Cystathionine Beta-synthase Enzyme Research Articles

Architecture and regulation of filamentous human cystathionine beta-synthase

Cystathionine beta-synthase (CBS) is an essential metabolic enzyme across all domains of life for the production of glutathione, cysteine, and hydrogen sulfide. Appended to the conserved catalytic domain of human CBS is a regulatory domain that modulates activity by S-adenosyl-L-methionine (SAM) and promotes oligomerisation. Here we show using cryo-electron microscopy that full-length human CBS in the basal and SAM-bound activated states polymerises as filaments mediated by a conserved regulatory domain loop. In the basal state, CBS regulatory domains sterically block the catalytic domain active site, resulting in a low-activity filament with three CBS dimers per turn. This steric block is removed when in the activated state, one SAM molecule binds to the regulatory domain, forming a high-activity filament with two CBS dimers per turn. These large conformational changes result in a central filament of SAM-stabilised regulatory domains at the core, decorated with highly flexible catalytic domains. Polymerisation stabilises CBS and reduces thermal denaturation. In PC-3 cells, we observed nutrient-responsive CBS filamentation that disassembles when methionine is depleted and reversed in the presence of SAM. Together our findings extend our understanding of CBS enzyme regulation, and open new avenues for investigating the pathogenic mechanism and therapeutic opportunities for CBS-associated disorders.

Classical homocystinuria presenting with transient basal ganglia pathology and dystonia.

Classical homocystinuria is caused by pathogenic variants in the CBS gene leading to a deficiency of the vitamin B6-dependent enzyme cystathionine beta synthase. The disease is typically associated with high blood homocysteine concentrations. Clinical features include developmental delay/intellectual disability, psychiatric problems, thromboembolism, lens dislocation, and marfanoid habitus. We report on a child with classical homocystinuria presenting with acute episodes of dystonia and symmetrical basal ganglia abnormalities mimicking a mitochondrial disease. After starting treatment with vitamin B6, homocysteine levels rapidly normalized and dystonic episodes did not re-occur. Moreover, brain-imaging findings almost completely disappeared. The case illustrates that homocystinuria should be considered as a treatable differential diagnosis of dystonia.

CTH/MPST double ablation results in enhanced vasorelaxation and reduced blood pressure via upregulation of the eNOS/sGC pathway.

Hydrogen sulfide (H2S), a gasotransmitter with protective effects in the cardiovascular system, is endogenously generated by three main enzymatic pathways: cystathionine gamma lyase (CTH), cystathionine beta synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MPST) enzymes. CTH and MPST are the predominant sources of H2S in the heart and blood vessels, exhibiting distinct effects in the cardiovascular system. To better understand the impact of H2S in cardiovascular homeostasis, we generated a double Cth/Mpst knockout (Cth/Mpst -/- ) mouse and characterized its cardiovascular phenotype. CTH/MPST-deficient mice were viable, fertile and exhibited no gross abnormalities. Lack of both CTH and MPST did not affect the levels of CBS and H2S-degrading enzymes in the heart and the aorta. Cth/Mpst -/- mice also exhibited reduced systolic, diastolic and mean arterial blood pressure, and presented normal left ventricular structure and fraction. Aortic ring relaxation in response to exogenously applied H2S was similar between the two genotypes. Interestingly, an enhanced endothelium-dependent relaxation to acetylcholine was observed in mice in which both enzymes were deleted. This paradoxical change was associated with upregulated levels of endothelial nitric oxide synthase (eNOS) and soluble guanylate cyclase (sGC) α1 and β1 subunits and increased NO-donor-induced vasorelaxation. Administration of a NOS-inhibitor, increased mean arterial blood pressure to a similar extent in wild-type and Cth/Mpst -/- mice. We conclude that chronic elimination of the two major H2S sources in the cardiovascular system, leads to an adaptive upregulation of eNOS/sGC signaling, revealing novel ways through which H2S affects the NO/cGMP pathway.

Current and Novel Therapeutical Approaches of Classical Homocystinuria in Childhood With Special Focus on Enzyme Replacement Therapy, Liver-Directed Therapy and Gene Therapy.

