Fine tuning the catalytic cycle of kinesin motors

微调驱动蛋白马达的催化循环

• 批准号: 8446463
• 负责人: Fredrick Jon Kull
• 金额: $ 28.97万
• 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 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8446463
• 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）：刺猬（Hh）信号是许多生物发育过程的基础，如黑胃果蝇和人类。Hh家族分泌的蛋白激活细胞内信号级联反应，参与多种细胞过程，并在胚胎模式和细胞类型规范中发挥重要作用。Hh的脊椎动物同源基因参与四肢、肺、毛囊和其他过程的模式，Hh信号基因的错误调控或突变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）的大蛋白复合物。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的其他两个组成部分是丝氨酸/苏氨酸蛋白激酶融合（Fu）和假定的马达/支架蛋白Costal-2 （Cos2）。Cos2与kinesin家族马达具有显著的序列同一性，提示Cos2的运动性可能对其功能起重要作用。当细胞暴露于Hh时，它似乎也以一种减弱的方式在微管上富集，这再次与它是Hh调节的激酶家族成员一致。然而，Cos2中缺乏一个关键的保守催化序列，这使得人们怀疑Cos2是否是一个功能性的运动蛋白马达，并且有人提出Cos2仅作为支架蛋白起作用，结合并富集各种其他Hh通路组分。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马达结构域同时具有atp酶和GTPase活性。这些研究将详细探讨Cos2的催化循环，并利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，我们已经开发了一种基于细菌的系统，用于高水平表达真核样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，并可能为调节Hedgehog信号传导提供新的治疗靶点。