A novel posttranslational modification of histone, H3 S-sulfhydration, is down-regulated in asthenozoospermic sperm

Oxidative stress is one of the major causes leading to male infertility including asthenozoospermia. Hydrogen sulfide (H2S) has been widely recognized to be a potent antioxidant whose role is partially implemented by protein S-sulfhydration. However, protein S-sulfhydration has not been reported in germ cells. Therefore, we investigated whether asthenozoospermia could be associated with sperm protein S-sulfhydration. S-sulfhydrated proteins in human sperm were enriched via biotin-switch assay and analyzed using LC-MS/MS spectrometry. Two hundred forty-four S-sulfhydrated proteins were identified. Importantly, we validated that sperm histones H3.1 and H3.3 were the S-sulfhydrated proteins. Their S-sulfhydrated amino acid residue was Cysteine111. Abundances of S-sulfhydrated H3 (sH3) and S-sulfhydrated H3.3 (sH3.3) were significantly down-regulated in asthenozoospermic sperm, compared with the fertile controls, and were significantly correlated with progressive motility. Retinoic acid (RA) up-regulated level of sH3.3 in primary round spermatids and the C18-4 cells (a mouse spermatogonial stem cell line). Overexpression of the mutant H3.3 (Cysteine111 was replaced with serine) affected expression of 759 genes and raised growth rate of C18-4 cells. For the first time, S-sulfhydration H3 and H3.3 were demonstrated in the present study. Our results highlight that aberrant S-sulfhydration of H3 is a new pathophysiological basis in male infertility.

eNOS-dependent S-nitrosylation of the NF-κB subunit p65 has neuroprotective effects

Cell death by glutamate excitotoxicity, mediated by N-methyl-d-aspartate (NMDA) receptors, negatively impacts brain function, including but not limited to hippocampal neurons. The NF-κB transcription factor (composed mainly of p65/p50 subunits) contributes to neuronal death in excitotoxicity, while its inhibition should improve cell survival. Using the biotin switch method, subcellular fractionation, immunofluorescence, and luciferase reporter assays, we found that NMDA-stimulated NF-κB activity selectively in hippocampal neurons, while endothelial nitric oxide synthase (eNOS), an enzyme expressed in neurons, is involved in the S-nitrosylation of p65 and consequent NF-κB inhibition in cerebrocortical, i.e., resistant neurons. The S-nitro proteomes of cortical and hippocampal neurons revealed that different biological processes are regulated by S-nitrosylation in susceptible and resistant neurons, bringing to light that protein S-nitrosylation is a ubiquitous post-translational modification, able to influence a variety of biological processes including the homeostatic inhibition of the NF-κB transcriptional activity in cortical neurons exposed to NMDA receptor overstimulation.

S-nitrosylation of CYR61 protein reduces triple-negative breast cancer metastasis ability

