Nitric oxide (NO) and NO-derived nitrosating agents (nitrosants) are signaling molecules. Protein and low mass thiols are components of signal transduction circuits subject to NO regulation. NO can also be cytotoxic when levels exceed a dangerous threshold. Under such conditions, nitros(yl)ation of cellular constituents imposes a nitrosative stress, which manifests in mutagenesis, cytostasis and cell death. Resistance to the toxic effects of NO-derived molecules is, thus, an indispensable requirement for normal cell function. In E. coli, S-nitros(yl)ation of the transcription factor OxyR activates antioxidant genes and, moreover, protects against a nitrosative threat. Part of the resistance is due to accelerated breakdown of S-nitrosothiols (SNOs). Additional protection is conferred by glutathione and the inducible flavohemoglobin HMP that metabolizes NO to nitrate. The proposed study uses this bacterial model system to further probe resistance mechanisms.
Aim1. Elucidates the pathway in E. coli that metabolizes SNOs and provides resistance to SNO-induced cytostasis. In this pathway, SNO is cleaved to release NO, and is also reduced to nitrous oxide. The free NO released is oxidized to nitrate by the SNO/NO-inducible HMP. Reaction mechanisms will be identified and the enzymes involved in SNO breakdown will be purified and characterized.
Aim 2. Studies the contributions of the antioxidant/antinitrosant glutathione, the transcription factor OxyR, and the 'denitrosolase' HMP in aerobic and anaerobic nitrosative stress resistance.
Aim 3. Explores the ability of antinitrosative enzymes to ameliorate nitrosative stress in mammalian systems, specifically, in septic shock and cancer models.
Aim 4. Employs circular dichroism and X-ray crystallography to reveal the functional redox-related switch that regulates OxyR. The reduced, oxidized, mixed disulfide and nitrosylated forms of purified OxyR are analyzed and their functional behaviors compared.
| Parsa, Cyrus J; Kim, Jihee; Riel, Ryan U et al. (2004) Cardioprotective effects of erythropoietin in the reperfused ischemic heart: a potential role for cardiac fibroblasts. J Biol Chem 279:20655-62 |
| Parsa, Cyrus J; Matsumoto, Akio; Kim, Jihee et al. (2003) A novel protective effect of erythropoietin in the infarcted heart. J Clin Invest 112:999-1007 |
| Stamler, J S; Lamas, S; Fang, F C (2001) Nitrosylation. the prototypic redox-based signaling mechanism. Cell 106:675-83 |
| Sun, J; Xu, L; Eu, J P et al. (2001) Classes of thiols that influence the activity of the skeletal muscle calcium release channel. J Biol Chem 276:15625-30 |
| Hausladen, A; Gow, A; Stamler, J S (2001) Flavohemoglobin denitrosylase catalyzes the reaction of a nitroxyl equivalent with molecular oxygen. Proc Natl Acad Sci U S A 98:10108-12 |
| Eu, J P; Sun, J; Xu, L et al. (2000) The skeletal muscle calcium release channel: coupled O2 sensor and NO signaling functions. Cell 102:499-509 |
