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

破译泛素密码用泛素对蛋白质进行翻译后修饰，调节着无数的生物过程。多功能性源于泛素形成结构独特的多泛素链的能力，根据连接(异肽和肽)的不同而不同，连接到目标蛋白质的选定赖氨酸残基上。最近的研究表明，这个‘泛素密码’可以通过额外的翻译后修饰来扩展，包括乙酰化和磷酸化。特定的效应蛋白(泛素结合蛋白)对多泛素链的非共价识别是各自被调控的生物过程的基础。(多)泛素的识别是由泛素结合域(UBD)介导的，UBD是在这些泛素结合蛋白中发现的，它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已知的由泛素控制的一系列生物学过程，到目前为止，只有少数泛素结合蛋白的功能得到了验证和表征。此外，能够区分磷酸化泛素和未修饰泛素的全序列UBD仍有待鉴定。我们假设，由于目前纯化方法和信息学方法的限制，存在许多非常规UBD，包括对磷酸化泛素的特异性UBD。在这个项目中，我们使用新的生化、噬菌体展示和序列分析相结合的方法来寻找和鉴定这些‘缺失的环节’(泛素结合蛋白和UBD)，它们必须存在，以便将泛素修饰与已知的生物输出联系起来。重点放在识别额外的翻译后修饰的UBD上，如磷酸化。对于选定的蛋白质，将通过使用最先进的结构生物学和生物物理方法表征它们与泛素的相互作用来探索它们作为效应器的能力的机制基础，以便将观察放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.