Physiological roles of hydrogen sulfide in mammalian cells, tissues, and organs.

Over the last two decades, hydrogen sulfide (H2S) has emerged as an endogenous regulator of a broad range of physiological functions. H2S belongs to the class of molecules known as gasotransmitters, which typically include nitric oxide (NO) and carbon monoxide (CO). Three enzymes are recognized as endogenous sources of H2S in various cells and tissues: cystathionine γ-lyase (CSE), cystathionine ß-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST). The present article reviews the regulation of these enzymes as well as the pathways of their enzymatic and nonenzymatic degradation and elimination. The multiple interactions of H2S with other labile endogenous molecules (e.g., NO) and reactive oxygen species are also outlined. Next, the various biological targets and signaling pathways are outlined, with special reference to H2S or oxidative posttranscriptional modification (persulfidation or sulfhydration) of proteins and the effect of H2S on various channels and intracellular second messenger pathways, the regulation of gene transcription and translation, and the regulation of cellular bioenergetics and metabolism. The pharmacological and molecular tools currently available to study H2S physiology are also reviewed, including their utility and limitations. In subsequent sections, the role of H2S in the regulation of various physiological and cellular functions is reviewed, including the regulation of membrane potential, endo- and exocytosis, regulation of various cell organelles (endoplasmic reticulum, Golgi, mitochondria), regulation of cell movement, cell cycle, cell differentiation, and physiological aspects of regulated cell death. Next, the physiological roles of H2S in various cell types and organ systems are overviewed, including the role of H2S in red blood cells, immune cells, the central and peripheral nervous systems (with focus on neuronal transmission, learning, and memory formation), and regulation of vascular function (including angiogenesis as well as its specialized roles in the cerebrovascular, renal, and pulmonary vascular beds) and the role of H2S in the regulation of special senses, vision, hearing, taste and smell, and pain-sensing. Finally, the roles of H2S in the regulation of various organ functions (lung, heart, liver, kidney, urogenital organs, reproductive system, bone and cartilage, skeletal muscle, and endocrine organs) are presented, with a focus on physiology (including physiological aging) but also extending to some common pathophysiological conditions. From these data, a wide array of significant roles of H2S in the physiological regulation of all organ functions emerges and the characteristic bell-shaped biphasic effects of H2S are highlighted. In addition, key pathophysiological aspects, debated areas, and future research and translational areas are identified.

Prp19/Pso4 Is an Autoinhibited Ubiquitin Ligase Activated by Stepwise Assembly of Three Splicing Factors.

Human nineteen complex (NTC) acts as a multimeric E3 ubiquitin ligase in DNA repair and splicing. The transfer of ubiquitin is mediated by Prp19-a homotetrameric component of NTC whose elongated coiled coils serve as an assembly axis for two other proteins called SPF27 and CDC5L. We find that Prp19 is inactive on its own and have elucidated the structural basis of its autoinhibition by crystallography and mutational analysis. Formation of the NTC core by stepwise assembly of SPF27, CDC5L, and PLRG1 onto the Prp19 tetramer enables ubiquitin ligation. Protein-protein crosslinking of NTC, functional assays in vitro, and assessment of its role in DNA damage response provide mechanistic insight into the organization of the NTC core and the communication between PLRG1 and Prp19 that enables E3 activity. This reveals a unique mode of regulation for a complex E3 ligase and advances understanding of its dynamics in various cellular pathways.

Mitochondrial sulfide promotes life span and health span through distinct mechanisms in developing versus adult treated Caenorhabditis elegans.

