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Electron Transfer Activity of Mitochondrial Outer Membrane Protein MitoNEET
439 The Nuclear Form of Heme Oxygenase (HO)-1 Preferentially Promotes Glycolysis in Hyperoxia But Fails to Protect Against Heme Toxicity Jennifer F. Carr1, Abigail L. Peterson1, Hongwei Yao1, and Phyllis A. Dennery1,2 1 Brown University, Providence, USA, 2Hasbro Children's Hospital, Providence, USA Heme oxygenase-1 (HO-1) is a stress-inducible enzyme that catalyzes the degradation of heme, generating carbon monoxide and antioxidant bile pigments. Cytoplasmic HO-1 is tethered to the smooth endoplasmic reticulum via a C-terminal trans-membrane segment. Cleavage of this anchor allows for localization of a truncated, catalytically inactive form of HO-1 to the nucleus, as has been observed in fetal lung cells in hyperoxia and in several cancer tissues. While cytoplasmic HO-1 is associated with cytoprotection directly via its antioxidant activities, nuclear HO-1 also plays a role in protection against oxidative injury via its interaction with other nuclear proteins and transcription factors, resulting in altered gene expression of several rate-limiting enzymes in key metabolic pathways including glycolysis. Interestingly, nuclear HO-1 is also associated with a proliferative phenotype. Although HO-1 has been shown to play a role in glucose metabolism and altered mitochondrial dynamics, the mechanism by which this occurs is not known. In an effort to investigate this, wild type and HO-1-/- mouse embryonic fibroblast (MEF) cells as well as MEF expressing only the nuclear, truncated HO (HO-TR), were evaluated for glycolysis and oxidative phosphorylation (oxphos) after hyperoxic exposure (95% O2/5% CO2 for 24 hours) and controls were exposed to air/5% CO2 for 24 hours. The HO-TR had increased glycolytic activity compared to HO+/+ or HO-/- cells. In contrast, oxphos, in particular spare respiratory capacity, was no different between any of the groups in hyperoxia. However, the presence of excess heme (0-50 micromolar) inhibited oxphos activity in the MEF lacking HO-1 catalytic activity, namely, HO-TR and HO-/-, in a dose dependent fashion. Our results suggest a role of nuclear HO-1 in enhancing glycolysis during hyperoxic stress thereby protecting against injury and promoting cell proliferation. They also suggest that catalytically active HO-1 is required for homeostasis of mitochondial function. Taken together our results suggest that nuclear HO-1 is cytoprotective under hyperoxic stress via a metabolic shift toward glycolysis and highlight a putative role of HO-1 in mitochondrial dynamics and metabolic regulation.
doi: 10.1016/j.freeradbiomed.2016.10.480 440 Electron Transfer Activity of Mitochondrial Outer Membrane Protein MitoNEET Yiming Wang1, Aaron P. Landry1, and Huangen Ding1 1 Louisiana State University, Baton Rouge, USA Mitochondrial outer membrane protein MitoNEET, a primary target of type II diabetes drug pioglitazone, is a key regulator of energy metabolism in mitochondria. The protein exists as a homodimer with each monomer containing a redox active [2Fe-2S] cluster. Here we report that mitoNEET has a specific interaction with flavin mononucleotide (FMN). In the presence of flavin reductase, FMNH2 reduced by NADH can rapidly reduce the mitoNEET [2Fe2S] clusters under anaerobic conditions. Under aerobic conditions,
however, the reduced mitoNEET [2Fe-2S] clusters are constantly re-oxidized by oxygen with concomitant oxidation of NADH in the presence of FMN and flavin reductase. The reduced mitoNEET [2Fe-2S] clusters can also reduce ubiquinone-2, a ubiquinone-10 analog, under aerobic or anaerobic conditions, indicating that mitoNEET has the ability to transfer electron from NADH to the membrane-bound ubiquinone in mitochondria. The [2Fe-2S] cluster is required for the electron transfer activity of mitoNEET, as apo-form mitoNEET has no activity to mediate oxidation of NADH or reduction of ubiquinone. The pioglitazone analog NL-1, which has a specific binding affinity for mitoNEET, effectively inhibits the electron transfer activity of the mitoNEET [2Fe-2S] clusters. The results suggest that mitoNEET is a novel electron transfer protein that may directly regulate energy metabolism in mitochondria.
