Cystathionine Β-synthase Research Articles

H2S-Prdx4 axis mitigates Golgi stress to bolster tumor-reactive T cell immunotherapeutic response.

The role of tumor microenvironment (TME)-associated inadequate protein modification and trafficking due to insufficiency in Golgi function, leading to Golgi stress, in the regulation of T cell function is largely unknown. Here, we show that disruption of Golgi architecture under TME stress, identified by the decreased expression of GM130, was reverted upon treatment with hydrogen sulfide (H2S) donor GYY4137 or overexpressing cystathionine β-synthase (CBS), an enzyme involved in the biosynthesis of endogenous H2S, which also promoted stemness, antioxidant capacity, and increased protein translation, mediated in part by endoplasmic reticulum-Golgi shuttling of Peroxiredoxin-4. In in vivo models of melanoma and lymphoma, antitumor T cells conditioned ex vivo with exogenous H2S or overexpressing CBS demonstrated superior tumor control upon adoptive transfer. Further, T cells with high Golgi content exhibited unique metabolic and glycation signatures with enhanced antitumor capacity. These data suggest that strategies to mitigate Golgi network stress or using Golgihi tumor-reactive T cells can improve tumor control upon adoptive transfer.

The Role of Hydrogen Sulfide in iNOS and APP Localization and Expression in Neurons and Glial Cells Under Traumatic Effects: An Experimental Study with Bioinformatics Analysis and Biomodeling

Hydrogen sulfide (H2S) donors are emerging as promising candidates for neuroprotective agents. However, H2S-dependent neuroprotective mechanisms are not yet fully understood. We have demonstrated that an H2S donor (sodium sulfide, Na2S) reduces the expression of inducible NO synthase (iNOS) and amyloid-beta precursor protein (APP) in damaged neural tissue at 24 h and 7 days following traumatic brain injury (TBI). The application of aminooxyacetic acid (AOAA), an inhibitor of cystathionine β-synthase (CBS), produced the opposite effect. Seven days after TBI, iNOS expression was observed not only in the cytoplasm but also in some neuronal nuclei, while APP was exclusively localized in the cytoplasm and axons of damaged neurons. It was also shown that iNOS and APP were present in the cytoplasm of mechanoreceptor neurons (MRNs) in the crayfish, in axons, as well as in certain glial cells 8 h after axotomy. Na2S and AOAA had opposing effects on axotomized MRNs and ganglia in the ventral nerve cord (VNC). Multiple sequence alignments revealed a high degree of identity among iNOS and APP amino acid residues in various vertebrate and invertebrate species. In the final stage of this study, biomodeling identified unique binding sites for H2S, hydrosulfide anion (HS−), and thiosulfate (S2O32−) with iNOS and APP.

CBS/CSE mediated H2S production induced AMPs expression through Toll pathway in crabs with black gill syndrome

The occurrence of black gill syndrome (BGS) is a serious threat to the healthy culture of Eriocheir sinensis. Studying the innate immune ability of E. sinensis with BGS can help develop new strategies for disease prevention and treatment. Antimicrobial peptides (AMPs) have crucial roles in crustacean humoral immunity. In this study, we found that the expression levels of two antilipopolysaccharide factor (EsALF7 and EsALF-L), one Toll receptor 3 (EsToll3), and one Pelle kinase (EsPelle) were upregulated in E. sinensis with BGS. Moreover, ALFs expressions in E. sinensis with BGS were positively regulated by EsToll3 and EsPelle. The content of hydrogen sulfide (H2S) in the gills of E. sinensis with BGS was increased. Further studies showed that the expressions of cystathionine β-synthase (EsCBS) and cystathionine γ-lyase (EsCSE) in the gills of E. sinensis with BGS were upregulated, which positively regulate the production of H2S. Whether there was a correlation between the upregulation of ALFs expression and changes in H2S content? Further studies showed that 1) the expressions of EsToll3, EsPelle, EsALF7, and EsALF-L in the gills of E. sinensis were upregulated under H2S exposure and 2) the knockdown of EsCBS and EsCSE in E. sinensis reduced the transcriptions of EsToll3, EsPelle, EsALF7, and EsALF-L. To sum up, these findings suggest that upregulation of H2S content induced by CBS/CSE promotes the expression of ALFs through the Toll pathway in E. sinensis suffering from BGS.

