The genome-wide transcriptional consequences of the deletions of sulfur metabolism genes in Drosophila melanogaster
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ABSTRACT: The gasotransmitter hydrogen sulfide (H2S), produced by transsulfuration pathway, in recent years has been recognized as a biological mediator playing important role under normal conditions and in various pathologies both in eukaryotes and prokaryotes. The transsulfuration pathway (TSP) includes the conversion of homocysteine to cysteine following the breakdown of methionine. In Drosophila melanogaster and other eukaryotes H2S is produced by cystathionine B-synthase (CBS), cystathionine y-lyase (CSE), and 3-mercaptopyruvate sulftransferase (MST). In the experiments reported here we were able exploring CRISPR technique to obtain single and double deletions of these three major genes responsible for H2S production. In most cases the deletion of one studied gene does not result in the compensatory induction of two other genes responsible for H2S production. Transcriptomic studies demonstrated that the deletions of the above genes alter the genome expression to different degree with more pronounced effect exerted by deletions of CBS gene. Furthermore, the double deletion of both CBS and CSE resulted in cumulative effect on the transcription in the resulted strains. Overall, we found that the obtained deletions affected numerous genes involved in various biological pathways. Characteristically, genes involved in oxidative reduction process, stress-response genes, house-keeping genes, genes participating in olfactory and reproduction were among the most affected. Furthermore, characteristic differences in the response to the deletions of the studied genes were apparently organ specific and have clear-cut gender character. Importantly, the deletions altered global methylation and chromatin state of the experimental strains by increasing the level of inactive chromatin of the genome. Single and double deletions of the three genes responsible for the production of H2S helped to understand new aspects of biological significance of this vital physiological mediator.
Project description:Hydrogen sulfide (H2S) is an endogenous gasotransmitter and is capable of regulating various endogenous signaling pathways, inculding inflamation and immune response. In mammals, H2S is mainly generated by two pyridoxal-5'-phosphate-dependent enzymes, termed cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). CBS-deficient mice showed autoimmune disorders. H2S play important roles in T cell development and differentiation, especially Treg cells development and differentiation. We used microarrays to detail the global gene expression of WT and CBS-deficient CD4+ T cells.
Project description:Hydrogen sulfide (H2S) is an endogenous gasotransmitter and is capable of regulating various endogenous signaling pathways, inculding inflamation and immune response. In mammals, H2S is mainly generated by two pyridoxal-5’-phosphate-dependent enzymes, termed cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE). CBS-deficient mice showed autoimmune disorders. H2S play important roles in T cell development and differentiation, especially Treg cells development and differentiation. We used microarrays to detail the global gene expression of WT and CBS-deficient CD4+ T cells. CD4+ T cells from WT and CBS-deficient mice were isolated for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain the homogeneous populations of mice at 6 weeks in order to increase the temporal resolution of expression profiles.
Project description:To reveal the role of sulfur metabolism genes in memory formation processes, transcriptome libraries were obtained from the heads of 5-day-old naive males. The libraries were generated from Drosophila strains created in our laboratory with deleted cbs genes ( CBS-/-(5) and CBS-/-(8), cse (CSE-/-) and strains with double deletion of cbs and cse genes (CBS-/-,CSE-/-(1) and (CBS-/-,CSE-/-(2). Strain 58492, in which deletions were introduced by the CRISP/CAS9 method, was used as a control strain.
