Stimulation and clustering of cytochrome b₅ reductase in caveolin-rich lipid microdomains is an early event in oxidative stress-mediated apoptosis of cerebellar granule neurons

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• 作者：Alejandro K. Samhan-Arias^a ; Dorinda Marques-da-Silva^a ; Naveena Yanamala^b ; Carlos Gutierrez-Merino^a ; ^{carlosgm@unex.es}
• 关键词：Cav1 ; caveolin isoform 1 ; Cav2 ; caveolin isoform 2 ; Cb5 ; cytochrome b5 ; Cb5R ; cytochrome b5 reductase ; DHE ; dihydroethidium ; DIV ; days in vitro ; DNP ; 2 ; 4-dinitrophenol ; FCCP ; carbonyl cyanide 4-(trifluoromethoxy)phenyl hydrazone ; FRET ; fluorescence resona
• 刊名：Journal of Proteomics
• 出版年：2012
• 期刊代码：P37_18743919
• 类别：ch
• 出版时间：6 June, 2012
• 卷：75
• 期：10
• 页码：2934-2949
• 文件大小：1248 K

摘要

The apoptosis of cerebellar granule neurons (CGN) induced by low potassium in the extracellular medium is a model of neuronal apoptosis where an overshot of reactive oxygen species (ROS) triggers the neuronal death. In this work, using dihydroethidium and L-012 as specific dyes for superoxide anion detection we show that this ROS overshot can be accounted by an increased release of superoxide anion to the extracellular medium. The amplitude and time course of the increase of superoxide anion observed early during apoptosis correlated with the increase of the content of soluble cytochrome b₅, a substrate of the NADH-dependent oxidase activity of the cytochrome b₅ reductase associated with lipid rafts in CGN. Western blotting and immunofluorescence microscopy approaches, including fluorescence energy transfer, pointed out an enhanced clustering of cytochrome b₅ reductase within caveolins-rich lipid rafts microdomains. Protein/protein docking analysis suggests that cytochrome b₅ reductase can form complexes with caveolins 1伪, 1尾 and 2, playing electrostatic interactions a major role in this association. In conclusion, our results indicate that overstimulation of cytochrome b₅ reductase associated with lipid rafts can account for the overshot of plasma membrane-focalized superoxide anion production that triggers the entry of CGN in the irreversible phase of apoptosis. This article is part of a Special Issue entitled: Proteomics: The clinical link.