Should I stay or should I go? Transsulfuration influences invasion and growth in glioblastoma.

A major challenge in treating patients with glioblastoma is the inability to eliminate highly invasive cells with chemotherapy, radiation, or surgical resection. As cancer cells face the issue of replicating or invading neighboring tissue, they rewire their metabolism in a concerted effort to support necessary cellular processes and account for altered nutrient abundance. In this issue of the JCI, Garcia et al. compared an innovative 3D hydrogel-based invasion device to regional patient biopsies through a comprehensive multiomics-based approach paired with a CRISPR knockout screen. Their findings elucidate a role for cystathionine γ-lyase (CTH), an enzyme in the transsulfuration pathway, as a means of regulating the cellular response to oxidative stress. CTH-mediated conversion of cystathionine to cysteine was necessary for regulating reactive oxygen species to support invasion. Meanwhile, inhibition of CTH suppressed the invasive glioblastoma phenotype. However, inhibiting CTH resulted in a larger overall tumor mass. These findings suggest that targeting the transsulfuration pathway may serve as a means of redirecting glioblastoma to proliferate or invade.

The role of the mitochondrial trans-sulfuration in cerebro-cardio renal dysfunction during trisomy down syndrome.

One in 700 children is born with the down syndrome (DS). In DS, there is an extra copy of X chromosome 21 (trisomy). Interestingly, the chromosome 21 also contains an extra copy of the cystathionine beta synthase (CBS) gene. The CBS activity is known to contribute in mitochondrial sulfur metabolism via trans-sulfuration pathway. We hypothesize that due to an extra copy of the CBS gene there is hyper trans-sulfuration in DS. We believe that understanding the mechanism of hyper trans-sulfuration during DS will be important in improving the quality of DS patients and towards developing new treatment strategies. We know that folic acid "1-carbon" metabolism (FOCM) cycle transfers the "1-carbon" methyl group to DNA (H3K4) via conversion of s-adenosyl methionine (SAM) to s-adenosyl homocysteine (SAH) by DNMTs (the gene writers). The demethylation reaction is carried out by ten-eleven translocation methylcytosine dioxygenases (TETs; the gene erasers) through epigenetics thus turning the genes off/on and opening the chromatin by altering the acetylation/HDAC ratio. The S-adenosyl homocysteine hydrolase (SAHH) hydrolyzes SAH to homocysteine (Hcy) and adenosine. The Hcy is converted to cystathionine, cysteine and hydrogen sulfide (H2S) via CBS/cystathioneγ lyase (CSE)/3-mercaptopyruvate sulfurtransferase (3MST) pathways. Adenosine by deaminase is converted to inosine and then to uric acid. All these molecules remain high in DS patients. H2S is a potent inhibitor of mitochondrial complexes I-IV, and regulated by UCP1. Therefore, decreased UCP1 levels and ATP production can ensue in DS subjects. Interestingly, children born with DS show elevated levels of CBS/CSE/3MST/Superoxide dismutase (SOD)/cystathionine/cysteine/H2S. We opine that increased levels of epigenetic gene writers (DNMTs) and decreased in gene erasers (TETs) activity cause folic acid exhaustion, leading to an increase in trans-sulphuration by CBS/CSE/3MST/SOD pathways. Thus, it is important to determine whether SIRT3 (inhibitor of HDAC3) can decrease the trans-sulfuration activity in DS patients. Since there is an increase in H3K4 and HDAC3 via epigenetics in DS, we propose that sirtuin-3 (Sirt3) may decrease H3K4 and HDAC3 and hence may be able to decrease the trans-sulfuration in DS. It would be worth to determine whether the lactobacillus, a folic acid producing probiotic, mitigates hyper-trans-sulphuration pathway in DS subjects. Further, as we know that in DS patients the folic acid is exhausted due to increase in CBS, Hcy and re-methylation. In this context, we suggest that folic acid producing probiotics such as lactobacillus might be able to improve re-methylation process and hence may help decrease the trans-sulfuration pathway in the DS patients.

