Fundamental Research Center, Shanghai YangZhi Rehabilitation Hospital (Shanghai Sunshine Rehabilitation Center), School of Life Sciences and Technology, Tongji University, Shanghai 200092, China.
Department of Biological Sciences, Boler-Parseghian Center for Rare and Neglected Diseases, Harper Cancer Research Institute, University of Notre Dame, Notre Dame, IN 46556, USA.
Cells. 2022 Jan 29;11(3):472. doi: 10.3390/cells11030472.
The inactivation of von Hippel-Lindau (VHL) is critical for clear cell renal cell carcinoma (ccRCC) and VHL syndrome. VHL loss leads to the stabilization of hypoxia-inducible factor α (HIFα) and other substrate proteins, which, together, drive various tumor-promoting pathways. There is inadequate molecular characterization of VHL restoration in VHL-defective ccRCC cells. The identities of HIF-independent VHL substrates remain elusive. We reinstalled VHL expression in 786-O and performed transcriptome, proteome and ubiquitome profiling to assess the molecular impact. The transcriptome and proteome analysis revealed that VHL restoration caused the downregulation of hypoxia signaling, glycolysis, E2F targets, and mTORC1 signaling, and the upregulation of fatty acid metabolism. Proteome and ubiquitome co-analysis, together with the ccRCC CPTAC data, enlisted 57 proteins that were ubiquitinated and downregulated by VHL restoration and upregulated in human ccRCC. Among them, we confirmed the reduction of TGFBI (ubiquitinated at K676) and NFKB2 (ubiquitinated at K72 and K741) by VHL re-expression in 786-O. Immunoprecipitation assay showed the physical interaction between VHL and NFKB2. K72 of NFKB2 affected NFKB2 stability in a VHL-dependent manner. Taken together, our study generates a comprehensive molecular catalog of a VHL-restored 786-O model and provides a list of putative VHL-dependent ubiquitination substrates, including TGFBI and NFKB2, for future investigation.
von Hippel-Lindau（VHL）失活对于透明细胞肾细胞癌（ccRCC）和 VHL 综合征至关重要。VHL 缺失导致缺氧诱导因子α（HIFα）和其他底物蛋白的稳定，这些蛋白共同驱动各种促进肿瘤的途径。VHL 缺陷型 ccRCC 细胞中 VHL 恢复的分子特征描述不足，HIF 非依赖性 VHL 底物的身份仍然难以捉摸。我们在 786-O 细胞中重新安装 VHL 表达，并进行转录组、蛋白质组和泛素组谱分析，以评估分子影响。转录组和蛋白质组分析表明，VHL 恢复导致缺氧信号、糖酵解、E2F 靶标和 mTORC1 信号下调，以及脂肪酸代谢上调。蛋白质组和泛素组联合分析，以及 ccRCC CPTAC 数据，确定了 57 种由 VHL 恢复导致的泛素化和下调，并在人类 ccRCC 中上调的蛋白质。其中，我们通过在 786-O 中重新表达 VHL 证实了 TGFBI（在 K676 泛素化）和 NFKB2（在 K72 和 K741 泛素化）的减少。免疫沉淀实验表明 VHL 与 NFKB2 之间存在物理相互作用。NFKB2 的 K72 以 VHL 依赖的方式影响 NFKB2 的稳定性。总之，我们的研究生成了一个全面的 VHL 恢复的 786-O 模型的分子目录，并提供了一个可能的 VHL 依赖性泛素化底物的列表，包括 TGFBI 和 NFKB2，以供未来研究。
