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

2003年，复杂蛋白Ras （ROCO）蛋白被确定为大的多结构域蛋白，由于它们与包括帕金森病和癌症在内的许多人类疾病有关，因此一直受到密切关注。ROCO蛋白在人类蛋白质组中是独一无二的，因为它们是唯一在相同的开放阅读框中配对GTPase结构域和激酶结构域的蛋白质。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，对人类ROCO蛋白的大部分研究都局限于LRRK2，它是帕金森病的关键角色，但仍缺乏普遍的认识。然而，人们正在积极研究ROCO蛋白作为人类疾病的潜在治疗靶点，这强调了生成该蛋白家族结构数据的重要性。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通路，以人类ROCO蛋白包括LRRK2， LRRK1和DAPK1为模型。为了实现这一目标，将采用多构建多宿主策略，在通过晶体学和/或低温电镜分析进行结构分析之前产生全长和蛋白质结构域组合。结构动力学分析将由一系列技术支持，包括同步辐射圆二色（SR-CD）， x射线衍射，低温电子显微镜和钻石光源溶液散射。此外，基于Lewis团队的经验，将生成功能突变，用于系统地评估这些突变对活性的作用/影响。同时，这些结构将用于细胞生物学分析，以识别和表征雷丁大学的功能相互作用。

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.