BackgroundTriple-negative breast cancer (TNBC) is the most difficult cancer to be treated. TNBC expresses high level of matricellular cysteine-rich protein CYR61/CCN1 that plays a key role in producing cancer metastases and is an important target for metastasis chemoprevention. Nitric oxide (NO) can covalently bind to the thiol group of cysteines (termed S-nitrosylation) resulting in regulation of the targeted protein functions. MethodsProtein S-nitrosylation were detected by biotin-switch assay and western blotting assay. CYR61 protein S-nitrosylated sites and 3D structure were determined by mass spectrometry and MODELLER software. Adhesion assay, cell morphology assay, wound healing assay and transwell invasion assay were used to evaluate effects of CYR61 S-nitrosylation on the cell metastatic ability. In vivo metastasis activity of CYR61 S-nitrosylation were tested by intravenous injection and mammary xenograft implantation mouse metastatic models.ResultsS-nitrosylation by GSNO of CYR61 reached a plateau quickly and was confirmed by spectroscopic analysis and biotin-switch assay. Mass-spectrometry proteomic analysis revealed that S-nitrosylation predominantly occurred at Cys100, Cys117, Cys229 and Cys239, resulting in CYR61 structure relaxed and unstable evidenced by protein structure modeling. S-nitrosylation of MDA-MB-231 cells, their CYR61-overexpressed and CYR61–silenced counterparts significantly attenuated the metastatic ability of these cells, including their ability of adhesion, mobility, invasion, and interplay with platelets, and made the adhered cells unattached. The attenuation in metastatic ability proportionally increased with the degree of S-nitrosylation to CYR61 naturally-expressed or genetically-manipulated cells, and was demonstrated in mice, where, S-nitrosylation of these cell lines not only inhibited their acute seeding to lungs after an intravenous injection, but also inhibited the late development of these cells into the metastatic nodes after mammary xenograft implantation. Furthermore, orthotopically-implanted MDA-MB-231 developed mammary tumors and later lung metastasis; whereas, the same cells with S-nitrosylation developed no tumor and metastasis at all. Conclusionwe present the first evidence that S-nitrosylation of CYR61 can significantly inhibit metastatic aggressiveness of the TNBC MDA-MB-231 cells. This conceptual creative study opens a new avenue to prevent the most aggressive TNBC from metastases by S-nitrosylation to CYR61.

Hydrogen sulphide reduces the activity of human endothelial cells

INTRODUCTION: The volatile endogenous mediator hydrogen sulfide (H2S) is known to impair thrombus formation by affecting the activity of human platelets. Beside platelets and coagulation factors the endothelium is crucial during thrombogenesis. OBJECTIVE: This study evaluates the effect of the H2S donor GYY4137 (GYY) on human umbilical vein endothelial cells (HUVECs) in vitro. METHODS: Flow cytometry of resting, stimulated or GYY-treated and subsequently stimulated HUVECs was performed to analyse the expression of E-selectin, ICAM-1 and VCAM-1. To study a potential reversibility of the GYY action, E-selectin expression was further assessed on HUVECs that were stimulated 24 h after GYY exposure. A WST-1 assay was performed to study toxic effects of the H2S donor. By using the biotin switch assay, protein S-sulfhydration of GYY-exposed HUVECs was assessed. Further on, the effects of GYY on HUVEC migration and von Willebrand factor (vWF) secretion were assessed. RESULTS: GYY treatment significantly reduced the expression of E-selectin and ICAM-1 but not of VCAM-1. When HUVECs were stimulated 24 h after GYY treatment, E-selectin expression was no longer affected. The WST-1 assay revealed no effects of GYY on endothelial cell viability. Furthermore, GYY impaired endothelial migration, reduced vWF secretion and increased protein S-sulfhydration. CONCLUSIONS: Summarizing, GYY dose dependently and reversibly reduces the activity of endothelial cells.

iNOS is not responsible for nitrosylation of RyR1 in mdx mice with truncated dystrophin

Background: Previous research indicated that nitric oxide synthase (NOS) is the key molecule for S-nitrosylation of ryanodine receptor 1 (RyR1) in DMD model mice (mdx mice) and that both neuronal NOS (nNOS) and inducible NOS (iNOS) might contribute to the reaction because nNOS is mislocalized in the cytoplasm and iNOS expression is higher in mdx mice. We investigated the effect of iNOS on RyR1 S-nitrosylation in mdx mice and whether transgenic expression of truncated dystrophin reduced iNOS expression in mdx mice or not.Methods: Three- to 4-month-old C57BL/6J, mdx, and transgenic mdx mice expressing exon 45-55-deleted human dystrophin (Tg/mdx mice) were used. We also generated two double mutant mice, mdx iNOS KO and Tg/mdx iNOS KO to reveal the iNOS contribution to RyR1 S-nitrosylation. nNOS and iNOS expression levels in skeletal muscle of these mice were assessed by immunohistochemistry (IHC), qRT-PCR, and Western blotting. Total NOS activity was measured by a citrulline assay. A biotin-switch method was used for detection of RyR1 S-nitrosylation. Statistical differences were assessed by one-way ANOVA with Tukey-Kramer post-hoc analysis.Results: mdx and mdx iNOS KO mice showed the same level of RyR1 S-nitrosylation. Total NOS activity was not changed in mdx iNOS KO mice compared with mdx mice. iNOS expression was undetectable in Tg/mdx mice expressing exon 45-55-deleted human dystrophin, but the level of RyR1 S-nitrosylation was the same in mdx and Tg/mdx mice.Conclusion: Similar levels of RyR1 S-nitrosylation and total NOS activity in mdx and mdx iNOS KO demonstrated that the proportion of iNOS in total NOS activity was low, even in mdx mice. Exon 45-55-deleted dystrophin reduced the expression level of iNOS, but it did not correct the RyR1 S-nitrosylation. These results indicate that iNOS was not involved in RyR1 S-nitrosylation in mdx and Tg/mdx mice muscles.