Living longer without simultaneously extending years spent in good health ("health span") is an increasing societal burden, demanding new therapeutic strategies. Hydrogen sulfide (H2S) can correct disease-related mitochondrial metabolic deficiencies, and supraphysiological H2S concentrations can pro health span. However, the efficacy and mechanisms of mitochondrion-targeted sulfide delivery molecules (mtH2S) administered across the adult life course are unknown. Using a Caenorhabditis elegans aging model, we compared untargeted H2S (NaGYY4137, 100 µM and 100 nM) and mtH2S (AP39, 100 nM) donor effects on life span, neuromuscular health span, and mitochondrial integrity. H2S donors were administered from birth or in young/middle-aged animals (day 0, 2, or 4 postadulthood). RNAi pharmacogenetic interventions and transcriptomics/network analysis explored molecular events governing mtH2S donor-mediated health span. Developmentally administered mtH2S (100 nM) improved life/health span vs. equivalent untargeted H2S doses. mtH2S preserved aging mitochondrial structure, content (citrate synthase activity) and neuromuscular strength. Knockdown of H2S metabolism enzymes and FoxO/daf-16 prevented the positive health span effects of mtH2S, whereas DCAF11/wdr-23 - Nrf2/skn-1 oxidative stress protection pathways were dispensable. Health span, but not life span, increased with all adult-onset mtH2S treatments. Adult mtH2S treatment also rejuvenated aging transcriptomes by minimizing expression declines of mitochondria and cytoskeletal components, and peroxisome metabolism hub components, under mechanistic control by the elt-6/elt-3 transcription factor circuit. H2S health span extension likely acts at the mitochondrial level, the mechanisms of which dissociate from life span across adult vs. developmental treatment timings. The small mtH2S doses required for health span extension, combined with efficacy in adult animals, suggest mtH2S is a potential healthy aging therapeutic.

Pan-inhibition of the three H2S synthesizing enzymes restrains tumor progression and immunosuppression in breast cancer.

BACKGROUND: Hydrogen sulfide (H2S) is a significant endogenous mediator that has been implicated in the progression of various forms of cancer including breast cancer (BC). Cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST) are the three principal mammalian enzymes responsible for H2S production. Overexpression of CBS, CSE and 3MST was found to be associated with poor prognosis of BC patients. Moreover, H2S was linked to an immune-suppressive tumor microenvironment in BC. Recently it was observed that BC cells, in response to single or dual inhibition of H2S synthesizing enzymes, develop an escape mechanism by overexpressing alternative sources of H2S generation. Thus, the aim of this work is to escape the H2S compensatory mechanism by pan repressing the three enzymes using microRNAs (miRNAs) and to investigate their impact on the oncogenic and immunogenic profile of BC cells. METHODS: BC female patients (n = 25) were recruited. In-silico analysis was used to identify miRNAs targeting CBS, CSE, and 3MST. MDA-MB-231 cells were cultured and transfected using oligonucleotides. Total RNA was extracted using Biazol, reverse transcribed and quantified using qRT-PCR. H2S levels were measured using AzMc assay. BC hallmarks were assessed using trans-well migration, wound healing, MTT, and colony forming assays. RESULTS: miR-193a and miR-548c were validated by eight different bioinformatics software to simultaneously target CBS, CSE and 3MST. MiR-193a and miR-548c were significantly downregulated in BC tissues compared to their non-cancerous counterparts. Ectopic expression of miR-193a and miR-548c in MDA-MB-231 TNBC cells resulted in a marked repression of CBS, CSE, and 3MST transcript and protein levels, a significant decrease in H2S levels, reduction in cellular viability, inhibition of migration and colony forming ability, repression of immune-suppressor proteins GAL3 GAL9, and CD155 and upregulation of the immunostimulatory MICA and MICB proteins. CONCLUSION: This study sheds the light onto miR-193a and miR-548c as potential pan-repressors of the H2S synthesizing enzymes. and identifies them as novel tumor suppressor and immunomodulatory miRNAs in TNBC.

Dietary restriction and life-history trade-offs: insights into mTOR pathway regulation and reproductive investment in Japanese quail.

