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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

基本信息

批准号：
10685519
负责人：
Christina Woo
金额：
$ 32.84万
依托单位：
HARVARD UNIVERSITY
依托单位国家：
美国
项目类别：
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/10685519
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 In Vitro Investigation Kinetics Label 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 biological systems chemoproteomics chromosome 5q loss glutarimide guided inquiry immune modulating agents 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

项目摘要

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.

项目总结免疫调节药物沙利度胺、来那度胺和泊马度胺通过剪裁Cereblon的底物专一性，泛素蛋白酶体系统的一种成分，导致改变底物募集、泛素化和降解。然而，天然底物的特异性和尽管小脑蛋白在不同物种之间都是保守的，但它的生物学功能是难以捉摸的，它与与神经发育的关系，以及参与治疗的雷伯龙的潜在影响在这些底物和路径上。解决更好地描述本地角色的迫切需要在生物调节中，我们采用了一种新的方法来发现底物识别。脑波龙的作用机制。我们使用了有针对性的蛋白质降解方法来系统地筛选与脑蛋白在细胞中的功能结合的生理相关的配体，并发现我们可以用含有C-末端戊二酰亚胺或天冬氨酰亚胺的翻译后多肽取代沙利度胺分别由谷氨酰胺或天冬酰胺环化而来的修饰。从内源性来看，这些C-末端环酰亚胺可以作为分子胶通过调整底物识别来将底物招募到Cereblon上，类似于沙利度胺，或在蛋白质老化和损伤过程中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，我们将从功能上刻画在两种模型的背景下，雷公藤多酚在细胞中识别这些C末端环酰亚胺：作为多肽- 改变底物识别的基本代谢物或作为蛋白质C末端发现的新降解子的一部分在蛋白质老化之后或在信号事件期间。我们将首先全面评估基于多肽的配体的识别在用于靶向蛋白质降解的双功能降解剂的背景下，以表征范围和多肽与脑白蛋白的配基能力及其作为底物募集分子胶能力的评价类似于沙利度胺和来那度胺。接下来，我们将评估C-末端环酰亚胺在生物中作为降解物直接促进对工程蛋白和内源性蛋白的底物识别系统。为了便于研究这些修饰，我们将开发正交化学标记策略以检测和映射这些修改发生在单元格中的位置和时间。最后，我们将对形成进行调查在细胞中的这些修饰，以表征产生C-末端的条件、途径和“写入者” 可被Cereblon识别的环酰亚胺。C-末端戊二酰亚胺和天冬氨酰亚胺修饰的定义为脑白蛋白内源底物募集的关键识别元件及其相关研究构成了一项重大进步，将开启蛋白质生物调节的新研究通过这些翻译后修饰以及在小脑参与过程中对这些通路的影响通过小分子，这将为临床上使用和开发使用小分子脑波龙的疗法提供信息。