OTU家族去泛素化酶的结构和功能研究

Protein ubiquitination is a post-translational modification that is involved in many cellular processes. Ubiquitin can form eight distinct chain linkage types (Met1, Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, Lys63), determining the fate of the modified protein. Deubiquitinating enzymes of the ovarian tumour (OTU) family regulate cellular signalling by targeting distinct linkage types within polyubiquitin. We cloned, purified and crystallized two proteins of this family OTUD6A and OTUD6B, which share very high levels of sequence identity but exert distinct functions. OTUD6A can cleave Lys11-, Lys27-, Lys29- and Lys33- linked ubiquitin chains. However, OTUD6B has no obvious activity to cleave diubiquitin chains while it can deubiquitinate Ub-Met-β-gal fusion protein. Therefore，it remains the question how OTUD6A recognizes these four atypical ubiquitylation and whether OTUD6B has deubiquitinase activity. We plan to solve crystal structures of OTUD6A/OTUD6B and their complexes with ubiquitin. Together with biochemistry, the study will help illustrate the molecular mechanism of Lys11-, Lys27-, Lys29- and Lys33-polyubiquitin specificity in the deubiquitinase OTUD6A and clarify whether OTUD6B has deubiquitinase activity as well as its structural basis.

蛋白质的泛素化是一种广泛参与生命活动过程调控的翻译后修饰。泛素通过七个赖氨酸残基（Lys6/11/27/29/33/48/63）或者N端的甲硫氨酸残基（Met1）形成八种不同连接类型的泛素链，决定着蛋白质的不同命运。OTU家族的去泛素化酶通过靶向不同类型的泛素链参与细胞信号通路的调控。申请人前期克隆、表达纯化以及结晶了此家族中序列一致性很高但功能不同的两个蛋白质OTUD6A与OTUD6B。OTUD6A能对四种非典型双泛素进行去泛素化，而OTUD6B只能水解模式底物Ub-Met-β-gal融合蛋白，对双泛素却不具有去泛素化酶活性，其具体机制尚不清楚。本项目将利用X射线晶体学解析出蛋白质OTUD6A与OTUD6B及其与泛素的复合物晶体结构，并在此基础上结合生物化学等研究手段揭示OTUD6A特异性识别四种非典型泛素链的内在机理，阐明OTUD6B是否具有去泛素化酶活性及其结构基础。

OTUD6A与OTUD6B是OTU家族中序列一致性很高但功能存在差异的两个蛋白质。此前的研究认为OTUD6A能对Lys11、Lys27、Lys29及Lys33四种非典型双泛素进行去泛素化，而对于OTUD6B是否具有去泛素化酶活性尚存在争议。本项目利用X射线晶体学解析出了蛋白质OTUD6A与OTUD6B的晶体结构以及OTUD6A与单泛素及双泛素的复合物晶体结构，并进一步通过体外的去泛素化实验揭示了OTUD6A能特异性去泛素化K63连接类型的泛素链。而OTUD6B具有独特的His loop构象，这使得它不具有明显的去泛素化酶活性。这为研究两者的生理功能提供了一定的结构基础。此外，通过突变活性位点152位半胱氨酸发现，半胱氨酸突变成丝氨酸的失活突变不改变它与泛素的结合能力，这对于后续体内功能研究如何选择失活突变提供了理论依据。