Mechanism of action and impact of thiol homeostasis on efficacy of an enzyme replacement therapy for classical homocystinuria

Homocystinuria (HCU) due to cystathionine beta-synthase (CBS) deficiency is characterized by elevated plasma and tissue homocysteine levels. There is no cure, but HCU is typically managed by methionine/protein restriction and vitamin B6 supplementation. Enzyme replacement therapy (ERT) based on human CBS has been developed and has shown significant efficacy correcting HCU phenotype in several mouse models by bringing plasma total homocysteine below the clinically relevant 100 μM threshold. As the reactive nature of homocysteine promotes disulfide formation and protein binding, and ERT is unable to normalize plasma total homocysteine levels, the mechanism of action of ERT in HCU remains to be further characterized. Here we showed that only a reduced homocysteine serves as a substrate for CBS and its availability restricts the homocysteine-degrading capacity of CBS. We also demonstrated that cells export homocysteine in its reduced form, which is efficiently metabolized by CBS in the culture medium. Availability of serine, a CBS co-substrate, was not a limiting factor in our cell-based model. Biological reductants, such as N-acetylcysteine, MESNA or cysteamine, increased the availability of the reduced homocysteine and thus promoted its subsequent CBS-based elimination. In a transgenic I278T mouse model of HCU, administration of biological reductants significantly increased the proportion of protein-unbound homocysteine in plasma, which improved the efficacy of the co-administered CBS-based ERT, as evidenced by significantly lower plasma total homocysteine levels. These results clarify the mechanism of action of CBS-based ERT and unveil novel pharmacological approaches to further increase its efficacy.

Increased hydrogen sulfide turnover serves a cytoprotective role during the development of replicative senescence

The mammalian gasotransmitter hydrogen sulfide (H2S) is produced by enzymes such as cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), 3-mercaptopyruvate sulfurtransferase (3-MST). Prior studies suggest that H2S may have cytoprotective and anti-aging effects. This project explores the regulation and role of endogenous H2S in a murine model of replicative senescence. H2S and polysulfide levels in RAW 264.7 murine macrophages (control cells: passage 5–10; senescent cells: passage 30–40) were measured using fluorescent probes. The expression of H2S-related enzymes and the activity of senescence marker beta-galactosidase (SA-β-Gal) were also analyzed. CBS, CSE, and 3-MST were inhibited using selective pharmacological inhibitors. Senescence led to a moderate upregulation of CBS and in a significant increase in CSE and 3-MST. H2S degradation enzymes were also elevated in senescence. Inhibition of H2S-producing enzymes reduced H2S levels but increased polysulfides. Inhibition of H2S production during senescence suppressed cell proliferation, and elevated SA-β-Gal and p21 levels. Comparing young and old mice spleens revealed downregulation of CBS and ETHE1 and upregulation of rhodanese and SUOX in older mice. The results demonstrate that increased reactive sulfur turnover occurs in senescent macrophages and that reactive sulfur species supports cell proliferation and regulate cellular senescence

Isorhamnetin Alleviates Renal Fibrosis by Inducing Endogenous Hydrogen Sulfide and Regulating Thiol-Based Redox State in Obstructed Kidneys.

