CCCP‐induced mitochondrial dysfunction – characterization and analysis of integrated stress response to cellular signaling and homeostasis

Abstract

Mitochondrial dysfunction mediated by CCCP (carbonyl cyanide m-chlorophenyl hydrazone), an inhibitor of mitochondrial oxidative phosphorylation, evokes the integrated stress response (ISR), which is analyzed here by eIF2a phosphorylation and expression profiles of ATF4 and CHOP proteins. Our findings suggest that the CCCP-induced ISR pathway is mediated by activation of HRI kinase, but not by GCN2, PERK, or PKR. Also, CCCP activates AMPK, a cellular energy sensor, and AKT, a regulator implicated in cell survival, and suppresses phosphorylation of mTORC1 substrates eIF4E-BP1 and S6K. CCCP also downregulates translation and promotes autophagy, leading to noncaspase-mediated cell death in HepG2 cells. All these events are neutralized by NAC, an anti-ROS, suggesting that CCCP-induced mitochondrial dysfunction promotes oxidative stress. ISRIB, an inhibitor of the ISR pathway, mitigates CCCP-induced expression of ATF4 and CHOP, activation of AKT, and autophagy, similar to NAC. However, it fails to reverse CCCP-induced AMPK activation, suggesting that CCCP-induced autophagy is dependent on ISR and independent of AMPK activation. ISRIB restores partly, inhibition in eIF4E-BP1 phosphorylation, promotes eIF2a phosphorylation, albeit slowly, and mitigates suppression of translation accordingly, in CCCP-treated cells. These findings are consistent with the idea that CCCP-induced oxidative stress leading to eIF2a phosphorylation and ATF4 expression, which is known to stimulate genes involved in autophagy, play a pro-survival role together with AKT activation and regulate mTOR-mediated eIF4E-BP1 phosphorylation.

A tRNA processing enzyme is a key regulator of the mitochondrial unfolded protein response

“…Interestingly, ATF4 and one of its targets, ATF5, are orthologs of ATFS-1 ( Fiorese et al, 2016 ). Moreover, GCN2 and ISR in general have been shown to be responsive to mitochondrial stress ( Baker et al, 2012 ; Fessler et al, 2020 ; Guo et al, 2020 ; Koncha et al, 2021 ). Thus, we questioned if UPR mt activation by hoe-1(ΔNES ) is mediated via GCN2 and eIF2α phosphorylation.…”

How to Use Respiratory Chain Inhibitors in Toxicology Studies—Whole-Cell Measurements

“…To validate the measurement of mitochondrial potential with CMXRos, we used CCCP, which is a recognized mitochondrial uncoupler that causes dissipation of mitochondrial membrane potential [ 58 ]. CCCP in effect lowers the fluorescence readout for CMXRos ( Figure 5 A), which suggests that the measurement with this dye is an effective tool with which to determine the mitochondrial potential in the presented test system.…”

eIf2α-regulated translation modulates the early adhesion of mesenchymal-like cells

“…The inactivation of eIF2α is caused by the a single phosphorylation event on Ser 51, which is catalysed by 4 kinases (GCN2, HRI, PERK and PKR) (Koncha et al, 2021). General control non-derepressible-2 (GCN2) was shown to be activated by amino acid starvation or UV irradiation (Dokladal et al, 2021, Jiang and Wek, 2005, Sood et al, 2000), while heme-regulated inhibitor (HRI) activated in response to heme deprivation, oxidative and mitochondrial stress (Chen and London, 1995, Fessler et al, 2020, Guo et al, 2020, Ranu and London, 1979, Zhang et al, 2018), whereas PKR-like endoplasmic reticulum kinase (PERK) is activated in response to endoplasmic reticulum stress or UV irradiation (Popovic et al, 2021, Wu et al, 2002, Harding et al, 2000b).…”