Cystathionine Beta-synthase Expression Research Articles

New insights into the regulation of Cystathionine beta synthase (CBS), an enzyme involved in intellectual deficiency in Down syndrome.

Down syndrome (DS), the most frequent chromosomic aberration, results from the presence of an extra copy of chromosome 21. The identification of genes which overexpression contributes to intellectual disability (ID) in DS is important to understand the pathophysiological mechanisms involved and develop new pharmacological therapies. In particular, gene dosage of Dual specificity tyrosine phosphorylation Regulated Kinase 1A (DYRK1A) and of Cystathionine beta synthase (CBS) are crucial for cognitive function. As these two enzymes have lately been the main targets for therapeutic research on ID, we sought to decipher the genetic relationship between them. We also used a combination of genetic and drug screenings using a cellular model overexpressing CYS4, the homolog of CBS in Saccharomyces cerevisiae, to get further insights into the molecular mechanisms involved in the regulation of CBS activity. We showed that overexpression of YAK1, the homolog of DYRK1A in yeast, increased CYS4 activity whereas GSK3β was identified as a genetic suppressor of CBS. In addition, analysis of the signaling pathways targeted by the drugs identified through the yeast-based pharmacological screening, and confirmed using human HepG2 cells, emphasized the importance of Akt/GSK3β and NF-κB pathways into the regulation of CBS activity and expression. Taken together, these data provide further understanding into the regulation of CBS and in particular into the genetic relationship between DYRK1A and CBS through the Akt/GSK3β and NF-κB pathways, which should help develop more effective therapies to reduce cognitive deficits in people with DS.

Dysregulated Sulfide Metabolism in Multiple Sclerosis: Serum and Vascular Endothelial Inflammatory Responses.

Multiple sclerosis (MS) is a leading cause of neurodegenerative disability in younger individuals. When diagnosed early, MS can be managed more effectively, stabilizing clinical symptoms and delaying disease progression. The identification of specific serum biomarkers for early-stage MS could facilitate more successful treatment of this condition. Because MS is an inflammatory disease, we assessed changes in enzymes of the endothelial hydrogen sulfide (H2S) pathway in response to inflammatory cytokines. Blotting analysis was conducted to detect Cystathionine γ-lyase (CSE), Cystathionine beta synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (MST) in human brain microvascular endothelial apical and basolateral microparticles (MPs) and cells following exposure to tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). CSE was increased in MPs and cells by exposure to TNF-α/IFN-γ; CBS was elevated in apical MPs but not in cells or basolateral MPs; MST was not significantly affected by cytokine exposure. To test how our findings relate to MS patients, we evaluated levels of CSE, CBS, and MST in serum samples from healthy control and MS patients. We found significantly decreased levels of CBS and MST (p = 0.0004, 0.009) in MS serum samples, whereas serum levels of CSE were marginally increased (p = 0.06). These observations support increased CSE and lower CBS and MST expression being associated with the vascular inflammation in MS. These changes in endothelial-derived sulfide enzymes at sites of inflammation in the brain may help to explain sulfide-dependent changes in vascular dysfunction/neuroinflammation underlying MS. These findings further support the use of serum samples to assess enzymatic biomarkers derived from circulating MPs. For example, “liquid biopsy” can be an important tool for allowing early diagnosis of MS, prior to the advanced progression of neurodegeneration associated with this disease.

Differential expression of cystathionine beta synthase in adolescents with Down syndrome: impact on adiposity.

Obesity is more prevalent among people with Down Syndrome (DS) compared to general population. In this pilot study, we investigated the effect of cystathionine beta-synthase (CBS) overdosage on the regulation of transsulfuration pathway and the obesity phenotype in fifty adolescents (25 obese/overweight and 25 lean) with trisomy 21. The transcriptional levels of CBS in leukocytes and its translational levels in plasma were quantified using real time polymerase chain reaction and enzyme-linked immunosorbent assay respectively. Meanwhile, ultra performance liquid chromatography tandem mass spectrometry was used to determine the plasma concentrations of methionine, homocysteine, cystathionine and cysteine. Fasting plasma lipid profiles were assessed by colorimetric assays. The anthropometric measurements and indices of all subjects were recorded. Both DS groups had comparable levels of CBS transcripts (p = 0.2734). The plasma levels of the enzyme were significantly higher in the lean DS cases (p = 0.0174) compared to the obese/overweight participants. Total cholesterol, triglycerides, high-density lipoprotein, low-density lipoprotein, methionine, homocysteine, cystathionine and cysteine showed similar plasma levels in both groups. However, the plasma cysteine levels exceeded the normal range in all DS cases. We reported a statistically significant inverse association between CBS enzyme levels and weight (r= - 0.3498, p = 0.0128), hip circumference (r= - 0.3584, p = 0.0106), body mass index (r= - 0.3719, p = 0.0078) and body adiposity index (r= - 0.3183, p = 0.0243). Our data suggests that the high concentrations of CBS enzyme together with cysteine modulate the DS obesity presumably through increased hydrogen sulfide production which has recently showed anti-adiposity effects.

