Mechanisms Underlying the Hydrogen Sulfide Actions: Target Molecules and Downstream Signaling Pathways.

Significance: As a new important gas signaling molecule like nitric oxide (NO) and carbon dioxide (CO), hydrogen sulfide (H2S), which can be produced by endogenous H2S-producing enzymes through l-cysteine metabolism in mammalian cells, has attracted wide attention for long. H2S has been proved to play an important regulatory role in numerous physiological and pathophysiological processes. However, the deep mechanisms of those different functions of H2S still remain uncertain. A better understanding of the mechanisms can help us develop novel therapeutic strategies. Recent Advances: H2S can play a regulating role through various mechanisms, such as regulating epigenetic modification, protein expression levels, protein activity, protein localization, redox microenvironment, and interaction with other gas signaling molecules such as NO and CO. In addition to discussing the molecular mechanisms of H2S from the above perspectives, this article will review the regulation of H2S on common signaling pathways in the cells, including the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt), mitogen-activated protein kinase (MAPK), Janus kinase (JAK)/signal transducer, and activator of transcription (STAT) signaling pathway. Critical Issues: Although there are many studies on the mechanism of H2S, little is known about its direct target molecules. This article will also review the existing reports about them. Furthermore, the interaction between direct target molecules of H2S and the downstream signaling pathways involved also needs to be clarified. Future Directions: An in-depth discussion of the mechanism of H2S and the direct target molecules will help us achieving a deeper understanding of the physiological and pathophysiological processes regulated by H2S, and lay a foundation for developing new clinical therapeutic drugs in the future. Innovation: This review focuses on the regulation of H2S on signaling pathways and the direct target molecules of H2S. We also provide details on the underlying mechanisms of H2S functions from the following aspects: epigenetic modification, regulation of protein expression levels, protein activity, protein localization, redox microenvironment, and interaction with other gas signaling molecules such as NO and CO. Further study of the mechanisms underlying H2S will help us better understand the physiological and pathophysiological processes it regulates, and help develop new clinical therapeutic drugs in the future. Antioxid. Redox Signal. 40, 86-109.

Cascade signal amplification strategy by coupling chemical redox-cycling and Fenton-like reaction: Toward an ultrasensitive split-type fluorescent immunoassay.

An ultrasensitive split-type fluorescent immunobiosensor has been reported based on a cascade signal amplification strategy by coupling chemical redox-cycling and Fenton-like reaction. In this strategy, Cu2+ could oxidize chemically o-phenylenediamine (OPD) to generate photosensitive 2, 3-diaminophenazine (DAP) and Cu+/Cu0. On one hand, the generated Cu0 in turn catalyzed the oxidation of OPD. On the other hand, the introduced H2O2 reacted with Cu + ion to produce hydroxyl radicals (·OH) and Cu2+ ion through a Cu + -mediated Fenton-like reaction. The produced ·OH and recycled Cu2+ ion could take turns oxidizing OPD to generate more photoactive DAP, which triggering a self-sustaining chemical redox-cycling reaction and leading to a remarkable fluorescent improvement. It was worth mentioning that the cascade reaction did not stop until OPD molecules were completely consumed. Based on the H2O2-triggered cascade signal amplification, the strategy was exploited for the construction of split-type fluorescent immunoassay by taking interleukin-6 (IL-6) as the model target. It was realized for the ultrasensitive determination of IL-6 in a linear ranging from 20 fg/mL to 10 pg/mL with a limit of detection of 5 fg/mL. The study validated the practicability of the cascade signal amplification on the fluorescent bioanalysis and the superior performance in fluorescent immunoassay. It is expected that the strategy would offer new opportunities to develop ultrasensitive fluorescent methods for biosensor and bioanalysis.

Glutathione-stabilized copper nanoclusters mediated-inner filter effect for sensitive and selective determination of p-nitrophenol and alkaline phosphatase activity.

A simple and highly selective fluorescence biosensor has been exploited for p-nitrophenol (p-NP) and alkaline phosphatase (ALP) activity detection based on the glutathione-stabilized copper nanoclusters (GSH-CuNCs) mediated-inner filter effect (IFE). The GSH-CuNCs were prepared by employing GSH as stabilizer and ascorbic acid (AA) as reductant. The obtained GSH-CuNCs exhibited a strong blue fluorescence emission at 420 nm with an excitation wavelength of 365 nm, which overlapped largely with the absorption spectra of p-nitrophenol (p-NP). Therefore, the luminescence of GSH-CuNCs could be quenched by p-NP through inner filter effect. In addition, ALP catalyzed the substrate p-nitrophenyl phosphate (p-NPP) to form p-nitrophenol (p-NP), which also leading to the fluorescence quenching of GSH-CuNCs. The fluorescent strategy was realized for the sensitive determination of p-NP and ALP activity with the promising limit of detection of 20 nM (for p-NP) and 0.003 mU⋅mL-1 (for ALP). Furthermore, the method could be applied to detect the p-NP content in river water samples and ALP activity in human serum samples.