Genetic and Pharmacological Modulation of Cellular Proteostasis Leads to Partial Functional Rescue of Homocystinuria-Causing Cystathionine-Beta Synthase Variants.

Homocystinuria (HCU), an inherited metabolic disorder caused by lack of cystathionine beta-synthase (CBS) activity, is chiefly caused by misfolding of single amino acid residue missense pathogenic variants. Previous studies showed that chemical, pharmacological chaperones or proteasome inhibitors could rescue function of multiple pathogenic CBS variants; however, the underlying mechanisms remain poorly understood. Using Chinese hamster DON fibroblasts devoid of CBS and stably overexpressing human WT or mutant CBS, we showed that expression of pathogenic CBS variant mostly dysregulates gene expression of small heat shock proteins HSPB3 and HSPB8 and members of HSP40 family. Endoplasmic reticulum stress sensor BiP was found upregulated with CBS I278T variant associated with proteasomes suggesting proteotoxic stress and degradation of misfolded CBS. Co-expression of the main effector HSP70 or master regulator HSF1 rescued steady-state levels of CBS I278T and R125Q variants with partial functional rescue of the latter. Pharmacological proteostasis modulators partially rescued expression and activity of CBS R125Q likely due to reduced proteotoxic stress as indicated by decreased BiP levels and promotion of refolding as indicated by induction of HSP70. In conclusion, targeted manipulation of cellular proteostasis may represent a viable therapeutic approach for the permissive pathogenic CBS variants causing HCU.

Activating transcription factor (ATF) 6 upregulates cystathionine ß synthetase (CBS) expression and hydrogen sulfide (H2S) synthesis to ameliorate liver metabolic damage.

Activating transcription factor 6 (ATF6) is an endoplasmic reticulum stress responsive gene. We previously reported that conditional knockout of hepatic ATF6 exacerbated liver metabolic damage by repressing autophagy through mTOR pathway. However, the mechanism by which ATF6 influence liver metabolism has not been well established. Hydrogen sulfide (H2S) is a gaseous signaling molecule that plays an important role in regulating inflammation, and suppress nonalcoholic fatty liver in mice. Based on the previous study, we assumed that ATF6 may regulate H2S production to participate in liver metabolism. In order to clarify the mechanism by which ATF6 regulates H2S synthesis to ameliorate liver steatosis and inflammatory environment, we conducted the present study. We used the liver specific ATF6 knockout mice and fed on high-fat-diet, and found that H2S level was significantly downregulated in hepatic ATF6 knockout mice. Restoring H2S by the administration of slow H2S releasing agent GYY4137 ameliorated the hepatic steatosis and glucose tolerance. ATF6 directly binds to the promoter of cystathionine ß synthetase (CBS), an important enzyme in H2S synthesis. Thus, ATF6 could upregulate H2S production through CBS. Sulfhydrated Sirtuin-1 (SIRT1) was downregulated in ATF6 knockout mice. The expression of pro-inflammatory factor IL-17A was upregulated and anti-inflammatory factor IL-10 was downregulated in ATF6 knockout mice. Our results suggest that ATF6 can transcriptionally enhance CBS expression as well as H2S synthesis. ATF6 increases SIRT1 sulfhydration and ameliorates lipogenesis and inflammation in the fatty liver. Therefore, ATF6 could be a novel therapeutic strategy for high-fat diet induced fatty liver metabolic abnormalities.

A role for the cystathionine-ß-synthase /H2S axis in astrocyte dysfunction in the aging brain.

