Ishemia-reperfusion enhances GAPDH nitration in aging skeletal muscle.

Aging and skeletal muscle ischemia/reperfusion (I/R) injury leads to decreased contractile force generation that increases severely with age. Our studies show that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein expression is significantly decreased at 3 and 5 days reperfusion in the young mouse muscle and at 1, 3, 5, and 7 days in the aged muscle. Using PCR, we have shown that GAPDH mRNA levels in young and old muscle increase at 5 days reperfusion compared to control, suggesting that the protein deficit is not transcriptional. Furthermore, while total tyrosine nitration did not increase in the young muscle, GAPDH nitration increased significantly at 1 and 3 days reperfusion. In contrast, total tyrosine nitration in aged muscle increased significantly at 1, 3, and 5 days of reperfusion, with increases in GAPDH nitration at the same time points. We conclude that GAPDH protein levels decrease following I/R, that this is not transcriptionally mediated, that the aged muscle experiences greater oxidative stress, protein modification and GAPDH degradation, possibly contributing to decreased muscle function. We propose that tyrosine nitration enhances GAPDH degradation following I/R and that the persistent decrease of GAPDH in aged muscle is due to the prolonged increase in oxidative modification in this age group.

A proteomics analysis of the effects of chronic hemiparetic stroke on troponin T expression in human vastus lateralis.

Stroke disability is attributed to upper motor neuron deficits resulting from ischemic brain injury. We have developed proteome maps of the Vastus lateralis to examine the effects of ischemic brain injury on paretic skeletal muscle myofilament proteins. Proteomics analyses from seven hemiparetic stroke patients have detected a decrease of three troponin T isoforms in the paretic muscle suggesting that myosin-actin interactions may be attenuated. We propose that ischemic brain injury may prevent troponin T participation in complex formation thereby affecting the protein interactions associated with excitation-contraction coupling. We have also detected a novel skeletal troponin T isoform that has a C-terminal variation. Our data suggest that the decreased slow troponin T isoform pools in the paretic limb may contribute to the gait deficit after stroke. The complexity of the neurological deficit on Vastus lateralis is suggested by the multiple changes in proteins detected by our proteomics mapping.

High-throughput liquid-liquid fractionation of multiple protein post-translational modifications.

Post-translational protein modifications have contributed significantly to the identification of macromolecular biomarkers of biological processes. We have modified a two-dimensional HPLC system (Beckman Coulter PF2D ProteomeLab) to create proteome maps of post-translational protein modifications. This system resolves complex protein mixtures by anion exchange chromatofocusing in the first dimension and hydrophobicity (reverse phase chromatography) in the second dimension. The simultaneous identification of multiple protein modifications, accomplished by incorporating a photo diode array (PDA) detector into the PF2D system, facilitates the simultaneous production of three-dimensional proteome maps and visualization of both unmodified and post-translationally modified (PTM) proteins at their signature wavelengths within the proteome. We describe procedures for the simultaneous resolution of proteome maps, the identification of proteins modified by nitration, carbonylation, and phosphorylation, and proteins with unique spectra such as the heme containing proteins.

Oxidative modification and aggregation of creatine kinase from aged mouse skeletal muscle.

Creatine kinase catalyzes the reversible transfer of the gamma phosphate from ATP to creatine forming the high energy compound creatine phosphate. Muscle creatine kinase (CKm) activity maintains energetic homeostasis as variations in energy requirements dictate that ATP be readily available. Recent studies suggest that CKm activity is altered during aging. Proteomic analyses have shown that CKm is 3-nitrotyrosine (3-NT) modified and carbonylated in aged rodent skeletal muscle. However, it remains unknown if these modifications affect its structure and activity. To address this we characterized oxidatively modified CKm from the quadriceps of young, middle-aged, and aged mice. Our data indicate that 3-NT modified and carbonylated CKm are found predominantly in aged muscle and that it exists in high molecular weight oligomers and insoluble protein aggregates. CKm from middle-aged and aged mouse quadriceps also exhibits structural instability that may account for its reduction in function. These structural and functional changes correlate with the differential protein modifications. Interestingly, the majority of the age-related changes in enzyme activity and protein stability occurred by middle age. Our studies indicate that the age-associated oxidative and nitrative modification of CKm results in a decrease in its activity and may cause structural changes that promote oligomerization and aggregation.