Fine tuning the catalytic cycle of kinesin motors

微调驱动蛋白马达的催化循环

• 批准号: 8640193
• 负责人: Fredrick Jon Kull
• 金额: $ 30.02万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8640193
• 关键词: ATP phosphohydrolase; Amino Acids; Architecture; Attenuated; Baculovirus Expression System; Binding; Biochemical; Calorimetry; Cell physiology; Cells; Complex; Defect; Development; Developmental Process; Disease; Distant; Drosophila melanogaster; Elements; Embryo; Engineering; Erinaceidae; Escherichia coli; Family; Family member; Foundations; Genes; Goals; Guanosine Triphosphate Phosphohydrolases; Hair follicle structure; Hedgehog signaling complex location; Homologous Gene; Human; Human Pathology; Hybrids; Hydrolysis; Integral Membrane Protein; Kinesin; Kinetics; Lead; Limb structure; Lung; Methods; Microtubules; Modeling; Motor; Mutation; N-terminal; Nucleotides; Organism; Pathway interactions; Pattern; Play; Process; Protein Binding; Protein Kinase; Proteins; Regulation; Relative (related person); Resolution; Role; Scaffolding Protein; Signal Pathway; Signal Transduction; Structure; Surface; System; Tail; Thermodynamics; Titrations; Tubulin; Uncertainty; base; cell motility; cell type; design; dimer; insight; member; mutant; new therapeutic target; protein B; protein complex; public health relevance; smoothened signaling pathway; therapeutic target; transcription factor

DESCRIPTION (provided by applicant): Hedgehog (Hh) signaling is fundamental to many developmental processes in organisms as divergent as Drosophila melanogaster and humans. The Hh family of secreted proteins activates intracellular signaling cascades for a variety of cellular processes and plays an essential role in embryonic patterning and cell type specification. Vertebrate homologues of Hh are involved in patterning of limbs, lungs, hair follicles and other processes and misregulation of or mutations in Hh signaling genes lead to a number of developmental defects and disorders. Critical to Hh activation is a large protein complex termed the Hedgehog Signaling Complex (HSC). The HSC interprets the level of Hh activation by regulating the levels and activity of one of its components, the transcription factor Ci. Two other components of the HSC are the Ser/Thr protein kinase Fused (Fu) and the putative motor/scaffolding protein Costal-2 (Cos2). Cos2 has significant sequence identity to kinesin family motors, suggesting that Cos2 motility might be important for its function. It also appears to be enriched on microtubules in a manner that is attenuated when cells are exposed to Hh, again consistent with it being an Hh regulated kinesin family member. However, the lack of a key conserved catalytic sequence in Cos2 raises doubts as to whether it is a functional kinesin motor and it has been proposed that Cos2 functions solely as a scaffolding protein, binding and enriching various other Hh pathway components. In order to clarify the role Cos2 plays in Hh signaling, we will perform a detailed mechanistic analysis of its activity. Our preliminary results show that the Cos2 motor domain has both ATPase and GTPase activities. The proposed studies will explore in detail the Cos2 catalytic cycle, as well as characterizing the Cos2 protein interactome using structural and mechanistic approaches to investigate the interaction of Cos2 with microtubules and other binding partners. As a step towards achieving these goals, we have developed a bacterial-based system for the high level expression of a eukaryotic-like nonpolymerizing tubulin dimer. The high-resolution crystal structures to be obtained here will provide key insight into the mode of action of Cos2 in Hh signaling and may lead to novel therapeutic targets for regulation of Hedgehog signaling.

描述（由申请人提供）：Hedgehog (Hh) 信号传导是黑腹果蝇和人类等不同生物体中许多发育过程的基础。 Hh 分泌蛋白家族可激活多种细胞过程的细胞内信号级联，并在胚胎模式和细胞类型规范中发挥重要作用。 Hh 的脊椎动物同源物参与四肢、肺、毛囊和其他过程的模式形成，Hh 信号基因的失调或突变会导致许多发育缺陷和疾病。 Hh 激活的关键是一种称为刺猬信号复合物 (HSC) 的大型蛋白质复合物。 HSC 通过调节其成分之一转录因子 Ci 的水平和活性来解释 Hh 激活水平。 HSC 的另外两个成分是 Ser/Thr 蛋白激酶融合蛋白 (Fu) 和假定的运动/支架蛋白 Costal-2 (Cos2)。 Cos2 与驱动蛋白家族马达具有显着的序列同一性，表明 Cos2 运动性可能对其功能很重要。它似乎还以一种当细胞暴露于 Hh 时减弱的方式富集在微管上，这再次与其作为 Hh 调节的驱动蛋白家族成员一致。然而，Cos2 中缺乏关键的保守催化序列引起了人们对它是否是功能性驱动蛋白马达的怀疑，并且有人提出 Cos2 仅作为支架蛋白发挥作用，结合并富集各种其他 Hh 途径组件。 为了阐明 Cos2 在 Hh 信号传导中的作用，我们将对其活性进行详细的机制分析。我们的初步结果表明 Cos2 运动结构域同时具有 ATPase 和 GTPase 活性。拟议的研究将详细探索 Cos2 催化循环，并使用结构和机制方法表征 Cos2 蛋白相互作用组，以研究 Cos2 与微管和其他结合伙伴的相互作用。作为实现这些目标的一步，我们开发了一种基于细菌的系统，用于高水平表达类真核非聚合微管蛋白二聚体。这里获得的高分辨率晶体结构将为了解 Cos2 在 Hh 信号传导中的作用模式提供关键见解，并可能产生调节 Hedgehog 信号传导的新治疗靶点。