Isorhamnetin (ISO) is an active flavonoid compound mainly isolated from the fruits of Hippophae rhamnoides L. and the leaves of Ginkgo biloba L. Previous studies have revealed the antifibrotic action of ISO in the liver and lungs, although its potential protective effects against renal fibrosis and the underlying mechanisms are still poorly understood. Given that many actions of ISO could be similarly attained by hydrogen sulfide (H2S), we speculated that ISO may work through the induction of endogenous H2S. To test the hypothesis, we established the unilateral ureteral obstruction (UUO) renal fibrosis rat model and transforming growth factor-β1(TGF-β1)-induced fibrosis in cultured renal tubular cells. ISO treatment inhibited epithelial-mesenchymal transition (EMT) formation, decreased extracellular matrix (ECM) deposition, and relieved renal fibrosis. Further analysis revealed that ISO stimulated the expression of the H2S-synthesizing enzyme cystathionine lyase (CSE) and cystathionine beta-synthase (CBS), and promoted H2S production in vivo and in vitro. The elevated H2S attenuated oxidative stress and elevated the thiol level. It induced Keap1 sulfhydration, disrupted Keap1-Nrf2 interaction, and promoted the entry of Nrf2 into the nucleus. Finally, we found that circulating H2S mainly derived from the liver, and not the kidney. Collectively, our study revealed that ISO alleviated renal fibrosis by inducing endogenous H2S and regulating Keap1-Nrf2 interaction through sulfhydration of Keap1. Endogenous H2S could be an important mediator underlying the pharmacological actions of ISO. Due to the multifunctional properties of H2S, the H2S-inducing nature of ISO could be exploited to treat various diseases.

CETZOLE Analogs as Potent Ferroptosis Inducers and Their Target Identification Using Covalent/Affinity Probes.

Ferroptosis is a recently discovered cell death mechanism triggered by iron-dependent elevation of reactive oxygen species leading to lipid membrane peroxidation. We previously reported the development of a new class of ferroptosis inducers referred to as CETZOLEs with CC50 values in the low micromolar range. Structure-activity relationship study of these compounds led to the development of more potent analogs with CC50 values in the nanomolar range. Cells exposed to these compounds displayed the hallmarks of ferroptosis including cell death through ROS accumulation. Cancer cells were found to be more sensitive to these compounds than normal cells. Proteomic studies using covalent and affinity probes led to the identification of cystathionine β-synthase, peroxiredoxins, ADP/ATP carriers, and glucose dehydrogenase as enriched proteins. The binding of CETZOLEs to these proteins as well as GPX4 was validated by Western blotting. This group of proteins is known to be associated with cellular antioxidant pathways.

REDOX SIGNALLING IN THE PANCREAS IN HEALTH AND DISEASE.

This review addresses oxidative stress and redox signaling in the pancreas under physiological conditions as well as in acute pancreatitis, chronic pancreatitis, pancreatic cancer, and diabetes. Physiological redox homeodynamics is maintained mainly by NRF2/KEAP1, NF-κB, protein tyrosine phosphatases, peroxisome proliferator-activated receptor-γ co-activator 1α (PGC1α), and normal autophagy. Depletion of reduced glutathione in the pancreas is a hallmark of acute pancreatitis and is initially accompanied by disulfide stress, which is characterized by protein cysteinylation without increased glutathione oxidation. A cross-talk between oxidative stress, MAPKs, and NF-κB amplifies the inflammatory cascade, acting PP2A and PGC1α as key redox regulatory nodes. In acute pancreatitis, nitration of cystathionine-β synthase causes blockade of the trans-sulfuration pathway leading to increased homocysteine levels, whereas p53 triggers necroptosis in the pancreas through downregulation of sulfiredoxin, PGC1α, and peroxiredoxin 3. Chronic pancreatitis exhibits oxidative distress mediated by NADPH oxidase 1 and/or CYP2E1, which promotes cell death, fibrosis, and inflammation. Oxidative stress cooperates with mutant KRAS to initiate and promote pancreatic adenocarcinoma. Mutant KRAS increases mitochondrial ROS, which trigger acinar-to-ductal metaplasia and progression to PanIN. ROS are maintained at sufficient level to promote cell proliferation, whilst avoiding cell death or senescence through formation of NADPH and GSH, and activation of NRF-2, HIF-1/2α, and CREB. Redox signalling also plays a fundamental role in differentiation, proliferation, and insulin secretion of β-cells. However, ROS overproduction promotes β-cell dysfunction and apoptosis in type 1 and type 2 diabetes.

Asynchronous changes of hydrogen sulfide and its generating enzymes in most tissues with the aging process.

