磷酸化调控Lys63连接的多聚泛素的结构与功能的研究

Ubiquitin is an important cellular signal, and ubiquitin molecules are covalently linked to form polyubiquitins. Lys63-linked poly-ubiquitin is involved in a variety of non-degradable functions. Applicants' previous work has found that Lys63-linked dimeric ubiquitin (K63-diUb) comprises three different, dynamically inter-convertible quaternary structures, each of which can bind to a target protein and initiate the corresponding signaling pathway. Protein aggresome is essential for maintaining the homeostasis of proteins in cells, and timely removal protects cells. It was found that Lys63-linked polyubiquitin recruits proteasome shuttle protein Ubiquilin, serine kinase PINK1 and other proteins, and promotes the removal of aggresome. Studies have also shown that PINK1 phosphorylates ubiquitin Ser65, and affects the tertiary structure of ubiquitin. Based on our findings, we propose that upon ubiquitin phosphorylation, the Lys63-linked polyubiquitin would morph to a different set of quaternary structures, and bind to Ubiquilin more tightly for the removal of the aggresome. To this end, the project will use single-molecule fluorescence, mass spectrometry, small angle scattering technology and solution NMR jointly, for a comprehensive and in-depth characterization of phosphorylated K63-diUb structure, from the ubiquitin dynamic structure changes in phosphate The effect of modification on its function, The study will reveal how phosphorylation affects ubiquitin function and provide new ideas for the treatment of related diseases.

泛素蛋白是重要的细胞信号，泛素之间以共价连接形成多聚泛素。申请人的前期工作发现，Lys63连接的二聚泛素同时存在于三种不同的、动态转化的四级结构，每一种结构都能与靶蛋白结合，启动相应的信号通路。蛋白质聚集体是维持细胞中蛋白质稳态的必要条件，及时清除对细胞起保护作用。Lys63连接的多聚泛素会招募转运蛋白Ubiquilin、激酶PINK1等蛋白，促进聚集体的降解。PINK1能磷酸化泛素的Ser65氨基酸，影响泛素蛋白的三级结构。基于这些研究也基于我们的前期工作，我们推测泛素被磷酸化后，Lys63连接的多聚泛素会发生四级结构的变化，进而改变与Ubiquilin的结合方式，加速聚集体的清除。为此，本项目将综合运用单分子荧光、质谱交联、小角散射技术和溶液核磁等技术，全面深入的表征Lys63连接的多聚泛素的结构如何受磷酸化调控。该研究将初步揭示磷酸化如何影响泛素功能，并为相关疾病的治疗提供新思路。

泛素蛋白是重要的细胞信号，泛素之间能够以不同形式的共价连接形成多聚泛素。PINK1能磷酸化泛素的S65氨基酸，影响泛素蛋白的结构，并进而调控泛素蛋白的功能。围绕项目目标，项目研究团队取得了一系列的成果和进展：解释了泛素蛋白能够被PINK1进行双磷酸化修饰，并且这种修饰对pH值的变化响应更加敏感；表征了蛋白酶体受体Rpn13识别K48连接的二聚泛素的结构基础；阐明了泛素S65磷酸化对泛素蛋白与UBQLN2相互作用的影响机制；围绕核磁共振发展了一系列研究蛋白质动态结构的新方法或整合方法，为蛋白质动态结构，蛋白质动态修饰的机制研究提供了参考和帮助。在本项目的资助下，共发表研究论文8篇，培养出站博士后1人，培养毕业博士研究生2名，培养毕业硕士研究生2名。