Classical homocystinuria is a hereditary defect of the enzyme cystathionine beta synthase, which is produced in the liver. If this enzyme fails, the synthesis pathway of cysteine from methionine is interrupted, leading to the accumulation of homocysteine in the blood plasma and homocysteine in the urine. After birth, the children are unremarkable except for the characteristic laboratory findings. Symptoms rarely appear before the second year of life. The most common symptom is a prolapse of the crystalline lens. This finding is seen in 70% of untreated 10-year-old affected individuals. As the earliest symptom, psychomotor retardation occurs in the majority of patients already during the first two years of life. Limiting factors in terms of life expectancy are thromboembolism, peripheral arterial disease, myocardial infarction, and stroke. These symptoms are due to the damage to the vessels caused by the elevated amino acid levels. About 30% suffer a thromboembolic event by the age of 20, about half by the age of 30. This review focus on present and new therapeutical approaches like the role of enzyme replacement with presentation of different novel targets in research like pegtibatinase, pegtarviliase, CDX-6512, erymethionase, chaperones, proteasome inhibitors and probiotic treatment with SYNB 1353. Furthermore, we analyze the role of liver-directed therapy with three dimensional (3D) bioprinting, liver bioengineering of liver organoids in vitro and liver transplantation. The role of different gene therapy options to treat and cure this extremely rare disease in childhood will be discussed.

Laser ablation combined with Nanoimprint Lithography technology as new surface engineering approach to produce novel polymer-based heteronucleants for recalcitrant protein crystallization

Macromolecule crystallography is currently the most powerful technique for determining the three dimensional structure of proteins at atomic resolution. However, despite its enormous level of technical development achieved, it is still limited by the great difficulty of obtaining crystals that, after exposure to X-rays, allow a high-resolution diffraction pattern from which the structure of the target protein can be elucidated. Even today, the crystallization process is a trial-and-error procedure that requires enormous experimental effort and high economic costs. In this research work, Laser Ablation technology in combination with NanoImprint Lithography was applied on Polycarbonate surfaces to produce novel heteronucleating agents containing multiscale surface topographies (in the micro- and submicro-scale) that promote the growth of protein crystals. Two human proteins, known to be recalcitrant to crystallization when using standard protocols, were used in the assays: (i) a construct of the CNNM4 magnesium transporter lacking amino acid residues 545–730 (HsCNNM4545-730) and (ii) cystathionine beta-synthase enzyme lacking residues 516–525 (HsCBSΔ516-525). The surface engineering solution was validated by comparing the number of novel crystallization conditions induced by those surfaces compared against the control and the use of the Naomi’s Nucleant, a commercially available heteronucleant made of mesoporous bioglass. The aspect ratio of the topography emerges as the topographical parameter that most contributes to protein growth on patterned surfaces. Additionally, the presence of a random distribution of micropores as well as a hierarchical (merging micro- and nano - scale features) surface topography also resulted in a more efficient crystallization process, showing an increment of between 38 and 68% in the number of new crystallization conditions, depending on the surface topography and protein selected. A conclusion is made that Laser Ablation with ultrashort pulses and the combination with Nanoimprint Lithography can be efficiently applied on polymeric substrates to produce a novel concept of heteronucleants that would bridge the gap to the development of universal nucleation agents.

Homocystinuria presents with generalized tonic-clonic seizure and Broca's aphasia

Homocystinuria follows an autosomal recessive pattern and transpires due to Cystathionine beta-synthase enzyme deficiency. This condition is associated with thrombotic and thromboembolic complications due to an increase in homocysteine and methionine levels in the plasma and urine. We present a case of an Indian adolescent boy who presented to us with generalized tonic clinic seizure and Broca's aphasia he was diagnosed as having homocystinuria type 1, and he responded well to treatment.Background: Stroke and seizure disorders in young adults have increased substantially and are a relatively common disorder that presents in the emergency department in all age groups. Secondary seizures are more common and manifest as a result of another underlying pathology. This case demonstrates the importance of considering rarer causes of thrombosis such as arterial thrombosis, even in younger patients, with no obvious risk factors or comorbidities. A detailed medical history and thorough investigations need to be taken to identify a potentially treatable underlying cause.Objective: To increase awareness about this rare etiology and help clinicians to have a higher level of suspicion to diagnose this condition and prevent future life-threatening complications in younger patients with thrombotic and thromboembolic events.  Â

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.

Modulation of Human Hydrogen Sulfide Metabolism by Micronutrients, Preliminary Data.

