Fine tuning the catalytic cycle of kinesin motors

微调驱动蛋白马达的催化循环

• 批准号: 8083250
• 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/8083250
• 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; 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）：Hedgehog（Hh）信号传导是生物体（如黑腹果蝇和人类）许多发育过程的基础。分泌蛋白的Hh家族激活细胞内信号级联反应，用于各种细胞过程，并在胚胎模式和细胞类型特化中起重要作用。Hh的脊椎动物同源物参与四肢、肺、毛囊和其他过程的模式化，并且Hh信号传导基因的失调或突变导致许多发育缺陷和障碍。Hh激活的关键是一个大的蛋白质复合物，称为刺猬信号复合物（HSC）。HSC通过调节其组分之一转录因子Ci的水平和活性来解释Hh活化的水平。HSC的另外两种组分是Ser/Thr蛋白激酶融合（Fu）和推定的马达/支架蛋白Costal-2（Cos 2）。Cos 2与驱动蛋白家族马达具有显著的序列同一性，表明Cos 2运动性对其功能可能很重要。它也似乎是丰富的微管的方式，是衰减时，细胞暴露于Hh，再次与它是一个Hh调节驱动蛋白家族成员一致。然而，缺乏一个关键的保守的催化序列中的Cos 2提出了怀疑，它是否是一个功能性的驱动蛋白电机，它已被提出，Cos 2功能仅作为一个支架蛋白，结合和丰富各种其他Hh途径的组件。 为了阐明Cos 2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，Cos 2运动域具有ATP酶和GT3酶活性。拟议的研究将详细探讨Cos 2催化循环，以及表征Cos 2蛋白质相互作用组使用结构和机制的方法来研究Cos 2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，我们已经开发了一个基于细菌的系统，用于高水平表达真核生物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Hh信号传导中Cos 2的作用模式提供关键见解，并可能导致Hedgehog信号传导调节的新治疗靶点。 公共卫生相关性：Hedgehog（Hh）家族的分泌蛋白激活细胞内信号级联以用于多种细胞过程，在发育中起重要作用，并且Hh通路的破坏导致越来越多的不同人类病理。Costal-2（Cos 2）是细胞运动蛋白的远亲，并且对于Hh途径中的适当信号传导是必需的。该提案旨在描述Cos 2的特征，以便准确地了解它是如何运作的，这是设计合理的预防和治疗策略以对抗与Hh信号通路故障相关的疾病的关键第一步。