Argininosuccinate Synthetase is Reversibly Inactivated by S-Nitrosylation in Vitro and in Vivo

Abstract

Prior studies have demonstrated that the substrate for NO synthesis, L-arginine, can be regenerated from the NOS co-product L-citrulline. This requires the sequential action of two enzymes, argininosuccinate synthetase (AS) and argininosuccinate lyase (AL). AS activity has been shown to be rate-limiting for high output NO synthesis by immunostimulant-activated cells and represents a potential site for metabolic control of NO synthesis. We now demonstrate that NO mediates reversible S-nitrosylation and inactivation of AS in vitro and in lipopolysaccharide-treated cells and mice. Using a novel mass spectrometry-based method, we show that Cys-132 in human AS is the sole target for S-nitrosylation among five Cys residues. Mutagenesis studies confirm that S-nitrosylation of Cys-132 is both necessary and sufficient for the inhibition of AS by NO donors. S-nitroso-AS content is regulated by cellular glutathione levels and selectively influences NO production when citrulline is provided to cells as a protosubstrate of NOS but not when L-arginine is provided. A phylogenetic comparison of AS sequences suggests that Cys-132 evolved as a site for post-translational regulation of activity in the AS in NOS-expressing species, endowing NO with the capacity to limit its own synthesis by restricting arginine availability.Nitric oxide (NO) 1 is a cell signaling molecule with diverse and important biological functions (1, 2). The mammalian genome encodes three distinct isoforms of NO synthase (NOS) that catalyze identical reactions-conversion of L-arginine (Arg) to NO and L-citrulline (Cit) (3). The immunostimulant-induced isoform of nitric-oxide synthase (iNOS) produces cytotoxic quantities of NO and NO-derived species that are used to fend off tumor cells and various invading organisms (4) at the expense of possible injury to itself (5). Regulation of iNOS activity occurs principally at the level of transcription, endowing cells with a relatively slow means to adapt high output NO production to mammalian cell needs. Once iNOS protein is resident in a cell, however, the availability of substrate Arg is thought to be the key determinant of NO synthesis rate. Importantly, Arg is synthesized within NO-producing cells from the NOS coproduct Cit (6) via the sequential action of two enzymes: argininosuccinate synthetase (AS) and argininosuccinate lyase (AL). AS catalyzes an ATP-dependent ligation of the NOS product Cit with Asp, yielding argininosuccinate, AMP, and pyrophosphate. Argininosuccinate is subsequently cleaved by argininosuccinate lyase (AL), yielding Arg and fumarate. Although AS and AL are generally considered in the context of their contribution to the urea cycle of the liver, where AS serves as the rate-limiting enzyme for ammonia detoxification (7), these enzymes also endow iNOS-expressing cells with an Arg/Cit cycle for continuous regeneration of Arg from Cit, providing iNOS with a sustained supply of substrate. Although AL was found to be constitutively expressed in NO-producing cells, AS mRNA, protein and activity ar...

Structural profiling of endogenous S-nitrosocysteine residues reveals unique features that accommodate diverse mechanisms for protein S-nitrosylation

“…The depth of this analysis represents a significant advancement versus present methodologies (17). The majority of the proteins identified in the current study, 186, corresponded to previously unidentified endogenous targets of S-nitrosylation in the mouse liver, whereas six proteins (GAPDH, hemoglobin, β-tubulin, argininosuccinate synthase, alcohol dehydrogenase, and catalase) have been previously identified as endogenously S-nitrosylated in hepatocytes and other organ systems (18)(19)(20)(21). Proteins were also distributed across a wide range of molecular weights (13-272 kDa) and cellular localization including membrane-associated proteins, demonstrating the efficacy of the method to identify S-nitrosocysteine independent of protein size and location.…”

“…S2), and were identified with high SEQUEST cross correlation (X c ) scores at ppm mass error. Cys132 has been previously reported as a target of S-nitrosylation in human argininosuccinate synthase (20), whereas the identification of Cys97 corresponded to a previously unidentified site of S-nitrosocysteine formation. (D) Colloidal blue protein staining of bound fractions from MRC and mPEGb enrichment of S-nitrosylated proteins from WT and eNOS −/− mouse livers.…”

Interactions between the NO-Citrulline Cycle and Brain-derived Neurotrophic Factor in Differentiation of Neural Stem Cells

“…Reactions were incubated at 37°C in 96-well microtiter plates and stopped after 30 min by the addition of an equal volume of molybdate buffer (10 mM ascorbic acid, 2.5 mM ammonium molybdate, 2% (v/v) sulfuric acid). Accumulation of phosphate was determined spectrophotometrically at 650 nm, and concentrations were extrapolated from a standard curve of inorganic phosphate (22).…”

Arginine Deprivation as a Targeted Therapy for Cancer

“…The implication of these findings to tumor cell survival/carcinogenesis has not been studied. On the other hand NO can reversibly inactivate ASS via Snitrosulation both in vitro and in vivo [41]. Thus the complex relationship of these parameters will need further investigation.…”