Project description:The ubiquitous gasotransmitter hydrogen sulfide (H2S) has been recognised to play a crucial role in human health. However, a role for host H2S in pathogenesis has not yet been demonstrated. Using cystathionine -lyase (CSE) deficient mice, we demonstrated an unexpected role of H2S in Mtb pathogenesis. We showed that Mtb-infected CSE-/- mice survive longer than wild-type mice, and support reduced pathology and lower bacterial burdens in the lung, spleen and liver. Similarly, in vitro Mtb infection of macrophages resulted in reduced colony forming units (CFUs) in CSE-/-cells. Chemical complementation of infected wild-type and CSE-/-macrophages using the slow H2S releaser GYY3147 and the CSE inhibitor DL-propargylglycine demonstrated that H2S is the effector molecule regulating Mtb survival in macrophages. Furthermore, we demonstrate that CSE promotes an excessive innate immune response, suppresses the adaptive immune response and reduces circulating IL1β, IL-6, TNFα, and IFN-γ levels in response to Mtb infection. Notably, Mtb infected CSE-/- macrophages show increased flux through glycolysis and the pentose phosphate pathway thereby establishing a critical link between H2S and central metabolism. Our data suggest that excessive H2S produced by the infected wild type mice reduce HIF-1α levels, thereby suppressing glycolysis and production of IL-1β, IL-6 and IL-12, and increasing bacterial burden. Clinical relevance was demonstrated by the spatial distribution of H2S producing enzymes in human necrotic, non-necrotic and cavitary pulmonary TB lesions. In summary, CSE exacerbates TB pathogenesis by altering immunometabolism in mice and inhibiting CSE or modulating glycolysis are potential targets for host-directed TB control.
Project description:Down syndrome (DS) is a genetic condition where the person is born with an extra chromosome 21. DS is associated with accelerated aging; people with DS are prone to age-related neurological conditions including an early-onset Alzheimer’s disease. Using the Dp(17)3Yey/ + mice, which overexpresses a portion of mouse chromosome 17, which encodes for the transsulfuration enzyme cystathionine β-synthase (CBS), we investigated the functional role of the CBS/hydrogen sulfide (H2S) pathway in the pathogenesis of neurobehavioral dysfunction in DS. The data demonstrate that CBS is higher in the brain of the DS mice than in the brain of wild-type mice, with primary localization in astrocytes. DS mice exhibited impaired recognition memory and spatial learning, loss of synaptosomal function, endoplasmic reticulum stress, and autophagy. Treatment of mice with aminooxyacetate, a prototypical CBS inhibitor, improved neurobehavioral function, reduced the degree of reactive gliosis in the DS brain, increased the ability of the synaptosomes to generate ATP, and reduced endoplasmic reticulum stress. H2S levels in the brain of DS mice were higher than in wild-type mice, but, unexpectedly, protein persulfidation was decreased. Many of the above alterations were more pronounced in the female DS mice. There was a significant dysregulation of metabolism in the brain of DS mice, which affected amino acid, carbohydrate, lipid, endocannabinoid, and nucleotide metabolites; some of these alterations were reversed by treatment of the mice with the CBS inhibitor. Thus, the CBS/H2S pathway contributes to the pathogenesis of neurological dysfunction in DS in the current animal model.
Project description:Ergothioneine (ET), a dietary thione/thiol, is receiving growing attention for its possible benefits in healthy aging and metabolic resilience. Our study investigates ET's effects on healthspan in aged animals, revealing extension of lifespan and enhanced mobility in Caenorhabditis elegans, accompanied by improved stress resistance and reduced age-associated biomarkers. In aged rats, ET administration enhances exercise endurance, muscle mass, and vascularization, concomitant with higher NAD+ levels in muscle. Mechanistically, ET acts as an alternative substrate for cystathionine gamma lyase (CSE), stimulating H2S production, which increases protein persulfidation of more than 300 protein targets. Among these, protein persulfidation-driven activation of cytosolic glycerol-3-phosphate dehydrogenase (cGPDH) primarily contributes to the ET-induced NAD+ increase. ET’s effects are abolished in models lacking CSE and cGPDH, highlighting essential role of H2S signaling and protein persulfidation. These findings elucidate ET's multifaceted actions and provide insights into its therapeutic potential for combating age-related muscle decline and metabolic perturbations.