YB-1 Recruits Drosha to Promote Splicing of pri-miR-192 to Mediate the Proangiogenic Effects of H2S.

Aims: The genes targeted by miRNAs have been well studied. However, little is known about the feedback mechanisms to control the biosynthesis of miRNAs that are essential for the miRNA feedback networks in the cells. In this present study, we aimed at examining how hydrogen sulfide (H2S) promotes angiogenesis by regulating miR-192 biosynthesis. Results: H2S promoted in vitro angiogenesis and angiogenesis in Matrigel plugs embedded in mice by upregulating miR-192. Knockdown of the H2S-generating enzyme cystathionine γ-lyase (CSE) suppressed in vitro angiogenesis, and this suppression was rescued by exogenous H2S donor NaHS. Plakophilin 4 (PKP4) served as a target gene of miR-192. H2S up-regulated miR-192 via the VEGFR2/Akt pathway to promote the splicing of primary miR-192 (pri-miR-192), and it resulted in an increase in both the precursor- and mature forms of miR-192. H2S translocated YB-1 into the nuclei to recruit Drosha to bind with pri-miR-192 and promoted its splicing. NaHS treatment promoted angiogenesis in the hindlimb ischemia mouse model and the skin-wound-healing model in diabetic mice, with upregulated miR-192 and downregulated PKP4 on NaHS treatment. In human atherosclerotic plaques, miR-192 levels were positively correlated with the plasma H2S concentrations. Innovation and Conclusion: Our data reveal a role of YB-1 in recruiting Drosha to splice pri-miR-192 to mediate the proangiogenic effect of H2S. CSE/H2S/YB-1/Drosha/miR-192 is a potential therapeutic target pathway for treating diseases, including organ ischemia and diabetic complications. Antioxid. Redox Signal. 36, 760-783. The Clinical Trial Registration number is 2016-224.

A Common Molecular Switch for H2S to Regulate Multiple Protein Targets.

Hydrogen sulfide, a small molecule, produced by endogenous enzymes, such as CTH, CBS, and MPST using L-cysteine as substrates, has been reported to have numerous protective effects. However, the key problem that the target of H2S and how it can affect the structure and activity of biological molecules is still unknown. Till now, there are two main theories of its working mechanism. One is that H2S can modify the free thiol in cysteine to produce the persulfide state of the thiol and the sulfhydration of cysteine can significantly change the structure and activity of target proteins. The other theory is that H2S, as an antioxidant molecule, can directly break the disulfide bond in target proteins, and the persulfide state of thiol can be an intermediate product during the reaction. Both phenomena exit for no doubt since they are both supported by large amounts of experiments. Here, we will summarize both theories and try to discuss which one is the more effective or direct mechanism for H2S and what is the relationship between them. Therefore, we will discover more protein targets of H2S with the mechanism and understand more about the effect of this small molecule.

Hydrogen sulfide promotes angiogenesis by downregulating miR-640 via the VEGFR2/mTOR pathway.

We previously found hydrogen sulfide (H2S) to be a new proangiogenic factor. However, the mechanisms underlying the cardiovascular effect of this small gas molecule remain largely unknown. The aim of the present study was to identify the essential microRNAs (miRNAs) involved in the transduction of H2S signals in vascular endothelial cells (ECs). The expression of miR-640 and its signaling elements, vascular endothelial growth factor receptor 2 (VEGFR2), hypoxia inducible factor 1-α (HIF1A), and mammalian target of rapamycin (mTOR), was measured using quantitative PCR and Western blotting. Overexpression and inhibition of miR-640 were performed to clarify their roles in mediating the effect of H2S. In addition, knockdown of VEGFR2, HIF1A, and mTOR was performed using siRNAs, dominant negative mutants, or inhibitors to examine their roles in the transduction of the H2S signals. miR-640 levels decreased in vascular ECs that were treated with H2S, whereas overexpression of miR-640 blunted the proangiogenic effect of H2S. Knockdown of either VEGFR2 or mTOR blunted the downregulation of miR-640 and the proangiogenic effect induced by H2S. In addition, miR-640 bound to the 3'-UTR of HIF1A mRNA and then inhibited the expression of HIF1A. The inhibition could be recovered by treating cells with H2S. Thus we concluded that miR-640 plays a pivotal role in mediating the proangiogenic effect of H2S; H2S acts through downregulation of the expression of miR-640 and increasing the levels of HIF1A through the VEGFR2-mTOR pathway.

