Protein persulfidation is a significant post-translational modification that involves addition of a sulfur atom to the cysteine thiol group and is facilitated by sulfide species. Persulfidation ...targets reactive cysteine residues within proteins, influencing their structure and/or function across various biological systems. This modification is evolutionarily conserved and plays a crucial role in preventing irreversible cysteine overoxidation, a process that becomes prominent with aging. While, persulfidation decreases with age, its levels in the aged heart and the functional implications of such a reduction in cardiac metabolism remain unknown. Here we interrogated the cardiac persulfydome in wild-type adult mice and age-matched mice lacking the two sulfide generating enzymes, namely cystathionine gamma lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST). Our findings revealed that cardiac persulfidated proteins in wild type hearts are less abundant compared to those in other organs, with a primary involvement in mitochondrial metabolic processes. We further focused on one specific target, NDUFB7, which undergoes persulfidation by both CSE and 3MST derived sulfide species. In particular, persulfidation of cysteines C80 and C90 in NDUFB7 protects the protein from overoxidation and maintains the complex I activity in cardiomyocytes. As the heart ages, the levels of CSE and 3MST in cardiomyocytes decline, leading to reduced NDUFB7 persulfidation and increased cardiac NADH/NAD+ ratio. Collectively, our data provide compelling evidence for a direct link between cardiac persulfidation and mitochondrial complex I activity, which is compromised in aging.
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•Cardiac proteins are differentially persulfidated by CSE and 3MST.•Endogenous cardiac sulfides modify mainly mitochondrial proteins.•Cardiac NDUFB7 persulfidation maintains complex I activity and preserves NAD + levels.•Reduced CSE and 3MST expression in aging results in impaired sulfide sensing and impacts on cardiac NAD + bioavailability.
3-mercaptopyruvate sulfurtransferase (3-MST) is an enzyme capable of synthesizing hydrogen sulfide (H
S) and polysulfides. In spite of its ubiquitous presence in mammalian cells, very few studies ...have investigated its contribution to homeostasis and disease development, thus the role of 3-MST remains largely unexplored. Here, we present a clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) induced
mutant zebrafish line, which will allow the study of 3-MST's role in several biological processes. The
zebrafish orthologue was identified using a bioinformatic approach and verified by its ability to produce H
S in the presence of 3-mercaptopyruvate (3-MP). Its expression pattern was analyzed during zebrafish early development, indicating predominantly an expression in the heart and central nervous system. As expected, no detectable levels of 3-Mst protein were observed in homozygous mutant larvae. In line with this, H
S levels were reduced in
zebrafish. Although the mutants showed no obvious morphological deficiencies, they exhibited increased lethality under oxidative stress conditions. The elevated levels of reactive oxygen species, detected following
deletion, are likely to drive this phenotype. In line with the increased ROS, we observed accelerated fin regenerative capacity in
deficient zebrafish. Overall, we provide evidence for the expression of
in zebrafish, confirm its important role in redox homeostasis and indicate the enzyme's possible involvement in the regeneration processes.
The molecular and cellular mechanisms underlying plaque destabilization remain obscure. We sought to elucidate the correlation between NO, H2S and CO-generating enzymes, nitro-oxidative stress and ...plaque stability in carotid arteries. Carotid atherosclerotic plaques were collected from 62 patients who had undergone endarterectomy due to internal artery stenosis. Following histological evaluation the plaques were divided into stable and unstable ones. To investigate the impact of simvastatin we divided patients with stable plaques, into those receiving and to those not receiving simvastatin. Expression and/or levels of p-eNOS/eNOS, pAkt/t-Akt, iNOS, cystathionine beta synthase (CBS), cystathionine gamma lyase (CSE), heme oxygenase-1(HO-1), soluble guanyl cyclase sGCα1, sGCβ1, NOX-4 and HIF-1α were evaluated. Oxidative stress biomarkers malondialdehyde (MDA) and nitrotyrosine (NT) were measured. NT levels were decreased in stable plaques with a concomitant increase of eNOS phosphorylation and expression and Akt activation compared to unstable lesions. An increase in HIF-1α, NOX-4, HO-1, iNOS, CBS and CSE expression was observed only in unstable plaques. 78% of patients under simvastatin were diagnosed with stable plaques whereas 23% of those not receiving simvastatin exhibited unstable plaques. Simvastatin decreased iNOS, HO-1, HIF-1α and CSE whilst it increased eNOS phosphorylation. In conclusion, enhanced eNOS and reduced iNOS and NOX-4 were observed in stable plaques; CBS and CSE positively correlated with plaque vulnerability. Simvastatin, besides its known effect on eNOS upregulation, reduced the HIF-1α and its downstream targets. The observed changes might be useful in developing biomarkers of plaque stability or could be targets for pharmacothepary against plaque vulnerability.