Background:Hydrogen sulfide (H2S) is a pivotal gasotransmitter networking with nitric oxide (NO) and carbon monoxide (CO) to regulate basic homeostatic functions. It is released by the alternative pathways of transulfuration by the enzymes Cystathionine Beta Synthase (CBS) and Cystathionine Gamma Lyase (CSE), and by Cysteine AminoTransferase (CAT)/ 3-Mercaptopyruvate Sulfur Transferase (3MPST). A non-enzymatic, intravascular release is also in place. We retrospectively investigated the possibility to modulate the endogenous H2S release and signaling in humans by a dietary manipulation with supplemented micronutrients (L-cystine, Taurine and pyridoxal 5-phopsphate/P5P).Methods:Patients referring for antiaging purposes underwent a 10-day supplementation. Blood was collected at baseline and after treatment and the metabolome was investigated by mass spectrometry to monitor the changes in the metabolites reporting on H2S metabolism and related pathways.Results:Data were available from 6 middle aged subjects (2 women). Micronutrients increased 3-mercaptopyruvate (P = .03), reporting on the activity of CAT that provides the substrate for H2S release within mitochondria by 3MPST, decreased lanthionine (P = .024), reporting the release of H2S from CBS, and had no significant effect of H2S release from CSE. This is compatible with a homeostatic balancing. We also recorded a strong increase of reporters of H2S-induced pathways including 5-MethylTHF (P = .001) and SAME (P = .022), reporting on methylation capacity, and of BH4 (P = .021) and BH2 (P = .028) reporting on nitric oxide metabolism. These activations may be explained by the concomitant induction of non-enzymatic release of H2S.Conclusions:Although the current evidences are weak and will need to be confirmed, the effect of micronutrients was compatible with an increase of the H2S endogenous release and signaling within the control of homeostatic mechanisms, further endorsing the role of feeding in health and disease. These effects might result in a H2S boosting effect in case of defective activity of pathologic origin, which should be checked in duly designed clinical trials.

57 The Saga of Just One QTL; From Laying Hen Bone Strength to One Carbon Metabolism

Abstract Skeletal damage is a challenge for laying hens, as the adaptations for egg laying make them susceptible to osteoporosis. We know that activity, growth and puberty influence skeletal quality genetics. However, in this story, we follow the process of resolving a QTL for skeletal quality to a single gene. Providing both a nutritional and a genetic solution and illustrating another level of complexity. Using divergently selected populations in an F2 cross, a QTL for skeletal quality was described. Utilising a series of experiments in the commercial founder line the QTL was fine mapped on chromosome 1 and the differences in physiology of hens that segregated for markers at the locus defined e.g. tibia breaking strength 200.4 vs 218.1 Newton (p< 0.002). Transcriptome profiling of tibia from the high and low bone strength genotype revealed a highly differentially expressed gene co localising to the QTL, the enzyme cystathionine beta synthase (CBS). CBS is a component of one carbon metabolism. Plasma homocysteine, the substrate of CBS had a higher concentration in the genotype associated with poor bone quality. Homocysteine interferes with crosslinking of the collagen matrix, critical to bone formation. Homocysteine can be re-methylated into methionine, a limiting amino acid in laying hens, using betaine as a methyl donor. Testing the hypothesis that reducing plasma homocysteine by feeding betaine, a widely available feed additive, would improve bone quality in laying hens proved successful.This is just one component of a complex trait and its elucidation took us along unexpected routes. However, I am optimistic that with modern tools and an open mind we can progress more quickly. Alongside understanding the factors that determine skeletal quality genetics, the discovery of the mechanisms behind complex traits, although rarely easy, offer the potential for novel interventions to improve animal health and production.

Highly recurrent CBS epimutations in gastric cancer CpG island methylator phenotypes and inflammation

