CCCP-induced mitochondrial dysfunction - characterization and analysis of integrated stress response to cellular signaling and homeostasis.

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 eIF2α 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 eIF2α 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 eIF2α 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.

An in silico analysis of troponin I mutations in hypertrophic cardiomyopathy of Indian origin.

Hypertrophic Cardiomyopathy (HCM) is an autosomal dominant disorder of the myocardium which is hypertrophied resulting in arrhythmias and heart failure leading to sudden cardiac death (SCD). Several sarcomeric proteins and modifier genes have been implicated in this disease. Troponin I, being a part of the Troponin complex (troponin I, troponin C, troponin T), is an important gene for sarcomeric function. Four mutations (1 novel) were identified in Indian HCM cases, namely, Pro82Ser, Arg98Gln, Arg141Gln and Arg162Gln in Troponin I protein, which are in functionally significant domains. In order to analyse the effect of the mutations on protein stability and protein-protein interactions within the Troponin complex, an in silico study was carried out. The freely available X-ray crystal structure (PDB ID: 1JIE) was used as the template to model the protein followed by loop generation and development of troponin complex for both the troponin I wild type and four mutants (NCBI ID: PRJNA194382). The structural study was carried out to determine the effect of mutation on the structural stability and protein-protein interactions between three subunits in the complex. These mutations, especially the arginine to glutamine substitutions were found to result in local perturbations within the troponin complex by creating/removing inter/intra molecular hydrogen bonds with troponin T and troponin C. This has led to a decrease in the protein stability and loss of important interactions between the three subunits. It could have a significant impact on the disease progression when coupled with allelic heterogeneity which was observed in the cases carrying these mutations. However, this can be further confirmed by functional studies on protein levels in the identified cases.