There are over 600 E3 ligase complexes reported, many of which are poorly characterized in terms of both their substrate scope and biological functions. In recent years, potent and selective small-molecule ligands have been discovered for a small number of E3 ligases that regulate proteostasis of important disease targets, including CRBN, VHL, MDM2, and IAP.(1) However, there is increasing evidence to suggest that E3 ligases are a broadly ligandable protein family, frequently targeted by covalent fragments.(2,3) There is an additional current interest in new E3 ligase characterization and ligand discovery as a means to expand the degradable proteome via the development of PROTACs and molecular glues that recruit E3 ligases beyond VHL, MDM2, CRBN, and IAP. Thus, a better understanding of E3 ligase endogenous function and substrates is an important endeavor and may help inform the selection of E3-ligase targets suitable for follow-up medicinal chemistry efforts toward the discovery of inhibitors or binders for incorporation into PROTACs. In a recent Biochemistry paper, Zhang et al. identified Spindlin-4 (SPIN4) as an endogenous substrate of the CRL4^DCAF16E3 ligase complex.(4) DCAF16 is localized in the cell nucleus and was previously reported to form an effective degrader complex with electrophilic PROTACs.(2) This characteristic permitted an additional level of degradation specificity by sparing target protein levels in the cytoplasm. In this work, a comparison of the global protein expression profiles of DCAF^WT and DCAF^–/– cells identified SPIN4 as the only target whose levels were upregulated in DCAF16 knockout cells. Rigorous follow-up experiments confirmed the proteomics results and further characterized the DCAF16 substrate interaction and specificity. The workflow described could be applied to other E3 ligases in order to elucidate their protein substrates, which has traditionally been a challenging endeavor in the field (Figure 1). Figure 1. Summary of the approach to identify the new E3-ligase complex: substrate pairs. Organisms use the ubiquitin proteasome pathway to identify and dispose of unwanted proteins. The process begins when a ubiquitin-activating enzyme (E1) adenylates and transfers a ubiquitin protein to a ubiquitin-conjugating enzyme (E2). A ubiquitin ligase enzyme (E3) recognizes the degradation target and catalyzes the transfer of adenylated ubiquitin from the E2 to a lysine residue on the unwanted protein. The action of ubiquitination promotes subsequent poly ubiquitination. The marked protein is finally shuttled to a large protein complex known as the proteasome, whereupon it is digested into its component amino acids. Zhang et al. identified SPIN4 as a substrate of the E3 ligase DCAF16. Using CRISPR/Cas9, they knocked out the DCAF16 gene in two human cancer cell lines and compared protein levels in knockout cells with wild-type cells using tandem mass tagging (TMT) proteomics, which indicated that SPIN4 was the only protein with increased abundance in knockout cells. Western blotting confirmed this result, and qPCR verified that this was not due to the upregulation of the gene responsible for SPIN4 expression. Treatment of wild-type cells with a neddylation inhibitor that inhibits cullin-ring ligases or a proteasome inhibitor both resulted in the elevation of SPIN4 levels comparable to those in knockout cells. Finally, wild-type cells possessed higher levels of polyubiquitinated SPIN4 versus the knockout cells. These control experiments further supported the case that DCAF16 was acting on SPIN4 through an E3 ligase and proteasome-dependent protein degradation pathway. Immunoprecipitation studies using cells expressing FLAG-tagged SPIN4 proteins confirmed that DCAF16 interacted with SPIN4. As the other SPIN family proteins SPIN1, SPIN2a/b, and SPIN3 have similar sequences with SPIN4, the authors subjected them to immunoprecipitation studies and discovered that none interacted with DCAF16. The paper noted that SPIN2a/b and 3 were not detected in the initial proteomics experiments, and this manual inclusion of related proteins by classification or sequence alignment allowed a more complete picture of DCAF16’s interactions and specificity. Sequence analysis of the SPIN protein family further pointed to the potentially important role of three lysine residues in SPIN4 that were not present in other SPIN proteins. Site-directed mutagenesis studies determined that lysine residue 70 on SPIN4 was essential for binding to DCAF16. As it was known that SPIN4 (like the other proteins in the SPIN family) contain domains that interact with methylated histones, the authors assayed FLAG-tagged SPIN4 cells against a variety of histone peptides and discovered that the strongest interaction occurred with biotinylated H3K4me3 peptide, which suggested SPIN4 may play a role in transcriptional regulation. However, SPIN4 knockout did not result in significant changes in protein levels, which the authors speculated could be due to cellular adaptation or functional redundancy, with other SPIN proteins compensating for the loss of SPIN4. It was concluded that the exact function of SPIN4 remains uncertain, despite it being highly conserved among mammalian species. A potential future approach for studying the immediate downstream effects of SPIN4 loss on the proteome could be the use of tag-based chemical-genetic degradation systems.(5) The identification of a single substrate in this study indicates that DCAF16 has a small number of endogenous targets for which it exclusively regulates proteostasis. Despite this, the authors’ previous work suggests DCAF16 can be readily hijacked by electrophilic PROTACs and redirected toward nonendogenous substrates such as FKBP12.(2) The authors present a well-designed example of how CRISPR/Cas-9 gene editing and mass spectrometry-based global proteomics analysis can be leveraged to identify substrates of poorly characterized E3 ligase complexes. In future studies, the methodology described in this paper could be broadly applied to map the exclusive E3 ligase: protein substrate pairs across the understudied E3 ligase-ome and complement other methods of ligase substrate discovery, such as substrate-trapping based strategies. Images were created with BioRender.com. This article references 5 other publications.