Cardiac H2S Generation Is Reduced in Ageing Diabetic Mice.

Aims. To examine whether hydrogen sulfide (H2S) generation changed in ageing diabetic mouse hearts. Results. Compared to mice that were fed tap water only, mice that were fed 30% fructose solution for 15 months exhibited typical characteristics of a severe diabetic phenotype with cardiac hypertrophy, fibrosis, and dysfunction. H2S levels in plasma, heart tissues, and urine were significantly reduced in these mice as compared to those in controls. The expression of the H2S-generating enzymes, cystathionine γ-lyase and 3-mercaptopyruvate sulfurtransferase, was significantly decreased in the hearts of fructose-fed mice, whereas cystathionine-ß-synthase levels were significantly increased. Conclusion. Our results suggest that this ageing diabetic mouse model developed diabetic cardiomyopathy and that H2S levels were reduced in the diabetic heart due to alterations in three H2S-producing enzymes, which may be involved in the pathogenesis of diabetic cardiomyopathy.

Hydrogen Sulfide Targets the Cys320/Cys529 Motif in Kv4.2 to Inhibit the Ito Potassium Channels in Cardiomyocytes and Regularizes Fatal Arrhythmia in Myocardial Infarction.

AIMS: The mechanisms underlying numerous biological roles of hydrogen sulfide (H2S) remain largely unknown. We have previously reported an inhibitory role of H2S in the L-type calcium channels in cardiomyocytes. This prompts us to examine the mechanisms underlying the potential regulation of H2S on the ion channels. RESULTS: H2S showed a novel inhibitory effect on Ito potassium channels, and this effect was blocked by mutation at the Cys320 and/or Cys529 residues of the Kv4.2 subunit. H2S broke the disulfide bridge between a pair of oxidized cysteine residues; however, it did not modify single cysteine residues. H2S extended action potential duration in epicardial myocytes and regularized fatal arrhythmia in a rat model of myocardial infarction. H2S treatment significantly increased survival by ∼1.4-fold in the critical 2-h time window after myocardial infarction with a protection against ventricular premature beats and fatal arrhythmia. However, H2S did not change the function of other ion channels, including IK1 and INa. INNOVATION AND CONCLUSION: H2S targets the Cys320/Cys529 motif in Kv4.2 to regulate the Ito potassium channels. H2S also shows a potent regularizing effect against fatal arrhythmia in a rat model of myocardial infarction. The study provides the first piece of evidence for the role of H2S in regulating Ito potassium channels and also the specific motif in an ion channel labile for H2S regulation.

VEGFR2 functions as an H2S-targeting receptor protein kinase with its novel Cys1045-Cys1024 disulfide bond serving as a specific molecular switch for hydrogen sulfide actions in vascular endothelial cells.

AIMS: The potential receptor for hydrogen sulfide (H2S) remains unknown. RESULTS: H2S could directly activate vascular endothelial growth factor receptor 2 (VEGFR2) and that a small interfering RNA (siRNA)-mediated knockdown of VEGFR2 inhibited H2S-induced migration of human vascular endothelial cells. H2S promoted angiogenesis in Matrigel plug assay in mice and this effect was attenuated by a VEGF receptor inhibitor. Using tandem mass spectrometry (MS), we identified a new disulfide complex located between Cys1045 and Cys1024 within VEGFR2 that was labile to H2S-mediated modification. Kinase activity of the mutant VEGFR2 (C1045A) devoid of the Cys1045-Cys1024 disulfide bond was significantly higher than wild-type VEGFR2. Transfection with vectors expressing VEGFR2 (C1045A) caused a significant increase in cell migration, while the migration-promoting effect of H2S disappeared in the cells transfected with VEGFR2 (C1045A). Therefore, the Cys1045-Cys1024 disulfide bond serves as an intrinsic inhibitory motif and functions as a molecular switch for H2S. The formation of the Cys1045-Cys1024 disulfide bond disrupted the integrity of the active conformation of VEGFR2. Breaking the Cys1045-Cys1024 disulfide bond recovered the active conformation of VEGFR2. This motif was prone to a nucleophilic attack by H2S via an interaction of their frontier molecular orbitals. siRNA-mediated knockdown of cystathionine γ-lyase attenuated migration of vascular endothelial cells induced by VEGF or moderate hypoxia. INNOVATION AND CONCLUSION: The study provides the first piece of evidence of a molecular switch in H2S-targeting receptor protein kinase in H2S-induced angiogenesis and that may be applicable to additional kinases containing functionally important disulfide bonds in mediating various H2S actions.