Astrocytic dysfunction is central to age-related neurodegenerative diseases. However, the mechanisms leading to astrocytic dysfunction are not well understood. We identify that among the diverse cellular constituents of the brain, murine and human astrocytes are enriched in the expression of CBS. Depleting CBS in astrocytes causes mitochondrial dysfunction, increases the production of reactive oxygen species (ROS) and decreases cellular bioenergetics that can be partially rescued by exogenous H2S supplementation or by re-expressing CBS. Conversely, the CBS/H2S axis, associated protein persulfidation and proliferation are decreased in astrocytes upon oxidative stress which can be rescued by exogenous H2S supplementation. Here we reveal that in the aging brain, the CBS/H2S axis is downregulated leading to decreased protein persulfidation, together augmenting oxidative stress. Our findings uncover an important protective role of the CBS/H2S axis in astrocytes that may be disrupted in the aged brain.

Body condition prepartum and its association with term placentome nutrient transporters, one­carbon metabolism pathway activity, and intermediate metabolites in Holstein cows.

We investigated linkages among BCS prior to calving and placentome concentrations of metabolites, proteins in one­carbon metabolism (OCM) and protein synthesis, and nutrient transport. Multiparous Holstein cows retrospectively divided by prepartal BCS at -4 weeks relative to parturition into high BCS (HBCS = 3.58 ± 0.23; n = 9) or normal BCS (NBCS = 3.02 ± 0.17; n = 13) were used. BCS was assessed using a 5-point scale (1 = thin, 5 = fat). Four placentomes per cow were collected at delivery and frozen in liquid N. Western blotting was used for protein abundance. Cystathionine-ß-synthase (CBS) and betaine-homocysteine-S-methyltransferase (BHMT) activity were measured via 14C assays. Amino acids (AA) and metabolites in OCM were measured by liquid chromatography mass spectrometry (LC-MS). Compared with NBCS cows, the cellular stress sensor p-eIF2α was more than 2-fold greater (P = 0.04) in HBCS. Abundance of the AA-catabolism enzyme branched-chain α-ketoacid dehydrogenase complex was lower (P = 0.05) in HBCS cows. Although BHMT activity did not differ, greater concentration of betaine (P = 0.01) and lower (P = 0.05) concentration of dimethylglycine in HBCS cows suggested reduced flux through the methionine cycle. Despite a lack of difference in CBS activity, lower concentrations of cystathionine (P = 0.03) and hypotaurine (P = 0.04) along with lower cysteine and the tendency for lower total GSH (P = 0.10) in HBCS cows suggested a decrease in transsulfuration. Overall, associations between OCM in placentomes and BCS at calving exist. Identifying mechanisms responsible for these effects merits further research.

Key substrate recognition residues in the active site of cystathionine beta-synthase from Toxoplasma gondii.

Cystathionine ß-synthase (CBS) catalyzes the condensation of l-serine and l-homocysteine to give l-cystathionine in the transsulfuration pathway. Recently, a few O-acetylserine (l-OAS)-dependent CBSs (OCBSs) have been found in bacteria that can exclusively function with l-OAS. CBS from Toxoplasma gondii (TgCBS) can efficiently use both l-serine and l-OAS to form l-cystathionine. In this work, a series of site-specific variants substituting S84, Y160, and Y246 with hydrophobic residues found at the same positions in OCBSs was generated to explore the roles of the hydroxyl moieties of these residues as determinants of l-serine/l-OAS preference in TgCBS. We found that the S84A/Y160F/Y246V triple mutant behaved like an OCBS in terms of both substrate requirements, showing ß-replacement activity only with l-OAS, and pH optimum, which is decreased by ~1 pH unit. Formation of a stable aminoacrylate upon reaction with l-serine is prevented by the triple mutation, indicating the importance of the H-bonds between the hydroxyl groups of Y160, Y246, and S84 with l-serine in formation of the intermediate. Analysis of the independent effect of each mutation on TgCBS activity and investigation of the protein-aminoacrylate complex structure allowed for the conclusion that the hydroxyl group of Y246 has a major, but not exclusive, role in controlling the l-serine preference by efficiently stabilizing its leaving group. These studies demonstrate that differences in substrate specificity of CBSs are controlled by natural variations in as few as three residue positions. A better understanding of substrate specificity in TgCBS will facilitate the design of new antimicrobial compounds.