Potentials of CCL21 and CBS as Therapeutic Approaches for Breast Cancer

Objective: The present research set out to ascertain the roles of CC chemokine ligand 21 (CCL21) and cystathionine beta-synthase (CBS) in breast cancer (BC) cell biological behaviors and the relationship of CCL21 and CBS expression with the clinicopathological features of patients with BC. Methods: Immunohistochemistry of CCL21 or CBS was performed in 18 intraductal cancer tissues, 124 invasive BC tissues, 50 paraneoplastic tissues, 50 lobular hyperplasia tissues, and 30 normal breast tissues. For cell experiments, two human BC cell lines (MDA-MB-231 and MCF-7) and a human breast epithelial cell line (MCF-10A) were utilized to detect the expression of CCL21 and CBS. After loss- and gain-of-function assays for CCL21 or CBS, the expression of CBS and CCL21 was measured by quantitative real-time polymerase chain reaction and western blot. Additionally, BC cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and 5-ethynyl-2′-deoxyuridine staining, and BC cell migration was determined by scratch test and Transwell assay. Results: In the clinical data, the positive rate of CCL21 or CBS was significantly higher in invasive BC tissues than in intraductal BC tissues, lobular hyperplasia tissues, paraneoplastic tissues, and normal breast tissues (p < 0.05 or p < 0.01). CBS or CCL21 expression shared close association with the clinicopathological characteristics and the poor prognosis of BC patients. In cell experiments, overexpression of CCL21 or CBS enhanced the proliferative and migratory abilities of BC cells. Conclusion: CCL21 and CBS promoted BC cell migration and proliferation. CCL21 or CBS expression was strongly related to the poor prognosis of BC patients.

Low CBS expression can identify patients who benefit from adjuvant chemotherapy in gastric cancer

ABSTRACT Aim: To explore the clinical significance of Cystathionine beta-synthase (CBS) expression in gastric cancer (GC). Research design and methods: CBS expression and clinicopathological/follow-up information of patients with gastric cancer undergoing operation were collected from The Cancer Genome Atlas (TCGA) database. The association of CBS expression with patients’ overall survival (OS) was determined in the entire cohort and different subgroups. Validation was performed in two external cohorts from NCBI Gene Expression Omnibus (GEO) database. The estimated drug response of the tumors with different CBS expressions was characterized. The potential CBS-related cellular pathways in chemoresistance were explored. Results: High CBS was associated with poor OS in patients receiving adjuvant chemotherapy (ACT) but not those without ACT. And ACT was associated with favorable OS in patients with low CBS expression but not those with high CBS expression. The results were verified in two external cohorts. Drug response prediction suggested that patients with low CBS expression showed high sensitivity to 5-Fluorouracil. Gene Set Enrichment Analysis (GSEA) suggested that CBS might contribute to GC chemoresistance via modulating many cellular pathways, including down-regulating apoptosis and P53 pathways while up-regulating DNA repair pathway. Conclusion: Low CBS expression can predict the benefit from ACT in GC.

Characterization of hyperglycemia due to sub-chronic administration of red ginseng extract via comparative global proteomic analysis

Ginseng (Panax ginseng Meyer) is commonly used as an herbal remedy worldwide. Few studies have explored the possible physiological changes in the liver although patients often self-medicate with ginseng preparations, which may lead to exceeding the recommended dose for long-term administration. Here, we analyzed changes in the hepatic proteins of mouse livers using quantitative proteomics after sub-chronic administration of Korean red ginseng (KRG) extract (control group and 0.5, 1.0, and 2.0 g/kg KRG) using tandem mass tag (TMT) 6‐plex technology. The 1.0 and 2.0 g/kg KRG groups exhibited signs of liver injury, including increased levels of aspartate transaminase (AST) and alanine aminotransferase (ALT) in the serum. Furthermore, serum glucose levels were significantly higher following KRG administration compared with the control group. Based on the upregulated proteins found in the proteomic analysis, we found that increased cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE) levels promoted greater hydrogen sulfide (H2S) synthesis in the liver. This investigation provides novel evidence that sub-chronic administration of KRG can elevate H2S production by increasing protein expression of CBS and CSE in the liver.