Aging is an inevitable and irreversible biological process that gradually heightens the risks of various diseases and death. As a newly discovered endogenous gasotransmitter, hydrogen sulfide (H2S) has been identified to exert multiple beneficial impacts on the regulation of aging and age-related pathologies. This study was aimed at systematically exploring the relationship between asynchronous aging processes and H2S concentrations in various tissues of aging mice. Samples of plasma and 13 tissues were collected from four cross-sectional age groups (3, 6, 12 and 18 months of age) covering the lifespan of male C57BL/6J mice. The H2S concentration was quantified by a reported liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with monobromobimane derivatization. Additionally, the expressions of cystathionine γ-lyase (CSE), cystathionine β-synthase and 3-mercaptopyruvate sulfurtransferase, in those tissues were analyzed by Western blotting. We discovered that the H2S concentrations decreased asynchronously with the aging process in plasma, heart, liver, kidney, spleen, subcutaneous fat and brown fat and increased in brain and lung. At least one of the three H2S-generating enzymes expressions was compensatorily up-regulated with the aging process in most tissues, among which the up-regulation of CSE was the most prominent.

Clinical, biochemical, molecular characteristics and clinical outcome of hyperhomocysteinemia in Malaysian children

BackgroundHyperhomocysteinemia can be due to various abnormalities of the complex interaction of methionine, folate and vitamin B12. It has been known to be a cardiovascular risk factor. This study aims to review the clinical presentation, underlying causes and clinical outcome in paediatric patients diagnosed with significant hyperhomocysteinemia in Malaysia. Design and methodsData were obtained from the medical records and the laboratory information system. Paediatric patients with significant hyperhomocysteinemia were identified from a selective high-risk screening of 96,721 patients, performed between 2010 and 2022. Inclusion criteria for the study were paediatric patients with significant hyperhomocysteinemia (>40 µmol/L). ResultsSixteen patients were identified. The average total homocysteine (tHcy) and methionine were 269 µmol/L and 499 µmol/L in cystathionine β-synthase deficiency (CBS), 127 µmol/L and 29 µmol/L in patients with remethylation defects and 390 µmol/L and 4 µmol/L in congenital B12 deficiency. We found c.609G>A as the most prevalent mutation in MMACHC gene and possible novel mutations for CBS (c.402del, c.1333C>T and c.1031T>G) and MTHFR genes (c.266T>A and c.1249del). Further subclassification revealed CBS was 5/16 patients (31 %), remethylation defects was 9/16 (56 %) and congenital B12 deficiency was 2/16 (13 %). All patients received standard treatment and regular monitoring of the main biomarkers. The average age at the time of diagnosis were 9.2 years (CBS) and 1.2 years (remethylation defects). Congenital B12 deficiency had slight delay in milestones, remethylation defects had mild to moderate learning disabilities, CBS had variable degree of intellectual disability, delayed milestones, ophthalmological abnormalities, and thrombosis at an early adolescent/adulthood. ConclusionsThe majority of significant hyperhomocysteinemia in Malaysian children was due to remethylation defects. Screening for hyperhomocysteinemia in Malaysian children is recommended for earlier treatment and improved clinical outcome.

Expression of Cystathionine-β-Synthase (CBS) Genes in Grape under Various Abiotic Stresses and Subcellular Localization of VvCBS27

The cystathionine-β-synthase (CBS) domain is present in the proteins of all living organisms and functions as an energy sensor, regulating protein activity through the binding capacity of its adenosine ligands. The role of the CBS gene in plant growth and development, as well as tolerance to abiotic stresses, remains largely unknown, especially in grapevine. In our study, 32 members of the CBS gene family were obtained that were distributed on 15 chromosomes. The results of the structural and evolutionary tree analyses indicated that the VvCBS gene family exhibits diverse intron-exon patterns and highly conserved motifs. Furthermore, the phylogenetic classification of the VvCBS genes revealed the presence of three subfamilies. Subcellular localization analyses showed that the VvCBS genes are mainly located in the plasma membrane region. The secondary structure of the VvCBS protein mainly consists of α-helices, extended strands, β-turns, and random coils. The VvCBS gene family exhibited four co-linear gene pairs, while the numbers for Arabidopsis thaliana and rice were 21 and 7, respectively. The promoter cis-acting element analysis revealed the presence of light-responsive, hormone-responsive, stress-responsive, and growth- and development-related elements in the VvCBS family. The expression characterization demonstrated that 12 VvCBS genes exhibited high expression levels in all grape tissues. Additionally, the RT-qPCR expression analyses showed that the 32 VvCBS exhibited different responses to a variety of abiotic stresses (cold, drought, salt), suggesting that they were functionally differentiated. VvCBS27 was cloned from ‘Pinot Noir’ of grapevine with a coding sequence of 624 bp. Subcellular localization showed that VvCBS27 protein was mainly located in the cytoplasm, cell membrane, and nucleus. This study lays a foundation for elucidating the function of grape CBS protein.