Cystathionine-γ-lyase attenuates inflammatory response and pain of osteoarthritis.

The chronic articular disease osteoarthritis (OA) is characterized by osteophyte generation, subchondral bone remodeling, and cartilage deterioration. Low levels of H2S catalyzed by cystathionine-γ-lyase (CSE) encoded by Cthhas neuroprotective, cardioprotective, anti-apoptotic, and anti-inflammatory effects thus, Cth is being developed as a potential therapy for the management of the pathogenesis and symptoms of osteoarthritis. Single-cell RNA sequencing (scRNA-seq) and immunohistochemistry of human cartilage revealed that the expression of CTH was decreased in OA patients. We found that Cthoverexpression decrease IL-1ß-induced overactivation of the NF-κB signaling pathway. In vivo, Cthoverexpression relieved pain response and cartilage damage in the anterior cruciate ligament transection (ACLT) rat model. In vitro, CSE alleviated chondrocytes catabolism, inflammation, apoptosis, and senescence, and suppressed the NF-κB pathway. We postulate that CSE has therapeutic effects in suppressing inflammation and degeneration in OA and should be further investigated clinically.

The role of rhoA/rho-kinase and PKC in the inhibitory effect of L-cysteine/H2S pathway on the carbachol-mediated contraction of mouse bladder smooth muscle.

We investigated the role of RhoA/Rho-kinase (ROCK) and PKC in the inhibitory effect of L-cysteine/hydrogen sulfide (H2S) pathway on the carbachol-mediated contraction of mouse bladder smooth muscle. Carbachol (10-8-10-4 M) induced a concentration-dependent contraction in bladder tissues. L-cysteine (H2S precursor; 10-2 M) and exogenous H2S (NaHS; 10-3 M) reduced the contractions evoked by carbachol by ~ 49 and ~ 53%, respectively, relative to control. The inhibitory effect of L-cysteine on contractions to carbachol was reversed by 10-2 M PAG (~ 40%) and 10-3 M AOAA (~ 55%), cystathionine-gamma-lyase (CSE) and cystathionine-ß-synthase (CBS) inhibitor, respectively. Y-27632 (10-6 M) and GF 109203X (10-6 M), a specific ROCK and PKC inhibitor, respectively, reduced contractions evoked by carbachol (~ 18 and ~ 24% respectively), and the inhibitory effect of Y-27632 and GF 109203X on contractions was reversed by PAG (~ 29 and ~ 19%, respectively) but not by AOAA. Also, Y-27632 and GF 109203X reduced the inhibitory responses of L-cysteine on the carbachol-induced contractions (~ 38 and ~ 52% respectively), and PAG abolished the inhibitory effect of L-cysteine on the contractions in the presence of Y-27632 (~ 38%). Also, the protein expressions of CSE, CBS, and 3-MST enzymes responsible for endogenous H2S synthesis were detected by Western blot method. H2S level was increased by L-cysteine, Y-27632, and GF 109203X (from 0.12 ± 0.02 to 0.47 ± 0.13, 0.26 ± 0.03, and 0.23 ± 0.06 nmol/mg respectively), and this augmentation in H2S level decreased with PAG (0.17 ± 0.02, 0.15 ± 0.03, and 0.07 ± 0.04 nmol/mg respectively). Furthermore, L-cysteine and NaHS reduced carbachol-induced ROCK-1, pMYPT1, and pMLC20 levels. Inhibitory effects of L-cysteine on ROCK-1, pMYPT1, and pMLC20 levels, but not of NaHS, were reversed by PAG. These results suggest that there is an interaction between L-cysteine/H2S and RhoA/ROCK pathway via inhibition of ROCK-1, pMYPT1, and pMLC20, and the inhibition of RhoA/ROCK and/or PKC signal pathway may be mediated by the CSE-generated H2S in mouse bladder.

