Hozumi Motohashi, Professor, Department of Medical Biochemistry, Tohoku University Graduate School of Medicine2019.5 ～ 2019.8, 2022.5 ～ 2024.2― 261 ―The KEAP1 (Kelch-like ECH-associated protein 1)-NRF2 (nuclear factor-erythroid 2-related factor 2) system is an important cytoprotective mechanism against oxidative and electrophilic stresses1). Under basal conditions, NRF2 is constitutively subjected to ubiquitination and proteasomal degradation through binding to KEAP1, a substrate adaptor protein of E3 ubiquitin ligase. NRF2 has been shown to impact mitochondrial function as a master regulator of cellular redox homeostasis2). However, the precise mechanism by which NRF2 enhances mitochondrial activity is still not fully understood.Recently, we found that mitochondrial sulfur metabolism makes an important contribution to cellular energy production3). Because NRF2 promotes cystine uptake by upregulating SLC7A11, which encodes the cystine transporter xCT4), we hypothesized that NRF2 enhances mitochondrial activity by increasing cellular cysteine availability to facilitate mitochondrial sulfur metabolism. In this study, we found that cystine availability is one of the major determinants of intracellular persulfides and that persulfide supplementation promotes mitochondrial function. Mitochondrial activation induced by NRF2 activation due to KEAP1 inhibition relies on cystine uptake, followed by persulfide production and sulfur oxidation. Hepa1c1c7 cells were cultured and maintained in low glucose Dulbecco’s Modified Eagle Medium (DMEM; Wako Pure Chemicals, Osaka, Japan) containing 10% fetal bovine serum (FBS; Biosera, Kansas, MO, USA) and 1% penicillin/streptomycin (Nacalai Tesque, Kyoto, Japan) under 5% CO2 at 37°C .Mitochondrial membrane potential assayThe mitochondrial membrane potential (MMP) was measured using a Cell Meter JC-10 Mitochondrion Membrane Potential Assay Kit (AAT Bioquest, Sunnyvale, CA, USA) according to the manufacturer’s IntroductionExperimental MethodsCell cultureContribution of NRF2-mediated transcriptional activation to sulfur metabolism and its impacton mitochondrial bioenergeticsMd. Morshedul Alam
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