Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, Scotland, United Kingdom; Center for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, Scotland, United Kingdom; Department of Molecular Physiology and Cell Signalling, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, United Kingdom.
Center for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, Scotland, United Kingdom; Department of Molecular Physiology and Cell Signalling, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, United Kingdom.
J Biol Chem. 2021 Aug;297(2):100910. doi: 10.1016/j.jbc.2021.100910. Epub 2021 Jun 24.
Von Hippel-Lindau (VHL) disease is characterized by frequent mutation of VHL protein, a tumor suppressor that functions as the substrate recognition subunit of a Cullin2 RING E3 ligase complex (CRL2). CRL2 plays important roles in oxygen sensing by targeting hypoxia-inducible factor-alpha (HIF-α) subunits for ubiquitination and degradation. VHL is also commonly hijacked by bifunctional molecules such as proteolysis-targeting chimeras to induce degradation of target molecules. We previously reported the design and characterization of VHL inhibitors VH032 and VH298 that block the VHL:HIF-α interaction, activate the HIF transcription factor, and induce a hypoxic response, which can be beneficial to treat anemia and mitochondrial diseases. How these compounds affect the global cellular proteome remains unknown. Here, we use unbiased quantitative MS to identify the proteomic changes elicited by the VHL inhibitor compared with hypoxia or the broad-spectrum prolyl-hydroxylase domain enzyme inhibitor IOX2. Our results demonstrate that VHL inhibitors selectively activate the HIF response similar to the changes induced in hypoxia and IOX2 treatment. Interestingly, VHL inhibitors were found to specifically upregulate VHL itself. Our analysis revealed that this occurs via protein stabilization of VHL isoforms and not via changes in transcript levels. Increased VHL levels upon VH298 treatment resulted in turn in reduced levels of HIF-1α protein. This work demonstrates the specificity of VHL inhibitors and reveals different antagonistic effects upon their acute versus prolonged treatment in cells. These findings suggest that therapeutic use of VHL inhibitors may not produce overt side effects from HIF stabilization as previously thought.
希佩尔-林道（VHL）病的特征是 VHL 蛋白频繁发生突变，VHL 蛋白是一种肿瘤抑制因子，作为 Cullin2 RING E3 连接酶复合物（CRL2）的底物识别亚基发挥作用。CRL2 通过靶向缺氧诱导因子-α（HIF-α）亚基进行泛素化和降解，在氧感应中发挥重要作用。VHL 也经常被双功能分子如蛋白水解靶向嵌合体劫持，以诱导靶分子的降解。我们之前报道了 VHL 抑制剂 VH032 和 VH298 的设计和表征，它们阻断了 VHL：HIF-α 相互作用，激活了 HIF 转录因子，并诱导了缺氧反应，这对治疗贫血和线粒体疾病可能是有益的。这些化合物如何影响全局细胞蛋白质组仍然未知。在这里，我们使用无偏定量 MS 来鉴定与缺氧或广谱脯氨酰-羟化酶结构域酶抑制剂 IOX2 相比，VHL 抑制剂引起的蛋白质组变化。我们的结果表明，VHL 抑制剂选择性地激活 HIF 反应，类似于缺氧和 IOX2 处理引起的变化。有趣的是，VHL 抑制剂被发现特异性地上调 VHL 本身。我们的分析表明，这是通过 VHL 异构体的蛋白稳定化而不是通过转录水平的变化发生的。VH298 处理后 VHL 水平的增加反过来导致 HIF-1α 蛋白水平降低。这项工作证明了 VHL 抑制剂的特异性，并揭示了它们在细胞中的急性和长期治疗中产生的不同拮抗作用。这些发现表明，以前认为 VHL 抑制剂的治疗用途不会因 HIF 稳定而产生明显的副作用。
