Transsulfuration Research Articles

Genes Responsible for H2S Production and Metabolism Are Involved in Learning and Memory in Drosophila melanogaster.

The gasotransmitter hydrogen sulfide (H2S) produced by the transsulfuration pathway (TSP) is an important biological mediator, involved in many physiological and pathological processes in multiple higher organisms, including humans. Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) enzymes play a central role in H2S production and metabolism. Here, we investigated the role of H2S in learning and memory processes by exploring several Drosophila melanogaster strains with single and double deletions of CBS and CSE developed by the CRISPR/Cas9 technique. We monitored the learning and memory parameters of these strains using the mating rejection courtship paradigm and demonstrated that the deletion of the CBS gene, which is expressed predominantly in the central nervous system, and double deletions completely block short- and long-term memory formation in fruit flies. On the other hand, the flies with CSE deletion preserve short- and long-term memory but fail to exhibit long-term memory retention. Transcriptome profiling of the heads of the males from the strains with deletions in Gene Ontology terms revealed a strong down-regulation of many genes involved in learning and memory, reproductive behavior, cognition, and the oxidation–reduction process in all strains with CBS deletion, indicating an important role of the hydrogen sulfide production in these vital processes.

Homocysteine Metabolism Pathway Is Involved in the Control of Glucose Homeostasis: A Cystathionine Beta Synthase Deficiency Study in Mouse.

Cystathionine beta synthase (CBS) catalyzes the first step of the transsulfuration pathway from homocysteine to cystathionine, and its deficiency leads to hyperhomocysteinemia (HHcy) in humans and rodents. To date, scarce information is available about the HHcy effect on insulin secretion, and the link between CBS activity and the setting of type 2 diabetes is still unknown. We aimed to decipher the consequences of an inborn defect in CBS on glucose homeostasis in mice. We used a mouse model heterozygous for CBS (CBS+/−) that presented a mild HHcy. Other groups were supplemented with methionine in drinking water to increase the mild to intermediate HHcy, and were submitted to a high-fat diet (HFD). We measured the food intake, body weight gain, body composition, glucose homeostasis, plasma homocysteine level, and CBS activity. We evidenced a defect in the stimulated insulin secretion in CBS+/− mice with mild and intermediate HHcy, while mice with intermediate HHcy under HFD presented an improvement in insulin sensitivity that compensated for the decreased insulin secretion and permitted them to maintain a glucose tolerance similar to the CBS+/+ mice. Islets isolated from CBS+/− mice maintained their ability to respond to the elevated glucose levels, and we showed that a lower parasympathetic tone could, at least in part, be responsible for the insulin secretion defect. Our results emphasize the important role of Hcy metabolic enzymes in insulin secretion and overall glucose homeostasis.

The ataxia-telangiectasia mutated gene product regulates the cellular acid-labile sulfide fraction

The ataxia-telangiectasia mutated (ATM) protein regulates cell cycle checkpoints, the cellular redox state, and double-stranded DNA break repair. ATM loss causes the disorder ataxia-telangiectasia (A-T), distinguished by ataxia, telangiectasias, dysregulated cellular redox and iron responses, and an increased cancer risk. We examined the sulfur pool in A-T cells, with and without an ATM expression vector. While free and bound sulfide levels were not changed with ATM expression, the acid-labile sulfide faction was significantly increased. ATM expression also increased cysteine desulfurase (NFS1), NFU1 iron-sulfur cluster scaffold homolog protein, and several mitochondrial complex I proteins’ expression. Additionally, ATM expression suppressed cystathionine β-synthase and cystathionine γ-synthase protein expression, cystathionine γ-synthase enzymatic activity, and increased the reduced to oxidized glutathione ratio. This last observation is interesting, as dysregulated glutathione is implicated in A-T pathology. As ATM expression increases the expression of proteins central in initiating 2Fe-2S and 4Fe-4S cluster formation (NFS1 and NFU1, respectively), and the acid-labile sulfide faction is composed of sulfur incorporated into Fe-S clusters, our data indicates that ATM regulates aspects of Fe-S cluster biosynthesis, the transsulfuration pathway, and glutathione redox cycling. Thus, our data may explain some of the redox- and iron-related pathologies seen in A-T.

