Institut Curie, PSL Research University, Laboratoire de Spectrométrie de Masse Protéomique, Paris, France.
Sorbonne Université, Inserm, Centre de Recherche Saint-Antoine, CRSA, Paris, France.
Mol Cell Proteomics. 2021;20:100173. doi: 10.1016/j.mcpro.2021.100173. Epub 2021 Nov 3.
RNF111/Arkadia is an E3 ubiquitin ligase that activates the transforming growth factor-β (TGF-β) pathway by degrading transcriptional repressors SKIL/SnoN and SKI. Truncations of the RING C-terminal domain of RNF111 that abolish its E3 function and subsequently activate TGF-β signaling are observed in some cancers. In the present study, we sought to perform a comprehensive analysis of RNF111 endogenous substrates upon TGF-β signaling activation using an integrative proteomic approach. In that aim, we carried out label-free quantitative proteomics after the enrichment of ubiquitylated proteins (ubiquitylome) in parental U2OS cell line compared with U2OS CRISPR engineered clones expressing a truncated form of RNF111 devoid of its C-terminal RING domain. We compared two methods of enrichment for ubiquitylated proteins before proteomics analysis by mass spectrometry, the diGlycine (diGly) remnant peptide immunoprecipitation with a K-ε-GG antibody, and a novel approach using protein immunoprecipitation with a ubiquitin pan nanobody that recognizes all ubiquitin chains and monoubiquitylation. Although we detected SKIL ubiquitylation among 108 potential RNF111 substrates with the diGly method, we found that the ubiquitin pan nanobody method also constitutes a powerful approach because it enabled the detection of 52 potential RNF111 substrates including SKI, SKIL, and RNF111. Integrative comparison of the RNF111-dependent proteome and ubiquitylomes enabled the identification of SKI and SKIL as the only targets ubiquitylated and degraded by RNF111 E3 ligase function in the presence of TGF-β. Our results indicate that lysine 343 localized in the SAND domain of SKIL constitutes a target for RNF111 ubiquitylation and demonstrate that RNF111 E3 ubiquitin ligase function specifically targets SKI and SKIL ubiquitylation and degradation upon TGF-β pathway activation.
RNF111/Arkadia 是一种 E3 泛素连接酶，通过降解转录抑制因子 SKIL/SnoN 和 SKI 来激活转化生长因子-β (TGF-β) 途径。在一些癌症中观察到 RNF111 的 RING C 末端结构域截断，该截断消除了其 E3 功能，随后激活了 TGF-β 信号。在本研究中，我们试图使用综合蛋白质组学方法在 TGF-β 信号激活后对 RNF111 的内源性底物进行全面分析。为此，我们在表达缺乏 C 端 RING 结构域的截断形式的 RNF111 的 U2OS CRISPR 工程克隆的亲本 U2OS 细胞系中，与 U2OS CRISPR 工程克隆相比，进行了泛素化蛋白（泛素组）的无标记定量蛋白质组学富集后进行了分析。我们在进行质谱分析之前，通过使用 K-ε-GG 抗体的二甘氨酸（diGly）残基肽免疫沉淀和一种新的使用泛素 pan 纳米抗体的蛋白质免疫沉淀方法，比较了两种泛素化蛋白的富集方法，该方法可识别所有泛素链和单泛素化。尽管我们在用 diGly 方法检测到 108 种潜在的 RNF111 底物中的 SKIL 泛素化，但我们发现泛素 pan 纳米抗体方法也是一种强大的方法，因为它能够检测到 52 种潜在的 RNF111 底物，包括 SKI、SKIL 和 RNF111。RNF111 依赖性蛋白质组和泛素组的综合比较鉴定出 SKI 和 SKIL 是在 TGF-β存在下由 RNF111 E3 连接酶功能泛素化和降解的唯一靶标。我们的结果表明，SKIL 的 SAND 结构域中定位的赖氨酸 343 是 RNF111 泛素化的靶标，并证明 RNF111 E3 泛素连接酶功能在 TGF-β 途径激活时特异性靶向 SKI 和 SKIL 的泛素化和降解。