Cystathionine-ß-synthase X proteins negatively regulate NADPH-thioredoxin reductase C activity.

Redox regulation is a posttranslational modification based on the redox reaction of protein thiols. A small ubiquitous protein thioredoxin (Trx) plays a central role in redox regulation, but a unique redox-regulatory factor called NADPH-Trx reductase C (NTRC) is also found in plant chloroplasts and some cyanobacteria. Several important functions of NTRC have been suggested, but the mechanism for controlling NTRC activity remains undetermined. Cystathionine-ß-synthase X (CBSX) proteins have been previously shown to interact with NTRC physically. Based on these observations, this study biochemically investigated the functional interaction between CBSX proteins and NTRC from Arabidopsis thaliana in vitro. Consequently, we concluded that CBSX proteins act as negative regulators of NTRC in the presence of AMP.

[The Effect of the Knockout of Major Transsulfuration Genes on the Pattern of Protein Synthesis in D. melanogaster].

The enzymes involved in the transsulfuration pathway and hydrogen sulfide production-cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) - play an important cytoprotective role in the functioning of the organism. Using CRISPER/Cas9 technology, we obtained Drosophila strains with deleted cbs, cse, and mst genes as well as with double deletion of cbs and cse genes. We analyzed the effect of these mutations on the pattern of protein synthesis in the salivary glands of third instar larvae and in the ovaries of mature flies. In the salivary glands of strains with cbs and cse deletions, a decrease was found in the accumulation of the FBP2 storage protein containing 20% methionine amino acid residues. In the ovaries, changes were detected in the level of expression and isofocusing points of proteins involved in cell protection against oxidative stress, hypoxia, and protein degradation. It was shown that in the strains with deletions of transsulfuration enzymes the proteins have a similar degree of oxidation to that of the control strain. A decrease in the total number of proteasomes and their activity was found in the strains with deletions of the cbs and cse genes.

Structural basis of the inhibition of cystathionine γ-lyase from Toxoplasma gondii by propargylglycine and cysteine.

Cystathionine γ-lyase (CGL) is a PLP-dependent enzyme that catalyzes the last step of the reverse transsulfuration route for endogenous cysteine biosynthesis. The canonical CGL-catalyzed process consists of an α,γ-elimination reaction that breaks down cystathionine into cysteine, α-ketobutyrate, and ammonia. In some species, the enzyme can alternatively use cysteine as a substrate, resulting in the production of hydrogen sulfide (H2 S). Importantly, inhibition of the enzyme and consequently of its H2 S production activity, makes multiresistant bacteria considerably more susceptible to antibiotics. Other organisms, such as Toxoplasma gondii, the causative agent of toxoplasmosis, encode a CGL enzyme (TgCGL) that almost exclusively catalyzes the canonical process, with only minor reactivity to cysteine. Interestingly, the substitution of N360 by a serine (the equivalent amino acid residue in the human enzyme) at the active site changes the specificity of TgCGL for the catalysis of cystathionine, resulting in an enzyme that can cleave both the CγS and the CßS bond of cystathionine. Based on these findings and to deepen the molecular basis underlying the enzyme-substrate specificity, we have elucidated the crystal structures of native TgCGL and the variant TgCGL-N360S from crystals grown in the presence of cystathionine, cysteine, and the inhibitor d,l-propargylglycine (PPG). Our structures reveal the binding mode of each molecule within the catalytic cavity and help explain the inhibitory behavior of cysteine and PPG. A specific inhibitory mechanism of TgCGL by PPG is proposed.

Dysregulated cystathionine-ß-synthase/hydrogen sulfide signaling promotes chronic stress-induced colonic hypermotility in rats.