Resources are needed for growth, reproduction and survival, and organisms must trade off limited resources among competing processes. Nutritional availability in organisms is sensed and monitored by nutrient-sensing pathways that can trigger physiological changes or alter gene expression. Previous studies have proposed that one such signalling pathway, the mechanistic target of rapamycin (mTOR), underpins a form of adaptive plasticity when individuals encounter constraints in their energy budget. Despite the fundamental importance of this process in evolutionary biology, how nutritional limitation is regulated through the expression of genes governing this pathway and its consequential effects on fitness remain understudied, particularly in birds. We used dietary restriction to simulate resource depletion and examined its effects on body mass, reproduction and gene expression in Japanese quails (Coturnix japonica). Quails were subjected to feeding at 20%, 30% and 40% restriction levels or ad libitum for 2 weeks. All restricted groups exhibited reduced body mass, whereas reductions in the number and mass of eggs were observed only under more severe restrictions. Additionally, dietary restriction led to decreased expression of mTOR and insulin-like growth factor 1 (IGF1), whereas the ribosomal protein S6 kinase 1 (RPS6K1) and autophagy-related genes (ATG9A and ATG5) were upregulated. The pattern in which mTOR responded to restriction was similar to that for body mass. Regardless of the treatment, proportionally higher reproductive investment was associated with individual variation in mTOR expression. These findings reveal the connection between dietary intake and the expression of mTOR and related genes in this pathway.

Physiological concentrations of cyanide stimulate mitochondrial Complex IV and enhance cellular bioenergetics.

In mammalian cells, cyanide is viewed as a cytotoxic agent, which exerts its effects through inhibition of mitochondrial Complex IV (Cytochrome C oxidase [CCOx]). However, the current report demonstrates that cyanide's effect on CCOx is biphasic; low (nanomolar to low-micromolar) concentrations stimulate CCOx activity, while higher (high-micromolar) concentrations produce the "classic" inhibitory effect. Low concentrations of cyanide stimulated mitochondrial electron transport and elevated intracellular adenosine triphosphate (ATP), resulting in the stimulation of cell proliferation. The stimulatory effect of cyanide on CCOx was associated with the removal of the constitutive, inhibitory glutathionylation on its catalytic 30- and 57-kDa subunits. Transfer of diluted Pseudomonas aeruginosa (a cyanide-producing bacterium) supernatants to mammalian cells stimulated cellular bioenergetics, while concentrated supernatants were inhibitory. These effects were absent with supernatants from mutant Pseudomonas lacking its cyanide-producing enzyme. These results raise the possibility that cyanide at low, endogenous levels serves regulatory purposes in mammals. Indeed, the expression of six putative mammalian cyanide-producing and/or -metabolizing enzymes was confirmed in HepG2 cells; one of them (myeloperoxidase) showed a biphasic regulation after cyanide exposure. Cyanide shares features with "classical" mammalian gasotransmitters NO, CO, and H2S and may be considered the fourth mammalian gasotransmitter.

Therapeutic reconditioning of damaged lungs by transient heat stress during ex vivo lung perfusion.

Ex vivo lung perfusion (EVLP) may serve as a platform for the pharmacologic repair of lung grafts before transplantation (LTx). We hypothesized that EVLP could also permit nonpharmacologic repair through the induction of a heat shock response, which confers stress adaptation via the expression of heat shock proteins (HSPs). Therefore, we evaluated whether transient heat application during EVLP (thermal preconditioning [TP]) might recondition damaged lungs before LTx. TP was performed during EVLP (3 hours) of rat lungs damaged by warm ischemia by transiently heating (30 minutes, 41.5 °C) the EVLP perfusate, followed by LTx (2 hours) reperfusion. We also assessed the TP (30 minutes, 42 °C) during EVLP (4 hours) of swine lungs damaged by prolonged cold ischemia. In rat lungs, TP induced HSP expression, reduced nuclear factor κB and inflammasome activity, oxidative stress, epithelial injury, inflammatory cytokines, necroptotic death signaling, and the expression of genes involved in innate immune and cell death pathways. After LTx, heated lungs displayed reduced inflammation, edema, histologic damage, improved compliance, and unchanged oxygenation. In pig lungs, TP induced HSP expression, reduced oxidative stress, inflammation, epithelial damage, vascular resistance, and ameliorated compliance. Collectively, these data indicate that transient heat application during EVLP promotes significant reconditioning of damaged lungs and improves their outcomes after transplantation.

Host cystathionine-γ lyase derived hydrogen sulfide protects against Pseudomonas aeruginosa sepsis.

