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

小分子泛素连接酶复合物底物特异性的分子基础

基本信息

批准号：
9763326
负责人：
Tyler B Faust
金额：
$ 6.16万
依托单位：
DANA-FARBER CANCER INST
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-01 至 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9763326
关键词：
Address Binding Biochemical Cell Line Cells Chemicals Complex Detection Development Disease Goals Human Immunomodulators In Vitro Knowledge Length Libraries Ligands Ligase Malignant Neoplasms Mass Spectrum Analysis Mediating Methods Modification Molecular Mus Mutation Post-Translational Protein Processing Protein Splicing Proteins Public Health RNA Recognition Motif RNA Splicing RNA-Binding Proteins Recruitment Activity Research Specificity Structural Models Structure Substrate Interaction Substrate Specificity Sulfonamides Surface System Thalidomide Therapeutic Intervention Ubiquitin Ubiquitination Work analog anticancer activity cell type design exhaustion experimental study in vivo insight lenalidomide multicatalytic endopeptidase complex mutant novel novel drug class novel therapeutics pomalidomide protein degradation protein expression receptor recruit small molecule therapeutic development transcription factor ubiquitin ligase ubiquitin-protein ligase

项目摘要

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.

项目概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-蛋白酶体系统中的底物特异性使它们泛素化。虽然细胞中的数千种蛋白质被 600 多种连接酶泛素化，泛素连接酶的直接靶标仍然难以确定。因此，底物的一般原则认识仍不清楚。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标的降解来实现特异性。最近，另一类化合物（磺胺类药物）已被证明通过诱导剪接因子 RBM39 的降解以类似的方式发挥作用。这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新底物的内源性靶标。该应用的目的是确定磺酰胺如何化合物 E7820 改变泛素连接酶底物受体 DCAF15 的靶标特异性。中央假设 E7820 与 DCAF15 结合产生了一种新型的小分子-蛋白质相互作用表面，为含有 RNA 识别基序 (RRM) 的底物（包括 RBM39）提供特异性。为了解决这一假设，目标 1 将结合生物化学和结构方法来提供 E7820 介导的 RBM39 募集到 CRL4DCAF15 进行降解的分子基础。可视化 CRL4DCAF15-E7820-RBM39 复合物将揭示 DCAF15 的底物相互作用表面如何被重新利用对于新的目标。将产生结构引导的突变体以支持体内的这种分子图景。目标2 将采用人类 RRM-GFP 文库的降解筛选，这将揭示 E7820 的全部功能- 依赖 CRL4DCAF15 底物。任何通过 E7820 治疗稳定的 RRM 都可能是该药物的自然目标 CRL4DCAF15 连接酶。这些研究的见解将为化合物的设计提供一个框架，这些化合物可以改变其他泛素连接酶的选择性并促进类似调节的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治疗途径开发，拟议的工作将加速新一类药物的设计，这些药物可以降解更广泛的目标，为目前无法治愈的疾病提供新的疗法。