BackgroundCIMP (CpG island methylator phenotype) is an epigenetic molecular subtype, observed in multiple malignancies and associated with the epigenetic silencing of tumor suppressors. Currently, for most cancers including gastric cancer (GC), mechanisms underlying CIMP remain poorly understood. We sought to discover molecular contributors to CIMP in GC, by performing global DNA methylation, gene expression, and proteomics profiling across 14 gastric cell lines, followed by similar integrative analysis in 50 GC cell lines and 467 primary GCs.ResultsWe identify the cystathionine beta-synthase enzyme (CBS) as a highly recurrent target of epigenetic silencing in CIMP GC. Likewise, we show that CBS epimutations are significantly associated with CIMP in various other cancers, occurring even in premalignant gastroesophageal conditions and longitudinally linked to clinical persistence. Of note, CRISPR deletion of CBS in normal gastric epithelial cells induces widespread DNA methylation changes that overlap with primary GC CIMP patterns. Reflecting its metabolic role as a gatekeeper interlinking the methionine and homocysteine cycles, CBS loss in vitro also causes reductions in the anti-inflammatory gasotransmitter hydrogen sulfide (H2S), with concomitant increase in NF-κB activity. In a murine genetic model of CBS deficiency, preliminary data indicate upregulated immune-mediated transcriptional signatures in the stomach.ConclusionsOur results implicate CBS as a bi-faceted modifier of aberrant DNA methylation and inflammation in GC and highlights H2S donors as a potential new therapy for CBS-silenced lesions.

Suspecting classical homocystinuria in an adolescent born before the newborn screening program

Introduction: Classical homocystinuria (HCU) is an autosomal recessive disorder caused by a deficiency in the cystathionine beta-synthase enzyme and associated with a high probability of vascular complications. Herein is presented the case of an adolescent diagnosed with HCU during cerebral venous sinus thrombosis (CVST) study. Case Report: A 14-year-old girl presented with thrombophilia screening tests suggestive of HCU during CVST study. After referral to an Inherited Metabolic Diseases Unit, she started supplementation with pyridoxine, folic acid, vitamin B12, betaine anhydrous, and cysteine and was advised to restrict natural proteins and methionine from diet. Genetic analysis revealed a homozygous CBS mutation (c.572C>T (p.T191M) with c.699C>T (p.Y233Y) polymorphism. Discussion: In adolescents born before 2004 (year of implementation of the Portuguese newborn screening program), HCU should be considered when studying hypercoagulability syndromes, as it is a treatable condition and treatment can prevent major morbidity and mortality causes.

The first Saudi baby with classic homocystinuria diagnosed by universal newborn screening

ABSTRACTClassic homocystinuria (CH) is an inborn error of metabolism caused by cystathionine beta-synthase enzyme deficiency. Affected patients present with intellectual disability and other comorbidities. If diagnosed early in infancy and started treatment, inevitable complications can be prevented. Newborn screening (NBS) uses tandem mass-spectroscopy (MSMS) to measure the amino acid levels. In CH, the first-tier screening test is the measurement of methionine by MSMS. If methionine remained elevated in the recall sample, plasma level for homocysteine is performed. A newborn infant underwent routine NBS in our institute that showed elevated methionine in the first and the recall sample. Thereafter, total serum homocysteine was found to be elevated, consistent with the diagnosis of CH. An early medical and dietary management was commenced for this first Saudi baby diagnosed with homocystinuria by universal NBS. This report demonstrates that NBS for CH is feasible and effective in preventing the disease burden.

When to measure plasma homocysteine and how to place it in context: The homocystinurias.

This review presents clinical patterns that should trigger homocysteine measurement in blood, as well as the further diagnostic work-up focused on inborn errors of metabolism and disorders of vitamin B12 (cobalamin) absorption and supply. The numerous conditions (e.g. cardiovascular disease, Alzheimer’s disease) for which mild-to-moderate hyperhomocysteinaemia caused by genetic polymorphisms or acquired reasons is considered a risk factor are beyond the scope of this review.Homocysteine is a sulphur-containing amino acid, which is derived from the amino acid methionine. Homocysteine is either trans-sulphurated to form cystathionine and then cysteine, or re-methylated to methionine. The trans-sulphuration reaction depends on the enzyme cystathionine beta synthase and its cofactor vitamin B6. The re-methylation reaction not only involves the enzymes methionine synthase and methionine synthase reductase but also depends on the cofactor cobalamin and on the provision of methyl groups from the folate cycle. Because the homocysteine–methionine cycle provides for the vast majority of methyl groups in the body, it is central to numerous pathways that depend on methyl group supply, such as creatine synthesis or DNA methylation. Based on this premise, the severity of clinical presentations of inborn errors of metabolism, such as classical homocystinuria or the cobalamin C (cblC) defect, affecting this pathway is unsurprising.