Hydrogen sulfide (H2S) has recently been recognized as a novel gaseous transmitter with several anti-inflammatory properties. The role of host- derived H2S in infections by Pseudomonas aeruginosa was investigated in clinical and mouse models. H2S concentrations and survival was assessed in septic patients with lung infection. Animal experiments using a model of severe systemic multidrug-resistant P. aeruginosa infection were performed using mice with a constitutive knock-out of cystathionine-γ lyase (Cse) gene (Cse-/-) and wild-type mice with a physiological expression (Cse+/+). Experiments were repeated in mice after a) treatment with cyclophosphamide; b) bone marrow transplantation (BMT) from a Cse+/+ donor; c) treatment with H2S synthesis inhibitor aminooxyacetic acid (ΑΟΑΑ) or propargylglycine (PAG) and d) H2S donor sodium thiosulfate (STS) or GYY3147. Bacterial loads and myeloperoxidase activity were measured in tissue samples. The expression of quorum sensing genes (QS) was determined in vivo and in vitro. Cytokine concentration was measured in serum and incubated splenocytes. Patients survivors at day 28 had significantly higher serum H2S compared to non-survivors. A cut- off point of 5.3 µΜ discriminated survivors with sensitivity 92.3%. Mortality after 28 days was 30.9% and 93.7% in patients with H2S higher and less than 5.3 µΜ (p = 7 x 10-6). In mice expression of Cse and application of STS afforded protection against infection with multidrug-resistant P. aeruginosa. Cyclophosphamide pretreatment eliminated the survival benefit of Cse+/+ mice, whereas BMT increased the survival of Cse-/- mice. Cse-/- mice had increased pathogen loads compared to Cse+/+ mice. Phagocytic activity of leukocytes from Cse-/- mice was reduced but was restored after H2S supplementation. An H2S dependent down- regulation of quorum sensing genes of P.aeruginosa could be demonstrated in vivo and in vitro. Endogenous H2S is a potential independent parameter correlating with the outcome of P. aeruginosa. H2S provides resistance to infection by MDR bacterial pathogens.

H2S biogenesis by cystathionine beta-synthase: mechanism of inhibition by aminooxyacetic acid and unexpected role of serine.

Cystathionine beta-synthase (CBS) is a pivotal enzyme of the transsulfuration pathway responsible for diverting homocysteine to the biosynthesis of cysteine and production of hydrogen sulfide (H2S). Aberrant upregulation of CBS and overproduction of H2S contribute to pathophysiology of several diseases including cancer and Down syndrome. Therefore, pharmacological CBS inhibition has emerged as a prospective therapeutic approach. Here, we characterized binding and inhibitory mechanism of aminooxyacetic acid (AOAA), the most commonly used CBS inhibitor. We found that AOAA binds CBS tighter than its respective substrates and forms a dead-end PLP-bound intermediate featuring an oxime bond. Surprisingly, serine, but not cysteine, replaced AOAA from CBS and formed an aminoacrylate reaction intermediate, which allowed for the continuation of the catalytic cycle. Indeed, serine rescued and essentially normalized the enzymatic activity of AOAA-inhibited CBS. Cellular studies confirmed that AOAA decreased H2S production and bioenergetics, while additional serine rescued CBS activity, H2S production and mitochondrial function. The crystal structure of AOAA-bound human CBS showed a lack of hydrogen bonding with residues G305 and Y308, found in the serine-bound model. Thus, AOAA-inhibited CBS could be reactivated by serine. This difference may be important in a cellular environment in multiple pathophysiological conditions and may modulate the CBS-inhibitory activity of AOAA. In addition, our results demonstrate additional complexities of using AOAA as a CBS-specific inhibitor of H2S biogenesis and point to the urgent need to develop a potent, selective and specific pharmacological CBS inhibitor.

Epigallocatechin gallate is a potent inhibitor of cystathionine beta-synthase: Structure-activity relationship and mechanism of action.

