Western Blot analysis of detoxifying enzymes, cytochrome c and sirtuin expression in patients suffering from mitochondrial complex I deficiency

Abstract

In recent years, a great deal of interest has been given to sirtuins and their role in mitochondrial metabolism, especially their implication in the process of aging. By activating detoxifying enzymes such as manganese-dependent superoxide dismutase (MnSOD) or copper-zinc superoxide dismutase (CuZnSOD), sirtuins are able to trigger enzymatic cascades leading to the reduction of cellular reactive oxygen species (ROS). This is of high importance as ROS is one of the main factors implicated in cellular aging.

In addition, it has been recently shown that mitochondrial complex I activity influences the expression and function of sirtuins. Complex I or the NADH dehydrogenase is the largest complex of the mitochondrial respiratory chain and a major regulator of energy production. Also, it is the main site for ROS production. That being said, it is important to determine not only the exact connection between sirtuins and ROS production, but also the way complex I affects sirtuin expression and activation.

In our study we analyzed selected cell lines carrying mitochondrial DNA mutations in genes encoding for complex I subunits. Our main goal was to identify potential correlations between sirtuin expression, ROS production and specific mitochondrial DNA mutations. This has been done by measuring antioxidant enzymes and sirtuin expression using Western Blot technique. The results demonstrate that complex I deficiencies do have an impact on ROS production and sirtuin expression. Moreover, we identified a particular correlation between SIRT3 and MnSOD.