DTX3L induced NLRP3 ubiquitination inhibit R28 cell pyroptosis in OGD/R injury
DTX3L induced NLRP3 ubiquitination inhibit R28 cell pyroptosis in OGD/R injury
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DTX3L 诱导 NLRP3 泛素化抑制 OGD/R 损伤中的 R28 细胞焦亡
DOI：
10.1016/j.bbamcr.2023.119433
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Kun Xiong
中科院分区：
文献类型：
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作者：
Ziyu Zhou;Lei Shang;Qi Zhang;Ximin Hu;Ju-fang Huang;Kun Xiong
缺血/再灌注（I/R）损伤是许多疾病中最常见的病因之一。视网膜I/R导致细胞因子风暴，导致组织损伤和细胞死亡。细胞凋亡是细胞I/R损伤后发生的一种新型的调节性细胞死亡。本研究建立了一种模拟视网膜I/R损伤的氧糖剥夺（OGD/R）细胞模型（R28）。我们进行了LDH测定，EthD-III和PI染色程序，以确认焦亡。利用质谱和生物信息学分析鉴定与NLRP 3相互作用的蛋白质。利用Co-IP和多种分子生物学技术研究了DTX 3L对NLRP 3的可能调控方式。EthD-III、PI染色和LDH测定证实OGD/R损伤诱导的细胞凋亡是通过NLRP 3途径介导的。质谱和生物信息学分析筛选出3个与NLRP 3相互作用的候选蛋白，进一步的Co-IP实验表明DTX-3L可能与NLRP 3相互作用，调节损伤后NLRP 3的蛋白水平。Co-IP实验和各种分子生物学方法表明，DTX 3L泛素化NLRP 3，导致R28 OGD/R损伤后的焦亡。此外，NLRP 3 LRR和DTX 3L RING结构域彼此相互作用。我们的研究表明，DTX 3L可能通过泛素化NLRP 3来调节OGD/R诱导的R28细胞凋亡。
Ischemia/reperfusion (I/R) injury is one of the most common etiologies in many diseases. Retinal I/R leads to cytokine storm, resulting in tissue damage and cell death. Pyroptosis, a novel type of regulated cell death, occurs after cellular I/R injury. In this study, we established an oxygen glucose deprivation (OGD/R) cellular model (R28) to simulate retinal I/R injury. We conducted an LDH assay, and EthD-III and PI staining procedures to confirm pyroptosis. Mass spectrometry and bioinformatics analysis were used to identify the possible proteins interacting with NLRP3. Co-IP and various molecular biology techniques were used to investigate the possible modes regulating NLRP3 by DTX3L. EthD-III, PI staining and LDH assays demonstrated pyroptosis induced by OGD/R injury, mediatedviaNLRP3 pathway. Mass spectrometry and bioinformatics analysis screened out three candidate proteins interacting with NLRP3, and further Co-IP experiment indicated that DTX-3L may interact with NLRP3 to regulate its protein levels after injury. Co-IP experiments and various molecular biology methods demonstrated that DTX3L ubiquitinates NLRP3 resulting in pyroptosis after R28 OGD/R injury. Further, NLRP3 LRR and DTX3L RING domains interact with each other. Our study demonstrated that DTX3L may ubiquitinate NLRP3 to regulate OGD/R-induced pyroptosis globally in R28 cells.