Methionine Deprivation Reveals the Pivotal Roles of Cell Cycle Progression in Ferroptosis That Is Induced by Cysteine Starvation.

Ferroptosis, a type of iron-dependent necrotic cell death, is triggered by the accumulation of excessive lipid peroxides in cells. Glutathione (GSH), a tripeptide redox molecule that contains a cysteine (Cys) unit in the center, plays a pivotal role in protection against ferroptosis. When the transsulfuration pathway is activated, the sulfur atom of methionine (Met) is utilized to generate Cys, which can then suppress Cys-starvation-induced ferroptosis. In the current study, we cultured HeLa cells in Met- and/or cystine (an oxidized Cys dimer)- deprived medium and investigated the roles of Met in ferroptosis execution. The results indicate that, in the absence of cystine or Met, ferroptosis or cell cycle arrest, respectively, occurred. Contrary to our expectations, however, the simultaneous deprivation of both Met and cystine failed to induce ferroptosis, although the intracellular levels of Cys and GSH were maintained at low levels. Supplementation with S-adenosylmethionine (SAM), a methyl group donor that is produced during the metabolism of Met, caused the cell cycle progression to resume and lipid peroxidation and the subsequent induction of ferroptosis was also restored under conditions of Met/cystine double deprivation. DNA methylation appeared to be involved in the resumption in the SAM-mediated cell cycle because its downstream metabolite S-adenosylhomocysteine failed to cause either cell cycle progression or ferroptosis to be induced. Taken together, our results suggest that elevated lipid peroxidation products that are produced during cell cycle progression are involved in the execution of ferroptosis under conditions of Cys starvation.

DNA Methylation Underlies the Long‐Term Association Between Periodontitis and Atherosclerotic Cardiovascular Disease

Periodontitis, one of the most common inflammatory conditions, and the leading cause of adult tooth loss, has been identified as an independent risk factor for cardiovascular disease (CVD). Long‐term studies in edentulous patients with a history of periodontitis showed persistence of increased CVD risk for years after edentulism, suggesting that clinical elimination of disease is not sufficient in decreasing periodontitis‐induced CVD risk. This is similar to other CVD risk factors, such as smoking and diabetes. We hypothesized that periodontitis induces epigenetic changes in hematopoietic stem cells in the bone marrow (BM), and such changes persist after clinical elimination of the disease and underlie the induced‐CVD risk.To simulate clinical elimination of periodontitis and persistence of the hypothesized epigenetic reprogramming, we used a BM transplant approach. Using the low density lipoprotein receptor (LDLR) KO atherosclerosis mouse model, BM donor mice were fed a high fat diet (HFD) to induce atherosclerosis, and orally inoculated with Porphyromonas gingivalis(Pg), a keystone periodontal pathogen, to induce periodontitis; a second group was sham inoculated. (We previously showed that HFD‐fed, Pginoculated mice developed more atherosclerosis than sham‐inoculated). Naïve LDLR KO mice were irradiated and transplanted with BM from one of the 2 donor groups.Recipients of BM from Pg‐inoculated donors developed significantly more atherosclerosis, accompanied by more pro‐inflammatory plasma and macrophage cytokine profiles. Using whole genome bisulfite sequencing, 375 differentially methylated regions (DMR), and a global hypomethylation in recipients of BM from Pg‐inoculated donors, were identified. Some DMRs pointed to involvement of enzymes with major roles in methylation and demethylation. In recipients of BM from Pg‐inoculated donors, methionine adenosyl transferase (MAT), which catalyzes the conversion of methionine to S‐denosylmethionine (SAM), the universal methyl‐donor, was hypermethylated. The gene for S‐adenosylhomocysteine hydrolase, which catalyzes the reversible conversion of the potent methylation inhibitor, S‐adenosylhomocysteine (SAH), to homocysteine and adenosine, was also hypermethylated. In contrast, the de‐methylation enzyme, Ten‐Eleven Translocase (TET) 2, was hypomethylated. In validation assays, we found a significant increase in activity of TET2 and a decrease in activity of DNA methyltransferases. Plasma SAH levels were significantly higher and the SAM to SAH ratio was decreased, both of which have been associated with CVD. Homocysteine, is at the intersection of the methionine cycle and the transsulphuration pathway, where it can either be re‐methylated to methionine and go through the methionine cycle, or it can synthesize cysteine through the transsulphuration pathway. Glutathione, a critical antioxidant, is the end‐product of the transsulphuration pathway. In conditions of oxidative stress, the transsulphuration pathway is favored over the methionine cycle. Periodontitis is associated with oxidative stress. These data suggest a paradigm shifting mechanism linking epigenetic changes in gene methylation with the long term association between periodontitis and atherosclerotic CVD.