BACKGROUND: Hydrogen sulfide (H2 S), an important endogenous gasotransmitter, is involved in the modulation of gastrointestinal motility, but whether it mediates the intestinal dysmotility in irritable bowel syndrome (IBS) is not known. This study explored the significance of cystathionine-ß-synthase (CBS)/H2 S signaling in stress-induced colonic dysmotility. METHODS: A rat model of IBS was established using chronic water avoidance stress (WAS). Colonic pathological alterations were detected histologically. Intestinal motility was determined by intestinal transit time (ITT) and fecal water content (FWC). Visceral sensitivity was assessed using the visceromotor response (VMR) to colorectal distension (CRD). Real-time PCR, Western blotting, and immunostaining were performed to identify the expression of CBS in the colon. The contractions of distal colon were studied in an organ bath system and H2 S content was measured by ELISA. The effects of SAM, a selective CBS activator, on colonic dysmotility were examined. MEK1 was tested as a potential upstream effector of CBS/H2 S loss. KEY RESULTS: After 10 days of WAS, the ITT was decreased and FWC was increased, and the VMR magnitude in response to CRD was enhanced. The colonic CBS expression and H2 S levels were significantly declined in WAS-exposed rats, and the density of CBS-positive enteric neurons in the myenteric plexus in WAS-treated rats was lower than that in controls. SAM treatment relieved WAS-induced colonic hypermotility via increased H2 S production. AZD6244, a selective inhibitor of MEK1, partially reversed CBS downregulation and colonic hypermotility in WAS-treated rats. CONCLUSIONS & INFERENCES: Decreased CBS/H2 S signaling through increased MEK1 signaling might be important in the pathogenesis of chronic stress-induced colonic hypermotility. SAM could be administered for disorders associated with intestinal hypermotility.

Plasma methionine metabolic profile is associated with longevity in mammals.

Methionine metabolism arises as a key target to elucidate the molecular adaptations underlying animal longevity due to the negative association between longevity and methionine content. The present study follows a comparative approach to analyse plasma methionine metabolic profile using a LC-MS/MS platform from 11 mammalian species with a longevity ranging from 3.5 to 120 years. Our findings demonstrate the existence of a species-specific plasma profile for methionine metabolism associated with longevity characterised by: i) reduced methionine, cystathionine and choline; ii) increased non-polar amino acids; iii) reduced succinate and malate; and iv) increased carnitine. Our results support the existence of plasma longevity features that might respond to an optimised energetic metabolism and intracellular structures found in long-lived species.

Water soluble selenometabolome of Cardamine violifolia.

Low molecular weight selenium containing metabolites in the leaves of the selenium hyperaccumulator Cardamine violifolia (261 mg total Se per kg d.w.) were targeted in this study. One dimensional cation exchange chromatography coupled to ICP-MS was used for purification and fractionation purposes prior to LC-Unispray-QTOF-MS analysis. The search for selenium species in full scan spectra was assisted with an automated mass defect based filtering approach. Besides selenocystathionine, selenohomocystine and its polyselenide derivative, a total number of 35 water soluble selenium metabolites other than selenolanthionine were encountered, including 30 previously unreported compounds. High occurrence of selenium containing hexoses was observed, together with the first assignment of N-glycoside derivatives of selenolanthionine. Quantification of the most abundant selenium species, selenolanthionine, was carried out with an ion pairing LC - post column isotope dilution ICP-MS setup, which revealed that this selenoamino acid accounted for 30% of the total selenium content of the leaf (78 mg (as Se) per kg d.w.).

Catalytic specificity of the Lactobacillus plantarum cystathionine γ-lyase presumed by the crystallographic analysis.

