Age-related alterations in oxidatively damaged proteins of mouse heart mitochondrial electron transport chain complexes

Abstract

Mitochondrial generated ROS increases with age and is a major factor that damages proteins by oxidative modification. Accumulation of oxidatively damaged proteins has been implicated as a causal factor in the age-associated decline in tissue function. Mitochondrial electron transport chain (ETC) complexes I and III are the principle sites of ROS production, and oxidative modifications to their complex subunits inhibit their in vitro activity. We hypothesize that mitochondrial complex subunits may be primary targets for modification by ROS which may impair normal complex activity. This study of heart mitochondria from young, middle-aged and old mice reveals that there is an agerelated decline in complex I and V activities that correlate with increased oxidative modification to their subunits. The data also show a specificity for modifications of the ETC complex subunits, i.e., several proteins have more than one type of adduct. We postulate that the electron leakage from ETC complexes causes specific damage to their subunits and increased ROS generation as oxidative damage accumulates, leading to further mitochondrial dysfunction, a cyclical process that underlies the progressive decline in physiologic function of aged mouse heart.

Age-related alterations in oxidatively damaged proteins of mouse skeletal muscle mitochondrial electron transport chain complexes

“…The covalent oxidative protein modifications caused by reactions with free radicals are more stable and easily detectable, and thus have been used as molecular markers of oxidative stress [17,[22][23][24]. The relative abundance of modified proteins has been used in our laboratory to indicate the level of oxidatively modified proteins that accumulate in aged tissues [22][23][24].…”

“…The relative abundance of modified proteins has been used in our laboratory to indicate the level of oxidatively modified proteins that accumulate in aged tissues [22][23][24]. Protein modifications caused by ROS include the formation of lipid peroxidation adducts (4-hydroxynonenal or HNE and malondialdehyde or MDA) on lysine, histidine, and cysteine, nitration of tyrosine and cysteine, and carbonylation of lysine, arginine, proline, and threonine [18,19,25,26].…”

“…Protein modifications caused by ROS include the formation of lipid peroxidation adducts (4-hydroxynonenal or HNE and malondialdehyde or MDA) on lysine, histidine, and cysteine, nitration of tyrosine and cysteine, and carbonylation of lysine, arginine, proline, and threonine [18,19,25,26]. Oxidatively modified proteins have been detected by immunoblotting using antibodies specific for these modifications and subsequently identified by mass spectrometry [17,[22][23][24]. In addition, such oxidative modifications to proteins can result in reduction of function associated with aging and age-associated diseases [18,19,[21][22][23][24][25][26][27].…”

“…Oxidatively modified proteins have been detected by immunoblotting using antibodies specific for these modifications and subsequently identified by mass spectrometry [17,[22][23][24]. In addition, such oxidative modifications to proteins can result in reduction of function associated with aging and age-associated diseases [18,19,[21][22][23][24][25][26][27]. These oxidative modifications are, therefore, molecular markers that provide insight into the cumulative effects of oxidative stress on the molecular mechanisms of aging and development of age-associated diseases.…”

“…Blue-native polyacrylamide gel electrophoresis (BN-PAGE) was used to resolve intact ETC complexes [28] followed by second-dimension denaturing SDS-PAGE to resolve individual complex subunits. Protein abundance of each complex was measured using antibodies to specific complex subunits to quantify age-related changes [22,23]. Using immunobloting with antibodies recognizing specific types of oxidative damage, we detected proteins that were carbonylated, and modified by HNE, MDA, and tyrosine nitration.…”