Biomarkers of the transsulfuration pathway and risk of renal cell carcinoma in the European Prospective Investigation into Cancer and Nutrition (EPIC) study.

Previous studies have suggested that components of one‐carbon metabolism, particularly circulating vitamin B6, have an etiological role in renal cell carcinoma (RCC). Vitamin B6 is a cofactor in the transsulfuration pathway. We sought to holistically investigate the role of the transsulfuration pathway in RCC risk. We conducted a nested case‐control study (455 RCC cases and 455 matched controls) within the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Plasma samples from the baseline visit were analyzed for metabolites of the transsulfuration pathway, including pyridoxal 5′‐phosphate (PLP, the biologically active form of vitamin B6), homocysteine, serine, cystathionine, and cysteine, in addition to folate. Bayesian conditional logistic regression was used to estimate associations of metabolites with RCC risk as well as interactions with established RCC risk factors. Circulating PLP and cysteine were inversely associated with RCC risk, and these associations were not attenuated after adjustment for other transsulfuration metabolites (odds ratio (OR) and 90% credible interval (CrI) per 1 SD increase in log concentration: 0.76 [0.66, 0.87]; 0.81 [0.66, 0.96], respectively). A comparison of joint metabolite profiles suggested substantially greater RCC risk for the profile representative of low overall transsulfuration function compared to high function (OR 2.70 [90% CrI 1.26, 5.70]). We found some statistical evidence of interactions of cysteine with body mass index, and PLP and homocysteine with smoking status, on their associations with RCC risk. In conclusion, we found evidence suggesting that the transsulfuration pathway may play a role in metabolic dysregulation leading to RCC development.

Down-Regulated Expression of Cystathionine β-Synthase and Cystathionine γ-Lyase in Varicocele, and Infertile Men: A Case-Control Study.

Objective Cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) are two important enzymes involved in One-Carbon metabolism. These enzymes play important roles in modulating oxidative stress and inflammation in male factor infertility through participating in the synthesis of glutathione (GSH) antioxidants in the trans-sulfuration pathway. Besides, the direct release of hydrogen sulfide (H2S) has anti-inflammatory and antioxidant effects. Therefore, the expression of CBS and CSE genes at mRNA levels in infertile and varicocele men was evaluated and compared to the healthy counterparts to clarify their possible role in the pathology of male infertility. Materials and Methods In this case-control study, semen parameter assessment (concentration, morphology, and motility of sperms) was performed on 28 men with varicocele, 43 infertile men with abnormal sperm parameters, and 19 fertile men. RNA was extracted from sperm samples followed by cDNA synthesis and real-time polymerase chain reaction (PCR) using CBS, CSE, and GAPDH primers. Results Sperm concentration and motility in infertile and varicocele groups were significantly lower (P=0.001), while spermatoza normal morphology was higher than fertile group (P=0.05). The expression levels of both CBS and CSE genes in infertile (P=0.04 and P=0.037 respectively) and varicocele (P=0.01 and P=0.046 respectively) groups were significantly lower than fertile group. Additionally, CBS gene expression indicated a positive correlation with expression of CSE gene (r=0.296, P=0.025) and sperm parameters. ConclusionIn light of our findings, there is a valid rationale to consider the primary role of CBS and CSE enzymes impairment in male factor infertility which specifically may point to a deficit in the release of essential antioxidants including the H2S as a molecular basis of infertility and warrants further investigation.

