Abstract
Protein S-nitrosylation mediates a large part of nitric oxide's influence on cellular function by providing a fundamental mechanism to control protein function across different species and cell types. At steady state, cellular S-nitrosylation reflects dynamic equilibria between S-nitrosothiols (SNOs) in proteins and small molecules (low-molecular-weight SNOs) whose levels are regulated by dedicated S-nitrosylases and denitrosylases. S-Nitroso-CoA (SNO-CoA) and its cognate denitrosylases, SNO-CoA reductases (SCoRs), are newly identified determinants of protein S-nitrosylation in both yeast and mammals. Because SNO-CoA is a minority species among potentially thousands of cellular SNOs, SCoRs must preferentially recognize this SNO substrate. However, little is known about the molecular mechanism by which cellular SNOs are recognized by their cognate enzymes. Using mammalian cells, molecular modeling, substrate-capture assays, and mutagenic analyses, we identified a single conserved surface Lys (Lys-127) residue as well as active-site interactions of the SNO group that mediate recognition of SNO-CoA by SCoR. Comparing SCoRK127Aversus SCoRWT HEK293 cells, we identified a SNO-CoA-dependent nitrosoproteome, including numerous metabolic protein substrates. Finally, we discovered that the SNO-CoA/SCoR system has a role in mitochondrial metabolism. Collectively, our findings provide molecular insights into the basis of specificity in SNO-CoA-mediated metabolic signaling and suggest a role for SCoR-regulated S-nitrosylation in multiple metabolic processes.
Highlights
Protein S-nitrosylation mediates a large part of nitric oxide’s influence on cellular function by providing a fundamental mechanism to control protein function across different species and cell types
We discovered that the SNO-CoA/SNO-CoA reductases (SCoRs) system has a role in mitochondrial metabolism
Docking of SNO-CoA to the SCoR active-site produced two possible binding modes (Fig. 1, A and B) with the SNO group oriented toward NADPH and the catalytic Tyr-50 [14]
Summary
Protein S-nitrosylation mediates a large part of nitric oxide’s influence on cellular function by providing a fundamental mechanism to control protein function across different species and cell types. Denitrosylases are likely fewer in number and fall into two categories: 1) direct protein denitrosylases, exemplified by thioredoxin-related proteins [6]; and 2) low-molecular weight (LMW) SNO reductases, including S-nitrosoglutathione (GSNO) reductases [7, 8] and S-nitroso-CoA (SNOCoA) reductases [9] The latter group of enzymes carry out NAD(P)H-dependent reduction of GSNO or SNO-CoA, thereby regulating coupled equilibria between SNO-proteins and LMW-SNOs to control SNO-protein levels [10]. There is a general lack of understanding of how denitrosylases recognize their substrates within the cellular milieu It remains unclear if these interactions depend on the R groups in RSNOs (e.g. GSH or CoA) and/or whether proteins may recognize the SNO moiety. We utilize mutant SCoR that is unable to bind SNO-CoA to identify novel targets of SNO-CoA–mediated S-nitrosylation in cellular systems, and to assess the role of SCoR in mitochondrial metabolism
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