doi: 10.1016/j.freeradbiomed.2016.10.481 441 Chronic Arsenic-Induced Metabolic Syndrome: A Role for Prolonged Nrf2 Activation and Mitochondrial Metabolism Matthew Dodson1 and Donna Zhang1 University of Arizona, Tucson, USA
1
Chronic exposure to inorganic arsenic, mainly via drinking water or contaminated food, is a global health problem linked to increased risk of cardiovascular disease, kidney disease, type II diabetes, and certain types of cancer. Among the pathologies associated with prolonged exposure to arsenic is metabolic syndrome, which is defined by a group of risk factors including dyslipidemia, insulin resistance, and inflammation. Two major contributors to metabolic syndrome are mitochondrial dysfunction and increased oxidative stress. One of the main antioxidant defense pathways against oxidative stress is the NRF2 pathway. However, emerging evidence also supports a role for NRF2 in mediating mitochondrial structure and function as well. While the role of NRF2 in metabolic syndrome is controversial, it has been shown that KEAP1knockdown (KEAP1-KD) mice, which have constitutively active NRF2, fed a high fat diet, exhibit increased markers of metabolic syndrome, thus inferring prolonged activation of NRF2 could play a key role in driving this syndrome. Previously, our group has shown that arsenic inhibits autophagy, resulting in the prolonged activation of NRF2. Here, we show that arsenic-induced activation of NRF2, or constitutive activation of NRF2 in KEAP1 knockout cells, results in fragmentation of the mitochondrial network. Interestingly, NRF2 knockout cells have fused mitochondria that are resistant to arsenic-induced fragmentation, indicating NRF2 may regulate changes to the mitochondrial network during stress. Arsenic treatment also prevents the mitophagic removal of fragmented mitochondria. Furthermore, pretreatment with rapamycin, an autophagy activator, prevented arsenic-mediated autophagy inhibition, and restored NRF2 to basal levels. These findings indicate that NRF2 levels can directly affect the mitochondrial network, which could play a key role in mediating mitochondrial function and cellular metabolism during prolonged exposure to arsenic. Furthermore, pharmacological activation of the autophagy pathway to restore NRF2 homeostasis, or pharmacological inhibition of mitochondrial fission could be viable therapeutic strategies to treat metabolic syndrome.
doi: 10.1016/j.freeradbiomed.2016.10.482
SfRBM / SFRRI 2016
S183
doi: 10.1016/j.freeradbiomed.2016.10.480 440 Electron Transfer Activity of Mitochondrial Outer Membrane Protein MitoNEET Yiming Wang1, Aaron P. Landry1, and Huangen Ding1 1 Louisiana State University, Baton Rouge, USA Mitochondrial outer membrane protein MitoNEET, a primary target of type II diabetes drug pioglitazone, is a key regulator of energy metabolism in mitochondria. The protein exists as a homodimer with each monomer containing a redox active [2Fe-2S] cluster. Here we report that mitoNEET has a specific interaction with flavin mononucleotide (FMN). In the presence of flavin reductase, FMNH2 reduced by NADH can rapidly reduce the mitoNEET [2Fe2S] clusters under anaerobic conditions. Under aerobic conditions,
however, the reduced mitoNEET [2Fe-2S] clusters are constantly re-oxidized by oxygen with concomitant oxidation of NADH in the presence of FMN and flavin reductase. The reduced mitoNEET [2Fe-2S] clusters can also reduce ubiquinone-2, a ubiquinone-10 analog, under aerobic or anaerobic conditions, indicating that mitoNEET has the ability to transfer electron from NADH to the membrane-bound ubiquinone in mitochondria. The [2Fe-2S] cluster is required for the electron transfer activity of mitoNEET, as apo-form mitoNEET has no activity to mediate oxidation of NADH or reduction of ubiquinone. The pioglitazone analog NL-1, which has a specific binding affinity for mitoNEET, effectively inhibits the electron transfer activity of the mitoNEET [2Fe-2S] clusters. The results suggest that mitoNEET is a novel electron transfer protein that may directly regulate energy metabolism in mitochondria.
doi: 10.1016/j.freeradbiomed.2016.10.481 441 Chronic Arsenic-Induced Metabolic Syndrome: A Role for Prolonged Nrf2 Activation and Mitochondrial Metabolism Matthew Dodson1 and Donna Zhang1 University of Arizona, Tucson, USA
1
Chronic exposure to inorganic arsenic, mainly via drinking water or contaminated food, is a global health problem linked to increased risk of cardiovascular disease, kidney disease, type II diabetes, and certain types of cancer. Among the pathologies associated with prolonged exposure to arsenic is metabolic syndrome, which is defined by a group of risk factors including dyslipidemia, insulin resistance, and inflammation. Two major contributors to metabolic syndrome are mitochondrial dysfunction and increased oxidative stress. One of the main antioxidant defense pathways against oxidative stress is the NRF2 pathway. However, emerging evidence also supports a role for NRF2 in mediating mitochondrial structure and function as well. While the role of NRF2 in metabolic syndrome is controversial, it has been shown that KEAP1knockdown (KEAP1-KD) mice, which have constitutively active NRF2, fed a high fat diet, exhibit increased markers of metabolic syndrome, thus inferring prolonged activation of NRF2 could play a key role in driving this syndrome. Previously, our group has shown that arsenic inhibits autophagy, resulting in the prolonged activation of NRF2. Here, we show that arsenic-induced activation of NRF2, or constitutive activation of NRF2 in KEAP1 knockout cells, results in fragmentation of the mitochondrial network. Interestingly, NRF2 knockout cells have fused mitochondria that are resistant to arsenic-induced fragmentation, indicating NRF2 may regulate changes to the mitochondrial network during stress. Arsenic treatment also prevents the mitophagic removal of fragmented mitochondria. Furthermore, pretreatment with rapamycin, an autophagy activator, prevented arsenic-mediated autophagy inhibition, and restored NRF2 to basal levels. These findings indicate that NRF2 levels can directly affect the mitochondrial network, which could play a key role in mediating mitochondrial function and cellular metabolism during prolonged exposure to arsenic. Furthermore, pharmacological activation of the autophagy pathway to restore NRF2 homeostasis, or pharmacological inhibition of mitochondrial fission could be viable therapeutic strategies to treat metabolic syndrome.
doi: 10.1016/j.freeradbiomed.2016.10.482
SfRBM / SFRRI 2016
S183