Simultaneous determination of serum homocysteine, cysteine, and methionine in patients with schizophrenia by liquid chromatography-tandem mass spectrometry.

Schizophrenia is a debilitating psychiatric disorder affecting approximately 1% of the population worldwide. Disturbances in the homocysteine metabolism are an important factor in the pathophysiology of schizophrenia. In this research, a novel validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification procedure was developed to investigate three significant compounds of homocysteine metabolism: homocysteine, cysteine, and methionine in patients with schizophrenia and healthy controls. Sample preparation involved a reduction with dithiothreitol followed by protein precipitation, and the chromatographic runtime was 2min. The LC-MS/MS method was validated according to CLSI C62-A and the Chinese Guidance for Liquid Chromatography and Mass Spectrometry Clinical Application. The performance of the method was excellent with a coefficient of variation for precision in the range of 0.5-6.9%, an accuracy of 90.4-101.6%. In addition, the practical applicability of the method was demonstrated by applying it in the routine sample analysis for a schizophrenic patient. Increased homocysteine levels and decreased cysteine levels were observed in the patient with schizophrenia. These results indicate that the activity of the transsulfuration pathway may play a key role in the pathogenesis of schizophrenia.

The critical role and molecular mechanisms of ferroptosis in antioxidant systems: a narrative review.

Background and ObjectiveFerroptosis is a recently discovered form of cell death which differs from other forms of cell death in terms of morphology, biochemistry, and regulatory mechanisms. Ferroptosis is regulated by a complex system and the precise molecular mechanisms are still being elucidated. Over the past few years, extensive research has revealed that the essence of ferroptosis is iron-dependent accumulation of lipid hydroperoxides induced by oxidative stress, and the System Xc-glutathione (GSH)-glutathione peroxidase 4 (GPX4) pathway is the main ferroptosis prevention system. Meanwhile, other antioxidant systems have also been implicated in regulating ferroptosis, including the transsulfuration pathway, mevalonate pathway, ferroptosis inhibitory protein 1 (FSP1)-Coenzyme Q10 (CoQ10) pathway, dihydroorotate dehydrogenase (DHODH)-dihydroubiquione (CoQH2) pathway, and GTP cyclohydrolase-1 (GCH1)-tetrahydrobiopterin (BH4) pathway. This article reviews the molecular mechanisms of ferroptosis and its critical role in antioxidant systems, aiming to reveal that antioxidation is an important method of inhibiting ferroptosis and to provide a new direction for the treatment of ferroptosis-related diseases.MethodsWe searched all original papers and reviews about the molecular mechanisms of ferroptosis in antioxidant systems using PubMed to November 2021. The search terms used included: ‘ferroptosis’, ‘ferroptosis inducers’, ‘ferroptosis inhibitors’, ‘ferroptosis and GSH’, ‘ferroptosis and GPX4’, ‘ferroptosis and System Xc-’, ‘SLC7A11’, ‘P53’, ‘NRF2 and ferroptosis’, ‘iron metabolism’, ‘lipid peroxidation’, ‘antioxidant systems’, ‘transsulfuration pathway’, ‘mevalonate pathway’, ‘FSP1-CoQ10’, ‘DHODH-CoQH2’, and ‘GCH1-BH4’.Key Content and FindingsWe first introduced the origin of ferroptosis and its common inhibitors and inducers. Next, we discussed the molecular mechanisms of ferroptosis and its role in antioxidant systems in existing studies. Finally, we briefly summarized the relationship between ferroptosis and diseases. It reveals that antioxidation is an important method of inhibiting ferroptosis.ConclusionsThis review discusses the recent rapid progress in the understanding of the molecular mechanisms of ferroptosis and its role in several antioxidant systems.

