Human Cystathionine Γ-lyase Research Articles

Discovery of a cystathionine γ-lyase (CSE) selective inhibitor targeting active-site pyridoxal 5′-phosphate (PLP) via Schiff base formation

D,L-Propargylglycine (PAG) has been widely used as a selective inhibitor to investigate the biological functions of cystathionine γ-lyase (CSE), which catalyzes the formation of reactive sulfur species (RSS). However, PAG also inhibits other PLP (pyridoxal-5′-phosphate)-dependent enzymes such as methionine γ-lyase (MGL) and L-alanine transaminase (ALT), so highly selective CSE inhibitors are still required. Here, we performed high-throughput screening (HTS) of a large chemical library and identified oxamic hydrazide 1 as a potent inhibitor of CSE (IC50 = 13 ± 1 μM (mean ± S.E.)) with high selectivity over other PLP-dependent enzymes and RSS-generating enzymes. Inhibitor 1 inhibited the enzymatic activity of human CSE in living cells, indicating that it is sufficiently membrane-permeable. X-Ray crystal structure analysis of the complex of rat CSE (rCSE) with 1 revealed that 1 forms a Schiff base linkage with the cofactor PLP in the active site of rCSE. PLP in the active site may be a promising target for development of selective inhibitors of PLP-dependent enzymes, including RSS-generating enzymes such as cystathionine β-synthase (CBS) and cysteinyl-tRNA synthetase 2 (CARS2), which have unique substrate binding pocket structures.

Mechanism-based and computational modeling of hydrogen sulfide biogenesis inhibition: interfacial inhibition

Hydrogen sulfide (H2S) is a gaseous signaling molecule that participates in various signaling functions in health and diseases. The tetrameric cystathionine γ-lyase (CSE) contributes to H2S biogenesis and several investigations provide evidence on the pharmacological modulation of CSE as a potential target for the treatment of a multitude of conditions. D-penicillamine (D-pen) has recently been reported to selectively impede CSE-catalyzed H2S production but the molecular bases for such inhibitory effect have not been investigated. In this study, we report that D-pen follows a mixed-inhibition mechanism to inhibit both cystathionine (CST) cleavage and H2S biogenesis by human CSE. To decipher the molecular mechanisms underlying such a mixed inhibition, we performed docking and molecular dynamics (MD) simulations. Interestingly, MD analysis of CST binding reveals a likely active site configuration prior to gem-diamine intermediate formation, particularly H-bond formation between the amino group of the substrate and the O3′ of PLP. Similar analyses realized with both CST and D-pen identified three potent interfacial ligand-binding sites for D-pen and offered a rational for D-pen effect. Thus, inhibitor binding not only induces the creation of an entirely new interacting network at the vicinity of the interface between enzyme subunits, but it also exerts long range effects by propagating to the active site. Overall, our study paves the way for the design of new allosteric interfacial inhibitory compounds that will specifically modulate H2S biogenesis by cystathionine γ-lyase.

ERα/ERβ-directed CBS transcription mediates E2β-stimulated hUAEC H2S production.

Elevated endogenous estrogens stimulate human uterine artery endothelial cell (hUAEC) hydrogen sulfide (H2S) production by selectively upregulating the expression of H2S synthesizing enzyme cystathionine β-synthase (CBS), but the underlying mechanisms are underdetermined. We hypothesized that CBS transcription mediates estrogen-stimulated pregnancy-dependent hUAEC H2S production. Estradiol-17β (E2β) stimulated CBS but not cystathionine γ-lyase (CSE) expression in pregnant human uterine artery ex vivo, which was attenuated by the estrogen receptor (ER) antagonist ICI 182,780. E2β stimulated CBS mRNA/protein and H2S production in primary hUAEC from nonpregnant and pregnant women, but with greater responses in pregnant state; all were blocked by ICI 182,780. Human CBS promoter contains multiple estrogen-responsive elements (EREs), including one ERE preferentially binding ERα (αERE) and three EREs preferentially binding ERβ (βERE), and one full ERE (α/βERE) and one half ERE (½α/βERE) binding both ERα and ERβ. Luciferase assays using reporter genes driven by human CBS promoter with a series of 5'-deletions identified the α/βEREs binding both ERα and ERβ (α/βERE and ½α/βERE) to be important for baseline and E2β-stimulated CBS promoter activation. E2β stimulated ERα/ERβ heterodimerization by recruiting ERα to α/βEREs and βERE, and ERβ to βERE, α/βEREs, and αERE. ERα or ERβ agonist alone trans-activated CBS promoter, stimulated CBS mRNA/protein and H2S production to levels comparable to that of E2β-stimulated, while ERα or ERβ antagonist alone abrogated E2β-stimulated responses. E2β did not change human CSE promoter activity and CSE mRNA/protein in hUAEC. Altogether, estrogen-stimulated pregnancy-dependent hUAEC H2S production occurs by selectively upregulating CBS expression via ERα/ERβ-directed gene transcription.

