Showing papers on "Nitrosylation published in 2023"

S-nitrosylation of the histone deacetylase HDA19 stimulates its activity to enhance plant stress tolerance in Arabidopsis.

Abstract: Arabidopsis histone deacetylase HDA19 is required for gene expression programs of a large spectrum of plant developmental and stress-responsive pathways. How this enzyme senses cellular environment to control its activity remains unclear. In this work, we show that HDA19 is post-translationally modified by S-nitrosylation at 4 Cysteine (Cys) residues. HDA19 S-nitrosylation depends on the cellular nitric oxide (NO) level which is enhanced under oxidative stress. We find that HDA19 is required for cellular redox homeostasis and plant tolerance to oxidative stress which in turn stimulates its nuclear enrichment, S-nitrosylation and epigenetic functions including binding to genomic targets, histone deacetylation, and gene repression. The Cys137 of the protein is involved in basal and stress-induced S-nitrosylation and is required for HDA19 functions in developmental, stress-responsive, and epigenetic controls. Together, these results indicate that S-nitrosylation regulates HDA19 activity and is a mechanism of redox-sensing for chromatin regulation of plant tolerance to stress.

Supplementary Figure 5 from Inducible Nitric Oxide Synthase Drives mTOR Pathway Activation and Proliferation of Human Melanoma by Reversible Nitrosylation of TSC2

Abstract: <p>PDF file - 135K, A375 melanoma cells were exposed for 6 hours or 24 Hours to ODQ (3 uM, 10 uM), or the vehicle control dimethylsulfoxide (DMSO). mTOR pathway activation was analyzed by 4EBP1 and p-S6RP western blot. Signals were quantified by densitometry, and the ratio of activated p-4EBP1 and p-S6RP was calculated, normalized against total protein (lower panel). ODQ even at extended time point (24 hours) and high dose (10 uM) had little effect on mTOR pathway activation.</p>

Transcription tuned by S-nitrosylation underlies a mechanism for Staphylococcus aureus to circumvent vancomycin killing

Abstract: Treatment of Staphylococcus aureus infections is a constant challenge due to emerging resistance to vancomycin, a last-resort drug. S-nitrosylation, the covalent attachment of a nitric oxide (NO) group to a cysteine thiol, mediates redox-based signaling for eukaryotic cellular functions. However, its role in bacteria is largely unknown. Here, proteomic analysis revealed that S-nitrosylation is a prominent growth feature of vancomycin-intermediate S. aureus. Deletion of NO synthase (NOS) or removal of S-nitrosylation from the redox-sensitive regulator MgrA or WalR resulted in thinner cell walls and increased vancomycin susceptibility, which was due to attenuated promoter binding and released repression of genes involved in cell wall metabolism. These genes failed to respond to H2O2-induced oxidation, suggesting distinct transcriptional responses to alternative modifications of the cysteine residue. Furthermore, treatment with a NOS inhibitor significantly decreased vancomycin resistance in S. aureus. This study reveals that transcriptional regulation via S-nitrosylation underlies a mechanism for NO-mediated bacterial antibiotic resistance.

NO and Heme Proteins: Cross-Talk between Heme and Cysteine Residues

Abstract: Heme proteins are a diverse group that includes several unrelated families. Their biological function is mainly associated with the reactivity of the heme group, which—among several other reactions—can bind to and react with nitric oxide (NO) and other nitrogen compounds for their production, scavenging, and transport. The S-nitrosylation of cysteine residues, which also results from the reaction with NO and other nitrogen compounds, is a post-translational modification regulating protein activity, with direct effects on a variety of signaling pathways. Heme proteins are unique in exhibiting this dual reactivity toward NO, with reported examples of cross-reactivity between the heme and cysteine residues within the same protein. In this work, we review the literature on this interplay, with particular emphasis on heme proteins in which heme-dependent nitrosylation has been reported and those for which both heme nitrosylation and S-nitrosylation have been associated with biological functions.

S-nitrosylation and S-glutathionylation of GAPDH: Similarities, differences, and relationships.