Project description:Ergothioneine (ET), a dietary thione/thiol, is receiving growing attention for its possible benefits in healthy aging and metabolic resilience. Our study investigates ET's effects on healthspan in aged animals, revealing extension of lifespan and enhanced mobility in Caenorhabditis elegans, accompanied by improved stress resistance and reduced age-associated biomarkers. In aged rats, ET administration enhances exercise endurance, muscle mass, and vascularization, concomitant with higher NAD+ levels in muscle. Mechanistically, ET acts as an alternative substrate for cystathionine gamma lyase (CSE), stimulating H2S production, which increases protein persulfidation of more than 300 protein targets. Among these, protein persulfidation-driven activation of cytosolic glycerol-3-phosphate dehydrogenase (cGPDH) primarily contributes to the ET-induced NAD+ increase. ET’s effects are abolished in models lacking CSE and cGPDH, highlighting essential role of H2S signaling and protein persulfidation. These findings elucidate ET's multifaceted actions and provide insights into its therapeutic potential for combating age-related muscle decline and metabolic perturbations. To better understand the role of ET the cells, we used thermal proteome profilling of cells treated or not with ergothionine, allowing us to find new targets for the compound including CSE.
Project description:Ginseng (Panax ginseng Meyer) is commonly used as an herbal remedy worldwide. Few studies have explored the possible physiological changes in the liver although patients often self-medicate with ginseng preparations, which may lead to exceeding the recommended dose for long-term administration. Here, we analyzed changes in the hepatic proteins of mouse livers using quantitative proteomics after sub-chronic administration of Korean red ginseng (KRG) extract (control group and 0.5, 1.0, and 2.0 g/kg KRG) using tandem mass tag (TMT) 6‐plex technology. The 1.0 and 2.0 g/kg KRG groups exhibited signs of liver injury, including increased levels of aspartate transaminase (AST) and alanine aminotransferase (ALT) in the serum. Furthermore, serum glucose levels were significantly higher following KRG administration compared with the control group. Based on the upregulated proteins found in the proteomic analysis, we found that increased cystathionin beta-synthase (CBS) and cystathionine gamma-lyase (CSE) levels promoted greater hydrogen sulfide (HRR2RRS) synthesis in the liver. This investigation provides novel evidence that14T sub-chronic administration of 4T14TKRG can elevate HRR2RRS production by increasing protein expression of CBS and CSE in the liver.
Project description:CIMP (CpG island methylator phenotype) is an epigenetic molecular subtype, observed in multiple malignancies and associated with the epigenetic silencing of tumour suppressors. Currently, for most cancers including gastric cancer [GC], mechanisms underlying CIMP remain poorly understood. We sought to discover molecular contributors to CIMP in GC, by performing global DNA methylation, gene expression, and proteomics profiling across 14 gastric cell lines, followed by similar integrative analysis in 50 GC cell lines and 467 primary GCs. We identify the cystathionine beta-synthase enzyme (CBS) as a highly recurrent target of epigenetic silencing in CIMP GC. Likewise, we show that CBS epimutations are significantly associated with CIMP in various other cancers, occurring even in premalignant gastroesophageal conditions and longitudinally linked to clinical persistence. Of note, CRISPR deletion of CBS in normal gastric epithelial cells induces widespread DNA methylation changes that overlap with primary GC CIMP patterns. Reflecting its metabolic role as a gatekeeper interlinking the methionine and homocysteine cycles, CBS loss in vitro also causes reductions in the anti-inflammatory gasotransmitter hydrogen sulphide (H2S), with concomitant increase in NF-κB activity. In a murine genetic model of CBS-deficiency, preliminary data indicate upregulated immune-mediated transcriptional signatures in the stomach. Our results implicate CBS as a bi-faceted modifier of aberrant DNA methylation and inflammation in GC and highlights H2S donors as a potential new therapy for CBS-silenced lesions.
Project description:We use stable expression of a cystathionine-beta synthase (CBS) variant library in HEK293T cells to identify CBS coding sequence variants that alter mRNA abundance