In its classical signaling role, NO is captured by the heme cofactor of soluble guanylate cyclase (sGC), activating sGC to produce the secondary messenger cyclic GMP (cGMP). However, mounting evidence points toward an alternative, cGMP-independent NO signaling pathway in which the S-nitrosation of cysteine residues regulates protein structure and activity. S-Nitrosation has been implicated in a broad spectrum of diseases, including cancer, diabetes, and other cardiovascular, pulmonary, and neurological disorders, yet the mechanism by which nitrosothiols are formed in vivo is unknown. In vitro, non-specific cysteine nitrosation occurs readily. In vivo nitrosation, however, is far more selective for specific cysteines and is driven by factors beyond thiol reactivity. We postulate that nitrosation selectivity is driven by protein-protein or protein-small molecule interactions that align a nitrosothiol with a free thiol for transnitrosation reactions.
Transnitrosation reactions require an “initiating” nitrosothiol. Nitric oxide synthases (NOSs) are potential candidates for the initial formation of nitrosothiols as all three mammalian NOS isoforms selectively form nitrosothiols at their Zn2+-tetrathiolate cysteines. We recently developed a kinetic model of NOS S-nitrosation. In this model, NO synthesized at the heme cofactor is partitioned between release into solution and NOS auto-S-nitrosation. The results suggested that NOS S-nitrosation is both a mechanism to control NOS activity and to generate physiological nitrosothiols.
The inducible NOS isoform (iNOS) has been shown to participate in protein–protein interaction-mediated S-nitrosation reactions with cyclooxygenase-2 (COX-2) and arginase-1. Furthermore, procaspase-3 and iNOS participate in an NO-dependent protein–protein interaction. As caspase-3 is known to be nitrosated on its active-site cysteine, iNOS might directly transnitrosate caspase-3. We are broadly interested in discovering and characterizing novel targets of NOS transnitrosation.
Recent genome sequencing revealed that many bacterial species also possess NOS-like proteins. While most of these bacterial NOS proteins contain only a single domain with high sequence homology to the oxygenase domain of mammalian NOS, a handful of recently-discovered bacterial NOS proteins are fused to reductase domains. We are generally interested in these novel bacterial NOS proteins. Do these bacterial NOS proteins produce NO? What substrates and cofactors are required for their activity? How do the mechanisms of NO synthesis compare and contrast to the mammalian NOS enzymes? What role do these bacterial NOS proteins play in signaling?
Together, these studies will provide compelling evidence for S-nitrosation as a signaling-competent, reversible post-translational modification driven by selective protein-protein and protein-small molecule interactions, and will set the stage for a molecular understanding of S-nitrosation in vivo.
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