Characterization of human cystathionine γ-lyase enzyme activities toward d-amino acids.

Various d-amino acids play important physiological roles in mammals, but the pathways of their production remain unknown except for d-serine, which is generated by serine racemase. Previously, we found that Escherichia coli cystathionine β-lyase possesses amino acid racemase activity in addition to β-lyase activity. In the present work, we evaluated the enzymatic activities of human cystathionine γ-lyase, which shares a relatively high amino acid sequence identity with cystathionine β-lyase. The enzyme did not show racemase activity toward various amino acids including alanine and lyase and dehydratase activities were highest toward l-cystathionine and l-homoserine, respectively. The enzyme also showed weak activity toward l-cysteine and l-serine but no activity toward d-amino acids. Intriguingly, the pH and temperature profiles of lyase activity were distinct from those of dehydratase activity. Catalytic efficiency was higher for lyase activity than for dehydratase activity.

Human Cystathionine γ-Lyase Is Inhibited by s-Nitrosation: A New Crosstalk Mechanism between NO and H2S.

The ‘gasotransmitters’ hydrogen sulfide (H2S), nitric oxide (NO), and carbon monoxide (CO) act as second messengers in human physiology, mediating signal transduction via interaction with or chemical modification of protein targets, thereby regulating processes such as neurotransmission, blood flow, immunomodulation, or energy metabolism. Due to their broad reactivity and potential toxicity, the biosynthesis and breakdown of H2S, NO, and CO are tightly regulated. Growing evidence highlights the active role of gasotransmitters in their mutual cross-regulation. In human physiology, the transsulfuration enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) are prominent H2S enzymatic sources. While CBS is known to be inhibited by NO and CO, little is known about CSE regulation by gasotransmitters. Herein, we investigated the effect of s-nitrosation on CSE catalytic activity. H2S production by recombinant human CSE was found to be inhibited by the physiological nitrosating agent s-nitrosoglutathione (GSNO), while reduced glutathione had no effect. GSNO-induced inhibition was partially reverted by ascorbate and accompanied by the disappearance of one solvent accessible protein thiol. By combining differential derivatization procedures and mass spectrometry-based analysis with functional assays, seven out of the ten protein cysteine residues, namely Cys84, Cys109, Cys137, Cys172, Cys229, Cys307, and Cys310, were identified as targets of s-nitrosation. By generating conservative Cys-to-Ser variants of the identified s-nitrosated cysteines, Cys137 was identified as most significantly contributing to the GSNO-mediated CSE inhibition. These results highlight a new mechanism of crosstalk between gasotransmitters.

S-3-Carboxypropyl-l-cysteine specifically inhibits cystathionine γ-lyase–dependent hydrogen sulfide synthesis

Hydrogen sulfide (H2S) is a gaseous signaling molecule, which modulates a wide range of mammalian physiological processes. Cystathionine γ-lyase (CSE) catalyzes H2S synthesis and is a potential target for modulating H2S levels under pathophysiological conditions. CSE is inhibited by propargylglycine (PPG), a widely used mechanism-based inhibitor. In this study, we report that inhibition of H2S synthesis from cysteine, but not the canonical cystathionine cleavage reaction catalyzed by CSE in vitro, is sensitive to preincubation of the enzyme with PPG. In contrast, the efficacy of S-3-carboxpropyl-l-cysteine (CPC) a new inhibitor described herein, was not dependent on the order of substrate/inhibitor addition. We observed that CPC inhibited the γ-elimination reaction of cystathionine and H2S synthesis from cysteine by human CSE with Ki values of 50 ± 3 and 180 ± 15 μm, respectively. We noted that CPC spared the other enzymes involved either directly (cystathionine β-synthase and mercaptopyruvate sulfurtransferase) or indirectly (cysteine aminotransferase) in H2S biogenesis. CPC also targeted CSE in cultured cells, inhibiting transsulfuration flux by 80-90%, as monitored by the transfer of radiolabel from [35S]methionine to GSH. The 2.5 Å resolution crystal structure of human CSE in complex with the CPC-derived aminoacrylate intermediate provided a structural framework for the molecular basis of its inhibitory effect. In summary, our study reveals a previously unknown confounding effect of PPG, widely used to inhibit CSE-dependent H2S synthesis, and reports on an alternative inhibitor, CPC, which could be used as a scaffold to develop more potent H2S biogenesis inhibitors.