Involvement of HAND1 and CBS in maintenance of cardiac micro-architecture following obesity-induced heart failure

Purpose: To study the role of heart and neural crest derivatives expressed 1 (HAND1) and cystathionine-beta-synthase (CBS) in the maintenance of cardiac architecture following high fat dietinduced obesity.&#x0D; Methods: Mouse models of initial and critical heart disease were established by continuous feeding of high fat diet for 7 and 12 months, respectively. The expression of HAND1 and CBS were assayed using immunohistochemistry and Western blotting.&#x0D; Results: Obesity led to mild and severe forms of heart disease which were confirmed through histological imaging. Initial obesity resulted in cardiac tissue remodeling along with initial degeneration, while critical obesity resulted in tissue hardening. The expression of HAND1 was upregulated 4.3 folds in the mild form of cardiac failure, relative to marginal expression pattern of HAND1 in control tissue. However, as the disease progressed, the expression of HAND1 was limited in serve form of cardiac failure. Moreover, the expression of cystathionine beta-synthase (CBS) was upregulated 3.7-fold in the initial form of heart failure, but was subsequently reduced in serve form of heart disease.&#x0D; Conclusion: These results reveal that in high fat diet-induced cardiac stress, the over-expressions of HAND1 and CBS at the initial stages induce extensive alterations in cardiac architecture.

BIMG-18. ELEVATED CYSTATHIONINE IN MEDULLOBLASTOMA DEMONSTRATES TUMOR-SPECIFIC METHIONINE METABOLISM

We investigated tumor-specific metabolism medulloblastoma using a non-biased MS-imaging screen and identified a pattern of methionine flux that may present a therapeutic opportunity. We studied brain tumors that form in mice genetically engineered to develop Sonic Hedgehog (SHH)-driven medulloblastoma. We subjected sagittal sections including brain and medulloblastoma to MS-imaging, generating concentration maps for hundreds of metabolites MW 100–400. We then confirmed results by analyzing tumor, brain and blood by LC-MS/MS, high-resolution NMR and 2D NMR-TOCSY, and used immunohistochemistry to determine the cellular localization of implicated enzymes. MS imaging, accomplished by matrix-assisted laser desorption electrospray ionization (MALDESI), detected cystathionine at an order of magnitude higher concentration in medulloblastomas compared to adjacent brain. No other metabolite showed such a strong, tumor-specific localization. LC-MS/MS and NMR methods confirmed cystathionine elevation. As cystathionine is the product of homocysteine and serine, catalyzed by cystathionine beta-synthase (CBS), we investigated CBS expression by IHC. Consistent with prior studies, we found that only astrocytes expressed CBS, both in the normal brain and within the tumors. ScRNA-seq confirmed Cbs only in astrocytes, and showed tumor cells express methionine-metabolizing enzymes Mat2a, Dnmt1, Ancy and Mtr. Together, these findings show that tumor cells generate and export homocysteine, which astrocytes convert to cystathionine. Tumor cystathionine generation responded to changes in methionine- cycle metabolites. In vivo, systemic administration of homocysteine increased tumor cystathionine which decreased in response to systemic folate, the methyl donor for homocysteine methyltransferase. Cystathionine itself was inert in tumors as tumor cells cultured in up to 8 mM cystathionine showed no change in cell cycle progression. Our studies show that medulloblastomas utilize methionine and generate homocysteine, but avoid folate-dependent homocysteine-methionine recycling by exporting homocysteine for detoxification by local astrocytes. This model suggests that treatments that impose methionine scarcity, folate scarcity or CBS inhibition may produce anti-tumor effects in medulloblastoma.