Abstract: The aim of this work was to compare the effect of reversible post-translational modifications, S-nitrosylation and S-glutathionylation, on the properties of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and to reveal the mechanism of the relationship between these modifications. Comparison of S-nitrosylated and S-glutathionylated GAPDH showed that both modifications inactivate the enzyme and change its spatial structure, decreasing the thermal stability of the protein and increasing its sensitivity to trypsin cleavage. Both modifications are reversible in the presence of dithiothreitol, however, in the presence of reduced glutathione and glutaredoxin 1, the reactivation of S-glutathionylated GAPDH is much slower (10% in 2 h) compared to S-nitrosylated GAPDH (60% in 10 min). This suggests that S-glutathionylation is a much less reversible modification compared to S-nitrosylation. Incubation of HEK 293 T cells in the presence of H2O2 or with the NO donor diethylamine NONOate results in accumulation of sulfenated GAPDH (by data of Western blotting) and S-glutathionylated GAPDH (by data of immunoprecipitation with anti-GSH antibodies). Besides GAPDH, a protein of 45 kDa was found to be sulfenated and S-glutathionylated in the cells treated with H2O2 or NO. This protein was identified as beta-actin. The results of this study confirm the previously proposed hypothesis based on in vitro investigations, according to which S-nitrosylation of the catalytic cysteine residue (Cys152) of GAPDH with subsequent formation of cysteine sulfenic acid at Cys152 may promote its S-glutathionylation in the presence of cellular GSH. Presumably, the mechanism may be valid in the case of beta-actin.

Comparison of the Nitric Oxide Synthase Interactomes and S-Nitroso-Proteomes: Furthering the case for enzymatic S-nitrosylation.

Abstract: AIMS S-nitrosylation of proteins is the main mechanism through which nitric oxide (NO) regulates cellular function and likely represents the archetype redox-based signaling system across aerobic and anaerobic organisms. How NO generated by different NOS isoforms leads to specificity of S-nitrosylation remains incompletely understood. This study aimed to identify proteins interacting with, and whose S-nitrosylation is mediated by, human NOS isoforms in the same cellular system, thereby illuminating the contribution of individual NOSs to specificity. RESULTS Of the hundreds of proteins interacting with each NOS, many were also S-nitrosylated. However, a large proportion of S-nitrosylated (SNO-) proteins did not associate with NOS. Moreover, most NOS interactors and SNO-proteins were unique to each isoform. The amount of NO produced by each NOS isoform was unrelated to numbers of S-nitrosylated proteins. Thus, NOSs promoted S-nitrosylation of largely distinct sets of target proteins. Different signaling pathways were enriched downstream of each NOS. INNOVATION AND CONCLUSION The interactomes and SNOomes of individual NOS isoforms were largely distinct. Only a small fraction of SNO-proteins interacted with their respective NOS. Amounts of S-nitrosylation were unrelated to amount of NO generated by NOSs. These data argue against free diffusion of NO or NOS interactions as being necessary or sufficient for S-nitrosylation and favor roles for additional enzymes and/or regulatory elements in imparting SNO-site specificity.

S-Nitrosylation of Tissue Transglutaminase in Modulating Glycolysis, Oxidative Stress, and Inflammatory Responses in Normal and Indoxyl-Sulfate-Induced Endothelial Cells

