Uncovering the substrate recognition mechanisms of the E3 ligase adaptor cereblon

揭示 E3 连接酶接头 cereblon 的底物识别机制

• 批准号: 10446251
• 负责人: Christina Woo
• 金额: $ 32.84万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10446251
• 关键词: Address; Aging; Amines; Amino Acid Sequence; Asparagine; Binding; Biological; Biological Assay; Biological Process; C-terminal; Cells; Chemicals; Clinic; Complex; Coupled; Cyclization; Data; Development; Dipeptides; Dysmyelopoietic Syndromes; Elements; Engineering; Enzymes; Evaluation; Event; Generations; Glues; Glutamine; Health; Hematopoietic Neoplasms; Human; Imides; Immunomodulators; In Vitro; Investigation; Kinetics; Label; Lead; Libraries; Ligands; Maps; Measurement; Modeling; Modification; Molecular; Multiple Myeloma; Neurologic; Pathway interactions; Patients; Peptides; Periodicity; Physiological; Play; Post-Translational Protein Processing; Proteins; Proteome; Proteomics; Reader; Regulation; Role; Signal Transduction; Site; Specificity; Substrate Specificity; System; Teratogenic effects; Thalidomide; Therapeutic; Therapeutic Effect; Tissue Sample; Ubiquitin; Work; Writing; analog; anti-cancer; base; biological systems; chemoproteomics; chromosome 5q loss; glutarimide; guided inquiry; inhibitor; insight; lenalidomide; multicatalytic endopeptidase complex; novel; novel strategies; nucleophilic addition; pomalidomide; protein degradation; recruit; response; small molecule; therapeutic development; tissue culture; ubiquitin-protein ligase

PROJECT SUMMARY The immunomodulatory drugs thalidomide, lenalidomide, and pomalidomide exert their therapeutic effects by tailoring the substrate specificity of cereblon, a component of the ubiquitin proteasome system, resulting in altered substrate recruitment, ubiquitylation, and degradation. However, the native substrate specificity and biological functions of cereblon are elusive, despite the conservation of cereblon across species, its association with neurological development, and the potential impact that therapeutic engagement of cereblon would have on these substrates and pathways. To address the urgent need for better characterization of the native role cereblon plays in biological regulation, we undertook a novel approach to discover the substrate recognition mechanism of cereblon. We used a targeted protein degradation approach to systematically screen physiologically relevant ligands for functional engagement of cereblon in cells and discovered that we could replace thalidomide with peptides bearing a C-terminal glutarimide or aspartimide, which are post-translational modifications derived from cyclization of glutamine or asparagine, respectively. Endogenously, these C-terminal cyclic imides may act as molecular glues to recruit substrates to cereblon by tailoring substrate recognition, analogous to thalidomide, or may be generated directly on the substrate during protein aging and damage response or in response to signaling events. To address these two hypotheses, we will functionally characterize the recognition of these C-terminal cyclic imides by cereblon in cells in the context of two models: as peptide- based metabolites that alter substrate recognition or as part of a novel degron found at the C-terminus of proteins after protein aging or during a signaling event. We will first fully evaluate the recognition of peptide-based ligands in the context of bifunctional degraders for targeted protein degradation to characterize the scope and ligandability of cereblon by peptides and evaluate their ability to act as molecular glues for substrate recruitment in a manner analogous to thalidomide and lenalidomide. Next, we will assess whether C-terminal cyclic imides act as degrons that promote substrate recognition directly on engineered and endogenous proteins in biological systems. To facilitate the study of these modifications, we will develop orthogonal chemical labeling strategies to detect and map where and when these modifications occur in cells. Finally, we will investigate the formation of these modifications in cells to characterize the conditions, pathways, and “writers” that generate the C-terminal cyclic imides recognized by cereblon. The definition of C-terminal glutarimide and aspartimide modifications as the key recognition elements used to recruit endogenous substrates to cereblon and the associated studies constitutes a significant advance that will open new investigations into the biological regulation of proteins through these post-translational modifications and the effects on these pathways during cereblon engagement by small molecules, which will inform the use and development of therapeutics that engage cereblon in the clinic.

项目概要 免疫调节药物沙利度胺、来那度胺和泊马度胺通过以下方式发挥治疗作用： 定制 cereblon（泛素蛋白酶体系统的一个组成部分）的底物特异性，从而产生 改变底物招募、泛素化和降解。然而，天然底物特异性和 尽管 cereblon 在跨物种中具有保守性，但 cereblon 的生物学功能仍难以捉摸，其关联性 与神经系统发育有关，以及 cereblon 治疗的潜在影响 在这些底物和途径上。满足更好地描述本地角色的迫切需要 cereblon 在生物调节中发挥作用，我们采取了一种新方法来发现底物识别 cereblon 的机制。我们使用靶向蛋白质降解方法来系统地筛选 细胞中大脑功能参与的生理相关配体，并发现我们可以 用带有 C 末端戊二酰亚胺或天冬酰亚胺的肽替代沙利度胺，这些肽是翻译后的 分别源自谷氨酰胺或天冬酰胺环化的修饰。内源性地，这些 C 末端 环状酰亚胺可以充当分子胶，通过定制底物识别将底物招募到 cereblon， 类似于沙利度胺，或者可能在蛋白质老化和损伤期间直接在底物上产生 响应或响应信号事件。为了解决这两个假设，我们将从功能上描述 在两种模型的背景下，细胞中的 cereblon 对这些 C 末端环状酰亚胺的识别：作为肽- 改变底物识别的代谢物或作为蛋白质 C 末端发现的新型降解决定子的一部分 蛋白质老化后或信号事件期间。我们将首先全面评估基于肽的配体的识别 在双功能降解剂的背景下，用于靶向蛋白质降解，以表征范围和 肽与 cereblon 的配体性并评估其作为底物招募分子胶的能力 以类似于沙利度胺和来那度胺的方式。接下来，我们将评估C端环状酰亚胺是否 作为降解决定子，促进生物中工程蛋白和内源蛋白直接识别底物 系统。为了促进这些修饰的研究，我们将开发正交化学标记策略 检测并绘制细胞中这些修饰发生的时间和地点。最后，我们将研究形成 细胞中的这些修饰来表征生成 C 末端的条件、途径和“作家” cereblon 识别的环状酰亚胺。 C端戊二酰亚胺和天冬酰亚胺修饰的定义为 用于招募内源性底物到 cereblon 的关键识别元件及相关研究 这是一项重大进展，将为蛋白质的生物调控开启新的研究 通过这些翻译后修饰以及大脑参与期间对这些途径的影响 通过小分子，这将为临床上使用 cereblon 的疗法的使用和开发提供信息。