Effect Of S-nitrosylation Research Articles

S-Nitrosylation-Mediated Reduction of CaV1.2 Surface Expression and Open Probability Underlies Attenuated Vasoconstriction Induced by Nitric Oxide.

L-type CaV1.2 calcium channel, the primary gateway for Ca2+ influx in smooth muscles, is widely regulated by multiple posttranslational modifications, such as protein kinase-mediated phosphorylation and nitric oxide-induced S-nitrosylation. However, the effect of S-nitrosylation on CaV1.2 channel function and its role in arterial contractility are not well understood. Electrophysiological recordings, Ca2+ and confocal imaging, and biochemical assays were used to functionally characterize S-nitrosylated CaV1.2 channels in vitro, while pressure myography and tail-cuff blood pressure measurement were conducted to evaluate the physiological effects of CaV1.2 S-nitrosylation ex vivo and in vivo. S-nitrosylation significantly reduced the CaV1.2 current density by promoting lysosomal degradation that leads to decreased levels of total and surface CaV1.2 channel proteins in a CaVβ-independent manner and reducing the open probability of CaV1.2 channel. Mechanistically, the Cys1180 and Cys1280 residues within CaV1.2 channel have been determined as the molecular targets for S-nitrosylation as substitution of either Cys1180 or Cys1280 for serine resulted in substantial reduction of S-nitrosylation levels. Of note, CaV1.2 S-nitrosylation levels were significantly reduced in arteries isolated from both spontaneously hypertensive rats and patients with pulmonary hypertension. Moreover, mouse resistance arteries incubated with S-nitrosocysteine displayed much lower contractility and spontaneously hypertensive rats injected with S-nitrosocysteine also showed significantly reduced blood pressure, suggesting that reduced S-nitrosylation contributes to the upregulation of CaV1.2 channel activity in hypertensive arteries. This study provides strong evidence that S-nitrosylation-mediated downregulation of CaV1.2 channels is via 2 distinctive mechanisms and the findings offer potential pathways for therapeutic inventions in hypertension.

The Relationship Between Protein S-Nitrosylation and Human Diseases: A Review.

S-nitrosylation (SNO) is a covalent post-translational oxidative modification. The reaction is the nitroso group (-NO) to a reactive cysteine thiol within a protein to form the SNO. In recent years, a variety of proteins in human body have been found to undergo thiol nitrosylation under specific conditions. Protein SNO, which is closely related to cardiovascular disease, Parkinson's syndrome, Alzheimer's disease and tumors, plays an important role in regulatory mechanism of protein function in both physiological and pathological pathways, such as in cellular homeostasis and metabolism. This review discusses possible molecular mechanisms protein SNO modification, such as the role of NO in vivo and the formation mechanism of SNO, with particular emphasis on mechanisms utilized by SNO to cause certain diseases of human. Importantly, the effect of SNO on diseases is multifaceted and multi-channel, and its critical value in vivo is not well defined. Intracellular redox environment is also a key factor affecting its level. Therefore, we should pay more attention to the equilibrium relationship between SNO and denitrosylation pathway in the future researches. These findings provide theoretical support for the improvement or treatment of diseases from the point of view of SNO.

Cysteine 343 in the substrate binding domain is the primary S-Nitrosylated site in protein disulfide isomerase

Abnormal protein accumulations are typical pathological features for neurodegenerative diseases. Protein disulfide isomerase (PDI) is a critical enzyme in oxidative protein folding. S-nitrosylated PDI has been detected in the postmortem brain in neurodegenerative disease patients, but the effect of S-nitrosylation on PDI function and developing neurodegeneration was not clarified in detail. In this study, we identified that in vitro and in vivo S-nitrosylation of C343 in the b’ domain of PDI occurs. Reduced recombinant human PDI (hPDI) reacted quickly with S-nitrosocompounds, with an observed increase in the expected S-nitrosylated species and the appearance of the disulfide state of the active sites. Both Mononitrosylated and dinitrosylated were observed from the S-nitrosylation of hPDI. Dinitrosylated species were S-nitrosylated both cysteines at active site. But, at least in part, mononitrosylated species were S-nitrosylated on cysteine 343 in the substrate binding b’ domain. Although active site S-nitrosylation is reversible by reduced glutathione, S-nitrosylation of C343 is comparative stable. S-nitrosylation of PDI in SH-SY5Y cells was observed after the S-nitrosocysteine (SNOC) treatment and S-nitrosylated PDI was still detected 24 h after removing SNOC. While wild-type PDI was S-nitrosylated, the level of S-nitrosylation of the C343S mutant in over-expressed cells was substantially lower and only wild-type PDI of S-nitrosylation remained 24 h after removing SNOC in over-expressed cells. Both of in vitro and in vivo results suggested that S-nitrosylation of C343 in PDI may be the causative effect on physiological changes in neurodegerenative disease patients, and may be useful for the drug development for neurodegenerative diseases.