Epigallocatechin gallate (EGCG) is the main bioactive component of green tea. Through screening of a small library of natural compounds, we discovered that EGCG inhibits cystathionine ß-synthase (CBS), a major H2S-generating enzyme. Here we characterize EGCG's mechanism of action in the context of CBS-derived H2S production. In the current project, biochemical, pharmacological and cell biology approaches were used to characterize the effect of EGCG on CBS in cellular models of cancer and Down syndrome (DS). The results show that EGCG binds to CBS and inhibits H2S-producing CBS activity almost 30-times more efficiently than the canonical cystathionine formation (IC50 0.12 versus 3.3 µM). Through screening structural analogs and building blocks, we identified that gallate moiety of EGCG represents the pharmacophore responsible for CBS inhibition. EGCG is a mixed-mode, CBS-specific inhibitor with no effect on the other two major enzymatic sources of H2S, CSE and 3-MST. Unlike the prototypical CBS inhibitor aminooxyacetate, EGCG does not bind the catalytic cofactor of CBS pyridoxal-5'-phosphate. Molecular modeling suggests that EGCG blocks a substrate access channel to pyridoxal-5'-phosphate. EGCG inhibits cellular H2S production in HCT-116 colon cancer cells and in DS fibroblasts. It also exerts effects that are consistent with the functional role of CBS in these cells: in HCT-116 cells it decreases, while in DS cells it improves viability and proliferation. In conclusion, EGCG is a potent inhibitor of CBS-derived H2S production. This effect may contribute to its pharmacological effects in various pathophysiological conditions.

Overproduction of H2S, generated by CBS, inhibits mitochondrial Complex IV and suppresses oxidative phosphorylation in Down syndrome.

Down syndrome (DS) is associated with significant perturbances in mitochondrial function. Here we tested the hypothesis that the suppression of mitochondrial electron transport in DS cells is due to high expression of cystathionine-ß-synthase (CBS) and subsequent overproduction of the gaseous transmitter hydrogen sulfide (H2S). Fibroblasts from DS individuals showed higher CBS expression than control cells; CBS localization was both cytosolic and mitochondrial. DS cells produced significantly more H2S and polysulfide and exhibited a profound suppression of mitochondrial electron transport, oxygen consumption, and ATP generation. DS cells also exhibited slower proliferation rates. In DS cells, pharmacological inhibition of CBS activity with aminooxyacetate or siRNA-mediated silencing of CBS normalized cellular H2S levels, restored Complex IV activity, improved mitochondrial electron transport and ATP synthesis, and restored cell proliferation. Thus, CBS-derived H2S is responsible for the suppression of mitochondrial function in DS cells. When H2S overproduction is corrected, the tonic suppression of Complex IV is lifted, and mitochondrial electron transport is restored. CBS inhibition offers a potential approach for the pharmacological correction of DS-associated mitochondrial dysfunction.

Farm to table: Residues of different pesticides in tomato and tomato juice - Food safety aspects.

During plant cultivation, the pesticides can get into the tissue of vegetables due to crop protection processes, and thus into the food chain. Therefore, they constitute a potential risk to the consumer's health. Depletion of pesticides [spirotetramat (Movento), azoxystrobin and difenoconazole (Amistar Top)] was monitored by testing tomatoes treated individually or simultaneously and tomato juices prepared from the treated tomatoes. The investigations aimed to reveal any kinetic interaction between the compounds tested and changes in their elimination, and thus to assess their compliance with the official Maximum Residue Limits (MRLs). The co-presence of pesticides prolonged the elimination of the individual compounds which reached significantly higher residue levels (P < 0.0001) in tomato, especially difenoconazole (45%) and azoxystrobin (50%) on day 8 after treatment that can cause food safety issues to the human consumers. However, the concentrations of pesticides applied alone or simultaneously were found to be below the corresponding MRL values after the withdrawal period in all investigated tomato and tomato juice samples. Accordingly, the investigated pesticides can be safely used simultaneously, their concentrations are in compliance with the legal regulations and thus their concomitant presence does not pose any risk to the consumers' health.

Pharmacological induction of mesenchymal-epithelial transition via inhibition of H2S biosynthesis and consequent suppression of ACLY activity in colon cancer cells.

