Structural and functional characterisation of human ROCO proteins - How multi-domain proteins work to integrate intracellular signalling pathways

人类 ROCO 蛋白的结构和功能表征 - 多结构域蛋白如何整合细胞内信号通路

• 批准号: 2108179
• 金额: --
• 依托单位: University of Reading
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2108179
• 关键词: Structural; functional; characterisation; human; ROCO

In 2003, the Ras of complex proteins (ROCO) proteins were identified as large multi-domain proteins and have been the subject of intense scrutiny, resulting from their association with a number of human diseases including Parkinson's and cancer. ROCO proteins are unique in the human proteome as they are the only proteins to pair a GTPase domain with a kinase domain in the same open reading frame. As both are key enzymatic domains involved in cell signalling, it is likely that ROCO proteins act as cellular signal transduction hubs. To date, much of the research of the human ROCO proteins has been limited to LRRK2, as a key player in Parkinson's disease, but a general understanding is still lacking. However, ROCO proteins are being actively investigated as potential therapeutic targets for human disease - emphasising the importance of generating structural data for this family of proteins.The aim of this project is to understand how the ROCO proteins integrate cell-signalling pathways at a structural level, using human ROCO proteins including LRRK2, LRRK1 and DAPK1 as models. To achieve this, a multi-construct multi-host strategy will be adopted to produce full-length and combinations of protein domains prior to structural analysis by crystallography and/or cryo-EM analysis. Analysis of structural dynamics will be supported by a range of techniques including Synchrotron Radiation Circular Dichroism (SR-CD), X-ray diffraction, cryoEM and solution scattering at Diamond Light Source. Additionally, built on the Lewis' group experience, functional mutations will be generated for assays to systematically assess the role/impact of these on activity. In parallel, the constructs will be used for cell biology assays to identify and characterise functional interactions based at the University of Reading.

2003 年，Ras 复合蛋白 (ROCO) 被鉴定为大型多结构域蛋白，并因其与帕金森病和癌症等多种人类疾病的关联而受到密切关注。 ROCO 蛋白在人类蛋白质组中是独一无二的，因为它们是唯一在同一开放阅读框中将 GTP 酶结构域与激酶结构域配对的蛋白质。由于两者都是参与细胞信号传导的关键酶结构域，因此 ROCO 蛋白很可能充当细胞信号转导枢纽。迄今为止，人类 ROCO 蛋白的大部分研究仅限于作为帕金森病关键参与者的 LRRK2，但仍缺乏总体了解。然而，ROCO 蛋白作为人类疾病的潜在治疗靶点正在被积极研究——强调为该蛋白家族生成结构数据的重要性。该项目的目的是了解 ROCO 蛋白如何在结构水平上整合细胞信号传导途径，使用包括 LRRK2、LRRK1 和 DAPK1 在内的人类 ROCO 蛋白作为模型。为了实现这一目标，在通过晶体学和/或冷冻电镜分析进行结构分析之前，将采用多构建体多宿主策略来产生全长和蛋白质结构域组合。结构动力学分析将得到一系列技术的支持，包括同步辐射圆二色性 (SR-CD)、X 射线衍射、冷冻电镜和钻石光源的溶液散射。此外，根据刘易斯小组的经验，将生成功能突变用于分析，以系统地评估这些突变对活性的作用/影响。与此同时，这些构建体将用于细胞生物学测定，以识别和表征雷丁大学的功能相互作用。