过程的空间和时间动力学的活性物质。 G-alpha 的新型 GoLoco 不敏感突变将用于解决 G-alpha/GoLoco 相互作用对这些过程的必要性。目标 3 是利用体外生物化学和细胞活力、MT 网络和细胞周期转运研究来绘制直接调节 MT 动力学的 RGS14 和 G-alpha-i1 蛋白的功能决定因素，从而检验我们最初的假设，即 G 的协调作用直接作用于微管蛋白和 MT 的 -α 和 G-α 调节剂可能代表有丝分裂期间有丝分裂纺锤体功能的力发生器。该研究项目将带来对 G 蛋白作用多样性及其对微管动力学影响的新认识，并进一步实现确定有丝分裂时微管力产生的精确分子机制的最终目标。这应该有助于抗癌治疗的新药发现，因为当前许多抗增殖药物都针对微管动力学。这些研究还将深入了解不对称细胞分裂的调节，以及细胞极性和细胞命运决定的机制，这些机制与神经规范、发育缺陷以及神经祖细胞干细胞治疗神经退行性疾病的潜在未来用途相关。失调。