Department of Biochemistry, Cancer Biology, Neuroscience and Pharmacology, School of Medicine, Meharry Medical College, Nashville, TN, USA.
Department of Microbiology, Immunology and Physiology, School of Medicine, Meharry Medical College, Nashville, TN, USA.
Cell Physiol Biochem. 2021 Mar 27;55(2):141-159. doi: 10.33594/000000351.
BACKGROUND/AIMS: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that binds to the antioxidant response element(s) (ARE) in target gene promoters, enabling oxidatively stressed cells to respond in order to restore redox homeostasis. Post-translational modifications (PTMs) that mediate activation of Nrf2, in the cytosol and its release from Keap1, have been extensively studied but PTMs that impact its biology after activation are beginning to emerge. In this regard, PTMs like acetylation, phosphorylation, ubiquitination and sumoylation contribute towards the Nrf2 subcellular localization, and its transactivation function. We previously demonstrated that Nrf2 traffics to the promyelocytic leukemia-nuclear bodies (PML-NB), where it is a target for modification by small ubiquitin-like modifier (SUMO) proteins (sumoylation), but the site(s) for SUMO conjugation have not been determined. In this study, we aim to identify SUMO-2 conjugation site(s) and explore the impact, sumoylation of the site(s) have on Nrf2 stability, nuclear localization and transcriptional activation of its target gene expression upon oxidative stress.
The putative SUMO-binding sites in Nrf2 for human isoform1 (NP_006155.2) and mouse homolog (NP_035032.1) were identified using a computer-based SUMO-predictive software (SUMOplot™). Site-directed mutagenesis, immunoblot analysis, and ARE-mediated reporter gene assays were used to assess the impact of sumoylation on these site(s) in vitro. Effect of mutation of these sumoylation sites of Nrf2 on expression of Heme Oxygenase1 (HO-1) was determined in HEK293T cell.
RESULTS: Eight putative sumoylation sites were identified by SUMOplot™ analysis. Out of the eight predicted sites only one LKDE of human (h) and its homologous LKDE of mouse (m) Nrf2, exactly matches the SUMO-binding consensus motif. The other high probability SUMO-acceptor site identified was residue K, in the motifs PKSD and PKQD of human and mouse Nrf2, respectively. Mutational analysis of putative sumoylation sites (human (h)/mouse (m) K, hK and mK) showed that these residues are needed for SUMO-2 conjugation, nuclear localization and ARE driven transcription of reporter genes and the endogenous HO-1 expression by Nrf2. These residues also stabilized Nrf2, as evident from shorter half-lives of the mutant protein compared to wild-type Nrf2.
CONCLUSION: Our findings indicate that SUMO-2 mediated sumoylation of K and K in human Nrf2 regulates in part its transcriptional activity by enhancing its stabilization and nuclear localization.
背景/目的：核因子红细胞 2 相关因子 2（Nrf2）是一种转录因子，可与靶基因启动子中的抗氧化反应元件（ARE）结合，使氧化应激细胞能够做出反应，以恢复氧化还原稳态。Nrf2 的翻译后修饰（PTMs）介导其在细胞质中的激活及其与 Keap1 的释放，已经得到了广泛的研究，但影响其激活后生物学的 PTMs 开始出现。在这方面，乙酰化、磷酸化、泛素化和 sumoylation 等 PTMs 有助于 Nrf2 的亚细胞定位及其转录激活功能。我们之前证明 Nrf2 易位到早幼粒细胞白血病核小体（PML-NB），在那里它是小泛素样修饰物（SUMO）蛋白（sumoylation）修饰的靶标，但 SUMO 缀合的位点尚未确定。在这项研究中，我们旨在确定 SUMO-2 缀合位点，并探讨这些位点的 sumoylation对 Nrf2 稳定性、核定位和其靶基因表达的转录激活的影响，在氧化应激下。
使用基于计算机的 SUMO 预测软件（SUMOplot™）鉴定人同种型 1（NP_006155.2）和小鼠同源物（NP_035032.1）Nrf2 的推定 SUMO 结合位点。定点突变、免疫印迹分析和 ARE 介导的报告基因测定用于评估体外 sumoylation 对这些位点的影响。在 HEK293T 细胞中，确定 Nrf2 的这些 sumoylation 位点突变对血红素加氧酶 1（HO-1）表达的影响。
结果：SUMOplot™ 分析鉴定了 8 个推定的 sumoylation 位点。在预测的 8 个位点中，只有人类（h）的一个 LKDE 和其同源的小鼠（m）Nrf2 的 LKDE，完全符合 SUMO 结合基序。鉴定的另一个高概率 SUMO 受体位点是人类和小鼠 Nrf2 的 PKSD 和 PKQD 基序中的残基 K。人（h）/鼠（m） K、hK 和 mK 假定的 sumoylation 位点的突变分析表明，这些残基是 SUMO-2 缀合、核定位和 ARE 驱动报告基因转录以及 Nrf2 驱动的内源性 HO-1 表达所必需的。这些残基还稳定了 Nrf2，从野生型 Nrf2 相比，突变蛋白的半衰期更短可以明显看出。
我们的研究结果表明，SUMO-2 介导的人 Nrf2 中 K 和 K 的 sumoylation 通过增强其稳定性和核定位，部分调节其转录活性。
