ROS generation and multiple forms of mammalian mitochondrial glycerol-3-phosphate dehydrogenase

Overproduction of reactive oxygen species (ROS) has been implicated in a range of pathologies. Mitochondrial flavin dehydrogenases glycerol-3-phosphate dehydrogenase (mGPDH) and succinate dehydrogenase (SDH) represent important ROS source, but the mechanism of electron leak is still poorly understood. To investigate the ROS production by the isolated dehydrogenases, we used brown adipose tissue mitochondria solubilized by digitonin as a model. Enzyme activity measurements and hydrogen peroxide production studies by Amplex Red fluorescence, and luminol luminescence in combination with oxygraphy revealed flavin as the most likely source of electron leak in SDH under in vivo conditions, while we propose coenzyme Q as the site of ROS production in the case of mGPDH. Distinct mechanism of ROS production by the two dehydrogenases is also apparent from induction of ROS generation by ferricyanide which is unique for mGPDH. Furthermore, using native electrophoretic systems, we demonstrated that mGPDH associates into homooligomers as well as high molecular weight supercomplexes, which represent native forms of mGPDH in the membrane. By this approach, we also directly demonstrated that isolated mGPDH itself as well as its supramolecular assemblies are all capable of ROS production.     

Respiratory chain components involved in the glycerophosphate dehydrogenase-dependent ROS production by brown adipose tissue mitochondria

Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H₂DCFDA and Amplex Red was used to determine the glycerophosphate- or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphate-dependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.     

mGPDH Deficiency leads to melanoma metastasis via induced NRF2

Oxidative stress critically influences carcinogenesis and the progression of melanoma, and aggressive malignant melanoma activity is due to its high metastatic ability. Some findings in several cancer cell lines have indicated that mGPDH, a component of the mitochondrial respiratory chain, also modulates oxidative stress. However, the role of mGPDH in melanoma remains elusive. Here, we report that the mGPDH protein level is decreased in human skin melanoma compared to normal skin and decreased in metastatic melanoma compared to primary melanoma. Our in vivo and in vitro experiments indicated that mGPDH depletion accelerated melanoma migration and invasion without affecting proliferation or apoptosis. Mechanistically, we found elevated NRF2 protein levels in human skin melanoma and mGPDH-knockout (ko) metastatic xenografts in the lungs of nude mice. Moreover, in A375 melanoma cells, the loss of mGPDH-induced NRF2 expression but did not affect NRF2 protein degradation. Additionally, melanoma metastasis induced by the loss of mGPDH was rescued by the further down-regulation of NRF2 in vivo and in vitro. Consistently, mGPDH overexpression (oe) depressed NRF2 expression and attenuated the malignant properties of melanoma cells. In conclusion, our findings suggest that mGPDH suppresses melanoma metastasis by inhibiting NRF2 and downstream oxidative signals, highlighting the therapeutic potential of mGPDH for melanoma treatment.