The cystathionine γ-lyase/hydrogen sulfide pathway mediates the trimetazidine-induced protection of H9c2 cells against hypoxia/reoxygenation-induced apoptosis and oxidative stress.

Objective:Trimetazidine is a piperazine-derived metabolic agent. It exerts cardioprotective effects against myocardial ischemia/reperfusion (I/R) injury. In addition, studies confirm that the cystathionine γ-lyase (CSE)/hydrogen sulfide (H2S) pathway serves a beneficent role in attenuating myocardial I/R injury. However, the underlying role of the CSE/H2S pathway in the trimetazidine-induced protection against myocardial I/R injury remains elusive. Therefore, this study investigated whether trimetazidine ameliorates hypoxia/reoxygenation (H/R)-induced H9c2 cardiomyocyte injuries in an in vitro cell model of myocardial I/R injury, by enhancing the CSE/H2S pathway.Methods:The H9c2 cell viability was determined with a cell counting Kit-8.Results:Trimetazidine significantly increased the cell viability and decreased lactate dehydrogenase (LDH) release in H/R-treated H9c2 cells. Additionally, trimetazidine increased the H2S levels and the CSE mRNA and protein levels, promoting the CSE/H2S pathway under H/R conditions. The inhibition of the CSE/H2S pathway, induced by transfection with specific siRNA against human CSE (si-CSE), eliminated the trimetazidine-induced upregulation of cell viability, downregulation of LDH release, increase of caspase-3 activity and apoptosis regulator BAX expression, and the decrease of apoptosis regulator Bcl-2 expression, which suggests involvement of the CSE/H2S pathway in trimetazidine-induced cardioprotection. Furthermore, trimetazidine mitigated the H/R-induced increase in reactive oxygen species production and NADPH oxidase 2 expression, and decrease in superoxide dismutase activity and glutathione level, in H9c2 cells. These effects were also reversed by si-CSE.Conclusion:This study revealed that the CSE/H2S pathway mediates the trimetazidine-induced protection of H9c2 cardiomyocytes against H/R-induced damage by inhibiting apoptosis and oxidative stress.

Simple and Novel Assay of the Host-Guest Complexation of Homocysteine with Cucurbit[7]uril.

This paper introduces three ways to determine host-guest complexation of cucurbit[7]uril (CB[7]) with homocysteine (Hcy). After preincubating Hcy and cysteine (Cys) with CB[7], Ellman's reagent (DTNB) was used to detect Hcy and Cys. Only Cys reacted with DTNB and Hcy gave a retarded color change. This suggests that the -SH group of Hcy is buried inside CB[7]. Human cystathionine γ-lyase (hCGL) decreased the level of Hcy degradation after preincubating Hcy and CB[7]. These results suggest that the amount of free Hcy available was decreased by the formation of a Hcy-CB[7] complex. The immunological signal of anti-Hcy monoclonal antibody was decreased significantly by preincubating CB[7] with Hcy. The ELISA results also show that ethanethiol group (-CH₂CH₂SH) of Hcy, which is an epitope of anti-Hcy monoclonal antibody, was blocked by the cavity in CB[7]. Overall, CB[7] can act as a host by binding selectively with Hcy, but not Cys. The calculated half-complexation formation concentration of CB[7] was 58.2 nmol using Ellman's protocol, 97.9 nmol using hCGL assay and 87.7 nmol using monoclonal antibody. The differing binding abilities of Hcy and Cys towards the CB[7] host may offer a simple and useful method for determining the Hcy concentration in plasma or serum.