Nitrous oxide abuse in the emergency practice, and Review of toxicity mechanisms and potential markers

Nitrous oxide (N2O) toxicity is a concern common to several medical fields. Here, retrospective study of four N2O abuses with neurological signs in the emergency practice provides a preliminary basis for a metabolic Discussion/Review. This latter highlights N2O abuse as pathology of DNA/RNA/protein methylations, for instance consistent with impairments of protein arginine methyltransferases involved in myelinogenesis and myelopathy in patients. Basically, pathogenesis starts with oxidation by N2O of coordinated cobalamine cobalt ions at enzyme sites with impairments of vitamin-B12-dependent pathways. Methionine synthase (methylcobalamine) and methymalonyl-CoA mutase (adenosylcobalamine) are inactivated and cofactor-depleted, respectively. The number of impacted pathways (folate cycle, methylation cycle, S-adenosylmethionine-dependent methyltransferases, transulfuration pathway, Krebs cycle fueling by methylmalonyl-CoA, glutathione synthesis) explains the variety of potential research/laboratory markers, and may provide new clues and future angles to explore N2O toxicity. Overall, homocysteine measurements obviously help diagnosis of N2O abuses. Additional markers may include vitamin-B12, methionine, methylmalonate, dimethylglycine, sarcosine, S-adenosylmethionine to S-adenosylhomocysteine ratio, various S-adenosylamino acids, S-adenosylmethionine-dependent cellular methylations, and additional analytes (propionylcarnitine, propionylglycine, cystathionine and derived metabolites, methylated amino acids [eg arginine], betaine).

MYCN upregulates the transsulfuration pathway to suppress the ferroptotic vulnerability in MYCN-amplified neuroblastoma.

Ferroptosis is an iron-dependent, oxidative form of cell death that is countered mainly by glutathione peroxidase 4 (GPX4) and the production of glutathione (GSH), which is formed from cysteine. The identification of the cancers that may benefit from pharmacological ferroptotic induction is just emerging. We recently demonstrated that inducing ferroptosis genetically or pharmacologically in MYCN-amplified neuroblastoma (NB) is a novel and effective way to kill these cells. MYCN increases iron metabolism and subsequent hydroxyl radicals through increased expression of the transferrin receptor 1 (TfR1) and low levels of the ferroportin receptor. To counter increased hydroxyl radicals, MYCN binds to the promoter of SLC3A2 (solute carrier family 3 member 2). SLC3A2 is a subunit of system Xc-, which is the cysteine-glutamate antiporter that exports glutamate and imports cystine. Cystine is converted to cysteine intracellularly. Here, we investigated other ways MYCN may increase cysteine levels. By performing metabolomics in a syngeneic NB cell line either expressing MYCN or GFP, we demonstrate that the transsulfuration pathway is activated by MYCN. Furthermore, we demonstrate that MYCN-amplified NB cell lines and tumors have higher levels of cystathionine beta-synthase (CBS), the rate-limiting enzyme in transsulfuration, which leads to higher levels of the thioether cystathionine (R-S-(2-amino-2-carboxyethyl)-l-homocysteine). In addition, MYCN-amplified NB tumors have high levels of methylthioadenosine phosphorylase (MTAP), an enzyme that helps salvage methionine following polyamine metabolism. MYCN directly binds to the promoter of MTAP. We propose that MYCN orchestrates both enhanced cystine uptake and enhanced activity of the transsulfuration pathway to counteract increased reactive oxygen species (ROS) from iron-induced Fenton reactions, ultimately contributing to a ferroptosis vulnerability in MYCN-amplified neuroblastoma.

