Bong-Jo Kim

JNK- and p38 Kinase-mediated Phosphorylation of Bax Leads to Its Activation and Mitochondrial Translocation and to Apoptosis of Human Hepatoma HepG2 Cells

Mitochondrial translocation of pro-apoptotic Bax prior to apoptosis is well established after treatment with many cell death stimulants or under apoptosis-inducing conditions. The mechanism of mitochondrial translocation of Bax is, however, still unknown. The aim of this work was to investigate the mechanism of Bax activation and mitochondrial translocation to initiate apoptosis of human hepatoma HepG2 and porcine kidney LLC-PK1 cells exposed to various cell death agonists. Phosphorylation of Bax by JNK and p38 kinase activated after treatment with staurosporine, H2O2, etoposide, and UV light was demonstrated by the shift in the pI value of Bax on two-dimensional gels and confirmed by metabolic labeling with inorganic [32P]phosphate in HepG2 cells. Specific inhibitors of JNK and p38 kinase significantly inhibited Bax phosphorylation and mitochondrial translocation and apoptosis of HepG2 cells. A specific small interfering RNA to MAPKK4 (the upstream protein kinase of JNK and p38 kinase) markedly decreased the levels of MAPKK4 and MAPKK3/6, blocked the activation of JNK or p38 kinase, and inhibited Bax phosphorylation. However, the negative control small interfering RNA did not cause these changes. Confocal microscopy of various Bax mutants showed differential rates of mitochondrial translocation of Bax before and after staurosporine treatment. Among the Bax mutants, T167D did not translocate to mitochondria after staurosporine exposure, suggesting that Thr167 is a potential phosphorylation site. In conclusion, our results demonstrate, for the first time, that Bax is phosphorylated by stress-activated JNK and/or p38 kinase and that phosphorylation of Bax leads to mitochondrial translocation prior to apoptosis.

Inactivation of oxidized and S-nitrosylated mitochondrial proteins in alcoholic fatty liver of rats†

Increased oxidative/nitrosative stress is a major contributing factor to alcohol‐mediated mitochondrial dysfunction. However, which mitochondrial proteins are oxidatively modified under alcohol‐induced oxidative/nitrosative stress is poorly understood. The aim of this study was to systematically investigate oxidized and/or S‐nitrosylated mitochondrial proteins and to use a biotin‐N‐maleimide probe to evaluate their inactivation in alcoholic fatty livers of rats. Binge or chronic alcohol exposure significantly elevated nitric oxide, inducible nitric oxide synthase, and ethanol‐inducible CYP2E1. The biotin‐N‐maleimide‐labeled oxidized and/or S‐nitrosylated mitochondrial proteins from pair‐fed controls or alcohol‐fed rat livers were subsequently purified with streptavidin‐agarose. The overall patterns of oxidized and/or S‐nitrosylated proteins resolved by 2‐dimensional polyacrylamide gel electrophoresis were very similar in the chronic and binge alcohol treatment groups. Seventy‐nine proteins that displayed differential spot intensities from those of control rats were identified by mass spectrometry. These include mitochondrial aldehyde dehydrogenase 2 (ALDH2), ATP synthase, acyl‐CoA dehydrogenase, 3‐ketoacyl‐CoA thiolase, and many proteins involved in chaperone activity, mitochondrial electron transfer, and ion transport. The activity of 3‐ketoacyl‐CoA thiolase involved in mitochondrial β‐oxidation of fatty acids was significantly inhibited in alcohol‐exposed rat livers, consistent with hepatic fat accumulation, as determined by biochemical and histological analyses. Measurement of activity and immunoblot results showed that ALDH2 and ATP synthase were also inhibited through oxidative modification of their cysteine or tyrosine residues in alcoholic fatty livers of rats. In conclusion, our results help to explain the underlying mechanism for mitochondrial dysfunction and increased susceptibility to alcohol‐mediated liver damage. (HEPATOLOGY 2006;44:1218–1230.)

Inhibition of mitochondrial aldehyde dehydrogenase by nitric oxide-mediated S-nitrosylation

Mitochondrial aldehyde dehydrogenase (ALDH2) is responsible for the metabolism of acetaldehyde and other toxic lipid aldehydes. Despite many reports about the inhibition of ALDH2 by toxic chemicals, it is unknown whether nitric oxide (NO) can alter the ALDH2 activity in intact cells or in vivo animals. The aim of this study was to investigate the effects of NO on ALDH2 activity in H4IIE‐C3 rat hepatoma cells. NO donors such as S‐nitrosoglutathione (GSNO), S‐nitroso‐N‐acetylpenicillamine, and 3‐morpholinosydnonimine significantly increased the nitrite concentration while they inhibited the ALDH2 activity. Addition of GSH‐ethylester (GSH‐EE) completely blocked the GSNO‐mediated ALDH2 inhibition and increased nitrite concentration. To directly demonstrate the NO‐mediated S‐nitrosylation and inactivation, ALDH2 was immunopurified from control or GSNO‐treated cells and subjected to immunoblot analysis. The anti‐nitrosocysteine antibody recognized the immunopurified ALDH2 only from the GSNO‐treated samples. All these results indicate that S‐nitrosylation of ALDH2 in intact cells leads to reversible inhibition of ALDH2 activity.

Post-translational modifications of mitochondrial aldehyde dehydrogenase and biomedical implications

Aldehyde dehydrogenases (ALDHs) represent large family members of NAD(P)+-dependent dehydrogenases responsible for the irreversible metabolism of many endogenous and exogenous aldehydes to the corresponding acids. Among 19 ALDH isozymes, mitochondrial ALDH2 is a low Km enzyme responsible for the metabolism of acetaldehyde and lipid peroxides such as malondialdehyde and 4-hydroxynonenal, both of which are highly reactive and toxic. Consequently, inhibition of ALDH2 would lead to elevated levels of acetaldehyde and other reactive lipid peroxides following ethanol intake and/or exposure to toxic chemicals. In addition, many East Asian people with a dominant negative mutation in ALDH2 gene possess a decreased ALDH2 activity with increased risks for various types of cancer, myocardial infarct, alcoholic liver disease, and other pathological conditions. The aim of this review is to briefly describe the multiple post-translational modifications of mitochondrial ALDH2, as an example, after exposure to toxic chemicals or under different disease states and their pathophysiological roles in promoting alcohol/drug-mediated tissue damage. We also briefly mention exciting preclinical translational research opportunities to identify small molecule activators of ALDH2 and its isozymes as potentially therapeutic/preventive agents against various disease states where the expression or activity of ALDH enzymes is altered or inactivated.

Exome sequencing as a tool for short stature gene discovery: analysis of a Korean family with pseudohypoparathyroidism

A 4xa0bp deletion in GNAS has known to be the causal mutation for pseudohypoparathyroidism. In this study, we performed sanger sequencing on exons of GNAS and identified the 4xa0bp causal deletion in Korean family with pseudohypoparathyroidism. Despite the finding of the deletion, underlying genetics of short stature in the family is unclear since paternal lineage showed history of short stature and mother didn’t show short stature. Therefore, we performed exome sequencing to explore putative causal mutations responsible for short stature phenotype in the Korean family with pseudohypoparathyroidism. Initially, we discovered 58,829 variants from four members of the family by exome sequencing. Subsequent variant filtering removed possible non-causal variants based on public databases including 1,000 genomes project and Exome Sequencing Project. After selecting dominant functional variants inherited from the father, we identified 40 putative candidates. Among them, the strongest possible candidate would be ALPL that has known to be related with hypophosphatasia. In conclusion, our results could provide additional insight into the understanding of genetics related with short stature phenotype.