How hydrogen peroxide (H2O2) produced by dual oxidases (DUOXs) contributes to the defense of mucosal surfaces against infection is incomplete, representing a critical gap in knowledge. The long-term goal of this re- search is to elucidate how ROS (reactive oxygen species) contribute to innate immunity in epithelial tissues. The overall objective of this application is to identify how H2O2 produced by DUOXs contributes to immune defense, using C. elegans as a model. The central hypothesis is that the H2O2 produced by BLI-3 (C. elegans DUOX) contributes to immune defense (1) as a signaling molecule that affects the redox state and therefore the activity of a cytoprotective transcription factor, and (2) as a substrate for immune-specific peroxidases. The rationale for the project is that a more complete understanding of how H2O2 production by DUOX enzymes during infection contributes to the immune response will potentially allow for its therapeutic modulation. The central hypothesis will be addressed by the following aims.
Specific Aim 1 : Determine how thioredoxin TRX-1 regulates the protective transcription factor SKN-1. We will test the working hypothesis that under reducing conditions TRX-1 inactivates the SKN-1 regulator NSY-1, by direct binding. Under oxidizing conditions, such as those that occur during the immune response, oxidation of redox-sensitive cysteines in TRX-1 releases NSY-1. The approaches used to test this hypothesis will include studying appropriate mutants of trx-1 and nsy-1 and in vivo and in vitro approaches to test for a direct interaction between TRX-1 and NSY-1.
Specific Aim 2 : Elucidate the mechanism(s) by which putative peroxidases contribute to pathogen resistance. We have identified putative peroxidases that contribute to immune defense, and we will test the postulate that they do so by utilizing H2O2 to generate more powerful oxidants. The approaches used to test the hypothesis will include characterization of animals with mutated versions of these genes and purification and enzymatic analysis of the proteins they encode.
Specific Aim 3 : Determine if BLI-3 directly interacts with the putative peroxidases. Our working hypothesis is that these peroxidases interact directly with BLI-3, the enzyme that produces their substrate. The approaches used to test this hypothesis will include localization of the peroxidases by genetic and cell-biological techniques and in vivo and in vitro approaches to test for a direct interaction with BLI-3. The research proposed is significant because knowledge of how the H2O2 produced by dual oxidases affects biological systems could lead to treatments that modulate its positive and negative effects. For example, pharmacologically increasing the amount of ROS production may lead to protection against infectious diseases, while decreasing ROS may help resolve inflammatory conditions. The proposed research is innovative be- cause it takes advantage of C. elegans unique feature of producing a single DUOX enzyme to understand the roles of DUOX-generated H2O2 during infection in the natural context of the whole organism. Not having to ac- count for the effects of other NADPH oxidases will facilitate the study of DUOX at the organismal level.
The research proposed in this application will lead to greater understanding of how an immune mechanism associated with the mucosal surfaces in the gastrointestinal and respiratory tracts works. Specifically, using a small animal called C. elegans, as a model, the mechanisms involved in generating and preventing self- damage from the response will be identified. Such knowledge is relevant to public health because it will lead to the potential manipulation of this immune response to the patient's advantage in the treatment of infections and autoimmune disorders associated with the mucosae.
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