Abstract: Circulating uremic toxin indoxyl sulfate (IS), endothelial cell (EC) dysfunction, and decreased nitric oxide (NO) bioavailability are found in chronic kidney disease patients. NO nitrosylates/denitrosylates a specific protein’s cysteine residue(s), forming S-nitrosothios (SNOs), and the decreased NO bioavailability could interfere with NO-mediated signaling events. We were interested in investigating the underlying mechanism(s) of the reduced NO and how it would regulate the S-nitrosylation of tissue transglutaminase (TG2) and its substrates on glycolytic, redox and inflammatory responses in normal and IS-induced EC injury. TG2, a therapeutic target for fibrosis, has a Ca2+-dependent transamidase (TGase) that is modulated by S-nitrosylation. We found IS increased oxidative stress, reduced NADPH and GSH levels, and uncoupled eNOS to generate NO. Immunoblot analysis demonstrated the upregulation of an angiotensin-converting enzyme (ACE) and significant downregulation of the beneficial ACE2 isoform that could contribute to oxidative stress in IS-induced injury. An in situ TGase assay demonstrated IS-activated TG2/TGase aminylated eNOS, NFkB, IkBα, PKM2, G6PD, GAPDH, and fibronectin (FN), leading to caspases activation. Except for FN, TGase substrates were all differentially S-nitrosylated either with or without IS but were denitrosylated in the presence of a specific, irreversible TG2/TGase inhibitor ZDON, suggesting ZDON-bound TG2 was not effectively transnitrosylating to TG2/TGase substrates. The data suggest novel roles of TG2 in the aminylation of its substrates and could also potentially function as a Cys-to-Cys S-nitrosylase to exert NO’s bioactivity to its substrates and modulate glycolysis, redox, and inflammation in normal and IS-induced EC injury.

Data from &lt;i&gt;S&lt;/i&gt;-nitrosylation of the Mitochondrial Chaperone TRAP1 Sensitizes Hepatocellular Carcinoma Cells to Inhibitors of Succinate Dehydrogenase

Abstract: <div>Abstract<p><i>S</i>-nitrosoglutathione reductase (GSNOR) represents the best-documented denitrosylase implicated in regulating the levels of proteins posttranslationally modified by nitric oxide on cysteine residues by <i>S</i>-nitrosylation. GSNOR controls a diverse array of physiologic functions, including cellular growth and differentiation, inflammation, and metabolism. Chromosomal deletion of GSNOR results in pathologic protein <i>S</i>-nitrosylation that is implicated in human hepatocellular carcinoma (HCC). Here we identify a metabolic hallmark of aberrant <i>S</i>-nitrosylation in HCC and exploit it for therapeutic gain. We find that hepatocyte GSNOR deficiency is characterized by mitochondrial alteration and by marked increases in succinate dehydrogenase (SDH) levels and activity. We find that this depends on the selective <i>S</i>-nitrosylation of Cys⁵⁰¹ in the mitochondrial chaperone TRAP1, which mediates its degradation. As a result, GSNOR-deficient cells and tumors are highly sensitive to SDH inhibition, namely to α-tocopheryl succinate, an SDH-targeting molecule that induced RIP1/PARP1-mediated necroptosis and inhibited tumor growth. Our work provides a specific molecular signature of aberrant <i>S</i>-nitrosylation in HCC, a novel molecular target in SDH, and a first-in-class therapy to treat the disease. <i>Cancer Res; 76(14); 4170–82. ©2016 AACR</i>.</p></div>

Supplementary Figure 4 from Inducible Nitric Oxide Synthase Drives mTOR Pathway Activation and Proliferation of Human Melanoma by Reversible Nitrosylation of TSC2

Abstract: <p>PDF file - 67K, A375 cells were treated with L-NIL (300 μM and 1000 μM) or the NO scavenger PTIO (300 μM) for 24 hours prior to harvest and preparation of protein lysates. Western Blot analyses of AKT phosphorylation at serine 473 and total AKT demonstrated lack of downregulation of AKT phosphorylation under conditions which strongly reduce downstream mTOR pathway activation.</p>

Supplementary Figure 4 from Inducible Nitric Oxide Synthase Drives mTOR Pathway Activation and Proliferation of Human Melanoma by Reversible Nitrosylation of TSC2

Abstract: <p>PDF file - 67K, A375 cells were treated with L-NIL (300 μM and 1000 μM) or the NO scavenger PTIO (300 μM) for 24 hours prior to harvest and preparation of protein lysates. Western Blot analyses of AKT phosphorylation at serine 473 and total AKT demonstrated lack of downregulation of AKT phosphorylation under conditions which strongly reduce downstream mTOR pathway activation.</p>