P44 Homocystinuria is a rare cause of fracture in young patients: a case report

Abstract Background Homocysteinuira is a rare autosomal recessive disorder in the metabolism of sulfur-containing aminoacid caused by mutations in the cystathionine beta-synthase gene which encodes the pyridoxine dependent enzyme cystathionine beta-synthase. It is characterised by significant elevations in plasma and urine homocysteine concentrations and fractures. Methods We describe a case of homocysteinuira in a patient who suffered from a low impact patellar fracture, along with a literature review highlighting the critical relationship between homocysteine level and bone health. Results We reported a 36-year-old female with homocysteinuira due to pyridoxine (B6) unresponsive severe Cystathionine Beta-Synthase deficiency. She developed a right patellar low-impact fracture with osteopenia on X-ray. Examination revealed severe scoliosis with bilateral aphakia (absence of the lens of the eye). Her labs showed, persistent high homocysteine above 100 umol/L (4-10), methionine 383 umol/L (10-42), vitamin D 12 ng/ml. Her spine X-ray revealed severe scoliosis with osteopenia but no fracture. Her DXA scan showed her Z-Score was within the expected range for her age in hip, spine and 1/3 radius areas, however her ultra-distal radius Z-Score was -4.0. Her homocysteine level was mostly higher than 100 due to non-compliance with dietary advice and treatment. The severity of complications primarily depends on the age at diagnosis and the residual cystathionine beta-synthase activity. Most homocysteinuira patients diagnosed in adulthood have severe osteoporosis. Notably, a thinning and lengthening of the long bones result in tall and thin individuals with an appearance like that of Marfan’s syndrome. However, osteoporosis, particularly of the spine, which is associated with scoliosis, is one of the distinguishing features of the disease. High homocysteine levels in homocysteinuric patients impair the function of bone cells that regulate bone remodeling as well as bone material properties such as collagen cross-linking. This imbalance between bone formation and resorption may lead to a low BMD and fracture in patients with. Interestingly, even in general population hyperhomocysteinemia with a plasma level of more than > 13 nmol/ml has been found to be associated with low BMD and an increased risk of fractures that is independent of BMD. Deficiencies in vitamin B6, B12, or folate can lead to increased serum levels of homocysteine as these vitamins act as co-factors for various enzymes involved in homocysteine metabolism. This can be easily rectified with dietary intervention. Conclusion In young patients with a fracture or low BMD, hyperhomocysteinemia could play an important role in the pathogenesis. Homocysteine level should be included in the work up for secondary osteoporosis at young age-group. Simple dietary measures and vitamin B6, folate and B12 supplement should be considered as adjuvant therapeutic option for lowm BMD patients if deficient. Possibly the BMD of the ultra-distal radius is the most sensitive to detect bone changes in hyperhomocysteinemic patients. Disclosures A. Al-Allaf None. T. Paul None. B. Awadh None.

A Putative Cystathionine Beta‐Synthase Homolog of Mycobacterium smegmatis is Involved in de novo Cysteine Biosynthesis

Mycobacteria include serious pathogens of humans and animals. Mycobacterium smegmatis is a non‐pathogenic model that is widely used to study core mycobacterial metabolism. Using M. smegmatis, we are genetically investigating mycobacterial pathways for biosynthesis of cysteine, a vital sulfur‐containing amino acid. Publishedin vitro biochemical studies had revealed three independent routes to cysteine synthesis in mycobacteria involving separate homologs of cysteine synthase, namely CysK1, CysK2, and CysM. However, in vivo data were lacking. The M. smegmatis genome encodes only a CysM homolog and lacks orthologs for CysK1 or CysK2. However, the genome encodes a putative cystathionine beta‐synthase (CBS) protein that has two domains ‐ an N‐terminal domain that shares weak sequence similarity with CysK1 and a C‐terminal domain that is specific to CBS enzymes. CBS is a metabolic enzyme that catalyzes the conversion of homocysteine to cystathionine in all the three domains of life (Bacteria, Archaea, and Eukarya). To dissect the roles of CysM and CBS proteins in cysteine biosynthesis in vivo, we generated a series of unmarked gene deletion mutants and gene complementation strains of M. smegmatis and analyzed them phenotypically. We found that neither the ΔcysM nor the Δcbs mutants of M. smegmatis were auxotrophic for cysteine. However, a ΔcbsΔcysM double mutant of M. smegmatis was auxotrophic for cysteine. Genetic complementation of the double mutant using either cbs or cysM genes rescued cysteine auxotrophy. Furthermore, the N‐terminal CysK1‐like domain of the putative CBS was sufficient to rescue cysteine auxotrophy. Thus, our in vivo data implicate a role for the putative CBS in cysteine biosynthesis and also suggest that the protein may have dual functions in mycobacteria. Multidrug resistant (MDR) strains of M. tuberculosis, the causative agent of Tuberculosis (TB), are becoming a global crisis. Mycobacterial sulfur metabolism has emerged as a vital target for developing novel drugs to treat MDR‐TB. Our findings reveal a potentially new target in mycobacterial sulfur metabolism relevant to strategic development of novel TB drugs.Support or Funding InformationThis project was supported by the Cell and Molecular Biology program and the Department of Biological Sciences at the University of Arkansas, Fayetteville, AR 72701.