H2S alleviated sepsis-induced acute kidney injury by inhibiting PERK/Bax-Bcl2 pathway

To investigate the protective mechanisms of hydrogen sulfide (H2S) in sepsis-induced acute kidney injury (SAKI), we conducted an in vivo study using a SAKI mouse model induced by intraperitoneal lipopolysaccharide (LPS) injection. Following 6 h of LPS injection, levels of tumor necrosis factor-alpha (TNF-α) and blood urea nitrogen (Bun) were significantly elevated in mouse plasma. In the kidneys of SAKI mice, expression of H2S-generating enzymes cysteinyl-tRNA synthetase (CARS), cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS) was markedly downregulated, while glucose-regulated protein 78 (GRP78), activating transcription factor 6 (ATF6), phosphorylated protein kinase R-like endoplasmic reticulum kinase/protein kinase R-like endoplasmic reticulum kinase (p-PERK/PERK), and B-cell lymphoma-2 recombinant protein X/B-cell lymphoma-2 (Bax/Bcl2) expression was significantly upregulated. H2S improved renal function and attenuated renal histopathological changes in SAKI mice, thereby alleviating LPS-induced endoplasmic reticulum stress (ERS). Additionally, it inhibited the expression of p-PERK/PERK and Bax/Bcl2. After inhibiting CSE activity with dl-propargylglycine (PPG i. p.), the renal tissue pathology in LPS-induced AKI mice was further exacerbated, leading to enhanced activation of the PERK/Bax-Bcl2 pathway. Our findings suggest that endogenous H2S influences the pathogenesis of SAKI, while exogenous H2S protects against LPS-induced AKI by inhibiting the PERK/Bax-Bcl2 pathway involved in ERS.

The role of cystathionine β-synthase in cancer

Cystathionine β-synthase (CBS) occupies a key position as the initiating and rate-limiting enzyme in the sulfur transfer pathway and plays a vital role in health and disease. CBS is responsible for regulating the metabolism of cysteine, the precursor of glutathione (GSH), an important antioxidant in the body. Additionally, CBS is one of the three enzymes that produce hydrogen sulfide (H2S) in mammals through a variety of mechanisms. The dysregulation of CBS expression in cancer cells affects H2S production through direct or indirect pathways, thereby influencing cancer growth and metastasis by inducing angiogenesis, facilitating proliferation, migration, and invasion, modulating cellular energy metabolism, promoting cell cycle progression, and inhibiting apoptosis. It is noteworthy that CBS expression exhibits complex changes in different cancer models. In this paper, we focus on the CBS synthesis and metabolism, tissue distribution, potential mechanisms influencing tumor growth, and relevant signaling pathways. We also discuss the impact of pharmacological CBS inhibitors and silencing CBS in preclinical cancer models, supporting their potential as targeted cancer therapies.

Hypoxia-inducible lipid droplet-associated protein (HILPDA) and cystathionine β-synthase (CBS) co-contribute to protecting intestinal epithelial cells from Staphylococcus aureus via regulating lipid droplets formation

Despite Staphylococcus aureus (S. aureus) being a highly studied zoontic bacterium, its enteropathogenicity remains elusive. Herein, our findings demonstrated that S. aureus infection led to the accumulation of lipid droplets (LDs) in intestinal epithelial cells, accompanied by marked elevation inflammatory response that ultimately decreases intracellular bacterial load. The aforestated phenomenon may be partly attributed to the up-regulation of hypoxia-inducible lipid droplet-associated protein (HILPDA) and the concomitant down-regulation of cystathionine β-synthase (CBS) protein. Moreover, S. aureus infection up-regulated the expression of HILPDA, thereby promoting LDs accumulation, and down-regulated that of CBS, consequently inhibiting microsomal triglyceride transfer protein (MTTP) expression. This process may suppress the transport of LDs to the extracellular environment, further contributing to the formation of intracellular LDs. In summary, the results of this study provide significant insights into the intricate mechanisms through which the host organism combats pathogens and maintains the balance of sulfur and lipid metabolism. These findings not only enhance our understanding of the host's defense mechanisms but also offer promising avenues for the development of novel strategies to combat intestinal infectious diseases.

