Differential Analysis of Cereblon Neosubstrates in Rabbit Embryos Using Targeted Proteomics.

IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Molecular & Cellular Proteomics Pub Date : 2024-07-01 Epub Date: 2024-06-10 DOI:10.1016/j.mcpro.2024.100797
Joel D Federspiel, Natasha R Catlin, William S Nowland, Christine M Stethem, Nagappan Mathialagan, Mireia Fernandez Ocaña, Christopher J Bowman
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Abstract

Targeted protein degradation is the selective removal of a protein of interest through hijacking intracellular protein cleanup machinery. This rapidly growing field currently relies heavily on the use of the E3 ligase cereblon (CRBN) to target proteins for degradation, including the immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide which work through a molecular glue mechanism of action with CRBN. While CRBN recruitment can result in degradation of a specific protein of interest (e.g., efficacy), degradation of other proteins (called CRBN neosubstrates) also occurs. Degradation of one or more of these CRBN neosubstrates is believed to play an important role in thalidomide-related developmental toxicity observed in rabbits and primates. We identified a set of 25 proteins of interest associated with CRBN-related protein homeostasis and/or embryo/fetal development. We developed a targeted assay for these proteins combining peptide immunoaffinity enrichment and high-resolution mass spectrometry and successfully applied this assay to rabbit embryo samples from pregnant rabbits dosed with three IMiDs. We confirmed previously reported in vivo decreases in neosubstrates like SALL4, as well as provided evidence of neosubstrate changes for proteins only examined in vitro previously. While there were many proteins that were similarly decreased by all three IMiDs, no compound had the exact same neosubstrate degradation profile as another. We compared our data to previous literature reports of IMiD-induced degradation and known developmental biology associations. Based on our observations, we recommend monitoring at least a major subset of these neosubstrates in a developmental test system to improve CRBN-binding compound-specific risk assessment. A strength of our assay is that it is configurable, and the target list can be readily adapted to focus on only a subset of proteins of interest or expanded to incorporate new findings as additional information about CRBN biology is discovered.

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利用靶向蛋白质组学对兔胚胎中的 Cereblon 新基质进行差异分析。
靶向蛋白质降解是通过劫持细胞内蛋白质清理机制,有选择性地清除感兴趣的蛋白质。这一迅速发展的领域目前主要依赖于使用 E3 连接酶 Cereblon(CRBN)来靶向降解蛋白质,包括沙利度胺、来那度胺和泊马度胺等免疫调节药物(IMiDs)。虽然 CRBN 招募可导致特定相关蛋白质(如药效)降解,但其他蛋白质(称为 CRBN 新基质)也会发生降解。在兔子和灵长类动物身上观察到的沙利度胺相关发育毒性中,一种或多种 CRBN 新底物的降解被认为起着重要作用。我们确定了一组与 CRBN 相关蛋白稳态和/或胚胎/胎儿发育有关的 25 种相关蛋白。我们针对这些蛋白质开发了一种结合肽免疫亲和富集和高分辨率质谱的靶向检测方法,并成功地将该检测方法应用于服用了三种 IMiDs 的怀孕兔子的胚胎样本。我们证实了之前报道的体内新底物(如 SALL4)的减少,并为之前仅在体外检测的蛋白质提供了新底物变化的证据。虽然有许多蛋白质在三种 IMiDs 的作用下都出现了类似的降解,但没有一种化合物与另一种化合物具有完全相同的新底物降解特征。我们将我们的数据与以前有关 IMiD 诱导降解的文献报告以及已知的发育生物学关联进行了比较。根据我们的观察结果,我们建议在发育测试系统中至少监测这些新基质的一个主要子集,以改进 CRBN 结合化合物特异性风险评估。我们的检测方法的优势在于它是可配置的,目标列表可随时调整,以便只关注感兴趣的蛋白质子集,或随着有关 CRBN 生物学的更多信息的发现而扩展,以纳入新的发现。
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来源期刊
Molecular & Cellular Proteomics
Molecular & Cellular Proteomics 生物-生化研究方法
CiteScore
11.50
自引率
4.30%
发文量
131
审稿时长
84 days
期刊介绍: The mission of MCP is to foster the development and applications of proteomics in both basic and translational research. MCP will publish manuscripts that report significant new biological or clinical discoveries underpinned by proteomic observations across all kingdoms of life. Manuscripts must define the biological roles played by the proteins investigated or their mechanisms of action. The journal also emphasizes articles that describe innovative new computational methods and technological advancements that will enable future discoveries. Manuscripts describing such approaches do not have to include a solution to a biological problem, but must demonstrate that the technology works as described, is reproducible and is appropriate to uncover yet unknown protein/proteome function or properties using relevant model systems or publicly available data. Scope: -Fundamental studies in biology, including integrative "omics" studies, that provide mechanistic insights -Novel experimental and computational technologies -Proteogenomic data integration and analysis that enable greater understanding of physiology and disease processes -Pathway and network analyses of signaling that focus on the roles of post-translational modifications -Studies of proteome dynamics and quality controls, and their roles in disease -Studies of evolutionary processes effecting proteome dynamics, quality and regulation -Chemical proteomics, including mechanisms of drug action -Proteomics of the immune system and antigen presentation/recognition -Microbiome proteomics, host-microbe and host-pathogen interactions, and their roles in health and disease -Clinical and translational studies of human diseases -Metabolomics to understand functional connections between genes, proteins and phenotypes
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