中文翻译：

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组装一个强大的工作流程来表征内源性 E3 连接酶底物

报告了 600 多种 E3 连接酶复合物，其中许多在其底物范围和生物学功能方面都没有得到很好的表征。近年来，已经发现了少量 E3 连接酶的有效和选择性小分子配体，这些连接酶可调节重要疾病靶点的蛋白质稳态，包括 CRBN、VHL、MDM2 和 IAP。(1) 然而，越来越多的证据表明E3 连接酶是一个广泛配体的蛋白质家族，经常被共价片段靶向。 (2,3) 目前对新的 E3 连接酶表征和配体发现有额外的兴趣，作为通过开发 PROTAC 和分子来扩展可降解蛋白质组的一种手段。在 VHL、MDM2、CRBN 和 IAP 之外招募 E3 连接酶的胶水。因此，更好地了解 E3 连接酶内源性功能和底物是一项重要的工作，可能有助于选择适合后续药物化学工作的 E3 连接酶靶标，以发现抑制剂或结合剂以纳入 PROTAC。在最近生物化学论文，Zhang 等。将 Spindlin-4 (SPIN4) 鉴定为 CRL4 ^DCAF16 E3 连接酶复合物的内源性底物。(4) DCAF16 位于细胞核中，之前有报道称其与亲电子 PROTAC 形成有效的降解复合物。(2) 这种特性允许通过保留细胞质中的靶蛋白水平来提高降解特异性。在这项工作中，比较 DCAF ^WT和 DCAF ^–/–的全局蛋白质表达谱细胞将 SPIN4 鉴定为在 DCAF16 敲除细胞中其水平上调的唯一靶标。严格的后续实验证实了蛋白质组学结果，并进一步表征了 DCAF16 底物相互作用和特异性。所描述的工作流程可应用于其他 E3 连接酶，以阐明其蛋白质底物，这在该领域历来是一项具有挑战性的工作（图 1）。图 1. 识别新 E3-连接酶复合物的方法总结：底物对。生物体使用泛素蛋白酶体途径来识别和处理不需要的蛋白质。当泛素激活酶 (E1) 腺苷酸化并将泛素蛋白转移到泛素结合酶 (E2) 时，该过程就开始了。泛素连接酶 (E3) 识别降解目标并催化腺苷酸化泛素从 E2 转移到不需要的蛋白质上的赖氨酸残基。泛素化的作用促进了随后的多泛素化。标记的蛋白质最终被运送到称为蛋白酶体的大型蛋白质复合物，然后被消化成其组成氨基酸。张等人。将 SPIN4 鉴定为 E3 连接酶 DCAF16 的底物。他们使用 CRISPR/Cas9 敲除两种人类癌细胞系中的 DCAF16 基因，并使用串联质量标记 (TMT) 蛋白质组学比较敲除细胞与野生型细胞中的蛋白质水平，这表明 SPIN4 是唯一在敲除细胞。蛋白质印迹证实了这一结果，qPCR 证实这不是由于负责 SPIN4 表达的基因的上调。用抑制 cullin 环连接酶的 neddylation 抑制剂或蛋白酶体抑制剂处理野生型细胞都导致 SPIN4 水平的升高，与敲除细胞中的水平相当。最后，与敲除细胞相比，野生型细胞具有更高水平的多泛素化 SPIN4。这些对照实验进一步支持 DCAF16 通过 E3 连接酶和蛋白酶体依赖性蛋白质降解途径作用于 SPIN4 的情况。使用表达 FLAG 标记的 SPIN4 蛋白的细胞进行的免疫沉淀研究证实 DCAF16 与 SPIN4 相互作用。由于其他 SPIN 家族蛋白 SPIN1、SPIN2a/b 和 SPIN3 与 SPIN4 具有相似的序列，作者对它们进行了免疫沉淀研究，发现没有一个与 DCAF16 相互作用。该论文指出，在最初的蛋白质组学实验中没有检测到 SPIN2a/b 和 3，这种通过分类或序列比对手动包含相关蛋白质的方法可以更完整地了解 DCAF16 的相互作用和特异性。SPIN 蛋白家族的序列分析进一步指出了其他 SPIN 蛋白中不存在的 SPIN4 中三个赖氨酸残基的潜在重要作用。定点诱变研究确定 SPIN4 上的第 70 位赖氨酸残基对于与 DCAF16 的结合至关重要。众所周知，SPIN4（与 SPIN 家族中的其他蛋白质一样）包含与甲基化组蛋白相互作用的结构域，作者针对多种组蛋白肽分析了带有 FLAG 标签的 SPIN4 细胞，发现与生物素化 H3K4me3 肽的相互作用最强，这表明 SPIN4 可能在转录调控中发挥作用。然而，SPIN4 敲除并没有导致蛋白质水平的显着变化，作者推测这可能是由于细胞适应或功能冗余，其他 SPIN 蛋白补偿了 SPIN4 的损失。结论是 SPIN4 的确切功能仍然不确定，尽管它在哺乳动物物种中高度保守。研究 SPIN4 丢失对蛋白质组的直接下游影响的一种潜在的未来方法可能是使用基于标签的化学遗传降解系统。(5) 本研究中对单一底物的鉴定表明 DCAF16 具有少量的内源性靶标，专门调节蛋白质稳态。尽管如此，作者之前的工作表明 DCAF16 可以很容易地被亲电 PROTAC 劫持并重定向到非内源性底物，如 FKBP12。(2) 作者展示了一个精心设计的例子，说明 CRISPR/Cas-9 基因编辑和质谱是如何-可以利用基于全局蛋白质组学的分析来识别特征不佳的 E3 连接酶复合物的底物。在未来的研究中，本文中描述的方法可以广泛应用于映射独特的 E3 连接酶：跨越未充分研究的 E3 连接酶组的蛋白质底物对，并补充其他连接酶底物发现方法，例如基于底物捕获的策略。图像是使用 BioRender.com 创建的。本文引用了 5 篇其他出版物。