Effect of S-Nitrosylation of RIP3 Induced by Cerebral Ischemia on its Downstream Signaling Pathway

• Published in: Journal of stroke and cerebrovascular diseases Vol. 31; no. 7; p. 106516
• Main Authors: Zhong, Yi, Peng, Peng, Zhang, Mengmeng, Han, Dong, Yang, Hongning, Yan, Xianliang, Hu, Shuqun
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.07.2022
• Subjects: Cerebral ischemia; Downstream signaling; RIP3; S-nitrosylation; GLUD1; Cerebral ischemia; MLKL; Downstream signaling; S-nitrosylation; Cl-Caspase8; PYGL; RIP3; OGD; GLUL
• ISSN: 1052-3057; 1532-8511
• DOI: 10.1016/j.jstrokecerebrovasdis.2022.106516

•Activated RIP3 further phosphorylate RIP1 and mixed lineage kinase-like domains (MLKL).•Activated RIP3 further promote the conversion of Caspase8 to active Cleaved-Caspase8 (Cl-Caspase8).•Activated RIP3 further enhance the activities of Glycogen phosphorylase (PYGL), Glutamate-ammonia ligase (GLUL) and Glutamate dehydrogenase (GLUD1). Our preliminary experiments indicate that receptor-interacting protein 3 (RIP3) is S-nitrosylated and contributes to its autophosphorylation (activation) after 3 h of rat brain ischemia/reperfusion mediated by activation of the N-methyl-D-aspartate receptor (NMDAR)-dependent neuronal NO synthase (nNOS) and is involved in the process of neuronal injury. Here, we will to demonstrate whether S-nitrosylation of RIP3 facilitates the activation of the downstream signaling pathway and finally exacerbates ischemic neuron death. Adult male Sprague-Dawley rat transient brain ischemia/reperfusion and cortical neurons oxygen and glucose deprivation (OGD)/reoxygenation models were performed. The hippocampal CA1 regions or cultured cells were homogenized and the cytosolic fraction were collected as tissue samples. Coimmunoprecipitation and western blot analysis were carried out for detecting phosphorylation of RIP1 and mixed lineage kinase-like domains (MLKL) and the Cleaved-Caspase8 (Cl-Caspase8). The activities of Glycogen phosphorylase (PYGL), Glutamate-ammonia ligase (GLUL) and Glutamate dehydrogenase (GLUD1) were detected with ultraviolet absorption method. This study showed that active RIP3 could phosphorylate RIP1 and MLKL through its kinase activity, promote the conversion of Caspase8 to active Cl-Caspase8, enhance the activities of PYGL, GLUL and GLUD1, and finally aggravate neuronal injury in cerebral ischemia/reperfusion. The inhibition of RIP3 S-nitrosylation inhibited the phosphorylation of RIP1 and MLKL, inhibited the activities of Caspase8, PYGL, GLUL, and GLUD1, and alleviated neuronal damage in cerebral ischemia/reperfusion. S-nitrosylation of RIP3 increased RIP1 and MLKL phosphorylation levels, Cl-Caspase8 content and PYGL, GLUL and GLUD1 activities and aggravated neuronal damage during cerebral ischemia/reperfusion and regulating the S-nitrosylation of RIP3 and its downstream signaling pathway might be a therapeutic target for stroke.