Colon cancer cells contain high levels of cystathionine-beta-synthase (CBS). Its product, hydrogen sulfide (H
S) promotes the growth and proliferation of colorectal tumor cells. In order to improve ...the antitumor efficacy of the prototypical CBS inhibitor aminooxyacetic acid (AOAA), we have designed and synthesized YD0171, a methyl ester derivative of AOAA. The antiproliferative effect of YD0171 exceeded the antiproliferative potency of AOAA in HCT116 human colon cancer cells. The esterase inhibitor paraoxon prevented the cellular inhibition of CBS activity by YD0171. YD0171 suppressed mitochondrial respiration and glycolytic function and induced G0/G1 arrest, but did not induce tumor cell apoptosis or necrosis. Metabolomic analysis in HCT116 cells showed that YD0171 affects multiple pathways of cell metabolism. The efficacy of YD0171 as an inhibitor of tumor growth was also tested in nude mice bearing subcutaneous HCT116 cancer cell xenografts. Animals were treated via subcutaneous injection of vehicle, AOAA (1, 3 or 9 mg/kg/day) or YD0171 (0.1, 0.5 or 1 mg/kg/day) for 3 weeks. Tumor growth was significantly reduced by 9 mg/kg/day AOAA, but not at the lower doses. YD0171 was more potent: tumor volume was significantly inhibited at 0.5 and 1 mg/kg/day. Thus, the
efficacy of YD0171 is 9-times higher than that of AOAA. YD0171 (1 mg/kg/day) attenuated tumor growth and metastasis formation in the intracecal HCT116 tumor model. YD0171 (3 mg/kg/day) also reduced tumor growth in patient-derived tumor xenograft (PDTX) bearing athymic mice. YD0171 (3 mg/kg/day) induced the regression of established HCT116 tumors
. A 5-day safety study in mice demonstrated that YD0171 at 20 mg/kg/day (given in two divided doses) does not increase plasma markers of organ injury, nor does it induce histological alterations in the liver or kidney. YD0171 caused a slight elevation in plasma homocysteine levels. In conclusion, the prodrug approach improves the pharmacological profile of AOAA; YD0171 represents a prototype for CBS inhibitory anticancer prodrugs. By targeting colorectal cancer bioenergetics, an emerging important hallmark of cancer, the approach exemplified herein may offer direct translational opportunities.
We have previously shown that the flavonoid luteolin inhibits the expression of pro‐inflammatory molecules induced by LPS. In the present study we tested the ability of luteolin to block signalling ...pathways implicated in LPS‐induced inflammatory gene expression in macrophages.
Exposure of the murine macrophage cell line RAW 264.7 to LPS increased phosphorylation of the mitogen‐activated protein kinase family members ERK1/2, p38 and JNK1/2 in a time‐dependent manner. Pretreatment of RAW 264.7 with luteolin inhibited the LPS‐induced ERK1/2 and p38, but not JNK1/2, phosphorylation, and blocked the LPS‐induced TNF‐α release.
To investigate which of these pathways contribute to the inhibitory effects of luteolin on TNF‐α release, cells were pretreated with pharmacological inhibitors of these pathways; PD98059 and SB203580 when used alone failed to inhibit TNF‐α release, whereas pretreatment with both agents attenuated TNF‐α release.
We have previously shown that luteolin blocks Akt phosphorylation in response to LPS in RAW 264.7 macrophages. To determine the role of Akt in TNF‐α release, cells were transiently transfected with a dominant negative form of Akt (K179M). Overexpression of K179M Akt did not alter LPS‐induced TNF‐α release, suggesting that inhibition of this kinase does not mediate the inhibitory action of luteolin.
In addition, DRB (a pharmacological inhibitor of CK2) blocked TNF‐α release in a concentration‐dependent manner, whereas co‐treatment of cells with luteolin and DRB did not have an additive effect.
We conclude that luteolin interferes with LPS signalling by reducing the activation of several MAPK family members and that its inhibitory action on TNF‐α release correlates with inhibition of ERK, p38 and CK2 activation.
