Fine tuning the catalytic cycle of kinesin motors

微调驱动蛋白马达的催化循环

• 批准号: 8245013
• 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 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8245013
• 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. PUBLIC HEALTH RELEVANCE: The Hedgehog (Hh) family of secreted proteins activates intracellular signaling cascades for a variety of cellular processes, plays an essential role in development, and disruption of the Hh pathway leads to an increasingly large number of different human pathologies. Costal-2 (Cos2) is a distant relative of cellular motor proteins, and is essential for proper signaling in the Hh pathway. This proposal seeks to characterize Cos2 in order to figure out exactly how it operates, a critical first step in designing rational preventative and curative strategies against diseases related to malfunctions in the Hh signaling pathway.

描述（由申请人提供）：刺猬（HH）信号传导是类似果蝇和人类等生物的许多发育过程的基础。分泌蛋白的HH家族激活细胞内信号传导级联反应，以进行多种细胞过程，并且在胚胎图案和细胞类型规范中起着至关重要的作用。 HH的脊椎动物同源物参与了四肢，肺，毛囊和其他过程的模式，以及HH信号基因中或突变的不调节导致许多发育缺陷和疾病。 HH激活至关重要的是一种称为刺猬信号复合物（HSC）的大蛋白质复合物。 HSC通过调节其成分之一的转录因子CI的水平和活动来解释HH激活的水平。 HSC的另外两个组件是Ser/Thr蛋白激酶融合（FU）和推定的运动/脚手架蛋白Costal-2（COS2）。 COS2对运动蛋白家族电机具有显着的序列身份，这表明COS2运动对其功能可能很重要。它似乎还以细胞暴露于HH时会减弱的方式富集在微管上，这再次与它是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运动的怀疑，并且已经提出COS2仅充当脚手架蛋白，结合和富集其他各种HH途径成分。 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械分析。我们的初步结果表明，COS2运动域具有ATPase和GTPase活动。拟议的研究将详细探讨COS2催化循环，并使用结构和机械方法来研究COS2蛋白质相互作用组，以研究COS2与微管和其他结合伴侣的相互作用。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，用于高水平的真核类非聚合微型小管蛋白二聚体。此处要获得的高分辨率晶体结构将为HH信号中COS2的作用方式提供关键的见解，并可能导致新的治疗靶标，以调节刺猬信号。 公共卫生相关性：分泌蛋白的刺猬（HH）家族激活各种细胞过程的细胞内信号传导级联，在发育中起着至关重要的作用，而HH途径的破坏会导致越来越多的不同人类病理。 Costal-2（COS2）是细胞运动蛋白的遥远亲戚，对于在HH途径中正确信号至关重要。该提案试图表征COS2，以确切弄清楚其运作方式，这是针对HH信号通路中与故障有关的疾病的理性预防和治疗策略的关键第一步。