Exercise intervention differentially induces the expression of cystathionine beta synthase (CBS) in African American with MCI: Implication for endogenous H2S generation and component of aging pathway

AbstractBackgroundDecline in metabolic fitness, accumulation of undesirable metabolic changes and cellular senescence are the hallmarks of aging [1,2]. The incorporation of lifespan‐extending platforms such as exercise can promote metabolic fitness and delay the process of aging [3]. However, the mechanisms involved are unclear. We hypothesized that exercise intervention can drive beneficial outcome by increasing expression of two enzymes; CBS and CTH involved in the production of H2S, glutathione and taurine as well as splice factors; SRSF2 and HNRNPD [4,5] in African American with MCI.MethodWe used TaqMan gene expression assay to investigate mechanistic link between exercise and components of aging pathway by evaluating CBS and CTH, enzymes metabolizing sulfur containing amino acid. We also evaluated if CBS and CTH expression correlate with SRSF2 and HNRNPD splice factors by virtue of endogenous H2S production.ResultComparison of CBS and CTH at baseline, 3‐ and 6‐month exercise time points revealed that 3‐month exercise intervention doubled the expression of CBS. However, CTH showed a decreasing trend, suggesting differential expression. Interestingly, expression of CBS correlated with SRSF2 splice factor whereas expression of CTH correlated with HNRNPD at all time points tested.ConclusionIn this study, we provided mechanistic insight through which exercise drive beneficial outcome by investigating component of aging pathway at baseline and exercise intervention in African American with MCI. Our data suggest differential effect of exercise on the expression of CBS, CTH; and the splice factors linked to delay pace of cellular senescence and aging by way of H2S [4,5]. Thus, our study implicates exercise‐induced CBS expression and endogenous H2S as drivers of benefit of fitness adaptation on metabolic fitness and life span with potential for clinical translation.

Abstract PR03: Metabolic control of Polycomb Repressive Complex 2 in Lung Disease and Lung Cancer

Abstract Dysregulated epigenetic control of lung lineage fate is believed to be a major driver of lung disease, including plasticity in lung cancer. Our laboratory has observed a marked decrease in Polycomb Repressive Complex 2 (PRC2) activity in bronchiolar epithelium of COPD patients, poorly differentiated adenocarcinomas and the majority of squamous cell carcinomas. Interestingly, the PRC2 methyltransferase EZH2 is still highly expressed in many of these tissues, suggesting a de-coupling of EZH2 and PRC2 activity. A mouse model of lineage switching from adenocarcinoma to squamous lung cancer clearly showed that loss of PRC2 activity was a catalyst to epigenetic reprogramming. This model also showed increased infiltration of neutrophils, which are known to produce large amounts of superoxide. Our current hypothesis is that oxidative stress, in part through neutrophil recruitment to lung tissue, drives the loss of PRC2 activity through metabolic changes. To support this hypothesis, we have shown that a redox-sensitive enzyme in the methionine pathway, cystathionine beta synthase (CBS), is expressed in COPD and lung cancer and modulates PRC2 activity. Enforced CBS expression drives global decrease in the PRC2-mediated histone marked, H3K27me3, and increased expression of genes expressed by basal cells of the lung, including KRT5, SOX2 and PD-L1. Furthermore, we have found that PRC2 activity can be stabilized through restriction of methionine levels or knock-down of CBS, leading to increased sensitivity of cells to chemotherapy and radiation treatments. PRC2-low epigenetic states can also be exploited – either pharmacological or genetic perturbation of EZH2 led to sensitivity to several classes of drugs, including immunotherapies. Our future studies include using Stable Isotope Resolved Metabolomics (SIRM) to understand how oxidative stress alters the methionine cycle and ultimately changes the stability and function of the PRC2 complex, and to use this information to design better therapeutic strategies. Work supported by AACR Innovation and Discovery Grant, American Cancer Society 133123-RSG-19-081-01-TBG and IRG-85-001-25, NCI R01 CA237643, NIGMS P20 GM121327-03, NIEHS T32 5T32ES007266-30, NCI T32 CA165990 and NHLBI F31 HL151111-01. Citation Format: Mojtaba Bakhtiari, Aria L. Byrd, Fan Chen, Alexsandr Lukyanchuk, Tanner J. DuCote, Christine Fillmore Brainson. Metabolic control of Polycomb Repressive Complex 2 in Lung Disease and Lung Cancer [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PR03.

Increased expression of cystathionine beta-synthase and chemokine ligand 21 is closely associated with poor prognosis in extrahepatic cholangiocarcinoma.