A recent advance in the intracellular and extracellular redox post-translational modification of proteins in plants.

Plants, as sessile organisms, have acquired through evolution sophisticated regulatory signal pathways to overcome external variable factors during each stage of the life cycle. Among these regulatory signals, two pathways in particular, reactive oxygen species and reactive nitrogen species, have become of significant interest in several aspects of plant biology, underpinning these molecules as critical regulators during development, cellular differentiation, and plant-pathogen interaction. Recently, redox posttranslational modifications (PTM), such as S-nitrosylation on cysteine residues and tyrosine nitration, have shed light on multiple protein targets, as they are associated with signal networks/downstream metabolic pathways, capable of transducing the imbalance of redox hemostasis and consequently redirecting the biochemical status under stress conditions. However, most of the redox PTM have been studied only in the intracellular compartment, providing limited information concerning redox PTM in the extracellular matrix of plant cells. Nevertheless, recent studies have indicated the plausibility of redox PTM in extracellular proteins, including cell wall associated proteins. Accordingly, in this review, we endeavor to examine evidence of redox PTM supported by mass spectrometry data in the intracellular and extracellular space in plant cells. As a further example, we focus the last section of this review on illustrating, using molecular dynamics simulation, the effect of S-nitrosylation on the structural conformation of well-known cell wall-associated proteins including pectin methylesterase and xyloglucan endo-transglycosylases.

Characterization of human triosephosphate isomerase S-nitrosylation

Triosephosphate isomerase (TPI), the glycolytic enzyme that catalyzes the isomerization of dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (G3P), has been frequently identified as a target of S-nitrosylation by proteomic studies. However, the effect of S-nitrosylation on its activity has only been explored in plants and algae. Here, we describe the in vitro S-nitrosylation of human TPI (hTPI), and the effect of the modification on its enzymatic parameters. NO-incorporation into the enzyme cysteine residues occurred by a time-dependent S-transnitrosylation from both, S-nitrosocysteine (CySNO) and S-nitrosoglutathione (GSNO), with CySNO being the more efficient NO-donor. Both X-ray crystal structure and mass spectrometry analyses showed that only Cys217 was S-nitrosylated. hTPI S-nitrosylation produced a 30% inhibition of the Vmax of the DHAP conversion to G3P, without affecting the Km for DHAP. This is the first study describing features of human TPI S-nitrosylation.

Human serine racemase is nitrosylated at multiple sites

Serine racemase is a pyridoxal 5′‑phosphate dependent enzyme responsible for the synthesis of d‑serine, a neuromodulator of the NMDA receptors. Its activity is modulated by several ligands, including ATP, divalent cations and protein interactors. The murine orthologue is inhibited by S-nitrosylation at Cys113, a residue adjacent to the ATP binding site. We found that the time course of inhibition of human serine racemase by S-nitrosylation is markedly biphasic, with a fast phase associated with the reaction of Cys113. Unlike the murine enzyme, two additional cysteine residues, Cys269, unique to the human orthologue, and Cys128 were also recognized as S-nitrosylation sites through mass spectrometry and site-directed mutagenesis. The effect of S-nitrosylation on the fluorescence of tryptophan residues and on that of the pyridoxal phosphate cofactor indicated that S-nitrosylation produces a partial interruption of the cross-talk between the ATP binding site and the active site. Overall, it appears that the inhibition results from a conformational change rather than the direct displacement of ATP.

