Using methodology developed herein, it is found that reactive persulfides and polysulfides are formed endogenously from both small molecule species and proteins in high amounts in mammalian cells and tissues. These reactive sulfur species were biosynthesized by two major sulfurtransferases: cystathionine β-synthase and cystathionine γ-lyase. Quantitation of these species indicates that high concentrations of glutathione persulfide (perhydropersulfide >100 μM) and other cysteine persulfide and polysulfide derivatives in peptides/proteins were endogenously produced and maintained in the plasma, cells, and tissues of mammals (rodent and human). It is expected that persulfides are especially nucleophilic and reducing. This view was found to be the case, because they quickly react with H₂O₂ and a recently described biologically generated electrophile 8-nitroguanosine 3′,5′-cyclic monophosphate. These results indicate that persulfides are potentially important signaling/effector species, and because H₂S can be generated from persulfide degradation, much of the reported biological activity associated with H₂S may actually be that of persulfides. That is, H₂S may act primarily as a marker for the biologically active of persulfide species. Significance Reactive sulfur-containing compounds, such as l-cysteine hydropersulfide (CysSSH), reportedly form in mammals. However, the biological relevance of these reactive sulfur species remains unclear. We determined that CysSSH was synthesized from cystine by cystathionine β-synthase and cystathionine γ-lyase, which in turn may contribute to high levels of glutathione hydropersulfide (>100 μM) and other CysSSH derivatives of peptides/proteins formed in cells, tissues, and plasma from mice and humans. Compared with glutathione and hydrogen sulfide, CysSSH derivatives were superior nucleophiles and reductants and capable of regulating electrophilic cell signaling mediated by 8-nitroguanosine 3′,5′-cyclic monophosphate. Altogether, it is proposed that reactive Cys persulfides and S-polythiolation have critical regulatory functions in redox cell signaling.

Cysteine hydropersulfide (CysSSH) occurs in abundant quantities in various organisms, yet little is known about its biosynthesis and physiological functions. Extensive persulfide formation is apparent in cysteine-containing proteins inEscherichia coliand mammalian cells and is believed to result from post-translational processes involving hydrogen sulfide-related chemistry. Here we demonstrate effective CysSSH synthesis from the substratel-cysteine, a reaction catalyzed by prokaryotic and mammalian cysteinyl-tRNA synthetases (CARSs). Targeted disruption of the genes encoding mitochondrial CARSs in mice and human cells shows that CARSs have a crucial role in endogenous CysSSH production and suggests that these enzymes serve as the principal cysteine persulfide synthases in vivo. CARSs also catalyze co-translational cysteine polysulfidation and are involved in the regulation of mitochondrial biogenesis and bioenergetics. Investigating CARS-dependent persulfide production may thus clarify aberrant redox signaling in physiological and pathophysiological conditions, and suggest therapeutic targets based on oxidative stress and mitochondrial dysfunction.

We have previously demonstrated that phosphorylation of neuronal nitric-oxide synthase (nNOS) at Ser(847) by Ca(2+)/calmodulin-dependent protein kinases (CaM kinases) attenuates the catalytic activity of the enzyme in vitro (Hayashi Y., Nishio M., Naito Y., Yokokura H., Nimura Y., Hidaka H., and Watanabe Y. (1999) J. Biol. Chem. 274, 20597-20602). In the present study we determined that CaM kinase IIalpha (CaM-K IIalpha) can directly phosphorylate nNOS on Ser(847), leading to a reduction of nNOS activity in cells. The phosphorylation abilities of purified CaM kinase Ialpha (CaM-K Ialpha), CaM-K IIalpha, and CaM-kinase IV (CaM-K IV) on Ser(847) were analyzed using the synthetic peptide nNOS-(836-859) (Glu-Glu-Arg-Lys-Ser-Tyr-Lys-Val-Arg-Phe-Asn-Ser-Val-Ser-Ser-Tyr-Ser- Asp-Ser-Arg-Lys-Ser-Ser-Gly) from nNOS as substrate. The relative V(max)/K(m) ratios of CaM kinases for nNOS-(836-859) were found to be as follows: CaM-K IIalpha, 100; CaM-K Ialpha, 54.5; CaM-K IV, 9.1. Co-transfection of constitutively active CaM-K IIalpha1-274 but not inactive CaM-K IIalpha1-274, generated by mutation of Lys(42) to Ala, with nNOS into NG108-15 cells, resulted in increased Ser(847) phosphorylation in the presence of okadaic acid, an inhibitor of protein phosphatase (PP)1 and PP2A, with a concomitant inhibition of NOS enzyme activity. In addition, this latter decrease could be reversed by treatment with exogenous PP2A. Cells expressing mutant nNOS (S847A) proved resistant to phosphorylation and a decrease of NOS activity. Thus, our results indicate that Ca(2+) triggers cross-talk signal transduction between CaM kinase and NO and CaM-K IIalpha phosphorylating nNOS on Ser(847), which in turn decreases the gaseous second messenger NO in neuronal cells.