The expression of Cystathionine beta-synthase (CBS) and Chemokine ligand 21 (CCL21) is associated with the tumorigenesis and progression of a variety of tumors, but whether alterations in their expression levels correlates with the carcinogenesis and progression of EHCC is still unknown. This study investigated the clinicopathological significance of CBS and CCL21 expression in EHCC.We investigated the correlations between the expression of CBS and CCL21 and clinicopathological characteristics in EHCC using EnVision immunohistochemistry.The expression of CBS and CCL21 was significantly higher in EHCC tumors than in nontumor tissues (P < .05 and P < .01). EHCC patients with CBS and CCL21 expression combined with lymph node metastasis, tumor cell invasion, and TNM III/IV stage had more severe conditions than those with no lymph node metastasis, distant invasion and TNM I/II stage (P < .01). Kaplan-Meier survival analysis showed that the overall survival rates for EHCC patients with negative CBS or CCL21 reaction were significantly higher than those for patients with positive CBS or CCL21 reaction((P < .01). CBS or CCL21 expression was revealed as an independent poor prognostic factor for EHCC patients by Cox multivariate analysis.The present study indicates that CBS and CCL21 expression is closely associated with the pathogenesis of clinical, pathological and biological behaviors and poor prognosis in EHCC.

Neuroprotective Effects of Early Brain Injury after Subarachnoid Hemorrhage in Rats by Calcium Channel Mediating Hydrogen Sulfide.

The present study explored the modulating apoptosis effect of hydrogen sulfide (H2S) in subarachnoid hemorrhage (SAH) rats and its exact mechanism. A rat SAH model established by intravascular puncturing was used for the present study. After giving NaHS (donor of H2S), an L-type calcium channel opener (Bay K8644), or a calcium channel agonist (nifedipine), the neurological function of the rats, associated pathological changes, and expression of apoptosis-related proteins (Bcl-2, Bax, and caspase-3) and microtubule-associated protein (MAP-2) were examined. The concentration of H2S and expression of cystathionine beta synthase in the hippocampus changed upon early brain injury (EBI) after SAH. Compared with the SAH group, the neurological function of the rats and microstructure observed by electron microscopy were better in the SAH + NaHS group and SAH + Bay K8644 group. It was observed that apoptosis was more obvious in the SAH group than in the control group and was alleviated in the SAH + NaHS group. Furthermore, the alleviating effect of NaHS was partially weakened by nifedipine, indicating that the effect of anti-apoptosis in H2S might be correlated with the calcium channel. The expression of Bax and caspase-3 was elevated, while the expression of Bcl-2 decreased in the SAH group but improved in the SAH + NaHS and SAH + Bay K8644 group. Compared with the SAH + NaHS group, the expression of pro-apoptotic proteins was higher in the SAH + NaHS + nifedipine group. Therefore, upon EBI following SAH, the H2S system plays an important neurological protective effect by modulating the function of the L-type calcium channel and inhibiting apoptosis.

H2S Donors Reverse Age-Related Gastric Malfunction Impaired Due to Fructose-Induced Injury via CBS, CSE, and TST Expression.

ObjectiveExcess of fructose consumption is related to life-treating conditions that affected more than a third of the global population. Therefore, to identify a newer therapeutic strategy for the impact prevention of high fructose injury in age-related malfunctions of the gastric mucosa (GM) in the animal model is important.MethodsAdult and aged male rats were divided into control groups (standard diet, SD) and high fructose diet (HFD) groups; acute water immersion restraint stress (WIRS) was induced for evaluation of GM adaptive response and effects of testing the therapeutic potential of H2S-releasing compounds (H2S donors). Histological examination of gastric damage was done on hematoxylin-eosin stained slides. Cystathionine beta-synthase (CBS), Cystathionine gamma-lyase (CSE), and Thiosulfate-dithiol sulfurtransferase (TST) activities and oxidative index were assessed during exogenous administration of H2S donors: sodium hydrosulfide (NaHS) and the novel hybrid H2S-releasing aspirin (ATB-340). The results showed that HFD increased gastric damage in adult and aged rats. HFD-associated malfunction characterized by low activities of H2S key enzymes, inducing increased oxidation. Pretreatment with NaHS, ATB-340 of aged rats in the models of HFD, and WIRS attenuated gastric damage in contrast to vehicle-treated group (p < 0.05). The effect of ATB-340 was characterized by reverse oxidative index and increased CBS, CSE, and TST activities. In conclusion, H2S donors prevent GM age-related malfunctions by enhancement of CBS, CSE, and TST expression against fructose excess injury though reduction of oxidative damage.