Alteration of the dietary methionine: Cysteine ratio modulates the inflammatory response to an inter-peritoneal injection of lipopolysaccharide in wistar rats

Sulphur amino acids (SAA) are essential for multiple physiological/metabolic processes, with the ratio of dietary methionine: cysteine (Met:Cys) being an important contributor to pro-inflammatory responses, including TNF-α activity. The current study was designed to determine the effect an altered dietary SAA ratio, and the resulting reliance on the transsulfuration pathway to supply Cys, will have on the inflammatory response. In the present study, 100 µg/kg of an intraperitoneal (IP) injection of lipopolysaccharide (LPS) was used as a model for systemic inflammation. Male Wistar rats were randomized to one of two amino acid-defined diets, (100Met:0Cys or 50Met:50Cys) and subdivided to receive either IP LPS or saline injections. LPS significantly increased total plasma Cys, homocysteine (Hcy) and glutathione (GSH) 240 min post-IP injection in rats fed a 50Met:50Cys ratio compared to other treatments. The TNF-α area under the curve for LPS-treated rats consuming a dietary 50Met:50Cys ratio was significantly higher (P < .004) compared to those consuming a dietary 100Met:0Cys ratio. A significant increase in the percentage of leukocytes that were neutrophils was observed in rats injected with LPS when compared to saline with no effect of diet. These results demonstrate that an alteration of the dietary Met:Cys ratio did not attenuate the inflammatory response to an IP injection of LPS in Wistar rats; however, a diet with a balanced Met:Cys ratio increased concentrations of Cys and GSH which may result in a more rapid response to an LPS challenge.

One-Carbon Metabolism: Pulling the Strings behind Aging and Neurodegeneration.

One-carbon metabolism (OCM) is a network of biochemical reactions delivering one-carbon units to various biosynthetic pathways. The folate cycle and methionine cycle are the two key modules of this network that regulate purine and thymidine synthesis, amino acid homeostasis, and epigenetic mechanisms. Intersection with the transsulfuration pathway supports glutathione production and regulation of the cellular redox state. Dietary intake of micronutrients, such as folates and amino acids, directly contributes to OCM, thereby adapting the cellular metabolic state to environmental inputs. The contribution of OCM to cellular proliferation during development and in adult proliferative tissues is well established. Nevertheless, accumulating evidence reveals the pivotal role of OCM in cellular homeostasis of non-proliferative tissues and in coordination of signaling cascades that regulate energy homeostasis and longevity. In this review, we summarize the current knowledge on OCM and related pathways and discuss how this metabolic network may impact longevity and neurodegeneration across species.

Cysteine is a limiting factor for glioma proliferation and survival.

Nutritional intervention is becoming more prevalent as adjuvant therapy for many cancers in view of the tumor dependence on external sources for some nutrients. However, little is known about the mechanisms that make cancer cells require certain nutrients from the microenvironment. Herein, we report the dependence of glioma cells on exogenous cysteine/cystine, despite this amino acid being nonessential. Using several 13C‐tracers and analysis of cystathionine synthase and cystathioninase levels, we revealed that glioma cells were not able to support glutathione synthesis through the transsulfuration pathway, which allows methionine to be converted to cysteine in cysteine/cystine‐deprived conditions. Therefore, we explored the nutritional deprivation in a mouse model of glioma. Animals subjected to a cysteine/cystine‐free diet survived longer, although this increase did not attain statistical significance, with concomitant reductions in plasma glutathione and cysteine levels. At the end point, however, tumors displayed the ability to synthesize glutathione, even though higher levels of oxidative stress were detected. We observed a compensation from the nutritional intervention revealed as the recovery of cysteine‐related metabolite levels in plasma. Our study highlights a time window where cysteine deprivation can be exploited for additional therapeutic strategies.

Profiling Cystathionine β/γ-Lyase in Complex Biosamples Using Novel Activatable Fluorogens.