Arecoline Increases the Production of Nitric Oxide and Post-Translational S-Nitrosoproteome in Endothelial Cells

Background: Arecoline is known as a carcinogenic toxicant. The refreshment effect of arecoline is mainly due to the increase in vasodilation and blood flow. This is essential to understand whether arecoline can induce the production of Nitric Oxide (NO•) and regulate the subsequent protein S-nitrosylation in Endothelial Cells (ECs). Objective: The present study is focused on the promotion effect of arecoline in NO• production and the subsequent regulation of S-nitrosoproteome. Method: The phosphorylation of endothelial nitric oxide synthase serine 1177 residue (peNOSSer1177) was investigated by western blot. By using a specific FA-OMe fluorescent probe, the NO• molecules could be observed by fluorescent microscopy or flow cytometry. S-nitrosylated proteins were purified by biotin switch and then subjected to the Isobaric Tag for Relative and Absolute Quantitation (iTRAQ)-labeled shotgun proteomic analysis. Results: Our study reveals that a lower concentration of arecoline can increase the phosphorylation of peNOSSer1177. Pretreatment of NG-nitro-L-arginine methyl ester (L-NAME) indicated that arecolineinduced NO• production was mediated by e-NOS. We identified 224 proteins with up-regulated S-nitrosylation and 159 proteins with down-regulated S-nitrosylation. The NO• binding sites of seven representative S-nitrosoproteins were illustrated. The effect of arecoline on the S-nitrosylation of HSP60 chaperonin and calnexin was verified. Conclusion: Our experimental results proved that a lower concentration of arecoline could modulate the production of NO• and the subsequent protein S-nitrosylation. Therefore, it is worthy for further investigation and discussion if these S-nitrosoproteomes are important in maintaining endothelium homeostasis.

iNOS is not responsible for RyR1 S-nitrosylation in mdx mice with truncated dystrophin.

Background: Previous research indicated that nitric oxide synthase (NOS) is the key molecule for S-nitrosylation of ryanodine receptor 1 (RyR1) in DMD model mice ( mdx mice) and that both neuronal NOS (nNOS) and inducible NOS (iNOS) might contribute to the reaction because nNOS is mislocalized in the cytoplasm and iNOS expression is higher in mdx mice. We investigated the effect of iNOS on RyR1 S-nitrosylation in mdx mice and whether transgenic expression of truncated dystrophin reduced iNOS expression in mdx mice or not. Methods: Three- to 4-month-old C57BL/6J, mdx , and transgenic mdx mice expressing exon 45-55-deleted human dystrophin (Tg/ mdx mice) were used. We also generated two double mutant mice, mdx iNOS KO and Tg/ mdx iNOS KO to reveal the iNOS contribution to RyR1 S-nitrosylation. nNOS and iNOS expression levels in skeletal muscle of these mice were assessed by immunohistochemistry (IHC), qRT-PCR, and Western blotting. Total NOS activity was measured by a citrulline assay. A biotin-switch method was used for detection of RyR1 S-nitrosylation. Statistical differences were assessed by one-way ANOVA with Tukey-Kramer post-hoc analysis. Results: mdx and mdx iNOS KO mice showed the same level of RyR1 S-nitrosylation. Total NOS activity was not changed in mdx iNOS KO mice compared with mdx mice. iNOS expression was undetectable in Tg/ mdx mice expressing exon 45-55-deleted human dystrophin, but the level of RyR1 S-nitrosylation was the same in mdx and Tg/ mdx mice. Conclusion: Similar levels of RyR1 S-nitrosylation and total NOS activity in mdx and mdx iNOS KO demonstrated that the proportion of iNOS in total NOS activity was low, even in mdx mice. Exon 45-55-deleted dystrophin reduced the expression level of iNOS, but it did not correct the RyR1 S-nitrosylation. These results indicate that iNOS was not involved in RyR1 S-nitrosylation in mdx and Tg /mdx muscles.

