Development and application of a novel fluorescent labeling strategy for the assessment of protein S-nitrosation

Nitric oxide (NO) is a diatomic free radical that has been the subject of extensive research in the two decades since it was discovered to be a physiological mediator of vascular tone. A functional role for NO in signal transduction was clearly established by the demonstration that endothelium-derived NO could relax vascular smooth muscle. While this response is initiated through direct binding of NO to the heme-iron of the enzyme soluble guanylyl cyclase, other NO-mediated protein modifications are thought to be involved in the transmission of cellular signals. Specifically, it has been postulated that the S-nitrosation of protein cysteine residues conveys a large part of the influence NO has on signal transduction. The reversible nature of this modification in addition to its potential to alter target protein function certainly makes for an attractive possible signal transduction mechanism. Whether protein S-nitrosation is extensively involved in physiological signal transduction or is simply a consequence of stress-induced elevations in NO synthesis is an area of significant interest. Since the basis for selective targeting of S-nitrosoprotein formation in biological systems is not clear and there is no reliable prediction scheme for protein or cysteine targets, a global analysis of potential targets should help uncover patterns that may define this selectivity. However, the low levels of S-nitrosoproteins (PrSNO) detected in biological systems combined with an incomplete understanding of both their formation and metabolism has hindered analyses of this dynamic sub-proteome in a cellular context. Clearly, there is a need for detection methodologies with increased sensitivity in order to study S-nitrosation in the context of physiological NO signaling using unbiased proteomic approaches.;A novel difference gel electrophoresis (DIGE) strategy whereby the S-nitroso functional group of modified cysteine residues is selectively replaced with a fluorescent tag was developed and optimized in simple systems and then used in a global screen for potential protein targets in human pulmonary epithelial cells exposed to nitrosative stress. Evolution of the S-nitrosoproteome (i.e. the entire population of S-nitrosated proteins) as a function of the total cellular PrSNO level revealed a hierarchy of target sensitivity and suggests that only a relatively select subset of proteins, including the critical adaptor signaling protein 14-3-3 sigma, may be modified in cells containing biologically relevant levels of PrSNO. Additional focused studies employing immunological strategies in conjunction with our fluorescent-based labeling methodology were used in order to confirm the identification of 14-3-3 sigma as a target for S-nitrosation and begin to quantitatively address the extent to which this protein is modified at given levels of total cellular PrSNO.