Hydrogen sulfide (H2S) is an important endogenous gaseous transmitter mediator, which regulates a variety of cellular functions in autocrine and paracrine manner. The enzymes responsible for the biological generation of H2S include cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST). Increased expression of these enzymes and overproduction of H2S has been implicated in essential processes of various cancer cells, including the stimulation of metabolism, maintenance of cell proliferation and cytoprotection. Cancer cell identity is characterized by so-called "transition states". The progression from normal (epithelial) to transformed (mesenchymal) state is termed epithelial-to-mesenchymal transition (EMT) whereby epithelial cells lose their cell-to-cell adhesion capacity and gain mesenchymal characteristics. The transition process can also proceed in the opposite direction, and this process is termed mesenchymal-to-epithelial transition (MET). The current project was designed to determine whether inhibition of endogenous H2S production in colon cancer cells affects the EMT/MET balance in vitro. Inhibition of H2S biosynthesis in HCT116 human colon cancer cells was achieved either with aminooxyacetic acid (AOAA) or 2-[(4-hydroxy-6-methylpyrimidin-2-yl)sulfanyl]-1-(naphthalen-1-yl)ethan-1-one (HMPSNE). These inhibitors induced an upregulation of E-cadherin and Zonula occludens-1 (ZO-1) expression and downregulation of fibronectin expression, demonstrating that H2S biosynthesis inhibitors can produce a pharmacological induction of MET in colon cancer cells. These actions were functionally reflected in an inhibition of cell migration, as demonstrated in an in vitro "scratch wound" assay. The mechanisms involved in the action of endogenously produced H2S in cancer cells in promoting (or maintaining) EMT (or tonically inhibiting MET) relate, at least in part, in the induction of ATP citrate lyase (ACLY) protein expression, which occurs via upregulation of ACLY mRNA (via activation of the ACLY promoter). ACLY in turn, regulates the Wnt-ß-catenin pathway, an essential regulator of the EMT/MET balance. Taken together, pharmacological inhibition of endogenous H2S biosynthesis in cancer cells induces MET. We hypothesize that this may contribute to anti-cancer / anti-metastatic effects of H2S biosynthesis inhibitors.

Arginine vasopressin receptor 2 activation promotes microvascular permeability in sepsis.

Methicillin-resistant Staphylococcus aureus (MRSA) sepsis is a severe condition associated with vascular leakage and poor prognosis. The hemodynamic management of sepsis targets hypotension, but there is no specific treatment available for vascular leakage. Arginine vasopressin (AVP) has been used in sepsis to promote vasoconstriction by activating AVP receptor 1 (V1R). However, recent evidence suggests that increased fluid retention may be associated with the AVP receptor 2 (V2R) activation worsening the outcome of sepsis. Hence, we hypothesized that the inhibition of V2R activation ameliorates the severity of microvascular hyperpermeability during sepsis. The hypothesis was tested using a well-characterized and clinically relevant ovine model of MRSA pneumonia/sepsis and in vitro assays of human lung microvascular endothelial cells (HMVECs). in vivo experiments demonstrated that the treatment of septic sheep with tolvaptan (TLVP), an FDA-approved V2R antagonist, significantly attenuated the sepsis-induced fluid retention and markedly reduced the lung water content. These pathological changes were not affected by the treatment with V2R agonist, desmopressin (DDAVP). Additionally, the incubation of cultured HMVECs with DDAVP, and DDAVP along with MRSA significantly increased the paracellular permeability. Finally, both the DDAVP and MRSA-induced hyperpermeability was significantly attenuated by TLVP. Subsequent protein and gene expression assays determined that the V2R-induced increase in permeability is mediated by phospholipase C beta (PLCß) and the potent permeability factor angiopoietin-2. In conclusion, our results indicate that the activation of the AVP-V2R axis is critical in the pathophysiology of severe microvascular hyperpermeability during Gram-positive sepsis. The use of the antagonist TLVP should be considered as adjuvant treatment for septic patients. The results from this clinically relevant animal study are highly translational to clinical practice.

The mitochondria-targeted hydrogen sulfide donor AP39 improves health and mitochondrial function in a C. elegans primary mitochondrial disease model.

