Comparative and integrative metabolomics reveal that S-nitrosation inhibits physiologically relevant metabolic enzymes

Joel J Bruegger,Brian C Smith,Sarah L Wynia-Smith,Michael A Marletta

doi:10.1074/jbc.m117.817700

Abstract

Cysteine S-nitrosation is a reversible post-translational modification mediated by nitric oxide (•NO)-derived agents. S-Nitrosation participates in cellular signaling and is associated with several diseases such as cancer, cardiovascular diseases, and neuronal disorders. Despite the physiological importance of this nonclassical •NO-signaling pathway, little is understood about how much S-nitrosation affects protein function. Moreover, identifying physiologically relevant targets of S-nitrosation is difficult because of the dynamics of transnitrosation and a limited understanding of the physiological mechanisms leading to selective protein S-nitrosation. To identify proteins whose activities are modulated by S-nitrosation, we performed a metabolomics study comparing WT and endothelial nitric-oxide synthase knockout mice. We integrated our results with those of a previous proteomics study that identified physiologically relevant S-nitrosated cysteines, and we found that the activity of at least 21 metabolic enzymes might be regulated by S-nitrosation. We cloned, expressed, and purified four of these enzymes and observed that S-nitrosation inhibits the metabolic enzymes 6-phosphogluconate dehydrogenase, Δ1-pyrroline-5-carboxylate dehydrogenase, catechol-O-methyltransferase, and d-3-phosphoglycerate dehydrogenase. Furthermore, using site-directed mutagenesis, we identified the predominant cysteine residue influencing the observed activity changes in each enzyme. In summary, using an integrated metabolomics approach, we have identified several physiologically relevant S-nitrosation targets, including metabolic enzymes, which are inhibited by this modification, and we have found the cysteines modified by S-nitrosation in each enzyme.

Highlights

Cysteine S-nitrosation is a reversible post-translational modification mediated by nitric oxide (1⁄7NO)-derived agents
The most thoroughly characterized 1⁄7NO-signaling pathway involves the enzyme soluble guanylate cyclase [6]. 1⁄7NO produced by nitric-oxide synthase (NOS) freely diffuses into adjacent cells where it activates sGC to increase the concentration of the secondary messenger cyclic guanosine monophosphate that activates downstream signaling pathways
To narrow the list of target enzymes to those whose activities are inhibited or activated by S-nitrosation, a metabolomics comparison of WT and eNOS knockout mice was carried out, and putative metabolite changes were cross-referenced with the eNOS-dependent metabolic enzyme S-nitrosation sites identified by the proteomics study of Ischiropoulos and co-workers [23]

Summary

To whom correspondence should be addressed

Nitric oxide (1⁄7NO) is an important signaling molecule in vertebrate tissue that controls physiological processes, including vasodilation, neurotransmission, and platelet aggregation [1,2,3]. 1⁄7NO is biosynthesized by the three mammalian isoforms of nitric-oxide synthase (NOS). Endothelial (eNOS) and neuronal NOS produce picomolar to nanomolar concentrations of 1⁄7NO for cellular signaling, whereas inducible NOS produces 1⁄7NO at cytotoxic concentrations in the low micromolar range at sites of infection [4, 5]. Targets of S-nitrosation identified while using exogenous NO donors are difficult to establish as physiologically relevant due to the high reactivity and low specificity of NO-derived agents This is complicated because the in vivo mechanisms of S-nitrosation and primary NO-donor sources are unknown (18 –22). S-Nitrosoglutathione (GSNO) was selected as the nitrosothiol donor because of its putative physiological role in transnitrosation signaling [18, 25, 26], but it is important to note that GSNO may not be the relevant nitrosating agent that led to the observed S-nitrosation of these enzymes in mice. This study integrates comparative proteomics published previously with metabolomics performed in this study and presents quantitative and functional analysis of metabolic enzymes with the primary goal of identifying physiologically relevant protein targets of S-nitrosation

Results

43 Urocanate hydratase

Discussion

Experimental procedures