Proteomic analysis of ubiquitin ligase KEAP1 reveals associated proteins that inhibit NRF2 ubiquitination.
Proteomic analysis of ubiquitin ligase KEAP1 reveals associated proteins that inhibit NRF2 ubiquitination.
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DOI：
10.1158/0008-5472.can-12-4400
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发表时间：
2013-04-01
期刊：
影响因子：
11.2
通讯作者：
Major MB
中科院分区：
文献类型：
--
作者：
Hast BE;Goldfarb D;Mulvaney KM;Hast MA;Siesser PF;Yan F;Hayes DN;Major MB
KEAP1 泛素连接酶或其底物 NRF2 (NFE2L2) 的体细胞突变通常发生在人类癌症中，导致 NRF2 介导的细胞保护基因的组成型转录。然而，许多肿瘤在没有突变的情况下表现出高 NRF2 活性，支持了存在替代途径激活机制的假设。此前，我们和其他人发现，通过竞争性结合机制，蛋白质 WTX (AMER1)、PALB2 和 SQSTM1 结合 KEAP1 以激活 NRF2。 KEAP1 蛋白相互作用网络的蛋白质组学分析揭示了含有 ETGE 氨基酸基序的相关蛋白显着富集，该基序与 NRF2 中发现的 KEAP1 相互作用基序相匹配。与 WTX、PALB2 和 SQSTM1 一样，我们发现二肽基肽酶 3 (DPP3) 蛋白通过“ETGE”基序结合 KEAP1 以取代 NRF2，从而抑制 NRF2 泛素化并驱动 NRF2 依赖性转录。将 KEAP1 相互作用蛋白谱与癌症基因组图谱表征的 178 例鳞状细胞肺癌的基因组图谱进行比较，发现在具有高 NRF2 活性但缺乏 NRF2 稳定突变的肿瘤中 DPP3 基因扩增和 mRNA 过表达。我们进一步表明，KEAP1 中的肿瘤衍生突变对于 NRF2 抑制而言是低等态的，并且在这些突变体存在下 DPP3 过度表达进一步促进 NRF2 激活。总的来说，我们的研究结果进一步支持了 NRF2 激活的竞争模型，并表明 DPP3 等含有“ETGE”的蛋白质有助于癌症中的 NRF2 活性。
Somatic mutations in the KEAP1 ubiquitin ligase or its substrate NRF2 (NFE2L2) commonly occur in human cancer, resulting in constitutive NRF2-mediated transcription of cytoprotective genes. However, many tumors display high NRF2 activity in the absence of mutation, supporting the hypothesis that alternative mechanisms of pathway activation exist. Previously, we and others discovered that via a competitive binding mechanism, the proteins WTX (AMER1), PALB2 and SQSTM1 bind KEAP1 to activate NRF2. Proteomic analysis of the KEAP1 protein interaction network revealed a significant enrichment of associated proteins containing an ETGE amino acid motif, which matches the KEAP1 interaction motif found in NRF2. Like WTX, PALB2, and SQSTM1, we found that the dipeptidyl peptidase 3 (DPP3) protein binds KEAP1 via an ‘ETGE’ motif to displace NRF2, thus inhibiting NRF2 ubiquitination and driving NRF2-dependent transcription. Comparing the spectrum of KEAP1 interacting proteins with the genomic profile of 178 squamous cell lung carcinomas characterized by The Cancer Genome Atlas revealed amplification and mRNA over-expression of the DPP3 gene in tumors with high NRF2 activity but lacking NRF2 stabilizing mutations. We further show that tumor-derived mutations in KEAP1 are hypomorphic with respect to NRF2 inhibition and that DPP3 over-expression in the presence of these mutants further promotes NRF2 activation. Collectively, our findings further support the competition model of NRF2 activation and suggest that ‘ETGE’-containing proteins like DPP3 contribute to NRF2 activity in cancer.