Effect of protein S-nitrosylation on autolysis and catalytic ability of μ-calpain

The effect of S-nitrosylation on the autolysis and catalytic ability of μ-calpain in vitro in the presence of 50μM Ca2 + was investigated. μ-Calpain was incubated with different concentrations of nitric oxide donor S-nitrosoglutathione (GSNO) and subsequently reacted with purified myofibrils. Results showed that the amount of 80kDa μ-calpain subunit significantly decreased as GSNO increased from 0 to 300μM, but increases of GSNO to 300, 500 and 1000μM did not result in further inhibition. The catalytic ability of nitrosylated μ-calpain to degrade titin, nebulin, troponin-T and desmin was significantly reduced when the GSNO concentration was higher than 300μM. The cysteine residues of μ-calpain at positions 49, 351, 384, and 592 in the catalytic subunit and at 142 in small subunit were S-nitrosylated, which could be responsible for decreased μ-calpain activity. Thus, S-nitrosylation can negatively regulate the activation of μ-calpain resulting in decreased proteolytic ability on myofibrils.

Abstract 154: Interaction Between Nitroso-Redox Balance and Temperature Influences SR Ca 2+ Leak In Cardiomyocytes: Role of Neuronal NOS Signaling

Sarcomeric reticulum (SR) Ca 2+ leak contributes to impaired Ca 2+ cycling and contractile dysfunction of the heart. Leak is thought to be regulated by post-translational modification of RyR2 associated with the nitroso-redox state. Ca 2+ cycling is affected by both nitric oxide (NO) signaling and temperature. We studied the effect of temperature and nitroso-redox state on Ca 2+ leak. We hypothesized that nitroso-redox balance modulates leak in a temperature–dependent manner. Tetracaine was used to estimate the leak in cardiomyocytes (CMs) in mouse models of nitroso-redox imbalance (NOS1 -/- ), hyper S-nitrosylation (GSNOR -/- ) and in WT mice. Reactive oxygen species (ROS) were measured. Experiments were carried out at 23°C – 37°C. In WT CMs, Ca 2+ leak was inversely proportional to temperature whereas, in NOS1 -/- CMs, the leak suddenly increased when the temperature surpassed 30°C. Supplementing the media with NO (1 microM SNAP), reduced leak in NOS1 -/- CMs at physiologic temperature while at low temperatures, there was a trend toward an increase, a pattern similar to that of GSNOR -/- rather than WT CMs. The xanthine oxidase inhibitor, oxypurinol (100 microM), diminished the leak in NOS1 -/- CMs. SR Ca 2+ content in NOS1 -/- CMs was reduced as the temperature decreased, whereas it was not affected in WT cells. Cooling from 37°C to 23 °C increased ROS generation in WT CMs; however there was an opposite effect observed in NOS1 -/- CMs, which was further reduced by oxypurinol. Expression of the NOS isoforms (by RT-PCR) was unaffected by temperature in WT. Therefore, Ca 2+ leak is temperature dependent, is affected by the absence of NOS1 activity and increases with the rise of ROS production. Defective denitrosylation activity keeps leak low, suggesting a protective effect of S-nitrosylation against the cooling-induced increase in SR Ca 2+ leak. These results suggest that Ca 2+ leak from the SR is regulated by a temperature-dependent balance between NO and ROS.

S‐nitrosylated proteins of a medicinal CAM plant Kalanchoe pinnata– ribulose‐1,5‐bisphosphate carboxylase/oxygenase activity targeted for inhibition

Nitric oxide (NO) is a signaling molecule that affects a myriad of processes in plants. However, the mechanistic details are limited. NO post-translationally modifies proteins by S-nitrosylation of cysteines. The soluble S-nitrosoproteome of a medicinal, crassulacean acid metabolism (CAM) plant, Kalanchoe pinnata, was purified using the biotin switch technique. Nineteen targets were identified by MALDI-TOF mass spectrometry, including proteins associated with carbon, nitrogen and sulfur metabolism, the cytoskeleton, stress and photosynthesis. Some were similar to those previously identified in Arabidopsis thaliana, but kinesin-like protein, glycolate oxidase, putative UDP glucose 4-epimerase and putative DNA topoisomerase II had not been identified as targets previously for any organism. In vitro and in vivo nitrosylation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), one of the targets, was confirmed by immunoblotting. Rubisco plays a central role in photosynthesis, and the effect of S-nitrosylation on its enzymatic activity was determined using NaH14CO3. The NO-releasing compound S-nitrosoglutathione inhibited its activity in a dose-dependent manner suggesting Rubisco inactivation by nitrosylation for the first time.