Using RNA-Seq Analysis to Select Key Genes Related to Seed Dormancy in ALS-Inhibiting Resistant Descurainia sophia with Pro-197-Thr Mutation.

Flixweed (Descurainia sophia) is a weed that seriously affects wheat fields in China. Over the past 20 years, it has evolved resistance to the herbicide tribenuron-methyl. In the present study, a resistant D. sophia population with a Pro-197-Thr mutation of acetolactate synthetase (ALS) was found to have a resistance index of 457.37 for tribenuron-methyl. Under the same growth conditions, the seeds of resistant (R) and susceptible (S) populations exhibited similar vitality but the germination rates of R seeds were higher than those of S seeds. This result demonstrated that seed dormancy periods were shorter in the R seeds. RNA-Seq transcriptome analysis was then used to choose candidate genes that could regulate seed dormancy pathways in the R population. A total of 504,976,046 clean reads were selected from nine RNA-Seq libraries and assembled into 79,729 unigenes. Among these, 33,476 unigenes were assigned to 51 GO subgroups, and 26,117 unigenes were assigned to 20 KEGG secondary metabolic pathways. Next, 2473 differentially expressed genes (DEGs) were divided into three groups, as follows: G-24 h (germinating seeds) vs. D (dormant seeds); G-48 h (germinated seeds) vs. D; and G-48 h vs. G-24 h. From these 2473 DEGs, 8 were selected as candidate dormancy unigenes for the R population if their expression levels continuously decreased during the seed germination progress and their functional annotations were related to plant seed dormancy. One candidate unigene was annotated as CYP707A2; two unigenes were annotated as the transcription factors TGA4 and TGA2; one unigene was annotated as the cystathionine beta-synthase gene; and four unigenes could not be annotated as any gene listed in the six public databases. However, qRT-PCR-validated results showed that, during the germination of R seeds, the expression of the three candidate unigenes first decreased and then increased, indicating that they may have other growth-regulating functions in R populations. In brief, the dormancy function of the eight candidate dormancy unigenes needs to be further studied.

Cystathionine gamma-lyase deficiency exaggerates diethylnitrosamine-induced liver damage in mice

Cystathionine gamma-lyase (CSE) is a key enzyme in reverse transsulfuration pathway and contributes to the majority of H2S generation in liver tissues via cysteine metabolism. Dysfunction of the CSE/H2S system is linked to both chronic and acute liver damage. This study investigated the regulatory role of CSE deficiency on diethylnitrosamine (DEN)-induced liver damage in mice. A single injection of DEN was administered into 4-week-old male CSE knockout (CSE-KO) mice and wild-type (WT) littermates, and the mice were sacrificed at 28 weeks of age. Compared to age-matched WT mice, CSE-KO mice spontaneously developed steatosis with increased oxidative stress and higher expressions of inflammation and fibrosis-related genes at 28-weeks of age. Following DEN injection, CSE-KO mice experienced more severe liver damage in comparison with the WT group as reflected by elevated levels of lipid accumulation, increased activities of alanine aminotransferase and aspartate aminotransferase, higher oxidative stress and fibrosis development, and increased expressions of inflammation and fibrosis-related genes. No visible tumors were observed in both types of mice with DEN treatment. In addition, the expression levels of the three H2S-generating proteins (CSE, cystathionine beta-synthase, and 3-mercaptopyruvate sulfurtransferase) and the H2S production rate in liver tissues were unaffected by DEN. Taken together, our study demonstrates that CSE provides a significant hepatoprotective effect and deficiency of CSE exaggerates DEN-induced liver damage in mice. Based on these findings, it can be suggested that targeting the CSE/H2S signaling pathway could be a potential therapeutic target for the treatment of liver diseases.

