王峥1, 董浩1,2, 李敏3, 梁秀彬1,*
1南京医科大学基础医学院病理生理学系，南京 211166；2安徽医科大学第一附属医院病理科，合肥 23002；3南京医科大学基础医学院病理学系，南京 211166
泛素-蛋白酶体系统在蛋白质降解时发挥着重要的作用。泛素化过程需要E1泛素激活酶、E2泛素结合酶、E3泛素连接酶协同完成。本研究组前期研究证明E3泛素连接酶HUWE1 (HECT, UBA and WWE domain containing 1)可降解表皮生长因子受体(epidermal growth factor receptor, EGFR)， 抑制肾小管间质纤维化。为了进一步明确HUWE1抑制肾小管间质纤维化的机制，本研究鉴定了参与HUWE1降解EGFR过程的E2泛素结合酶。通过real-time PCR观察可能与HUWE1发生相互作用的候选E2泛素结合酶在肾脏损伤模型中的表达变化。用小鼠单侧输尿管结扎(unilateral ureteral obstruction, UUO)模型的肾组织和转化生长因子-β (transforming growth factor-β, TGF-β)刺激的人肾脏近端小管上皮细胞(HK-2细胞)来检测候选E2泛素结合酶的表达量变化。结果显示，与对照组相比，E2泛素结合酶UBE2Q2在UUO术后小鼠肾脏组织中mRNA与蛋白水平均显著下调，在TGF-β刺激的HK-2细胞中UBE2Q2 mRNA和蛋白表达水平也显著下调，与HUWE1表达变化趋势一致，表明在肾脏损伤时E2泛素结合酶UBE2Q2表达水平与HUWE1具有协同性。免疫共沉淀(co-immunoprecipitation, Co-IP)和细胞免疫荧光染色结果验证了HUWE1与UBE2Q2的相互作用。在HK-2细胞中敲低UBE2Q2后，HUWE1、EGFR与泛素的结合均显著降低。上述结果提示，UBE2Q2可能是与HUWE1相互作用的E2泛素结合酶，参与HUWE1对肾小管间质纤维化的抑制作用。
关键词： UBE2Q2; HUWE1; 表皮生长因子受体; 泛素化修饰; 肾小管间质纤维化
Ubiquitin-conjugating enzyme UBE2Q2 participates in HUWE1-mediated protection on renal tubulointerstitial fibrosis
WANG Zheng1, DONG Hao1,2, LI Min3, LIANG Xiu-Bin1,*
1Department of Pathophysiology, Nanjing Medical University, Nanjing 211166, China；2Department of Pathology, the First Affiliated Hospital of Anhui Medical University, Hefei 230022, China；3Department of Pathology, Nanjing Medical University, Nanjing 211166, China
The ubiquitin-proteasome system plays an important role in protein degradation. The process of ubiquitination requires ubiquitin activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3 to complete the coordination. Our previous studies have shown that HUWE1 (HECT, UBA and WWE domain containing 1), as an E3 ubiquitin ligase, can degrade epidermal growth factor receptor (EGFR) to inhibit renal tubulointerstitial fibrosis. However, E2 ubiquitin-conjugating enzymes binding to HUWE1 are still unclear. The aim of the present study was to identify E2 ubiquitin-conjugating enzymes of HUWE1. Real-time PCR was used to identify E2 ubiquitin-conjugating enzyme that may interact with HUWE1. The expression of E2 ubiquitin-conjugating enzyme was detected in kidney of unilateral ureteral obstruction (UUO) mice and HK-2 cells treated with transforming growth factor-β (TGF-β). The results showed that the expressions of E2 ubiquitin-conjugating enzyme UBE2Q2 were significantly down-regulated at both RNA and protein levels in UUO kidneys. The expression of UBE2Q2 was also down-regulated in HK-2 cells stimulated with TGF-β, which was consistent with the change in the expression of HUWE1. These findings indicated that UBE2Q2 expression was synergistic with HUWE1 in the injured kidney. Co-immunoprecipitation (Co-IP) experiments showed that HUWE1 interacted with UBE2Q2 in HK-2 cells. The co-localization of UBE2Q2 and HUWE1 was confirmed by cell immunofluorescence staining. After knocking down UBE2Q2 by siRNA, ubiquitin binding to HUWE1 and EGFR was decreased. In sum, our results demonstrated that UBE2Q2, ubiquitin-conjugating enzyme, works with HUWE1 to mediate ubiquitination and degradation of target protein in kidney.
Key words: UBE2Q2; HUWE1; epidermal growth factor receptor; ubiquitination; renal tubulointerstitial fibrosis
通讯作者：梁秀彬 E-mail: email@example.com
王峥, 董浩, 李敏, 梁秀彬. 泛素结合酶UBE2Q2参与HUWE1对肾小管间质纤维化的抑制作用[J]. 生理学报 2022; 74 (1): 117-124.
WANG Zheng, DONG Hao, LI Min, LIANG Xiu-Bin. Ubiquitin-conjugating enzyme UBE2Q2 participates in HUWE1-mediated protection on renal tubulointerstitial fibrosis. Acta Physiol Sin 2022; 74 (1): 117-124 (in Chinese with English abstract).