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Research Highlights

December 2004

Cellular Response to Protein Nitration in C2C12 Myocytes Detected with Complementary Approaches: Cell Biology and Global Proteomic Screens

Scientists at Pacific Northwest National Laboratory (PNNL) have identified 3-nitrotyrosine-modified proteins from gel-free global proteome analyses of skeletal muscle myocytes in culture. This proteomic screen is a critical step toward understanding cellular dysfunction when nitrated proteins accumulate in heart and skeletal muscle in aging, cardiovascular disease, and environmentally induced inflammation. Nitration of proteins by 3-nitrotyrosine modification signals the presence of cellular peroxynitrite (ONOO-), a potent and reactive oxygen species. Increased levels of nitration have been observed in more than 80 different pathologies, implicating nitrotyrosine modification in cellular damage. Recent observations of reversible nitration-denitration of metabolic enzymes also suggest a role for nitrotyrosine modification in redox-sensitive regulatory processes.

The PNNL team recently detected the ability of myocytes to undergo a rapid loss of protein nitration after exposure to high doses of peroxynitrite (Figure 1). This denitration is 75% complete within 2 hours followed by a slower phase of recovery to basal levels. Treated myocytes remain attached to the substrate and fully viable up to 72 hours after treatment, suggesting that the observed denitration is a normal survival response to nitration rather than a pathological response leading to cell death. Lactacystin, an inhibitor of the proteasome, inhibits only about one-third of this denitration indicating that multiple cellular mechanisms, including proteasome-dependent pathways, are involved. Current efforts are aimed at defining these additional processes.


Figure 1. Loss of nitrated proteins in differentiated C2C12 myotubes after exposure to 250-μM peroxynitrite (PN). Myocytes were lysed at various times after treatment; proteins were separated by SDS-PAGE and immunoblotted with anti-nitrotyrosine antibody (left); lanes from left to right represent increasing times after PN addition: 0, 10 min, 12 h, 24 h, 36 h, 48 h, and 72 h. Center panel: The density associated with the extent of nitrotyrosine immunoreactivity plotted as a function of time after PN addition (arrow). The right panel shows PN treated myotubes (10 min after treatment) fixed and immunostained with anti-nitrotyrosine antibody and secondary antibody conjugated with Alexa-488 dye (green). Nitration is diffuse in the cytoplasm with nucleolar concentration.

This observation of an immediate cellular response to nitration of proteins has not been previously reported, but it likely represents mechanisms that are critical for the ability of heart and skeletal muscle to maintain protein function under the highly aerobic metabolic conditions accompanying contractile activity. In all likelihood, these denitration processes represent a second line of defense that operates under conditions of high oxidative stress after the first line, the antioxidant defenses, are overwhelmed. Elucidating the molecular mechanisms of denitration will provide insights into the steady-state accumulation of nitrated proteins in pathologies.

Appropriate cellular response to either acute or chronic oxidative stress is likely to be mediated by the modification of sensitive proteins that may act as sensors. Current methods for detecting nitrated proteins are limited to high-abundance proteins, primarily with 2-D gels, which provide remarkably poor resolution of membrane proteins. Therefore, we have tested the applicability of global proteomic screens using the ultra-high-sensitivity mass spectrometry facility at PNNL to identify proteins most sensitive to nitration. Samples were prepared by exposing myocytes to the precursor molecule SIN-1, which continuously generates peroxynitrite, under conditions that result in the slow accumulation of levels of nitrated proteins slightly over baseline levels of nitration. The resulting treated and control cells were lysed and treated identically to reduce and alkylate cysteines. These samples were subjected to tryptic digestion with subsequent removal of detergent and salts with strong cation exchange chromatography prior to microliter liquid chromatography tandem mass spectrometric (LC-MS/MS) analyses. Ten LC-MS/MS runs were performed per sample, each focusing on a portion of the total 400 to 2000 mass charge (m/z) range covered. The SEQUEST algorithm was configured to search for both native and nitrotyrosine-containing peptides.

These preliminary screens identified a total of 1742 unique proteins, and of these, 68 (or 4%) were nitrated. To mine this wealth of information, the data were organized using the Gene Ontology program that groups identified proteins by both sub-cellular location and associated biological processes. This analysis shows protein recovery from most cellular compartments; nitrated proteins show no preferential distribution with respect to sub-cellular location. However, with respect to biological processes, the nitrated proteins show more than a two-fold preference for the category "response to stimulus," which includes subcategories of 1) response to stress, 2) response to endogenous stimuli, and 3) response to external stimuli. Among these, the top five nitration targets are all associated with inflammatory and antioxidant responses.

The unanticipated presence of nitration-sensitive proteins within the inflammatory pathway suggests that nitration may provide a way to inhibit this signaling pathway, which up-regulates the production of nitric oxide, a precursor of peroxynitrite. Moreover, previous studies show that an inflammatory response is up-regulated in aging muscle, suggesting that nitration may provide an additional dimension of regulation that has not been considered to date. This work represents development of new approaches testing the applicability of global proteomic screens for identification of post-translationally modified proteins of low abundance, which are likely to be important for elucidating biological stress responses in vivo.

The work is funded by the National Institutes of Health's National Institute on Aging and PNNL's Biomolecular Systems Initiative. The research team, headed by Diana Bigelow, includes biochemists and cell biologists Colette Sacksteder, David Stenoien, Tanya Knyushko, and Monica Londono; and bioinformatician Banu Gopalan, working in collaboration with David Camp and Richard Smith of the High Performance Mass Spectrometry Lab. These results were presented at two recent invited conferences: the 4th International Conference on Peroxynitrite and Reactive Nitrogen Species in Biology and Medicine, July 27-31, 2004, University of Konstanz, Germany; and the Oxidative Post-Translational Modifications in the Cardiovascular System (OPTM) Symposium, October 6-8, 2004, Boston University School of Medicine. Two manuscripts are in preparation.


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