Structure-function investigations of protein partnerships governing neuronal signaling

控制神经信号传导的蛋白质伙伴关系的结构功能研究

• 批准号: RGPIN-2018-05838
• 负责人: Donaldson, Logan
• 金额: $ 2.33万
• 依托单位: York University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=684440
• 关键词: Structure; function; investigations; protein; partnerships

Chemical and electrical signals are transmitted between neurons through a specialized junction called a synapse. For each side of the synapse to function efficiently, hundreds of receptors, intracellular messages and cytoskeletal proteins need to be brought together in highly regulated, yet dynamic way. This fluid state of communication is achieved by scaffolding and adaptor proteins. I use high resolution structural biology methods to determine how scaffolds and adaptors use protein-protein interactions and post-translational modifications to help neurons make connections and respond to external stimuli at the molecular level.******In this proposal, previously published structures from by my group on the adaptor protein HACS1 and the scaffolding protein CASKIN2 are extended to investigations of unconventional protein partnerships mediated by them.******HACS1 is a modular protein whose architecture helps it form partnerships with signal transduction and cytoskeletal proteins. Aim 1 builds upon a previously determined high resolution structure of the HACS1 Src homology-3 (SH3) domain from my group and the discovery that it interacts with the Paired receptor (PIRB) cytosolic region in a novel way. The interaction is believed to be novel because the complement of amino acids at the binding surface of the SH3 domain are atypical as well as the sequence in PIRB that is recognized. The long-term goal of this research is to provide a structural explanation for how negative signals from PIRB inhibit the maturation of neurons through HACS1. In addition to its SH3 domain, HACS1 also has a sterile alpha motif (SAM) domain that is also well known for its ability to support a wide range of protein-protein interactions. Aim 2 focuses upon discovering new partners for the HACS1 SAM domain. The identification of these partners represents an essential first step towards establishing a complete functional description of HACS1. As I have determined many SAM domain structures throughout my career, I can work with my research group to propel the most interesting partners towards high-resolution structural studies. ******Compared to HACS1, the organization of CASKIN2 is more complex thereby earning it the designation of a molecular scaffold, rather than an adaptor. CASKIN2, and CASKIN1 by similarity, are recognized for their roles in vesicle trafficking and creating proper connections between motor neurons and muscle cells. In Aim 3, I will determine the structural basis of how the SAM domain of CASKIN2 interacts with a known, yet poorly defined region, of the LAR receptor phosphatase. The CASKIN2/LAR partnership was selected because the SAM domain is predicted to interact with LAR in a manner that is distinct from other SAM domain structures that I have solved. The biochemical and structural insights from this aim can be extended to the Liprin class of neuronal scaffolds that also use SAM domains to bind LAR.

化学和电信号通过称为突触的特殊连接在神经元之间传递。为了让突触的每一侧都能有效发挥作用，数百种受体、细胞内信息和细胞骨架蛋白需要以高度调控但又动态的方式结合在一起。这种流体状态的通讯是通过支架和接头蛋白实现的。我使用高分辨率的结构生物学方法来确定支架和适配器如何利用蛋白质-蛋白质相互作用和翻译后修饰来帮助神经元在分子水平上建立连接和对外部刺激做出反应。*在这个建议中，我的团队以前发表的关于适配器蛋白HACS1和支架蛋白CASKIN2的结构被扩展到由它们介导的非传统蛋白质伙伴关系的研究。*HACS1是一种模块化蛋白质，其结构有助于它与信号转导和细胞骨架蛋白形成伙伴关系。目的1建立在本课题组先前确定的HACS1 Src Homology-3(SH3)结构域的高分辨率结构的基础上，并发现它以一种新的方式与配对受体(PirB)胞浆区域相互作用。这种相互作用被认为是新的，因为SH3结构域结合表面的氨基酸互补是不典型的，以及PirB中被识别的序列。这项研究的长期目标是为PirB的负面信号如何通过HACS1抑制神经元成熟提供结构性解释。除了SH3结构域，HACS1还有一个不育的α基序(SAM)结构域，该结构域也以支持广泛的蛋白质-蛋白质相互作用而闻名。目标2专注于为HACS1 SAM域寻找新的合作伙伴。确定这些合作伙伴是朝着建立HACS1的完整功能说明迈出的重要的第一步。由于我在整个职业生涯中确定了许多SAM领域结构，我可以与我的研究团队合作，推动最有趣的合作伙伴进行高分辨率结构研究。*与HACS1相比，CASKIN2的组织结构更加复杂，因此它被指定为分子支架，而不是接头。CASKIN2和CASKIN1的相似之处在于，它们在囊泡运输和运动神经元和肌肉细胞之间建立适当的连接方面起着重要作用。在目标3中，我将确定CASKIN2的SAM结构域如何与LAR受体磷酸酶的已知但未明确定义的区域相互作用的结构基础。之所以选择CASKIN2/LAR伙伴关系，是因为预计SAM域将以不同于我解决的其他SAM域结构的方式与LAR交互。来自这一目标的生化和结构洞察可以扩展到Liprin类神经支架，这种支架也使用SAM结构域来结合LAR。