Author
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Elsasser, Theodore |
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Kahl, Stanislaw |
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MARTINEX, ALFREDO - Crilar-Conicet: Regional Research Centre La Rioja |
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Miska, Kate |
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Fetterer, Raymond |
Submitted to: Meeting Abstract
Publication Type: Abstract Only Publication Acceptance Date: 3/1/2017 Publication Date: N/A Citation: N/A Interpretive Summary: Technical Abstract: Host cellular responses to coccidiosis infection are consistent with elements of apoptosis, autophagy, and necrosis. These processes are enhanced in the cell through cell-directed signaling or repressed through parasite-derived inhibitors of these processes favoring the survival of the parasite. Across all three of these cell-based responses one common factor at the origins of response-initiating signaling is the perturbation of the mitochondria and mitochondria-derived release of superoxide anion and nitric oxide. These reactants can interact to form the potent tyrosine-nitrating reactant peroxynitrite. The objective of our investigation was to determine how mitochondrial dysfunction (MD) might arise during the infection-response process and how endogenous biochemical reactions derived from this MD might affect the cell. In the mitochondria, ATP is generated by Complex V ATP-synthase, specifically in what is termed the P-loop, a highly ordered hydrophilic pocket. Based on literature citations suggesting that mitochondrial proteins are dysfunctionally nitrated during oxidative stress, we accessed the 3-D structure of the P-loop (PMID: 11509182) and used a computational chemistry algorithm (HyperProtein ver 1.0) to model how the nitration of a tyrosine residue (Y-345), significant to the orientation of the ADP, Pi and Mg+2, might affect the spatial structure of the domain. Analysis revealed that nitration of this specific residue reordered hydrogen bonding and electrostatic charges in the P-loop where the ADP, Pi and Mg+2 were disfavored from interacting. To test if this nitration could be identified during an oxidative stress in vivo, we developed a highly specific mouse monoclonal anti-nitroY-345 P-loop antibody and performed high resolution fluorescent imaging on duodenal tissues obtained from broilers infected with coccidiosis parasite Eimeria acervulina. Imaging revealed that this specific nitration event did occur (P < 0.05 vs. non-infected chickens) in villus enterocytes as early as 1 d (1d) post-infection (PI). This specific P-loop nitration colocalized directly with mitochondria (rabbit-anti-Complex V, C subunit) on d1 PI but became more generalized throughout the affected enterocytes as the infection progressed and mitochondria appeared to decrease (P < 0.04) in numbers and condense into tight clumps. The nitration mirrored the progression from d1 to d3 to d6 PI of the parasites down the villus to the crypts with the nuclei of both the trophozoites and the schizonts showing evidence of strong generalized protein nitration (P < 0.01, detected with anti-nitrotyrosine antibody) and membrane disruption. The data suggest that post-translational mitochondrial autonitration of Complex V P-loop may participate in cellular responses to coccidiosis. |