Fine tuning the catalytic cycle of kinesin motors

描述(由申请人提供)：刺猬(HH)信号是许多发育过程的基础，在像果蝇、黑腹果蝇和人类这样不同的生物体中。HH分泌蛋白家族在多种细胞过程中激活细胞内信号级联反应，在胚胎构型和细胞类型确定中起着重要作用。HH的脊椎动物同源物参与了肢体、肺、毛囊和其他过程的模式形成，HH信号基因的错误调节或突变会导致许多发育缺陷和疾病。HH激活的关键是一种被称为Hedgehog信号复合体(HSC)的大型蛋白质复合体。HSC通过调节其组成部分之一转录因子Ci的水平和活性来解释HH的激活水平。HSC的另外两个组成部分是丝氨酸/苏氨酸蛋白激酶融合蛋白(FU)和推测的运动/支架蛋白Costal-2(Cos2)。Cos2与Kinesin家族的运动蛋白有显著的序列同源性，提示Cos2的运动性可能对其功能起重要作用。它似乎也富含在微管上，当细胞暴露在HH中时，这种方式会减弱，这也与它是HH调节的Kinesin家族成员一致。然而，由于COS2中缺乏一个关键的保守催化序列，人们怀疑它是否是一种功能性的运动蛋白，因此有人提出，COS2仅作为一种支架蛋白发挥作用，结合和丰富其他HH途径的各种成分。为了阐明Cos2在HH信号转导中的作用，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运动区同时具有ATPase和GTPase活性。拟议的研究将详细探索COS2催化循环，以及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，我们开发了一种基于细菌的系统，用于高水平表达类真核生物样的非聚合微管蛋白二聚体。将在这里获得的高分辨率晶体结构将为深入了解Cos2在HH信号中的作用模式提供关键的见解，并可能导致调节Hedgehog信号的新的治疗靶点。

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.