Copyright © 2005, European Society of Cardiology
Room for both tyrosine nitration and cysteine nitrosylation to regulate NF
B activity, but perhaps only one modification at a time
aDepartment of Pharmacology, Weill Medical College of Cornell University, USA
bDepartment of Endocrinology & Metabolism, Dokkyo University School of Medicine, Japan
* Corresponding author. Tel.: +1 212 746 6257; fax: +1 212 746 8258. Email address: ssgross{at}med.cornell.edu
Received 15 June 2005;
In their letter to the editor, Biswas and Lopes de Faria expressed concern that a recent report by Park et al. in Molecular and Cellular Proteomics [1] appears to contradict our earlier report showing that NO, but not peroxynitrite, inhibits NF
B-mediated gene activation [2]. Careful consideration of the respective studies should allay the reader's concern–there is no disagreement between data presented in these two reports. Whereas our study probes the physiological process by which NO prevents NFB from becoming activated in vascular cells (i.e., in response to immunostimulants), Park examines how an NO-donor might turn off NFB in a pathophysiological setting where it is already activated. The rationale for the Park et al. study of NFB deactivation is that some cancer cells use this as a strategy to escape apoptosis. Accordingly, the Park study uses aberrant cell lines where NFB is constitutively active (for undefined reasons), as well as HEK cells that have been pretreated with TNF
to elicit NFB activation. We have no disagreement whatsoever with the elegant study of Park et al.; however we take this opportunity to suggest an alternative contributing interpretation. Additionally, we would like to point out an unappreciated relationship that can lead to cross-talk between Tyr nitration and S-nitrosylation in NFB modifications that are preferentially engendered by NO and NO-derived peroxynitrite, respectively.
The ability of endothelium-derived NO to constitutively suppress NFB activation in blood vessels has been established by a growing body of literature [3]. Loss of this activity explains the observed pro-inflammatory phenotype of eNOS-null mice [4]. The mechanism by which endogenous NO suppresses NFB activation has been shown to involve S-nitrosylation of specific Cys residues on proteins of the NFB signaling cascade. Notably, Reynaert et al. [5] demonstrated that constitutive S-nitrosylation of C179 on IB inactivates the kinase activity, thereby protecting IB from phosphorylation-triggered proteosomal degradation and preserving the p50/p65 heterodimer in its inactive cytosolic complex with IB. If NFB was to arrive in the nucleus, Marshall and Stamler [6] reported that S-nitrosylation at a single site on p50 (Cys62) would dampen its binding to consensus elements on NFB-regulated genes. In accord with these reports, we showed that NO, but not peroxynitrite, protects against immunostimulant-evoked nuclear translocation of NFB and consequently suppresses the transcription of NFB-dependent genes. The failure of peroxynitrite to protect against NFB activation, in contrast with NO, may be explained by the broader chemical reactivity of peroxynitrite with cellular targets.
Park et al. described the NFB-deactivating action of an NO donor, sodium nitroprusside (SNP), and came to the surprising conclusion that Tyr nitration rather than Cys nitrosylation was causal. They showed that SNP promoted the nitration of tyrosine residues on both p55 and p65 subunits of NFB, elicited nuclear efflux of p65, and attenuated the expression of NFB-driven genes [1]. Mass spectrometry demonstrated nitration on both Y66 and Y152 of peroxynitrite-treated p65 subunits and mutation of these residues decreased the peroxynitrite-mediated deactivation of NFB (in TNF-pretreated cells). It was also shown that deferoxamine, a peroxynitrite scavenger, could attenuate NFB deactivation. Together, these findings were interpreted to indicate that peroxynitrite-elicited Tyr nitration can deactivate NFB. Such reactions of peroxynitrite could have biological relevance in some pathophysiological settings.
An explanation that Park et al. do not consider is that SNP also suppresses activation of NFB in their cell-based model (as we have shown for vascular cells) and contributes to the apparent NFB deactivation. Notably, the basis for constitutive NFB activity in the cultured cell lines is not established, but it is likely to arise from overproduction of superoxide and/or peroxide, the prototypic activators of NFB. If so, diffusion-limited reaction of SNP-derived NO with constitutively produced superoxide would yield peroxynitrite and account for the otherwise paradoxical nitration of Tyr residues following SNP treatment (SNP decomposition yields NO, not peroxynitrite). Loss of superoxide as a consequence of this reaction may reduce the drive on NFB activation and be misconstrued as NFB inactivation. If amounts of SNP-derived NO exceed those required for effective neutralization of superoxide, then the NO excess may additionally suppress NFB activation by S-nitrosylation, as we and others showed previously. It is notable that deferoxamine, used as a peroxynitrite scavenger by Park et al., also scavenges superoxide [7] and hence its actions may not be simple–by scavenging superoxide it can potentially increase the quantity of SNP-derived NO that is available for S-nitrosylation, thereby decreasing both NFB activation and the amount of peroxynitrite available for nitration reactions. Notwithstanding, Park et al. clearly show that chemical nitration can be elicited on Y66 and Y152 of p65 in vitro and mutagenesis of these sites attenuates peroxynitrite-induced nuclear efflux of p65 in TNF-treated cells.
An intriguing possibility is that Tyr nitration and S-nitrosylation on NFB are interrelated. Electrostatic interaction between Cys thiols (with partial negative charge) and aromatic ring-edges (with partial positive charges) constitute a common structural motif in proteins [8]. We recently described such an electrostatic interaction of a Tyr residue in human argininosuccinate synthetase with C132, a residue that undergoes regulatory S-nitrosylation in vivo [9]. Inspection of the atomic environment of Y64, the p50 homolog of Y66 in p65, reveals a similar electrostatic interaction with C62, (<5.5 Å, see Fig. 1). Remarkably, C62 is the residue reported by Marshall and Stamler [6] to undergo S-nitrosylation in p50, resulting in inhibited DNA binding and NFB-mediated gene transcription. An electrostatic interaction of the aromatic ring-edge of Y66 in p65 (a nitrated Tyr that Park et al. attributes to NFB inactivation) is similarly apparent with another Cys thiol (C109, not shown). It is tempting to speculate that cross-talk between Tyr nitration and Cys nitrosylation will prove to be a common structural link between these alternative NO-dependent protein modifications and, in this regard, NFB may be exemplary.
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