Identification, Quantification, and Functional Aspects of Skeletal Muscle Protein-Carbonylation in Vivo during Acute Oxidative Stress

Abstract

Reactive oxidative species (ROS) play important roles in cellular signaling but can also modify and often functionally inactivate other biomolecules. Thus, cells have developed effective enzymatic and nonenzymatic strategies to scavenge ROS. However, under oxidative stress, ROS production is able to overwhelm the scavenging systems, increasing the levels of functionally impaired proteins. A major class of irreversible oxidative modifications is carbonylation, which refers to reactive carbonyl-groups. In this investigation, we have studied the production and clearance rates for skeletal muscle proteins in a rat model of acute oxidative stress over a time period of 24 h using a gel-based proteomics approach. Optimized ELISA and Western blots with 10-fold improved sensitivities showed that the carbonylation level was stable at 4 nmol per mg protein 3 h following ROS induction. The carbonylation level then increased 3-fold over 6 h and then remained stable. In total, the oxidative stress changed the steady state levels of 20 proteins and resulted in the carbonylation of 38 skeletal muscle proteins. Carbonylation of these proteins followed diverse kinetics with some proteins being highly carbonylated very quickly, whereas others peaked in the 9 h sample or continued to increase up to 24 h after oxidative stress was induced.

Profiling Carbonylated Proteins in Heart and Skeletal Muscle Mitochondria from Trained and Untrained Mice

“…Several proteins (Ckme2, Atp5a1, Dlat, Dlst, Vdac1, Atp5a1, and Atp5b) were found carbonylated in previous studies. 22,40,41 Some proteins in H resulted more carbonylated than in skeletal muscle, in particular Vdac1, Vdac2, Ckme2, and Atp5a1 that are situates in mitochondria membrane. Interestingly, the impact of training on H and TA protein oxidation appears different: in fact, in H mitochondria we observe a decrease of carbonylation level (with the exception of aconitate hydratase and ATP synthase subunit alpha) while in TA the general trend is represented by an increase of oxidation.…”

Identification of protein carbonylation sites by two-dimensional liquid chromatography in combination with MALDI- and ESI-MS

“…Peroxidase-conjugated donkey anti-goat Ab (Dianova GmbH, Hamburg, Germany) dissolved in PBS containing 5% milk powder and 0.1% Tween-20 (0.16 μg/mL, 10 mL) was then added to the membrane. After 1 h the membrane was washed with PBS containing Tween-20 (0.1% v/v, 2 × 10 min) and then with PBS (10 min) before being visualized with the ECL Advance Western Blotting Detection Kit (Amersham Bioscience) [30].…”

“…In order to confirm the extent of carbonylation after in vitro oxidation, protein samples were separated by SDS-PAGE, blotted onto a PVDF-membrane and carbonylated proteins were identified with an anti-DNP antibody (oxy blot) (Fig. 3) [30]. Both oxidized proteins displayed more bands with lower and higher mobilities in the protein stain than the native proteins, indicating oxidative cross-linking and degradation (Fig.…”

Ameliorative Effect of Salicin Against Gamma Irradiation Induced Electrophoretic Changes in Brain Tissue in Male Rats

“…The most important consequences of OS are lipid peroxidation, protein oxidation and depletion of antioxidants 17,18 . The latter authors showed thatthe increase of MDA level is probably due to the interaction of •OH resulting as a bi-product of water radiolysis with the polyunsaturated fatty acids presents in the phospholipids portion of cellular membranes.…”