Robarts Research Institute, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada.
Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada.
FASEB J. 2021 Sep;35(9):e21825. doi: 10.1096/fj.202100664R.
Ubiquitination is an essential post-translational modification that regulates protein stability or function. Its substrate specificity is dictated by various E3 ligases. The human C-terminal to LisH (CTLH) complex is a newly discovered multi-subunit really interesting new gene (RING) E3 ligase with only a few known ubiquitination targets. Here, we used mass spectrometry-based proteomic techniques to gain insight into CTLH complex function and ubiquitination substrates in HeLa cells. First, global proteomics determined proteins that were significantly increased, and thus may be substrates targeted for degradation, in cells depleted of CTLH complex member RanBPM. RanBPM-dependent ubiquitination determined using diGLY-enriched proteomics and the endogenous RanBPM interactome further revealed candidate ubiquitination targets. Three glycolysis enzymes alpha-enolase, L-lactate dehydrogenase A chain (LDHA), and pyruvate kinase M1/2 (PKM) had decreased ubiquitin sites in shRanBPM cells and were found associated with RanBPM in the interactome. Reduced polyubiquitination was validated for PKM2 and LDHA in cells depleted of RanBPM and CTLH complex RING domain subunit RMND5A. PKM2 and LDHA protein levels were unchanged, yet their activity was increased in extracts of cells with downregulated RanBPM. Finally, RanBPM deficient cells displayed enhanced glycolysis and deregulated central carbon metabolism. Overall, this study identifies potential CTLH complex ubiquitination substrates and uncovers that the CTLH complex inhibits glycolysis via non-degradative ubiquitination of PKM2 and LDHA.
泛素化是一种重要的翻译后修饰，可调节蛋白质的稳定性或功能。其底物特异性由各种 E3 连接酶决定。人类 C 端到 LisH（CTLH）复合物是一种新发现的多亚基真正有趣的新基因（RING）E3 连接酶，只有少数已知的泛素化靶标。在这里，我们使用基于质谱的蛋白质组学技术深入了解 CTLH 复合物在 HeLa 细胞中的功能和泛素化底物。首先，全局蛋白质组学确定了在 CTLH 复合物成员 RanBPM 耗尽的细胞中显着增加的蛋白质，因此可能是靶向降解的底物。使用富含二甘氨酸的蛋白质组学和内源性 RanBPM 相互作用组确定的 RanBPM 依赖性泛素化进一步揭示了候选泛素化靶标。三种糖酵解酶α-烯醇酶、L-乳酸脱氢酶 A 链（LDHA）和丙酮酸激酶 M1/2（PKM）在 shRanBPM 细胞中的泛素化位点减少，并在相互作用组中与 RanBPM 相关。RanBPM 和 CTLH 复合物 RING 结构域亚基 RMND5A 耗尽的细胞中证实了 PKM2 和 LDHA 的多泛素化减少。PKM2 和 LDHA 蛋白水平不变，但在 RanBPM 下调的细胞提取物中其活性增加。最后，RanBPM 缺陷细胞显示出增强的糖酵解和中央碳代谢失调。总体而言，这项研究确定了潜在的 CTLH 复合物泛素化底物，并揭示了 CTLH 复合物通过非降解性泛素化 PKM2 和 LDHA 抑制糖酵解。
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