Cellular turnover and degradation of the most common missense cystathionine beta-synthase variants causing homocystinuria.

Homocystinuria (HCU) due to cystathionine beta-synthase (CBS) deficiency is the most common inborn error of sulfur amino acid metabolism. Recent work suggests that missense pathogenic mutations-regardless of their topology-cause instability of the C-terminal regulatory domain, which likely translates into CBS misfolding, impaired assembly, and loss of function. However, it is unknown how instability of the regulatory domain translates into cellular CBS turnover and which degradation pathways are involved in CBS proteostasis. Here, we developed a human HEK293-based cellular model lacking intrinsic CBS and stably overexpressing wild-type (WT) CBS or its 10 most common missense HCU mutants. We found that HCU mutants, except the I278T variant, expressed similarly or better than CBS WT, with some of them showing impaired oligomerization, activity and response to allosteric activator S-adenosylmethionine. Cellular stability of all HCU mutants, except P49L and A114V, was significantly lower than the stability of CBS WT, suggesting their increased degradation. Ubiquitination analysis of CBS WT and two representative CBS mutants (T191M and I278T) showed that proteasomal degradation is the major pathway for CBS disposal, with a minor involvement of lysosomal-autophagic and endoplasmic reticulum-associated degradation (ERAD) pathways for HCU mutants. Proteasomal inhibition significantly increased the half-life and activity of T191M and I278T CBS mutants. Lysosomal and ERAD inhibition had only a minor impact on CBS turnover, but ERAD inhibition rescued the activity of T191M and I278T CBS mutants similarly as proteasomal inhibition. In conclusion, the present study provides new insights into proteostasis of CBS in HCU.

A cystathionine beta-synthase domain containing protein, OsCBSCBS4, interacts with OsSnRK1A and OsPKG and functions in abiotic stress tolerance in rice.

The Cystathionine-β-Synthase (CBS) domain-containing proteins (CDCPs) constitute a functionally diverse protein superfamily, sharing an evolutionary conserved CBS domain either in pair or quad. Rice genome (Oryza sativa subsp. indica) encodes 42 CDCPs; their functions remain largely unexplored. This study examines OsCBSCBS4, a quadruple CBS domain containing protein towards its role in regulating the abiotic stress tolerance in rice. Gene expression analyses revealed upregulation of OsCBSCBS4 in response to diverse abiotic stresses. Further, the cytoplasm-localised OsCBSCBS4 showed interaction with two different kinases, a cytoplasmic localised cGMP-dependant protein kinase (OsPKG) and the nucleo-cytoplasmic catalytic subunit of sucrose-nonfermentation 1-related protein kinase 1 (OsSnRK1A). The interaction with the latter assisted in trafficking of OsCBSCBS4 to the nucleus as well. Overexpression of OsCBSCBS4 in rice resulted in enhanced tolerance to drought and salinity stress, via maintaining better physiological parameters and antioxidant activity. Additionally, OsCBSCBS4-overexpressing rice plants exhibited reduced yield penalty under stress conditions. The in silico docking and in vitro binding analyses of OsCBSCBS4 with ATP suggest its involvement in cellular energy balance. Overall, this study provides novel insight into the unexplored functions of OsCBSCBS4 and demonstrates it as a new promising target for augmenting crop resilience.

Real-time detection of enzymatically formed hydrogen sulfide by pathogenic variants of cystathionine beta-synthase using hemoglobin I of Lucina pectinata as a biosensor