eNOS-dependent S-nitrosylation of the NF-κB subunit p65 has neuroprotective effects

Cell death by glutamate excitotoxicity, mediated by N-methyl-D-aspartate (NMDA) receptors, negatively impacts brain function, including but not limited to hippocampal neurons. The NF-κB transcription factor (composed mainly of p65/p50 subunits) contributes to neuronal death in excitotoxicity, while its inhibition should improve cell survival. Using the biotin switch method, subcellular fractionation, immunofluorescence and luciferase reporter assays, we found that NMDA stimulated NF-κB activity selectively in hippocampal neurons, while endothelial nitric oxide synthase (eNOS), an enzyme expressed in neurons, is involved in the S-nitrosylation of p65 and consequent NF-κB inhibition in cerebrocortical, i.e., resistant neurons. The S-nitro proteomes of cortical and hippocampal neurons revealed that different biological processes are regulated by S-nitrosylation in susceptible and resistant neurons, bringing to light that protein S-nitrosylation is a ubiquitous post-translational modification, able to influence a variety of biological processes including the homeostatic inhibition of the NF-κB transcriptional activity in cortical neurons exposed to NMDA receptor overstimulation.

Proteomic Investigation of S-Nitrosylated Proteins During NO-Induced Adventitious Rooting of Cucumber

Nitric oxide (NO) acts an essential signaling molecule that is involved in regulating various physiological and biochemical processes in plants. However, whether S-nitrosylation is a crucial molecular mechanism of NO is still largely unknown. In this study, 50 μM S-nitrosoglutathione (GSNO) treatment was found to have a maximum biological effect on promoting adventitious rooting in cucumber. Meanwhile, removal of endogenous NO significantly inhibited the development of adventitious roots implying that NO is responsible for promoting the process of adventitious rooting. Moreover, application of GSNO resulted in an increase of intracellular S-nitrosothiol (SNO) levels and endogenous NO production, while decreasing the S-nitrosoglutathione reductase (GSNOR) activity during adventitious rooting, implicating that S-nitrosylation might be involved in NO-induced adventitious rooting in cucumber. Furthermore, the identification of S-nitrosylated proteins was performed utilizing the liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) and biotin-switch technique during the development of adventitious rooting. Among these proteins, the activities and S-nitrosylated level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), tubulin alpha chain (TUA), and glutathione reductase (GR) were further analyzed as NO direct targets. Our results indicated that NO might enhance the S-nitrosylation level of GAPDH and GR, and was found to subsequently reduce these activities and transcriptional levels. Conversely, S-nitrosylation of TUA increased the expression level of TUA. The results implied that S-nitrosylation of key proteins seems to regulate various pathways through differential S-nitrosylation during adventitious rooting. Collectively, these results suggest that S-nitrosylation could be involved in NO-induced adventitious rooting, and they also provide fundamental evidence for the molecular mechanism of NO signaling during adventitious rooting in cucumber explants.