DTX3L induced NLRP3 ubiquitination inhibit R28 cell pyroptosis in OGD/R injury

Ziyu Zhou; Lei Shang; Qi Zhang; Ximin Hu; Ju-Fang Huang; Kun Xiong

doi:10.1016/j.bbamcr.2023.119433

DTX3L induced NLRP3 ubiquitination inhibit R28 cell pyroptosis in OGD/R injury

Biochim Biophys Acta Mol Cell Res. 2023 Mar;1870(3):119433. doi: 10.1016/j.bbamcr.2023.119433. Epub 2023 Jan 24.

Authors

Ziyu Zhou¹, Lei Shang², Qi Zhang³, Ximin Hu⁴, Ju-Fang Huang⁵, Kun Xiong⁶

Affiliations

¹ Department of Anatomy and Neurobiology, School of Basic Medical Science, Central South University, Changsha 410008, China; The Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital), Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Sciences, Hangzhou 310022, China.
² Jiangxi Research Institute of Ophthalmology and Visual Sciences, Affiliated Eye Hospital of Nanchang University, Nanchang 330006, China. Electronic address: shanglei1986@ncu.edu.cn.
³ Department of Anatomy and Neurobiology, School of Basic Medical Science, Central South University, Changsha 410008, China. Electronic address: zhangqi2014@csu.edu.cn.
⁴ Department of Anatomy and Neurobiology, School of Basic Medical Science, Central South University, Changsha 410008, China.
⁵ Department of Anatomy and Neurobiology, School of Basic Medical Science, Central South University, Changsha 410008, China. Electronic address: huangjufang@csu.edu.cn.
⁶ Department of Anatomy and Neurobiology, School of Basic Medical Science, Central South University, Changsha 410008, China; Hunan Key Laboratory of Ophthalmology, Changsha 410008, China; Key Laboratory of Emergency and Trauma, Ministry of Education, College of Emergency and Trauma, Hainan Medical University, Haikou 571199, China. Electronic address: xiongkun2001@163.com.

PMID: 36706922
DOI: 10.1016/j.bbamcr.2023.119433

Abstract

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, mediated via NLRP3 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.

Keywords: DTX3L; NLRP3; OGD/R; Pyroptosis; Ubiquitination.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cell Death
Cell Line
Humans
NLR Family, Pyrin Domain-Containing 3 Protein / genetics
NLR Family, Pyrin Domain-Containing 3 Protein / metabolism
Pyroptosis*
Reperfusion Injury* / metabolism
Ubiquitin-Protein Ligases

Substances

NLR Family, Pyrin Domain-Containing 3 Protein
DTX3L protein, human
Ubiquitin-Protein Ligases