Amelioration of glomerulosclerosis with all-trans retinoic acid is linked to decreased plasminogen activator inhibitor-1 and α-smooth muscle actin.

AIM: To examine the effects of all-trans retinoic acid (atRA) on renal morphology and function as well as on renal plasminogen activator inhibitor-1 (PAI-1) expression and plasmin activity in rats with 5/6 nephrectomy. METHODS: Adult male Sprague Dawley rats were given 5/6 nephrectomy or sham operation. Renal function was measured 2 weeks later. The nephrectomized rats were assigned to groups matched for proteinuria and treated with vehicle or atRA (5 or 10 mg/kg by gastric gavage once daily) for the next 12 weeks. Rats with sham operation were treated with vehicle. At the end of the treatments, kidneys were collected for histological examination, Western blot analysis, and enzymatic activity measurements. RESULTS: The 5/6 nephrectomy promoted hypertension, renal dysfunction, and glomerulosclerosis. These changes were significantly reduced in the atRA-treated group. The expressions of PAI-1 and α-smooth muscle actin (α-SMA) were significantly increased in the vehicle-treated nephrectomized rats. Treatment with atRA significantly reduced the expressions of PAI-1 and α-SMA. However, plasmin activity remained unchanged following atRA treatment. CONCLUSION: Treatment with atRA ameliorates glomerulosclerosis and improves renal function in rats with 5/6 nephrectomy. This is associated with a decrease in PAI-1 and α-SMA, but not with a change in plasmin activity.

Survivin is involved in the anti-apoptotic effect of edaravone in PC12 cells.

Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a newly developed hydroxy radical scavaging agent which has been widely used for protection against ischemia-reperfusion injury is highly effective in preventing cell apoptosis. However, the exact intracellular mechanism(s) underlying the protective action of edaravone is not clear. We observed that in PC12 cells cultured under serum deprivation (DEPV) condition, the levels of survivin were positively correlated with the anti-apoptotic action of edaravone. Survivin RNA interference (RNAi) increased DEPV-induced PC12 cell apoptosis, whereas the anti-apoptotic effect of edaravone was blunted by survivin RNAi. Moreover, survivin overexpression provided protection against DEPV-induced PC12 cell apoptosis. Inhibition of ERK and PI(3)-K/AKT prevented edaravone's ability to decrease apoptosis and increase survivin. In conclusion, the present study provides the first direct evidence that survivin involves in the anti-apoptotic effects of edaravone via a pathway involving ERK and PI(3)-K/AKT.

Protein phosphatase activity of PTEN inhibited the invasion of glioma cells with epidermal growth factor receptor mutation type III expression.

PTEN is a major tumor suppressor gene that has been shown to inhibit cell invasion. Its mutation has been found in 20-40% of malignant gliomas. Meanwhile, the type III EGFR mutation (EGFRvIII), which was frequently found in gliomas, promoted cell invasion. In the present study, the effects of PTEN on cell invasion were investigated in U87DeltaEGFR glioblastoma cells with EGFRvIII expression but missing PTEN. The cell invasion was downregulated by transfection of phosphatase-active forms of PTEN (wild-type and G129E) but not by PTEN (C124A) with an inactive phosphatase domain; the effects were correlated with decreased tyrosine phosphatase levels of FAK at Tyr397, which was increased by EGFRvIII. Overexpression of FAK mutant (Y397F) could partially mimic the effect of PTEN on cell invasion. Although EGFRvIII increased the levels of P-Akt and PTEN eliminated it, PI-3K inhibitors, wortmannin or Ly294002, could not decrease the cell invasion. In conclusion, PTEN could inhibit cell invasion even in the presence of the constitutively active EGFR; this inhibition depended on its protein phosphatase activity, partially by dephosphorylating FAK, but not depended on its lipid phosphatase activity.