Investigations into ubiquitin binding proteins using structure guided reactivity

项目摘要 泛素（Ubiquitin，Ub）是真核生物中重要的翻译后修饰蛋白。Ub系统中继一个 通过其在底物蛋白上的附着和分离对细胞过程进行复杂的控制， 一个“泛素密码”这种编码的失调会产生严重的后果，主要是在疾病的发病机制中。在 与其他翻译后修饰的单一性不同，泛素化有许多不同的形式。 随后将一个Ub部分的C-末端连接到底物赖氨酸残基或八个赖氨酸残基之一上， 另一个Ub部分的胺（Met 1、Lys 6、Lys 11、Lys 27、Lys 29、Lys 33、Lys 48、Lys 63）产生不同的Ub 由“泛素密码”的解码器或阅读器识别的连接。这些Ub受体促进 重要的功能，如炎症反应和DNA损伤修复。过去几年的研究 几十年来，已经提供了关键的洞察力，作家建立和橡皮擦，删除“泛素代码”， 然而，我们对这些Ub受体和Ub相互作用蛋白质的理解是有限的。这项建议 描述了基于Ub的探针用于分析UBD和Ub依赖性蛋白质-蛋白质相互作用的用途 使用扩展的蛋白质化学。在两个目标中，我们利用遗传密码扩展和液体 色谱-串联质谱，以解决这个项目的目标：开发一种手段，以确定 并在结构上表征了Ub依赖的蛋白质-蛋白质相互作用。目标1。识别瞬时相互作用 使用光亲和生物素化Ub探针的真核生物来源的UBD， 候选人使用化学选择性连接和X射线晶体学。目标2.开发一种方法来执行 使用无痕施陶丁格连接进行化学（多聚）泛素化以获得同质（多聚）Ub探针 和（聚）泛素化底物蛋白。这将应用于26 S的非典型Lys 29泛素化， 蛋白酶体受体Rpn 13，E3连接酶UBE 3C的调节作用。Ub化学的这些方法 生物学将使我们能够研究依赖于Ub的蛋白质-蛋白质相互作用及其生物物理对应物， 重组化学捕获非共价相互作用所需的元素，这是目前限制生物相容性的一个特征。 研究UB系统。总之，我们试图描述这些基于UB的相互作用如何在生物化学上 减弱功能，并使用结构证据证实这些发现。

PROJECT SUMMARY Ubiquitin (Ub) is a principal post-translational modifier crucial to eukaryotic biology. The Ub system relays an intricate control over cellular processes through its attachment and detachment on substrate protein, giving rise to a “ubiquitin code”. Deregulation in this code has severe consequences, primarily in disease pathogenesis. In contrast to other post-translational modifications being singular, ubiquitination comes in many different flavors. Subsequent attachment of the C-terminus of one Ub moiety onto a substrate lysine residue or one of eight amines (Met1, Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, Lys63) of another Ub moiety gives rise to distinct Ub linkages that are recognized by the decoders or readers of the “ubiquitin code”. These Ub receptors facilitate important functions such as inflammatory response and DNA damage repair. Research over the past few decades has provided critical insight into the writers that establish and erasers that remove the “ubiquitin code”, yet our understanding of these Ub-receptors and Ub-interacting proteins is limited in comparison. This proposal describes the use of Ub-based probes for profiling UBDs and Ub-dependent protein-protein interactions using expanded protein chemistry. In two aims, we leverage genetic code expansion and liquid chromatography-tandem mass spectrometry to address the goal of this project: to develop a means to identify and structurally characterize Ub-dependent protein-protein interactions. Aim 1. Identify transiently interacting UBDs of eukaryotic origin using photoaffinity biotinylated Ub probes and structurally characterize promising candidates using chemo-selective ligation and X-ray crystallography. Aim 2. Develop a means to perform chemical (poly)ubiquitination using the traceless Staudinger ligation for access to homogenous (poly)Ub probes and (poly)ubiquitinated substrate protein. This will be applied to the atypical Lys29 ubiquitination of the 26S proteasome receptor Rpn13, a regulatory effect of the E3 ligase UBE3C. These approaches in Ub chemical biology will allow us to study Ub-dependent protein-protein interactions and their biophysical counterpart by reconstituting the elements needed to chemically trap noncovalent interactions, a feature currently limiting the study of the Ub system. Taken together, we seek to describe how these Ub-based interactions biochemically attenuate function and corroborate these findings using structural evidence.