Hydrogen sulfide formation in experimental model of acute pancreatitis.

Acute pancreatitis (AP) is a disease defined as acute or chronic inflammatory process of the pancreas characterized by premature activation of digestive enzymes within the pancreatic acinar cells and causing pancreatic auto-digestion. In mammalian tissues, H2S is synthesized endogenously from L-cysteine in regulated enzymatic pathways catalyzed by pyridoxal phosphate-dependent enzymes: cystathionine beta - synthase (CBS), gamma - cystathionase (CTH) and cysteine aminotransferase (CAT) coupled with 3-mercaptopyruvate sulfurtransferase (MPST). In the mitochondria, hydrogen sulfide is oxidized to sulfite, which is then converted to thiosulfate (sulfane sulfur-containing compound) by thiosulfate sulfurtransferase (rhodanese; TST). The activity and the expression of CBS, CTH, MPST, and TST have been determined in vivo in pancreas of control rats, rats with acute pancreatitis and sham group. Levels of low-molecular sulfur compounds such as reduced and oxidized glutathione, cysteine, cystine and cystathionine were also determined. The study showed the significant role of MPST in H2S metabolism in pancreas. Stress caused by the surgery (sham group) and AP cause a decrease in H2S production associated with a decrease of MPST activity and expression. Markedly higher level of cysteine in the AP pancreas may be caused by a reduced rate of cysteine consumption in reaction catalyzed by MPST but it can also be a sign of the processes of proteolysis occurring in the changed tissue.

Hydrogen sulfide biosynthesis is impaired in the osteoarthritic joint.

Osteoarthritis (OA) is the most common form of arthritis and it is a leading cause of disability in the elderly. Its complete etiology is not known although there are several metabolic, genetic, epigenetic, and local contributing factors involved. At the moment, there is no cure for this pathology and treatment alternatives to retard or stop its progression are intensively being sought. Hydrogen sulfide (H2S) is a small gaseous molecule and is present in sulfurous mineral waters as its active component. Data from recent clinical trials shows that balneotherapy (immersion in mineral and/or thermal waters from natural springs) in sulfurous waters can improve OA symptoms, in particular, pain and function. Yet, the underlying mechanisms are poorly known. Hydrogen sulfide is also considered, with NO and CO, an endogenous signaling gasotransmitter. It is synthesized endogenously with the help of three enzymes, cystathionine gamma-lyase (CTH), cystathionine beta-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MPST). Here, the expression of these three enzymes was demonstrated by quantitative real-time polymerase chain reaction (qRT-PCR) and their protein abundance [by immunohistochemistry and Western blot (WB)] in human articular cartilage. No significant differences were found in CBS or CTH expression or abundance, but mRNA and protein levels of 3-MPST were significantly reduced in cartilage form OA donors. Also, the biosynthesis of H2S from OA cartilage, measured with a specific microelectrode, was significantly lower than in OA-free tissue. Yet, no differences were found in H2S concentration in serum from OA patients and OA-free donors. The current results suggest that reduced levels of the mitochondrial enzyme 3-MPST in OA cartilage might be, at least in part, responsible for a reduction in H2S biosynthesis in this tissue and that impaired H2S biosynthesis in the joint might be a contributing factor to OA. This could contribute to explain why exogenous supplementation of H2S, for instance with sulfurous thermal water, has positive effects in OA patients.

A diet rich in taurine, cysteine, folate, B12 and betaine may lessen risk for Alzheimer’s disease by boosting brain synthesis of hydrogen sulfide

