Aral 2018 MiP2018
LETM1 is an inner membrane protein of the mitochondria which is a gene found to be missing in Wolf-Hirschhorn syndrome patients. It has been suggested that cellular damage caused by deletion of LETM1 may be related to oxidative stress. It is well known that melatonin provides antioxidant enzyme activation to protect the cell and mitochondrion from oxidative stress-induced damage. In our study, in order to investigate the effects of LETM1 loss on oxidative stress and melatonin on mitochondrial function in immortalized mouse embryonic fibroblast cells, to suppress the LETM1 gene siRNA transfection, SDS-PAGE and BN-PAGE to decompose OXPHOS complexes and immunoblotting methods were used, to determine oxidative stress in cells, fluorescence microscopy, aconitase enzyme activity and protein oxidation determined, and oxygen consumption was measured in the presence of appropriate substrates and inhibitors in the cells. The expression of LETM1 was suppressed by approximately 50% with siRNA transfection in MEFs and the application of melatonin to these cells did not alter the level of LETM1. It has been determined that MnSOD expression and aconitase activity are decreased and total protein oxidation is increased in transfected cells. After administration of melatonin, MnSOD expression, aconitase activity and protein oxidation were normalized. It was determined that LETM1 suppression did not alter the expression and formation of OXPHOS complexes as determined by BN-PAGE, but the oxygen consumption rates decreased significantly. The change in oxygen consumption rate is not related to complex I but carried out on complex II shown for the first time in the literature and on complex IV in accordance with the literature. It was also determined that the reduced oxygen consumption rate was normalized by melatonin administration. These results provide contribution to the literature to clarify the effects of LETM1 loss in relation to oxidative stress and melatonin on mitochondrial function.
- Namık Kemal Univ, Arts Sciences Fac, Dept Molecular Biology & Genetics, Tekirdağ, Turkey. - [email protected]