Primary mitochondrial diseases (PMD) are inherited diseases that cause dysfunctional mitochondrial oxidative phosphorylation, leading to diverse multisystem diseases and substantially impaired quality of life. PMD treatment currently comprises symptom management, with an unmet need for therapies targeting the causative mitochondrial defects. Molecules which selective target mitochondria have been proposed as potential treatment options in PMD but have met with limited success. We have previously shown in animal models that mitochondrial dysfunction caused by the disease process could be prevented and/or reversed by selective targeting of the "gasotransmitter" hydrogen sulfide (H2 S) to mitochondria using a novel compound, AP39. Therefore, in this study we investigated whether AP39 could also restore mitochondrial function in PMD models where mitochondrial dysfunction was the cause of the disease pathology using C. elegans. We characterised several PMD mutant C. elegans strains for reduced survival, movement and impaired cellular bioenergetics and treated each with AP39. In animals with widespread electron transport chain deficiency (gfm-1[ok3372]), AP39 (100 nM) restored ATP levels, but had no effect on survival or movement. However, in a complex I mutant (nuo-4[ok2533]), a Leigh syndrome orthologue, AP39 significantly reversed the decline in ATP levels, preserved mitochondrial membrane potential and increased movement and survival. For the first time, this study provides proof-of-principle evidence suggesting that selective targeting of mitochondria with H2 S could represent a novel drug discovery approach to delay, prevent and possibly reverse mitochondrial decline in PMD and related disorders.

Poly(ADP-Ribose) Polymerase Inhibition in Acute Lung Injury. A Reemerging Concept.

PARP1, the major isoform of a family of ADP-ribosylating enzymes, has been implicated in the regulation of various biological processes including DNA repair, gene transcription, and cell death. The concept that PARP1 becomes activated in acute lung injury (ALI) and that pharmacological inhibition or genetic deletion of this enzyme can provide therapeutic benefits emerged over 20 years ago. The current article provides an overview of the cellular mechanisms involved in the pathogenetic roles of PARP1 in ALI and provides an overview of the preclinical data supporting the efficacy of PARP (poly[ADP-ribose] polymerase) inhibitors. In recent years, several ultrapotent PARP inhibitors have been approved for clinical use (for the therapy of various oncological diseases): these newly-approved PARP inhibitors were recently reported to show efficacy in animal models of ALI. These observations offer the possibility of therapeutic repurposing of these inhibitors for patients with ALI. The current article lays out a potential roadmap for such repurposing efforts. In addition, the article also overviews the scientific basis of potentially applying PARP inhibitors for the experimental therapy of viral ALI, such as coronavirus disease (COVID-19)-associated ALI.

Cystathionine γ Lyase Sulfhydrates the RNA Binding Protein Human Antigen R to Preserve Endothelial Cell Function and Delay Atherogenesis.

BACKGROUND: Hydrogen sulfide (H2S), generated by cystathionine γ lyase (CSE), is an important endogenous regulator of vascular function. The aim of the present study was to investigate the control and consequences of CSE activity in endothelial cells under physiological and proatherogenic conditions. METHODS: Endothelial cell CSE knockout mice were generated, and lung endothelial cells were studied in vitro (gene expression, protein sulfhydration, and monocyte adhesion). Mice were crossed onto the apolipoprotein E-deficient background, and atherogenesis (partial carotid artery ligation) was monitored over 21 days. CSE expression, H2S bioavailability, and amino acid profiling were also performed with human material. RESULTS: The endothelial cell-specific deletion of CSE selectively increased the expression of CD62E and elevated monocyte adherence in the absence of an inflammatory stimulus. Mechanistically, CD62E mRNA was more stable in endothelial cells from CSE-deficient mice, an effect attributed to the attenuated sulfhydration and dimerization of the RNA-binding protein human antigen R. CSE expression was upregulated in mice after partial carotid artery ligation and in atheromas from human subjects. Despite the increase in CSE protein, circulating and intraplaque H2S levels were reduced, a phenomenon that could be attributed to the serine phosphorylation (on Ser377) and inhibition of the enzyme, most likely resulting from increased interleukin-1ß. Consistent with the loss of H2S, human antigen R sulfhydration was attenuated in atherosclerosis and resulted in the stabilization of human antigen R-target mRNAs, for example, CD62E and cathepsin S, both of which are linked to endothelial cell activation and atherosclerosis. The deletion of CSE from endothelial cells was associated with the accelerated development of endothelial dysfunction and atherosclerosis, effects that were reversed on treatment with a polysulfide donor. Finally, in mice and humans, plasma levels of the CSE substrate l-cystathionine negatively correlated with vascular reactivity and H2S levels, indicating its potential use as a biomarker for vascular disease. CONCLUSIONS: The constitutive S-sulfhydration of human antigen R (on Cys13) by CSE-derived H2S prevents its homodimerization and activity, which attenuates the expression of target proteins such as CD62E and cathepsin S. However, as a consequence of vascular inflammation, the beneficial actions of CSE-derived H2S are lost owing to the phosphorylation and inhibition of the enzyme.