Classical homocystinuria is a rare disease caused by mutations in cystathionine β-synthase (CBS) gene (OMIM 613381). CBS catalyzes the first step of the transsulfuration pathway that converts homocysteine (Hcy) into cystathionine (Cysta) via a number of co-substrates and mechanisms. Formation of Cysta by condensation of Hcy and cysteine (Cys) produces a molar equivalent of hydrogen sulfide (H2S). H2S plays important roles in cognitive and vascular functions. Clinically, patients with CBS deficiency present with vascular, ocular, neurological and skeletal impairments. Biochemically, CBS deficiency manifests with elevated Hcy and reduced concentration of Cysta in plasma and urine. A number of pathogenic variants of human CBS have been characterized by their residual enzymatic activity, but very few studies have examined H2S production by pathogenic CBS variants, possibly due to technical hurdles in H2S detection and quantification. We describe a method for the real-time, continuous quantification of H2S formed by wild-type and pathogenic variants of human recombinant CBS, as well as by fibroblast extracts from healthy controls and patients diagnosed with CBS deficiency. The method takes advantage of the specificity and high affinity of hemoglobin I of the clam Lucina pectinata toward H2S and is based on UV–visible spectrophotometry. Comparison with the gold-standard, end-point H2S quantification method employing monobromobimane, as well as correlations with CBS enzymatic activity determined by LC-MS/MS showed agreement and correlation, and permitted the direct, time-resolved determination of H2S production rates by purified human recombinant CBS and by CBS present in fibroblast extracts. Rates of H2S production were highest for wild-type CBS, and lower for pathogenic variants. This method enables the examination of structural determinants of CBS that are important for H2S production and its possible relevance to the clinical outcome of patients.

Effects of L-Methionine and DL-Methionine on Growth Performance, Methionine-Metabolizing Enzyme Activities, Feather Traits, and Intestinal Morphology of Medium-Growing, Yellow-Feathered Chickens between 1 and 30 Days of Age.

This experiment investigated the effects of L-Methionine (L-Met) on growth performance, Met-metabolizing enzyme activity, feather traits, and small intestinal morphological characteristics, and compared these with DL-Methionine (DL-Met) for medium-growing, yellow-feathered broilers during the starter phase. Furthermore, the aim was to provide recommendations for the appropriate dietary Met levels in feed. A total of 1584 1-d broilers were randomly divided into 11 treatment groups with six replicates of 24 birds each: basal diet (CON, Met 0.28%), basal diet + L-Met (0.04%, 0.08%, 0.12%, 0.16%, 0.20%), and basal diet + DL-Met (0.04%, 0.08%, 0.12%, 0.16%, 0.20%). The total trial period was 30 days. Compared with broilers fed the basal diet, those fed 0.04 to 0.20% supplemental Met had higher final body weight (FBW), average daily feed intake (ADFI), average daily gain (ADG), and lower feed-to-gain ratio (F: G) (p < 0.05). Compared with DL-Met groups, the L-Met group had higher FBW and ADG (p < 0.05). The relative bioavailability (RBV) of L-Met in ADG of 1-30 d was 142.5%. Chicks fed diets supplemented with L-Met had longer fourth primary feather lengths compared to birds fed the control and diets supplemented with DL-Met (p < 0.05). Compared to the control, birds supplemented with DL-Met or L-Met had an increased moulting score (p ≤ 0.05). Chicks fed diets supplemented with L-Met had lower activities of methionine adenosyl transferase (MAT) compared to those fed the basal diet or supplemented with DL-Met (p < 0.05). Chicks supplemented with either DL-Met or L-Met had higher activities of cystathionine β-synthase (CBS) than those fed the basal diet (p < 0.05). Compared with the control, chicks fed diets supplemented with either DL-Met or L-Met had an enhanced level of albumin in plasma (p < 0.05). There were no obvious differences in the plasma content of uric acid and total protein among the treatments (p > 0.05). Chicks fed diets supplemented with either DL-Met or L-Met had higher villus height and V/C in the duodenal than chicks fed the basal diet (p < 0.05). The jejunum morphology was not affected by either L-Met or DL-Met supplementation (p > 0.05). Therefore, dietary supplementation with DL-Met or L-Met improved the growth performance, feather traits, and intestinal morphological characteristics of medium-growing, yellow-feathered broiler chickens aged 1 to 30 d by decreasing the enzyme activities of Met methylation (MAT) and increasing the enzyme activities of the sulfur transfer pathway (CBS), and supplementation with L-Met showed a better improvement compared with DL-Met. The relative efficacy of L-Met to DL-Met was 142.5% for ADG of yellow-feathered broilers. The appropriate Met levels for medium-growing, yellow-feathered broilers are between 0.36~0.38% (supplementation with DL-Met) or 0.32~0.33% (supplementation with L-Met) when based on ADG and feed-to-gain ratio.