Investigations into ubiquitin binding proteins using structure guided reactivity

项目总结 泛素(Ub)是一种对真核生物至关重要的翻译后修饰因子。Ub系统中继器 通过其对底物蛋白质的附着和分离，对细胞过程进行复杂的控制，从而产生 一种“泛素密码”。这一法规中的放松管制会带来严重的后果，主要是在疾病发病机制方面。在……里面 与其他单一的翻译后修饰不同，泛素化有许多不同的味道。 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(MET1、Lys6、Lys11、Lys27、Lys29、Lys33、Lys48、Lys63)产生不同的Ub “泛素代码”的解码者或阅读器所识别的链接。这些Ub受体使 具有炎症反应和DNA损伤修复等重要功能。过去几年的研究 几十年来，人们对那些建立和擦除“泛素密码”的作家提供了批判性的见解， 然而，相比之下，我们对这些Ub受体和Ub相互作用蛋白的了解是有限的。这项建议 描述了基于Ub的探针用于分析UBD和Ub依赖的蛋白质-蛋白质相互作用 使用扩展蛋白质化学。在两个目标中，我们利用遗传密码扩展和液体 色谱-串联质谱学解决这个项目的目标：开发一种方法来鉴定 并从结构上表征了Ub依赖的蛋白质-蛋白质相互作用。目标1.确定瞬时互动 光亲和生物素化Ub探针用于真核来源的UBD及其结构表征 使用化学选择性结扎和X射线结晶学的候选者。目标2.开发一种执行任务的方法 使用无迹Staudinger连接的化学(多)泛素化获得均一(多)Ub探针 和(多)泛素化底物蛋白。这将应用于26S的非典型Lys29泛素化 蛋白酶体受体Rpn13，E3连接酶UBE3C的调节作用。Ub化学中的这些方法 生物学将允许我们通过以下方式研究依赖于Ub的蛋白质-蛋白质相互作用及其生物物理对应 重组化学捕获非共价相互作用所需的元素，这一特征目前限制了 Ub系统的研究。综上所述，我们试图描述这些基于Ub的相互作用是如何在生物化学上 减弱功能，并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.