Meta-analysis of metabolites involved in bioenergetic pathways reveals a pseudohypoxic state in Down syndrome.

Clinical observations and preclinical studies both suggest that Down syndrome (DS) may be associated with significant metabolic and bioenergetic alterations. However, the relevant scientific literature has not yet been systematically reviewed. The aim of the current study was to conduct a meta-analysis of metabolites involved in bioenergetics pathways in DS to conclusively determine the difference between DS and control subjects. We discuss these findings and their potential relevance in the context of pathogenesis and experimental therapy of DS. Articles published before July 1, 2020, were identified by using the search terms "Down syndrome" and "metabolite name" or "trisomy 21" and "metabolite name". Moreover, DS-related metabolomics studies and bioenergetics literature were also reviewed. 41 published reports and associated databases were identified, from which the descriptive information and the relevant metabolomic parameters were extracted and analyzed. Mixed effect model revealed the following changes in DS: significantly decreased ATP, CoQ10, homocysteine, serine, arginine and tyrosine; slightly decreased ADP; significantly increased uric acid, succinate, lactate and cysteine; slightly increased phosphate, pyruvate and citrate. However, the concentrations of AMP, 2,3-diphosphoglycerate, glucose, and glutamine were comparable in the DS vs. control populations. We conclude that cells of subjects with DS are in a pseudo-hypoxic state: the cellular metabolic and bio-energetic mechanisms exhibit pathophysiological alterations that resemble the cellular responses associated with hypoxia, even though the supply of the cells with oxygen is not disrupted. This fundamental alteration may be, at least in part, responsible for a variety of functional deficits associated with DS, including reduced exercise difference, impaired neurocognitive status and neurodegeneration.

Potential role of the 3-mercaptopyruvate sulfurtransferase (3-MST)-hydrogen sulfide (H2S) pathway in cancer cells.

Hydrogen sulfide (H2S), produced by various endogenous enzyme systems, serves various biological regulatory roles in mammalian cells in health and disease. Over recent years, a new concept emerged in the field of H2S biology, showing that various cancer cells upregulate their endogenous H2S production, and utilize this mediator in autocrine and paracrine manner to stimulate proliferation, bioenergetics and tumor angiogenesis. Initial work identified cystathionine-beta-synthase (CBS) in many tumor cells as the key source of H2S. In other cells, cystathionine-gamma-lyase (CSE) has been shown to play a pathogenetic role. However, until recently, less attention has been paid to the third enzymatic source of H2S, 3-mercaptopyruvate sulfurtransferase (3-MST), even though several of its biological and biochemical features - e.g. its partial mitochondrial localization, its ability to produce polysulfides, which, in turn, can induce functionally relevant posttranslational protein modifications - makes it a potential candidate. Indeed, several lines of recent data indicate the potential role of the 3-MST system in cancer biology. In many cancers (e.g. colon adenocarcinoma, lung adenocarcinoma, urothelial cell carcinoma, various forms of oral carcinomas), 3-MST is upregulated compared to the surrounding normal tissue. According to in vitro studies, 3-MST upregulation is especially prominent in cancer cells that recover from oxidative damage and/or develop a multidrug-resistant phenotype. Emerging data with newly discovered pharmacological inhibitors of 3-MST, as well as data using 3-MST silencing approaches suggest that the 3-MST/H2S system plays a role in maintaining cancer cell proliferation; it may also regulate bioenergetic and cell-signaling functions. Many questions remain open in the field of 3-MST/cancer biology; the last section of current article highlights these open questions and lays out potential experimental strategies to address them.

Hydrogen sulfide: An endogenous regulator of the immune system.

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 ß-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.