The molecular basis of substrate specificity for a small molecule ubiquitin ligase complex

项目摘要 泛素对蛋白质的翻译后修饰导致其被蛋白酶体降解。泛素 连接酶通过结合靶蛋白赋予泛素-蛋白酶体系统底物特异性， 将它们呈现为泛素化。虽然细胞中成千上万的蛋白质被600多种连接酶泛素化， 遍在蛋白连接酶的直接靶标仍然难以鉴定。因此，基质的一般原理 承认仍然不清楚。值得注意的是，一类称为IMiD（免疫调节药物）的小分子， 包括沙利度胺、来那度胺和泊马度胺，可以结合泛素连接酶并改变其底物 通过诱导新靶点的降解来提高特异性。最近，另一类化合物（磺胺类） 已经显示通过诱导剪接因子RBM 39的降解以类似的方式起作用。的 该提案的长期目标是了解小分子如何重新连接泛素连接酶的选择性， 内源性靶向新底物。该应用程序的目的是确定磺胺如何 化合物E7820改变泛素连接酶底物受体DCAF 15的靶特异性。中央 假设E7820与DCAF 15结合产生了一种新的小分子-蛋白质相互作用表面， 对含有RNA识别基序（RRM）的底物（包括RBM 39）具有特异性。 为了解决这一假设，目标1将利用生物化学和结构方法的组合，以提供一个 E7820介导的RBM 39募集至CRL 4DCAF 15进行降解的分子基础。可视化 CRL 4DCAF 15-E7820-RBM 39复合物将揭示DCAF 15的底物相互作用表面如何被重新利用 寻找新的目标将产生结构引导的突变体以支持这种体内分子图像。目的2 将采用人RRM-GFP文库的降解筛选，这将揭示E7820的全部库- 依赖性CRL 4DCAF 15底物。E7820治疗稳定的任何RRM都可能是E7820治疗的天然靶点。 CRL 4DCAF 15连接酶。从这些研究中获得的见解将为设计化合物提供一个框架， 改变其他泛素连接酶的选择性，并促进天然配体的发现， 连接酶-底物相互作用。最后，靶向蛋白质降解是一种令人兴奋的新的治疗途径， 发展，拟议的工作将加速设计新的药物类别，可以降解 更广泛的目标，使目前无法治愈的疾病的新疗法。

Project Summary Post-translational modification of proteins with ubiquitin leads to their degradation by the proteasome. Ubiquitin ligases confer substrate specificity in the ubiquitin-proteasome system by binding target proteins and presenting them for ubiquitination. While thousands of proteins in the cell are ubiquitinated by over 600 ligases, the direct targets of ubiquitin ligases are still difficult to identify. As such, the general principles of substrate recognition remain unclear. Remarkably, a class of small molecules termed IMiDs (immunomodulatory drugs), including thalidomide, lenalidomide and pomalidomide, can bind ubiquitin ligases and alter their substrate specificity by inducing the degradation of novel targets. Recently, another class of compounds (sulfonamides) has been shown to act in an analogous manner by inducing the degradation of the splicing factor RBM39. The long-term goal of the proposal is to understand how small molecules can rewire ubiquitin ligase selectivity from endogenous targets to neo-substrates. The objective of the application is to determine how the sulfonamide compound E7820 alters the target specificity of the ubiquitin ligase substrate receptor DCAF15. The central hypothesis is that E7820 binding to DCAF15 generates a novel small molecule-protein interaction surface that provides specificity for substrates containing an RNA recognition motif (RRM), including RBM39. To address this hypothesis, Aim 1 will utilize a combination of biochemical and structural methods to provide a molecular basis for E7820-mediated recruitment of RBM39 to CRL4DCAF15 for degradation. Visualizing the CRL4DCAF15-E7820-RBM39 complex will reveal how the substrate interaction surface of DCAF15 is repurposed for novel targets. Structure-guided mutants will be generated to support this molecular picture in vivo. Aim 2 will employ a degradation screen of a human RRM-GFP library, which will uncover the full repertoire of E7820- dependent CRL4DCAF15 substrates. Any RRMs stabilized by E7820 treatment are likely natural targets of the CRL4DCAF15 ligase. Insight from these studies will provide a framework for the design of compounds that can alter selectivity of other ubiquitin ligases and facilitate the discovery of natural ligands that similarly regulate ligase-substrate interactions. Finally, targeted protein degradation is an exciting new avenue of therapeutic development, and the proposed work will accelerate the design of new classes of drugs that can degrade a broader range of targets, enabling novel therapies for currently incurable diseases.