Phosphorylation of neuronal nitric-oxide synthase (nNOS) by Ca²⁺/calmodulin (CaM)-dependent protein kinases (CaM kinases) including CaM kinase Iα (CaM-K Iα), CaM kinase IIα (CaM-K IIα), and CaM kinase IV (CaM-K IV), was studied. It was found that purified recombinant nNOS was phosphorylated by CaM-K Iα, CaM-K IIα, and CaM-K IV at Ser⁸⁴⁷ in vitro. Replacement of Ser⁸⁴⁷ with Ala (S847A) prevented phosphorylation by CaM kinases. Phosphorylated recombinant wild-type nNOS at Ser⁸⁴⁷ (≈0.5 mol of phosphate incorporation into nNOS) exhibited a 30% decrease ofV _max with little change of both theK _m for l-arginine andK _act for CaM relative to unphosphorylated enzyme. The activity of mutant S847D was decreased to a level 50–60% as much as the wild-type enzyme. The decreased NOS enzyme activity of phosphorylated nNOS at Ser⁸⁴⁷ and mutant S847D was partially due to suppression of CaM binding, but not to impairment of dimer formation which is thought to be essential for enzyme activation. Inactive nNOS lacking CaM-binding ability was generated by mutation of Lys⁷³²-Lys-Leu to Asp⁷³²-Asp-Glu (Watanabe, Y., Hu, Y., and Hidaka, H. (1997) FEBS Lett. 403, 75–78). It was phosphorylated by CaM kinases, as was the wild-type enzyme, indicating that CaM-nNOS binding was not required for the phosphorylation reaction. We developed antibody NP847, which specifically recognize nNOS in its phosphorylated state at Ser⁸⁴⁷. Using the antibody NP847, we obtained evidence that nNOS is phosphorylated at Ser⁸⁴⁷ in rat brain. Thus, our results suggest that CaM kinase-induced phosphorylation of nNOS at Ser⁸⁴⁷ alters the activity control of this enzyme.

Dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) plays a central role in medium spiny neurons in the neostriatum in the integration of various neurotransmitter signaling pathways. In its Thr-34-phosphorylated form, it acts as a potent protein phosphatase-1 inhibitor, and, in its Thr-75-phosphorylated form, it acts as a cAMP-dependent kinase inhibitor. Here, we investigated glutamate-dependent signaling cascades in mouse neostriatal slices by analyzing the phosphorylation of DARPP-32 at Thr-34 and Thr-75. Treatment with glutamate (5 mM) caused a complex change in DARPP-32 Thr-34 phosphorylation. An initial rapid increase in Thr-34 phosphorylation was NMDA/α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/metabotropic glutamate-5 receptor-dependent and was mediated through activation of a neuronal nitric oxide synthase/nitric oxide/cGMP/cGMP-dependent kinase signaling cascade. A subsequent decrease in phosphorylation was attributable to activation of an NMDA/AMPA receptor/Ca ²⁺ /protein phosphatase-2B signaling cascade. This decrease was followed by rephosphorylation via a pathway involving metabotropic glutamate-5 receptor/phospholipase C and extracellular receptor kinase signaling cascade. Treatment with glutamate initially decreased Thr-75 phosphorylation through activation of NMDA/AMPA receptor/Ca ²⁺ /protein phosphatase-2A signaling. Thereafter, glutamate slowly increased Thr-75 phosphorylation through activation of metabotropic glutamate-1 receptor/phospholipase C signaling. Our analysis of DARPP-32 phosphorylation in the neostriatum revealed that glutamate activates at least five different signaling cascades with different time dependencies, resulting in complex regulation of protein kinase and protein phosphatase activities.