Cystathionine lyase, the key enzyme in transsulfuration and reverse transsulfuration pathways, is involved in a wide array of physiological and pathophysiological processes in both mammals and nonmammals. Though the biological significance of the hydrogen sulfide/cystathionine lyase system in disease states is extensively discussed, the absence of molecular methods for direct monitoring of cystathionine lyase in complex biosamples renders the result unreliable and perplexing. Here, we present the first attempt at designing and developing effective activatable fluorescent probes for cystathionine lyase based on the naphthylamide scaffold. CBLP and CSEP were designed based on the catalytic preference of cystathionine β-lyase (CBL) and cystathionine γ-lyase (CSE). Briefly, incorporation of cysteine/homocysteine as the recognition moiety and a carbamate ethyl sulfide group as a self-immolated linker proved to be an effective strategy for cystathionine lyase fluorescence reporting. CBLP exhibits high selectivity and sensitivity in vitro in semiquantifying CBL levels in roots of wild-type Arabidopsis thaliana and cbl mutants (cbl knockout: SALK_014740C, overexpressed: OE-CBL). Meanwhile, CSEP successfully detected CSE levels in HCC-LM3 cells, zebrafish models, and upregulated CSE in frozen section slides from the liver tissue of cecal ligation and puncture (CLP)-induced septic rats, which was also validated by Western blotting and immunohistochemical analysis. In summary, the practical design strategy facilitates profiling of cystathionine lyase activity in biological processes. It may pave the way for the development of accurate and efficient methods for the direct estimation of cystathionine lyase.

Altered transsulfuration pathway enzymes and redox homeostasis in inherited retinal degenerative diseases

Retinal degenerative diseases result from apoptotic photoreceptor cell death. As endogenously produced gaseous molecules such as hydrogen sulfide (H2S) and nitric oxide (NO) play a key role in apoptosis, we compared the expression levels of genes and proteins involved in the production of these molecules in the retina of normal dogs and three canine models (rcd1, crd2, and xlpra2) of human inherited retinal degeneration (IRD). Using qRT-PCR, Western blot, and immunohistochemistry (IHC), we showed that mRNA and protein levels of cystathionine β–synthase (CBS), an enzyme that produces H2S in neurons, are increased in retinal degeneration, but those of cystathionine γ-lyase (CSE), an enzyme involved in the production of glutathione (GSH), an antioxidant, are not. Such findings suggest that increased levels of H2S that are not counterbalanced by increased antioxidant potential may contribute to disease in affected retinas. We also studied the expression of neuronal and inducible nitric oxide synthase (nNOS and iNOS), the enzymes responsible for NO production. Western blot and IHC results revealed increased levels of nNOS and iNOS, resulting in increased NO levels in mutant retinas. Finally, photoreceptors are rich in polyunsaturated fatty acids (PUFAs) that can make these cells vulnerable to oxidative damage through reactive oxygen species (ROS). Our results showed increased levels of acrolein and hydroxynonenal (4HNE), two main toxic products of PUFAs, surrounding the membranes of photoreceptors in affected canines. Increased levels of these toxic products, together with increased NO and ROS, likely render these cells susceptible to an intrinsic apoptotic pathway involving mitochondrial membranes. To assess this possibility, we measured the levels of BCL2, an anti-apoptotic protein in the mitochondrial membrane. Western blot results showed decreased levels of BCL2 protein in affected retinas. Overall, the results of this study identify alterations in the expression of enzymes directly involved in maintaining the normal redox status of the retina during retinal degeneration, thereby supporting future studies to investigate the role of H2S and NO in retinal degeneration and apoptosis.

Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2) Is Upregulated in Breast Epithelial–Mesenchymal Transition and Responds to Oxidative Stress