The gaseous physiological modulator hydrogen sulfide (H2S) has recently been shown to exert a variety of neuroprotective effects. In particular, the treatment of transgenic mouse models of Alzheimer’s disease (AD) with agents that release H2S aids preservation of cognitive function, suppresses brain production of amyloid beta, and decreases tau phosphorylation. The possible physiological relevance of these findings is suggested by the finding that brain and plasma levels of H2S are markedly lower in AD patients than matched controls. Hence, nutraceutical strategies which boost brain synthesis or levels of H2S may have potential for prevention of AD. The chief enzyme which synthesizes H2S in brain parenchyma, cystathionine beta-synthase (CBS), employs cysteine as its rate-limiting substrate, and is allosterically activated by S-adenosylmethionine (SAM). Supplemental taurine has been shown to boost expression of this enzyme, as well as that of another H2S source, cystathionine gamma-lyase, in vascular tissue, and to enhance plasma H2S levels; in rats subjected to hemorrhagic stroke, co-administration of taurine has been shown to blunt a marked reduction in brain CBS expression. Brain levels of SAM are about half as high in AD patients as in controls, and this is thought to explain the reduction of brain H2S in these patients. These considerations suggest that supplementation with cysteine, taurine, and agents which promote methyl group availability – such as SAM, folate, vitamin B12, and betaine – may have potential for boosting brain synthesis of H2S and thereby aiding AD prevention. Indeed, most of these agents have already demonstrated utility in mouse AD models – albeit the extent to which increased H2S synthesis contributes to this protection remains unclear. Moreover, prospective epidemiology has associated low dietary or plasma levels of folate, B12, and taurine with increased dementia risk. Rodent studies suggest that effective nutraceutical strategies for boosting brain H2S synthesis may in fact have broad neuroprotective utility, possibly aiding prevention and/or control not only of AD but also Parkinson’s disease and glaucoma, while diminishing the neuronal damage associated with brain trauma or stroke.

Abstract 952: Determining the contribution of cystathionine beta synthase to lung cancer lineage fate

Abstract The two major pathologies of non-small cell lung cancer, adenocarcinoma (ADC) and squamous cell carcinoma (SCC) are historically treated as separate diseases, even though mounting evidence shows that epigenetic reprogramming from ADC to a more SCC fate allows lung cancers to evade therapies. Our team recently developed an isogenic lung cancer model in which lineage switching from ADC to SCC was epigenetically controlled through loss of Polycomb Repressive Complex 2 (PRC2) gene repression of squamous genes. Levels of S-adenosyl methionine (SAM), the methyl donor for the complex, influence PRC2 stability and activity. Steady state metabolism showed that SAM levels were decreased significantly and cystathionine levels were increased significantly in SCC cells relative to ADC cells. Our hypothesis is that increased activity of the enzyme cystathionine beta synthase (CBS) drives epigenetic reprogramming of lung ADC to an aggressive and therapy-resistant state through reduction of SAM pools and destabilization of PRC2, leading to expression of squamous genes. First, we stained a series of human lung cancer tissues for CBS and H3K27me3 and queried TCGA data to validate that CBS is up-regulated in squamous tumors. In addition, we leveraged 3-dimensional organoid cultures and traditional 2-dimensional human non-small cell lung cancer cell lines to explore the contribution of CBS to lung cancer lineage fate. With both of these systems, we used lentiviral small hairpins targeting Cbs/CBS and a lentivirus encoding a full length Cbs/CBS cDNA to modulate gene and protein levels. Lastly, we tested differential sensitivities to the common chemotherapeutic carboplatin on matched isogenic cultures that differ only in lineage state. In human tumors, SCCs have significantly higher levels of CBS and significantly lower levels of H3K27me3 than ADCs. Knock-down of CBS decreased cystathionine and increased both homocysteine and S-adenosyl methionine levels in 2-dimensional human NSCLC cultures. We also observed a global increase in H3K27me3 mark in shCBS cultures relative to shGFP controls. In contrast, CBS over-expression decreased global levels of H3K27me3. Importantly, we observed that squamous organoids are significantly less sensitive to the common chemotherapeutic carboplatin than are ADC organoids, suggesting that lineage switching driven by increased CBS activity will drive chemotherapy resistance. We are now characterizing changes in lineage fate and carboplatin sensitivity that are caused by these genetic manipulations of CBS. Our data suggest that lung cancer lineage fate may be governed in part through expression and activity of the enzyme CBS. Because CBS is a redox-sensitive enzyme, this could be the missing link between increased oxidative stress and ultimate epigenetic reprogramming to a therapy-resistant squamous state in lung cancer. Funded by ACS IRG-85-001-25, K22 CA201036 and KY LCRP to CFB Citation Format: Mojtaba Bakhtiari, Christine Fillmore Brainson. Determining the contribution of cystathionine beta synthase to lung cancer lineage fate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 952.