Homocystinuria a rare cause of low BMD in young patients: A case report with literature review

Background: Homocystinuira is a rare autosomal recessive disorder in the metabolism of sulfur-containing amino acid caused by mutations in the cystathionine beta-synthase gene which encodes the pyridoxine (Vitamin B6) dependent enzyme cystathionine beta-synthase. It is characterized by significant elevations in plasma and urine homocysteine concentrations, which could be associated with increased risk of fracture. Methods: We describe a case of Homocystinuria who suffered from a low impact patellar fracture with a literature review to highlight the critical relationship between homocysteine level and bone health. Results: We reported a 36-year-old female with a diagnosis of Homocystinuria due to pyridoxine (B6) unresponsive severe Cystathionine Beta-Synthase deficiency. After a minor knee injury she developed a right patellar fracture and her X-ray revealed osteopenia. On examination, she has severe scoliosis in the spine with bilateral aphakia (absence of the lens of the eye). Her labs showed, persistent high Homocysteine above 100 umol/L, Methionine: 383 umol/L (10-42), Vitamin D 12 ng/ml. Her spine X-ray revealed very severe scoliosis with osteopenia but no vertebra fracture. Her DXA scan showed her Z-Score was within the expected range for her age in hip, spine and 1/3 radius areas, however her ultra-distal radius Z-Score was -4.0. Her Homocystine level was mostly higher than 100 due to non-compliance with dietary advice and treatment. High homocysteine levels in Homocystinuric patients impair the function of bone cells that regulate bone remodeling as well as bone material properties such as collagen cross-linking. This imbalance between bone formation and resorption may lead to a low BMD and fracture in patients with homocystinuria. Interestingly, even in general population hyperhomocystinemia with a plasma level of more than > 13 nmol/ml has been found to be associated with low BMD and an increased risk of fractures that is independent of BMD. Deficiencies in vitamin B6, B12, or folate can lead to increased serum levels of Homocysteine as these vitamins act as co-factors for various enzymes involved in homocysteine metabolism. This can be easily rectified with dietary intervention. Conclusion: In young patients with a fracture or low bone mineral density, hyperhomocystinemia could play an important role in the pathogenesis. This should be included in the work up for secondary causes for osteoporosis for this age-group. Simple dietary measures and vitamin B6, folate and B12 supplement should be considered as adjuvant therapeutic option for any patient with osteoporosis and fractures, if deficient. Possibly the BMD of the ultra-distal radius is the most sensitive to detect bone changes in these patients.

The H2S-Nrf2-Antioxidant Proteins Axis Protects Renal Tubular Epithelial Cells of the Native Hibernator Syrian Hamster from Reoxygenation-Induced Cell Death.

