Editors' ChoicePosttranslational Modifications

Knowing Where to S-Nitrosylate

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Science Signaling  11 Nov 2014:
Vol. 7, Issue 351, pp. ec314
DOI: 10.1126/scisignal.aaa2540

Modification of proteins at cysteine residues by nitric oxide (NO), a process called S-nitrosylation, is a reversible regulatory event that can control protein localization, stability, and function. NO is a highly reactive molecule and how site selectivity is mediated is an open question. Jia et al. identified a protein complex that mediates the site-specific S-nitrosylation of proteins in peripheral blood monocytes (PBMs) exposed to oxidized low density lipoprotein (LDLox) and interferon-γ (IFN-γ). Exposure of PBMs to both LDLox and IFN-γ (LDLox/IFN-γ), but not either stimulus alone, triggered S-nitrosylation of glyceraldehyde 3-phosphate dehydrogenase (GADPH) on Cys247 (the other two possible target cysteines were mutated to serine). S-nitrosylation of GADPH at Cys247 prevents its interaction with components of the GAIT translational repressor complex, thereby destabilizing and inhibiting the activity of this complex. S-nitrosylation of GAPDH and inhibition of GAIT function was blocked in bone marrow-derived macrophages from iNOS-knockout mice. Mass spectrometry analysis of proteins from LDLox/IFN-γ–exposed PBMs that coimmunoprecipitated with both an antibody recognizing iNOS and an antibody recognizing GAPDH identified the inflammatory mediators S100A9 and S100A8, calcium-binding proteins that form a heterodimer. LDLox/IFN-γ stimulated the formation of a complex between iNOS, GAPDH, S100A8, and S100A9 without affecting the expression of the genes encoding S100A8 and S100A9. S100A9 functioned as an S-nitrosylase that transferred NO to GADPH in vitro. LDLox alone stimulated the interaction between iNOS and S100A8 and S100A9, which required the LDLox-stimulated increase in cytosolic calcium. Knockdown of S100A9 prevented the interaction between iNOS and GADPH and S100A8. GADPH has three cysteines that can be potentially be S-nitrosylated. Although PBMs expressing the C247S mutant of GADPH did not form S-nitrosylated GADPH in response to LDLox/INF-γ, cells expressing this mutant of GADPH and in which S100A8 was knocked down did, suggesting that S100A8 was necessary for site-selective S-nitrosylation at Cys247. Proteomic analysis identified ~100 candidates that were S-nitrosylated in an S100A9-dependent manner in response to LDLox/INF-γ, of which 5 were explored in detail. Analysis of the sequence surrounding the modified cysteines in these candidates along with the sequence surrounding Cys247 of GAPDH identified a conserved motif—I/L-X-C-X-X-D/E. The first hydrophobic residue appeared necessary for recognition of the motif by S100A8 and the charged residues at the end of the motif appeared necessary for recognition by iNOS and S100A9: S-nitrosylation of Cys247 of GAPDH was blocked when leucine or glutamate were mutated and only occurred for the leucine mutant when S100A8 was depleted. Introduction of this motif near a helical motif that is surface accessible in moesin resulted in LDLox/INF-γ- stimulated S-nitrosylation at the cysteine residue in the engineered motif. Thus, S100A8 appears to direct the complex to the proper site so that NO generated by iNOS becomes transferred from S100A9 to the proper cysteine residue in the target protein.

J. Jia, A. Arif, F. Terenzi, B. Willard, E. F. Plow, S. L. Hazen, P. L. Fox, Target-selective protein S-nitrosylation by sequence motif recognition. Cell 159, 623–634 (2014). [Online Journal]

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