Nitration of Tryptophan 372 in Succinyl-CoA:3-Ketoacid CoA Transferase during Aging in Rat Heart Mitochondria

Abstract

The main objective of this study was to test the hypothesis that in vivo post-translational modifications in proteins, induced by the endogenously generated reactive oxygen and nitrogen molecules, can alter protein function and thereby have an effect on metabolic pathways during the aging process. Succinyl-CoA:3-ketoacid coenzyme A transferase (SCOT), the mitochondrial enzyme involved in the breakdown of ketone bodies in the extrahepatic tissues, was identified in rat heart to undergo age-associated increase in a novel, nitro-hydroxy, addition to tryptophan 372, located in close proximity ( approximately 10 A) of the enzyme active site. Between 4 and 24 months of age, the molar content of nitration was more than doubled while specific enzyme activity increased significantly. The amount of SCOT protein, however, remained unchanged. In vitro treatment of heart mitochondrial soluble proteins with relatively low concentrations of peroxynitrite enhanced the nitration as well as specific activity of SCOT. Results of this study identify tryptophan to be a specific target of nitration in vivo, for the first time. We hypothesize that increases in tryptophan nitration of SCOT and catalytic activity constitute a plausible mechanism for the age-related metabolic shift toward enhanced ketone body consumption as an alternative source of energy supply in the heart.

Metallothionein prevents diabetes-induced cardiac pathological changes, likely via the inhibition of succinyl-CoA:3-ketoacid coenzyme A transferase-1 nitration at Trp374

“…A similar phenomenon was also observed in the alloxan-induced mouse model of diabetes (26). The role of nitration in decreased SCOT activity in humans was confirmed by Rebrin et al (21). By investigating the posttranslational modifications that occurred in proteins during the aging process, tryptophan was identified as a specific in vivo target of nitration and shown to increase SCOT activity.…”

Aging might augment reactive oxygen species (ROS) formation and affect reactive nitrogen species (RNS) level after myocardial ischemia/reperfusion in both humans and rats

“…ONOO − has been shown to be highly reactive with a wide variety of molecules, including deoxyribose, cellular lipids, and protein sulfhydryl moieties, causing direct oxidative tissue damage. Several animal experiments tested the effects of aging upon in vivo RNS formation and demonstrated that aging resulted in increased nitration (Rebrin et al 2007;Zhang et al 2007). Currently, with the clinical trial, we did not find any significant difference in NOx and ONOO − formation between the adult and elderly populations.…”

“…The deleterious effects of RNS are further exacerbated with increased ambient ROS. However, nearly all studies concerning aging, myocardial ischemia/reperfusion (MI/R) injury, ROS, and RNS exacerbating MI/R injury (Rebrin et al 2007;Zhang et al 2007) utilize animal experiments. Aging-related effects with clinical applicability to humans are unknown.…”

Protein 3-Nitrotyrosine in Complex Biological Samples: Quantification by High-Pressure Liquid Chromatography/Electrochemical Detection and Emergence of Proteomic Approaches for Unbiased Identification of Modification Sites

“…Although immunoprecipitation can be used for effective enrichment of 3-NT-containing proteins, it is notable that a significant number of nonnitrated proteins coimmunoprecipitate. This apparent lack of specificity may arise from undesired interactions of anti-3-NT antibodies with 3-NT-free proteins (Aulak et al, 2004), such as those which contain 5-hydroxynitrotryptophan (Rebrin et al, 2007), and the predicted pull down of proteins that associate with 3-NT-containing proteins.…”