Design of bioinspired tandem ubiquitin binding domains as tools to investigate the ubiquitin-modified proteome

翻译后用泛素修饰蛋白质调节无数的生物过程。泛素的多功能性源于泛素形成结构独特的多泛素链的能力，这些多泛素链根据不同的链接（异肽和肽）而变化，这些链附着在目标蛋白的选定赖氨酸残基上。最近的研究表明，这种“泛素密码”通过额外的翻译后修饰（包括乙酰化和磷酸化）得到扩展。特异性效应蛋白（泛素结合蛋白）对多泛素链的非共价识别是各自受调节的生物过程的基础。（多）泛素的识别是由泛素结合结构域（UBDs）介导的，这些泛素结合蛋白中发现了泛素结合结构域（UBDs），它们识别泛素上不同的表面斑块。然而，尽管泛素修饰的复杂性和一系列已知由泛素控制的生物过程，迄今为止只有少数泛素结合蛋白在功能上得到了验证和表征。此外，能够区分磷酸化泛素和未修饰泛素的全范围UBDs仍有待鉴定。我们假设存在许多非常规的ubd，包括磷酸化泛素的特异性，由于当前纯化方法和信息学方法的限制，这些ubd被完全忽视了。在这个项目中，我们正在使用新的生化，噬菌体展示和序列分析方法的组合来搜索和识别这些“缺失环节”（泛素结合蛋白和UBDs），它们必须存在，以便将泛素修饰连接到已知的生物输出。重点放在识别额外翻译后修饰（如磷酸化）的UBDs上。对于选定的蛋白质，将利用最先进的结构生物学和生物物理学方法，通过表征它们与泛素的相互作用，来探索它们作为效应物的能力的机制基础，以便将观察结果建立在坚实的分子和定量基础上。

Deciphering the ubiquitin codePost-translational modification of proteins with ubiquitin regulates a myriad of biological processes. Versatility arises from ubiquitin's ability to form structurally unique polyubiquitin chains, varying according to linkage (isopeptide and peptide), which become attached to selected lysine residues of a target protein. Recent studies show that this 'ubiquitin code' is expanded by additional post-translation modifications including acetylation and phosphorylation.Non-covalent recognition of polyubiquitin chains by specific effector proteins (ubiquitin-binding proteins) underlies the respective biological process that is regulated. Recognition of (poly)ubiquitin is mediated by ubiquitin-binding domains (UBDs), found within these ubiquitin-binding proteins, which recognise distinct surface patches on ubiquitin. However despite the vast complexity of ubiquitin modifications and the array of biological processes that are known to be controlled by ubiquitin, only a handful of ubiquitin-binding proteins have been functionally verified and characterised to date. Further, the full range UBDs capable of discriminating phosphorylated ubiquitin from unmodified ubiquitin remain to be identified.We hypothesise that there are many unconventional UBDs, including with specificity for phosphorylated ubiquitin, that have been completely overlooked due to the limitations of current purification methods and informatics approaches. In this project we are using a combination of novel biochemical, phage display and sequence analysis approaches to search for and identify these 'missing links' (ubiquitin-binding proteins and UBDs) which must exist in order to connect ubiquitin modifications to known biological outputs. Emphasis is placed on UBDs that recognise additional post-translational modifications such as phosphorylation. For selected proteins, the mechanistic basis for their abilities to act as effectors will be probed by characterising their interactions with ubiquitin using state-of-the-art structural biology and biophysical approaches, in order to place observations on a firm molecular and quantitative footing.