Uncovering the substrate recognition mechanisms of the E3 ligase adaptor cereblon

项目摘要 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其治疗作用： 定制cereblon的底物特异性，cereblon是泛素蛋白酶体系统的组分，导致 改变的底物募集、泛素化和降解。然而，天然底物特异性和 cereblon的生物学功能是难以捉摸的，尽管cereblon在物种间是保守的，它的关联 与神经系统发育的关系，以及cereblon治疗的潜在影响 在这些基质和途径上。解决更好地描述土著人作用的迫切需要 cereblon在生物调节中的作用，我们采用了一种新的方法来发现底物识别 cereblon的机制我们使用靶向蛋白降解方法系统地筛选 生理学上相关的配体的cereblon在细胞中的功能参与，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代替沙利度胺，这些肽是翻译后的 分别衍生自谷氨酰胺或天冬酰胺的环化的修饰。内源性地，这些C末端 环状酰亚胺可以作为分子胶通过定制底物识别来将底物补充到cereblon， 类似于沙利度胺，或者可以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，我们将从功能上描述 在两种模型的背景下，这些C-末端环状酰亚胺在细胞中被cereblon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 在蛋白质老化之后或在信号传导事件期间。我们将首先全面评估肽基配体的识别 在用于靶向蛋白质降解的双功能降解剂的背景下， 通过肽的cereblon的可配位性，并评估它们作为底物募集的分子胶的能力 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-末端环状酰亚胺是否 作为降解决定子，直接促进生物学中工程化和内源性蛋白质上的底物识别 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 以检测和绘制这些修饰在细胞中何时何地发生。最后，我们将研究 这些修饰在细胞中表征的条件，途径，和“作家”，产生C-末端 Cereblon识别的环状酰亚胺。C-末端戊二酰亚胺和天冬酰亚胺修饰的定义为 用于募集内源性底物到cereblon的关键识别元件及相关研究 构成了一个重大的进步，将打开新的研究，生物调节蛋白质 通过这些翻译后修饰以及在cereblon参与过程中对这些通路的影响， 这将为临床上使用cereblon的治疗方法的使用和开发提供信息。

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.