Short tandem repeats and methylation in the promoter region affect expression of cystathionine beta-synthase gene in the laying hen

Homocysteine can be converted to cysteine via the transsulfuration pathway where cystathionine beta-synthase (CBS) is a rate-limiting enzyme. Homocysteine is thought to play a role in bone remodelling and strength. Previous results indicated that some of the difference in bone strength of end-of-lay hens may be associated with CBS expression level. To investigate if differences in the promoter region of the CBS gene might be responsible for observed differences in gene expression between CBS alleles post mortem- and in-vitro expression studies have been undertaken. Transfection of the DF-1 avian cell line with a series of deletion fragments of the 5′ promoter, or constructs containing three CBS allele sequences, with a luciferase reporter revealed that a core region of −155 to +131 bp in the CBS promoter was essential for mRNA expression. We found that a variable number of short tandem repeats (7 nucleotide motif and 6 nucleotide repeats) in the core region of the promoter affecting the transcriptional activity and a strong effect for gene expression. However, methylation of the 6 nucleotide repeats varied between allelic variants and these maybe responsible for differences in promoter activity. Our findings indicated variable short tandem repeats and the differentially methylated sites in the promoter region may be responsible for CBS expression differences in the bone of laying hens.

Alteration of dietary cysteine affects activities of genes of the transsulfuration and glutathione pathways, and development of skin tissues and feather follicles in chickens

The dietary requirement for cysteine is not determined in poultry since it is not an essential amino acid. The cysteine need is expected to be met through the transsulfuration pathway where homocysteine, a precursor of methionine, is converted to cysteine. Cysteine is a major component of plumage, and the degree to which cysteine is involved in plumage and other keratized proteins are unknown. We randomly assigned chicks to control and treatment (deficient in cysteine) diets for 49 d. The thickness of the skin layers, feather follicle length, and thickness were measured at days 10, 24, 34, and 49. We also measured the hepatic mRNA expressions of cystathionine beta synthase (CBS), cystathionine γ-lyase (CTL), cysteine dioxygenase (CDO), and glutathione synthetase (GSS). Chickens fed the treatment diet had reduced epidermis thickness and shorter feather follicles compared with the controls. The chicken fed the treatment diet also had increased mRNA expression of CBS and CTL indicating a disruption of the transsulfuration pathway. The treatment chickens also had a decreased hepatic CDO and increased GSS mRNA expressions which are in concordance with the homeostatic regulation of cysteine. Compromised cysteine metabolism could affect thermoregulation and subsequently affect feed efficiency and welfare of the birds.

Neuroprotective effects of lentivirus-mediated cystathionine-beta-synthase overexpression against 6-OHDA-induced parkinson's disease rats.

Parkinson's disease (PD) is age-related neurodegenerative disorder by a progressive loss of dopaminergic(DA) neurons in the substantia nigra (SN) and striatum, which is at least partly associated with α-synuclein protein accumulation in these neurons. Hydrogen sulfide (H2S) plays an important role in the nervous system. Studies have shown that H2S has a protective effect on PD. However, as a kind of gas molecules, H2S is lively, volatile, and not conducive to scientific research and clinical application. Cystathionine-beta-synthase(CBS) is the main enzymes of synthesis of H2S in the brain. In order to examine the neuroprotective effects of CBS on PD, we detected the effects of CBS overexpression on 6-Hydroxydopamine (6-OHDA)-lesioned PD rats using lentivirus-mediated gene transfection techniques. In the injured SN of 6-OHDA-induced PD rats, the CBS expression and the endogenous H2S level markedly decreased, while administration of lentivirus-mediated CBS overexpression increased the CBS expression and the endogenous H2S production.CBS overexpression dramatically reversed apomorphine-induced rotation of the 6-OHDA model rats, decreased the number of TUNEL-positive neurons and the loss of the nigral DA neurons，specifically inhibited 6-OHDA-induced oxidase stress injury, and down-regulated the expression of α-synuclein(α-SYN) in the injured SN. NaHS (an H2S donor) had similar effects to CBS overexpression, while Amino-oxyacetate(AOAA, a CBS inhibitor) had opposite effects on PD rats. In summary, we demonstrated that CBS overexpression was able to provide neuroprotective on PD rats and improving the expression of CBS may be a potential therapeutic method for PD.