During hibernation, repeated cycles of ischemia-reperfusion (I-R) leave vital organs without injury. Studying this phenomenon may reveal pathways applicable to improving outcomes in I-R injury-induced human diseases. We evaluated whether the H2S–nuclear factor erythroid 2-like 2 (Nrf2)–antioxidant proteins axis protects renal proximal tubular epithelial cells (RPTECs) of the native hibernator, the Syrian hamster, from reperfusion-induced cell death. To imitate I-R, the hamsters’, and control mice’s RPTECs were subjected to warm anoxia, washed, and then subjected to reoxygenation in fresh culture medium. Whenever required, the H2S-producing enzymes inhibitor aminooxyacetate or the lipid peroxidation inhibitor α-tocopherol were used. A handmade H2S detection methylene blue assay, a reactive oxygen species (ROS) detection kit, a LDH release cytotoxicity assay kit, and western blotting were used. Reoxygenation upregulated the H2S-producing enzymes cystathionine beta-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase in the hamster, but not in mouse RPTECs. As a result, H2S production increased only in the hamster RPTECs under reoxygenation conditions. Nrf2 expression followed the alterations of H2S production leading to an enhanced level of the antioxidant enzymes superoxide dismutase 3 and glutathione reductase, and anti-ferroptotic proteins ferritin H and cystine-glutamate antiporter. The upregulated antioxidant enzymes and anti-ferroptotic proteins controlled ROS production and rescued hamster RPTECs from reoxygenation-induced, lipid peroxidation-mediated cell death. In conclusion, in RPTECs of the native hibernator Syrian hamster, reoxygenation activates the H2S–Nrf2–antioxidant proteins axis, which rescues cells from reoxygenation-induced cell death. Further studies may reveal that the therapeutic activation of this axis in non-hibernating species, including humans, may be beneficial in I-R injury-induced diseases.

Analysis of the Qatari R336C cystathionine β-synthase protein in mice.

Classical homocystinuria is a recessive inborn error of metabolism caused by mutations in the cystathionine beta-synthase (CBS) gene. The highest incidence of CBS deficiency in the world is found in the country of Qatar due to the combination of high rates of consanguinity and the presence of a founder mutation, c.1006C>T (p.R336C). This mutation does not respond to pyridoxine and is considered severe. Here we describe the creation of a mouse that is null for the mouse Cbs gene and expresses human p.R336C CBS from a zinc-inducible transgene (Tg-R336C Cbs -/- ). Zinc-treated Tg-R336C Cbs -/- mice have extreme elevation in both serum total homocysteine (tHcy) and liver tHcy compared with control transgenic mice. Both the steady-state protein levels and CBS enzyme activity levels in liver lysates from Tg-R336C Cbs -/- mice are significantly reduced compared to that found in Tg-hCBS Cbs -/- mice expressing wild-type human CBS. Treatment of Tg-R336C Cbs -/- mice with the proteasome inhibitor bortezomib results in stabilization of liver CBS protein and an increase in activity to levels found in corresponding Tg-hCBS Cbs -/- wild type mice. Surprisingly, serum tHcy did not fully correct even though liver enzyme activity was as high as control animals. This discrepancy is explained by in vitro enzymatic studies of mouse liver extracts showing that p.R336C causes reduced binding affinity for the substrate serine by almost 7-fold and significantly increased dependence on pyridoxal phosphate in the reaction buffer. These studies demonstrate that the p.R336C alteration effects both protein stability and substrate/cofactor binding.

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.

Age‐dependence of Methylation, Transsulfuration and Glutathione Synthesis Pathways in Human Frontal Cortex

Neuronal synthesis of the antioxidant glutathione (GSH) depends upon availability of cysteine, provided either by cellular uptake or by transsulfuration of homocysteine generated by the methionine cycle of methylation (Fig. 1). To evaluate age‐dependent changes in the relative contribution of these two cysteine sources, we measured redox and methylation metabolite levels in postmortem human frontal cortex from subjects across the lifespan. Levels of the transsulfuration intermediate cystathionine were significantly decreased in 60–80 yr‐old subjects, indicating that the balance between these two cysteine sources shifts to transsulfuration in older age. This shift was accompanied by decreased methionine and S‐adenosylmethionine, and an increase in homocysteine, indicating lower activity of the folate and B12‐dependent enzyme methionine synthase (MS), consistent with earlier finding of decrease MS expression with age. Cystathionine levels in 10 yr‐old autistic subjects were decreased, similar to those of 60 yr‐old subjects. In vitro studies with human neuronal cells showed that the pro‐inflammatory cytokine TNF‐alpha increased transsulfuration and inhibited MS activity in association with decreased cysteine uptake. Transsulfuration pathway enzymes cystathionine beta synthase (CBS) and cystathionine gamma lyase (CGL) both produce hydrogen sulfide, which inhibits oxidative respiration and increases glycolysis. This action decreases production of reactive oxygen species (ROS), limiting the demand for antioxidant. Together these observations illustrate how an age‐dependent increase in transsulfuration links antioxidant synthesis to methylation status in human brain. TNF‐alpha, whose levels increase with age, may be a regulator of transsulfuration, with important implications for epigenetic regulation during early brain development.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.