British Journal of Pharmacology (2002) 136, 1058–1064. doi:10.1038/sj.bjp.0704803
Cystathionine
-synthase (CBS) is a key enzyme in the production of the signaling molecule hydrogen sulfide, deregulation of which is known to contribute to a range of serious pathological states. ...Involvement of hydrogen sulfide in pathways of paramount importance for cellular homeostasis renders CBS a promising drug target. An in-house focused library of heteroaromatic compounds was screened for CBS modulators by the methylene blue assay and a pyrazolopyridine derivative with a promising CBS inhibitory potential was discovered. The compound activity was readily comparable to the most potent CBS inhibitor currently known, aminoacetic acid, while a promising specificity over the related cystathionine
-lyase was identified. To rule out any possibility that the inhibitor may bind the enzyme regulatory domain due to its high structural similarity with cofactor s-adenosylmethionine, differential scanning fluorimetry was employed. A sub-scaffold search guided follow-up screening of related compounds, providing preliminary structure-activity relationships with respect to requisites for efficient CBS inhibition by this group of heterocycles. Subsequently, a hypothesis regarding the exact binding mode of the inhibitor was devised on the basis of the available structure-activity relationships (SAR) and a deep neural networks analysis and further supported by induced-fit docking calculations.
Poly(ADP-ribose) polymerase-1 (PARP-1) activation is a hallmark of oxidative stress-induced cellular injury that can lead to energetic failure and necrotic cell death via depleting the cellular ...nicotinamide adenine dinucleotide (NAD(+)) and ATP pools. Pharmacological PARP-1 inhibition or genetic PARP-1 deficiency exert protective effects in multiple models of cardiomyocyte injury. However, the connection between nuclear PARP-1 activation and depletion of the cytoplasmic and mitochondrial energy pools is poorly understood. By using cultured rat cardiomyocytes, here we report that ring finger protein 146 (RNF146), a cytoplasmic E3-ubiquitin ligase, acts as a direct interactor of PARP-1. Overexpression of RNF146 exerts protection against oxidant-induced cell death, whereas PARP-1-mediated cellular injury is augmented after RNF146 silencing. RNF146 translocates to the nucleus upon PARP-1 activation, triggering the exit of PARP-1 from the nucleus, followed by rapid degradation of both proteins. PARP-1 and RNF146 degradation occurs in the early phase of myocardial ischemia-reperfusion injury; it precedes the induction of heat shock protein expression. Taken together, PARP-1 release from the nucleus and its rapid degradation represent newly identified steps of the necrotic cell death program induced by oxidative stress. These steps are controlled by the ubiquitin-proteasome pathway protein RNF146. The current results shed new light on the mechanism of necrotic cell death. RNF146 may represent a distinct target for experimental therapeutic intervention of oxidant-mediated cardiac injury.
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Hydrogen sulfide (H2S) is now recognized as an endogenous signaling gasotransmitter in mammals. It is produced by mammalian cells and tissues by various enzymes - predominantly ...cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) - but part of the H2S is produced by the intestinal microbiota (colonic H2S-producing bacteria). Here we summarize the available information on the production and functional role of H2S in the various cell types typically associated with innate immunity (neutrophils, macrophages, dendritic cells, natural killer cells, mast cells, basophils, eosinophils) and adaptive immunity (T and B lymphocytes) under normal conditions and as it relates to the development of various inflammatory and immune diseases. Special attention is paid to the physiological and the pathophysiological aspects of the oral cavity and the colon, where the immune cells and the parenchymal cells are exposed to a special “H2S environment” due to bacterial H2S production. H2S has many cellular and molecular targets. Immune cells are “surrounded” by a “cloud” of H2S, as a result of endogenous H2S production and exogenous production from the surrounding parenchymal cells, which, in turn, importantly regulates their viability and function. Downregulation of endogenous H2S producing enzymes in various diseases, or genetic defects in H2S biosynthetic enzyme systems either lead to the development of spontaneous autoimmune disease or accelerate the onset and worsen the severity of various immune-mediated diseases (e.g. autoimmune rheumatoid arthritis or asthma). Low, regulated amounts of H2S, when therapeutically delivered by small molecule donors, improve the function of various immune cells, and protect them against dysfunction induced by various noxious stimuli (e.g. reactive oxygen species or oxidized LDL). These effects of H2S contribute to the maintenance of immune functions, can stimulate antimicrobial defenses and can exert anti-inflammatory therapeutic effects in various diseases.
Thioglycine and l-thiovaline are stable under acidic and basic conditions but in the presence of bicarbonate they liberate the gasotransmitter H2S. In cells both thioamino acids were proven to ...enhance cGMP formation and promote vasorelaxation in mouse aortic rings. Given that H2S is known to lower arterial hypertension, reduce oxidative stress and exhibit cardioprotective effects in preclinical models, H2S donors hold promise as novel treatments for cardiovascular diseases.
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