The reverse transsulfuration pathway, which is composed of cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CGL), plays a role to synthesize L-cysteine using L-serine and the sulfur atom in L-methionine. A plant-derived lactic acid bacterium Lactobacillus plantarum SN35N has been previously found to harbor the gene cluster encoding the CBS- and CGL-like enzymes. In addition, it has been demonstrated that the L. plantarum CBS can synthesize cystathionine from O-acetyl-L-serine and L-homocysteine. The aim of this study is to characterize the enzymatic functions of the L. plantarum CGL. We have found that the enzyme has the high γ-lyase activity toward cystathionine to generate L-cysteine, together with the ß-lyase activity toward L-cystine to generate L-cysteine persulfide. By the crystallographic analysis of the inactive CGL K194A mutant complexed with cystathionine, we have found the residues which recognize the distal amino and carboxyl groups of cystathionine or L-cystine. The PLP-bound substrates at the active site may take either the binding pose for the γ- or ß-elimination reaction, with the former being the major reaction in the case of cystathionine.

A hypomorphic cystathionine ß-synthase gene contributes to cavefish eye loss by disrupting optic vasculature.

Vestigial structures are key indicators of evolutionary descent, but the mechanisms underlying their development are poorly understood. This study examines vestigial eye formation in the teleost Astyanax mexicanus, which consists of a sighted surface-dwelling morph and multiple populations of blind cave morphs. Cavefish embryos initially develop eyes, but they subsequently degenerate and become vestigial structures embedded in the head. The mutated genes involved in cavefish vestigial eye formation have not been characterized. Here we identify cystathionine ß-synthase a (cbsa), which encodes the key enzyme of the transsulfuration pathway, as one of the mutated genes responsible for eye degeneration in multiple cavefish populations. The inactivation of cbsa affects eye development by increasing the transsulfuration intermediate homocysteine and inducing defects in optic vasculature, which result in aneurysms and eye hemorrhages. Our findings suggest that localized modifications in the circulatory system may have contributed to the evolution of vestigial eyes in cavefish.

Fermentative production of sulfur-containing amino acid with engineering putative l-cystathionine and l-cysteine uptake systems in Escherichia coli.

Here, proteins involved in sulfur-containing amino acid uptake in Escherichia coli strains were investigated with the aim of applying the findings in fermentative amino acid production. A search of genes in an l-methionine auxotrophic strain library suggested YecSC as the putative transporter of l-cystathionine. l-Methionine production increased by 15% after amplification of yecSC in producer strains. A candidate protein responsible for l-cysteine uptake was also found by experimentation with multicopy suppressor E. coli strains that recovered from growth defects caused by l-cysteine auxotrophy. Based on the results of an uptake assay, growth using l-cysteine as a sole sulfur source, and sensitivity to l-cysteine toxicity, we proposed that YeaN is an l-cysteine transporter. l-Cysteine production increased by 50% as a result of disrupting yeaN in producer strain. The study of amino acid transporters is valuable to industrialized amino acid production and also sheds light on the role of these transporters in sulfur assimilation.

Blockade of the trans-sulfuration pathway in acute pancreatitis due to nitration of cystathionine ß-synthase.

Acute pancreatitis is an inflammatory process of the pancreatic gland that may lead to dysregulation of the trans-sulfuration pathway. The aims of this work were firstly to study the methionine cycle as well as the trans-sulfuration pathway using metabolomic and proteomic approaches identifying the causes of this dysregulation in an experimental model of acute pancreatitis; and secondly to reveal the effects of S-adenosylmethionine administration on these pathways. Acute pancreatitis was induced by cerulein in mice, and a group of animals received S-adenosylmethionine treatment. Cerulein-induced acute pancreatitis rapidly caused marked depletion of methionine, S-adenosylmethionine, 5'-methylthioadenosine, cystathionine, cysteine, and glutathione levels in pancreas, but S-adenosylhomocysteine and homocysteine remained unchanged. Protein steady-state levels of S-adenosylhomocysteine-hydrolase and cystathionine gamma-lyase diminished but methylthioadenosine phosphorylase levels increased in pancreas with acute pancreatitis. Although cystathionine ß-synthase protein levels did not change with acute pancreatitis, Nos2 mRNA and protein levels were markedly up-regulated and caused tyrosine nitration of cystathionine ß-synthase in pancreas. S-adenosylmethionine administration enhanced Nos2 mRNA expression and cystathionine ß-synthase nitration and triggered homocysteine accumulation in acute pancreatitis. Furthermore, S-adenosylmethionine administration promoted enrichment of the euchromatin marker H3K4me3 in the promoters of Tnf-α, Il-6, and Nos2 and enhanced the mRNA up-regulation of these genes. Accordingly, S-adenosylmethionine administration increased inflammatory infiltrate and edema in pancreas with acute pancreatitis. In conclusion, tyrosine-nitration of cystathionine ß-synthase blockades the trans-sulfuration pathway in acute pancreatitis promoting homocysteine accumulation upon S-adenosylmethionine treatment.

