无标记交互作用分析揭示了CUL3-KEAP1复合体在介导PHD2泛素化和降解中的重要作用。,Journal of Proteome Research

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无标记交互作用分析揭示了CUL3-KEAP1复合体在介导PHD2泛素化和降解中的重要作用。
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2019-12-06 , DOI: 10.1021/acs.jproteome.9b00513
Ang Luo ₁ , Yue Chen ₁

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含有脯氨酰羟化酶结构域的蛋白质2（PHD2 / EGLN1）是一种关键的调节酶，在细胞低氧反应途径中起着基本作用，介导脯氨酸羟化依赖性蛋白质对选定目标蛋白质的降解。然而，对PHD2动态平衡在蛋白质水平上的调节尚不十分了解。在这里，我们通过免疫沉淀与质谱分析进行无标记的定量相互作用组分析。为了最大程度地减少由异位过表达引起的副作用，我们在HeLa细胞中稳定表达了带有Flag标签的PHD2，同时通过使用靶向其3'UTR区的shRNA抑制了内源性PHD2。我们鉴定并验证了Cullin 3作为体内新型PHD2相互作用子。通过候选人筛选，我们进一步确定了CUL3-KEAP1 E3泛素连接酶复合物是调节PHD2降解的主要酶。CUL3，KEAP1或两者的过度表达会显着增加PHD2泛素化并降低PHD2蛋白的丰度。敲低CUL3或KEAP1降低PHD2泛素化并抑制PHD2降解。因此，缺氧下CUL3-KEAP1复合物的缺失促进了PHD2的稳定，并导致PHD2靶标（低氧诱导因子1A（HIF1A））的丰度大大降低。因此，CUL3-KEAP1是调节细胞中PHD2泛素化和降解的重要途径。敲低CUL3或KEAP1降低PHD2泛素化并抑制PHD2降解。因此，缺氧下CUL3-KEAP1复合物的缺失促进了PHD2的稳定，并导致PHD2靶标（低氧诱导因子1A（HIF1A））的丰度大大降低。因此，CUL3-KEAP1是调节细胞中PHD2泛素化和降解的重要途径。敲低CUL3或KEAP1降低PHD2泛素化并抑制PHD2降解。因此，缺氧下CUL3-KEAP1复合物的缺失促进了PHD2的稳定，并导致PHD2靶标（低氧诱导因子1A（HIF1A））的丰度大大降低。因此，CUL3-KEAP1是调节细胞中PHD2泛素化和降解的重要途径。

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Label-Free Interactome Analysis Revealed an Essential Role of CUL3-KEAP1 Complex in Mediating the Ubiquitination and Degradation of PHD2.

Prolyl hydroxylase domain-containing protein 2 (PHD2/EGLN1) is a key regulatory enzyme that plays a fundamental role in the cellular hypoxic response pathway, mediating proline hydroxylation-dependent protein degradation of selected target proteins. However, the regulation of PHD2 homeostasis at the protein level is not well understood. Here, we perform label-free quantitative interactome analysis through immunoprecipitation coupled with mass spectrometry analysis. To minimize the side effects caused by ectopic overexpression, in HeLa cells, we stably overexpressed Flag-tagged PHD2 while suppressing the endogenous PHD2 by using an shRNA targeting its 3' UTR region. We identified and validated Cullin 3 as a novel PHD2 interactor in vivo. Through candidate screening, we further identified CUL3-KEAP1 E3 ubiquitin ligase complex as the major enzyme that regulates PHD2 degradation. Overexpression of either CUL3, KEAP1, or both significantly increases PHD2 ubiquitination and reduces PHD2 protein abundance. The knockdown of CUL3 or KEAP1 decreased PHD2 ubiquitination and inhibited PHD2 degradation. Accordingly, loss of the CUL3-KEAP1 complex under hypoxia promoted PHD2 stabilization and led to significantly reduced abundance of the PHD2 target, hypoxia-inducible factor 1A (HIF1A). Thus, CUL3-KEAP1 is an essential pathway that regulates PHD2 ubiquitination and degradation in cells.

更新日期：2019-12-06

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