Breast cancer cells that have undergone partial epithelial–mesenchymal transition (EMT) are believed to be more invasive than cells that have completed EMT. To study metabolic reprogramming in different mesenchymal states, we analyzed protein expression following EMT in the breast epithelial cell model D492 with single-shot LFQ supported by a SILAC proteomics approach. The D492 EMT cell model contains three cell lines: the epithelial D492 cells, the mesenchymal D492M cells, and a partial mesenchymal, tumorigenic variant of D492 that overexpresses the oncogene HER2. The analysis classified the D492 and D492M cells as basal-like and D492HER2 as claudin-low. Comparative analysis of D492 and D492M to tumorigenic D492HER2 differentiated metabolic markers of migration from those of invasion. Glutamine-fructose-6-phosphate transaminase 2 (GFPT2) was one of the top dysregulated enzymes in D492HER2. Gene expression analysis of the cancer genome atlas showed that GFPT2 expression was a characteristic of claudin-low breast cancer. siRNA-mediated knockdown of GFPT2 influenced the EMT marker vimentin and both cell growth and invasion in vitro and was accompanied by lowered metabolic flux through the hexosamine biosynthesis pathway (HBP). Knockdown of GFPT2 decreased cystathionine and sulfide:quinone oxidoreductase (SQOR) in the transsulfuration pathway that regulates H2S production and mitochondrial homeostasis. Moreover, GFPT2 was within the regulation network of insulin and EGF, and its expression was regulated by reduced glutathione (GSH) and suppressed by the oxidative stress regulator GSK3-β. Our results demonstrate that GFPT2 controls growth and invasion in the D492 EMT model, is a marker for oxidative stress, and associated with poor prognosis in claudin-low breast cancer.

Molecular characterization and immune protection by cystathionine β-synthase from Eimeria tenella.

Eimeria tenella is an obligate intracellular apicomplexan parasite that causes avian coccidiosis and leads to severe economic losses in the global poultry industry. Cystathionine β-synthase (CBS) and cystathionine γ-lyase (CGL) act together to generate H2S in the reverse transsulfuration pathway. In this study, E.tenella Cystathionine β-synthase (EtCBS) was cloned using rapid amplification of cDNA 5'-ends (5'RACE) and characterized, and its immunoprotective effects were evaluated. The recombinant EtCBS protein (rEtCBS) was expressed and successfully recognized by anti-sporozoites (Spz) protein rabbit serum. EtCBS mRNA levels were highest in Spz by qPCR, and the protein expression levels were higher in unsporulated oocysts (UO) than in other stages by Western blot. Indirect immunofluorescence showed that EtCBS protein was found on the surface of Spz and second-generation merozoites (Mrz). The invasion inhibition assays showed that rabbit anti-rEtCBS polyclonal antibodies effectively inhibited parasite invasion host cells. Chickens immunized with rEtCBS protein showed prominently increased weight gains and decreased oocyst output compared to nonimmunized and infected control group. The results suggest that EtCBS could be a potential vaccine candidate against E.tenella.

The betaine-dependent remethylation pathway is a homocysteine metabolism pathway associated with the carnivorous feeding habits of spiders.

Homocysteine (Hcy) is a sulfur-containing amino acid derived from the essential amino acid methionine (Met). Circulating levels of Hcy in animals can be increased by feeding on Met-enriched diets, which is generally considered harmful. Spiders are one of the largest groups of obligate carnivores and feed on animals high in protein and Met. We analyzed the Hcy metabolism pathways in 18 species of 3 taxa (Mammalia, Insecta, and Arachnida) and found that the betaine-dependent remethylation pathway (BRP) was present in all carnivorous arachnid species and mammals but absent in insects and red spider mites. We then studied the Hcy metabolism pathway in Pardosa pseudoannulata. In P. pseudoannulata, Hcy is metabolized through the transsulfuration pathway, BRP, and S-methylmethionine-dependent remethylation pathway. Because of a prior duplication event of the betaine homocysteine S-methyltransferase (BHMT) gene in the BRP, BHMTa and BHMTb are present in tandem in the genome of P. pseudoannulata. The high expression levels of BHMTa and its high abundance in detoxification tissues indicate that it plays an important role in the BRP; the ability of BHMTa and BHMTb to remethylate Hcy using betaine as substrate was similar. Compared with other Hcy metabolic enzyme genes, BHMT responded quickly to the application of Hcy or betaine. In sum, the BRP is important in Hcy metabolism in P. pseudoannulata and in other spider species.

S-Adenosyl methionine administration enhances nitrosative stress in acute pancreatitis leading to blockade of the trans-sulfuration pathway and accumulation of homocysteine

S-Adenosyl methionine administration enhances nitrosative stress in acute pancreatitis leading to blockade of the trans-sulfuration pathway and accumulation of homocysteine