[Expression of nicotinamide adenine dinucleotide phosphate-reduced oxidase-4/reactive oxygen species and cystathionine-γ-lyase/hydrogen sulfide in patients with chronic obstructive pulmonary disease-related pulmonary hypertension].

Objective: To observe the levels of serum reactive oxygen species (ROS) and hydrogen sulfide(H(2)S) in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD), and nicotinamide adenine dinucleotide phosphate-reduced (NADPH) oxidase 4 (NOX4) and cystathionine-γ-lyase (CSE) in lung tissue of patients with stable chronic obstructive pulmonary disease (COPD). Methods: (1) A total of 60 patients with AECOPD admitted to the Department of Respiratory Medicine at Ningxia Hui People's Hospital from November 2015 to December 2016 were recruited. According to the results of pulmonary function and echocardiography, the participants were divided into AECOPD-related pulmonary hypertension (PH) group(A) and AECOPD non-PH group (B).Other 30 healthy subjects were selected as the control group (C).Serum ROS and H(2)S of group A, B and C were detected by enzyme-linked immunosorbent assay (ELISA).(2)The lung tissues of patients undergoing lobectomy for lung cancer from November 2012 to April 2017 were collected, who were divided into COPD-related PH group (D), COPD non-PH group (E) and negative control (F). The expression of NOX4 and CSE protein in lung tissue was detected by immunohistochemistry and the thickness of pulmonary arteriole wall was measured. Results: (1)The serum ROS level in group A was higher than group B and C which were (613.52±69.66)IU/ml,(565.76±71.33)IU/ml, (294.63±60.39)IU/ml, respectively with that in group B higher than that in group C (P<0.05). Serum H(2)S level in group A was lower than group B and C, with that in group B lower than group C [(18.59±5.50) nmol/ml, (20.49±4.97) nmol/ml, (38.03±4.43) nmol/ml, respectively P<0.05]. ROS level was positively correlated with pulmonary systolic pressure (PASP) (r=0.59, P<0.05), H(2)S level was negatively correlated with PASP(r=-0.62, P<0.05).(2)The lung tissue expression of NOX4 in group D was higher than group E and F (P<0.05), which were 0.08±0.01,0.06±0.01,0.03±0.01, respectively,while the level of NOX4 in group E was higher than group F (P<0.05). The expression of CSE between group D, E and F were all significantly different (P<0.05),which were 0.03±0.01, 0.07±0.02,0.12±0.02, respectively.(3)Smooth muscle thickness of pulmonary arterioles as a percentage of vascular diameter (WT%) between group D, E and F was all different(P<0.05), which were (40.58±6.63)%,(36.87±5.60)%,(31.27±6.24)%, respectively; so was smooth muscle area of pulmonary arterioles as a percentage of total vascular area(WA%) with (32.33±6.27)%, (30.20±5.28)%, (25.20±4.31)%, respectively (P<0.05). (4)The expression of NOX4 was positively correlated with WT% and WA%, r was 0.81 and 0.66, respectively (P<0.05). The expression of CSE was negatively correlated with WT% and WA%, r was -0.55 and -0.39 respectively (P<0.05). Conclusions: NOX4/ROS and CSE/H(2)S signaling pathways may play an important role in the pathogenesis of COPD related PH.