51 - CSE derived sulfur metabolites regulates autophagy in ischemic endothelial cells

Background Peripheral arterial disease (PAD) causes narrowing of blood vessels resulting in restriction of blood flow to lower limbs. Recently, autophagy has progressed as an emerging therapeutic strategy targeted to minimize the risk of PAD. Autophagy is a catabolic process aimed at recycling damaged proteins and organelles. Cystathionine-γ-lyase (CSE) is a major H2S producing enzyme in the vasculature, which is vasoprotective. However, the role of CSE in regulating autophagy remains unknown. Hypothesis CSE derived sulfide metabolites protect endothelial cells from ischemic injury by inducing autophagy. Methods WT and CSE knockout mice were subjected to femoral artery ligation (FAL). Mouse aortic endothelial cells (MAECs) isolated from WT and CSE-/- mice served as in vitro model. Autophagic flux was measured with and without chloroquine (50mg/kg) and bafilomycin (50nM) in animals and cells respectively. Trypan blue assay was used to evaluate cell viability. Annexin V/ PI staining and the cleaved caspase-3 assay were used to evaluate apoptosis. Results In WT and CSE-/- mice subjected to FAL, CSE-/- mice showed defective autophagy. MAECs deficient in CSE-/- also showed impaired autophagy. Transfection with mRFP-GFP tandem fluorescent-tagged LC3 plasmid showed increased autolysosomes in WT compared to CSE-/- MAEC’s. Interestingly, re-expression of human CSE in CSE-/- endothelial cells restored autophagy. Exogenous sulfide treatment, including per/polysulfides were not able to restore autophagy in CSE-/- cells. However, lanthionine ketimine (LK), a novel sulfur metabolite of the transulfuration pathway, restored autophagy in its cell permeable ester form. Lastly, we observed that deficiency of CSE blocks autophagy leading to apoptosis in endothelial cells. Conclusion We reveal for the first time that LK, a CSE derived sulfur metabolite, could regulate autophagy in ischemic endothelial cells. These findings reveal a potential new experimental approach to protect against ischemic injury by activating autophagy during PAD.

Selectivity of commonly used pharmacological inhibitors for cystathionine β synthase (CBS) and cystathionine γ lyase (CSE)

Background and PurposeHydrogen sulfide (H2S) is a signalling molecule that belongs to the gasotransmitter family. Two major sources for endogenous enzymatic production of H2S are cystathionine β synthase (CBS) and cystathionine γ lyase (CSE). In the present study, we examined the selectivity of commonly used pharmacological inhibitors of H2S biosynthesis towards CSE and CBS.Experimental ApproachTo address this question, human CSE or CBS enzymes were expressed and purified from Escherichia coli as fusion proteins with GSH-S-transferase. After purification, the activity of the recombinant enzymes was tested using the methylene blue method.Key Resultsβ-cyanoalanine (BCA) was more potent in inhibiting CSE than propargylglycine (PAG) (IC50 14 ± 0.2 μM vs. 40 ± 8 μM respectively). Similar to PAG, L-aminoethoxyvinylglycine (AVG) only inhibited CSE, but did so at much lower concentrations. On the other hand, aminooxyacetic acid (AOAA), a frequently used CBS inhibitor, was more potent in inhibiting CSE compared with BCA and PAG (IC50 1.1 ± 0.1 μM); the IC50 for AOAA for inhibiting CBS was 8.5 ± 0.7 μM. In line with our biochemical observations, relaxation to L-cysteine was blocked by AOAA in aortic rings that lacked CBS expression. Trifluoroalanine and hydroxylamine, two compounds that have also been used to block H2S biosynthesis, blocked the activity of CBS and CSE. Trifluoroalanine had a fourfold lower IC50 for CBS versus CSE, while hydroxylamine was 60-fold more selective against CSE.Conclusions and ImplicationsIn conclusion, although PAG, AVG and BCA exhibit selectivity in inhibiting CSE versus CBS, no selective pharmacological CBS inhibitor is currently available.

P18 Application of fluorescence probe ’HSip-1’ to inhibitor screening for H2S producing enzyme

Recently, we have reported a novel fluorescence probe for H 2 S, HSip-1, based on azamacrocyclic Cu 2+ complex chemistry (J. Am. Chem. Soc., 133 (2011) 18003) (Figure A). HSip-1 can selectively detect H 2 S in aqueous solution with high selectivity over biothiols, inorganic sulfur compounds, ROS and RNS, and has excellent photophysical properties for biological applications (Φ fl = 0.019 and 0.78 in the absence or the presence of H 2 S, respectively), deriving from its fluorescein scaffold. We also confirmed that HSip-1 could be used both for detection of pseudoenzymatic H 2 S release in a cuvette and of H 2 S production by 3-mercaptopyruvate sulfurtransferase (3MST) or lysate of 3MST-expressing cells, and 3-mercaptopyruvate, i.e., H 2 S-producing enzyme and its substrate. So, we anticipated that HSip-1 would be useful for not only fluorescence imaging, but also high-throughput screening of CBS (cystathionine β -synthase), CSE (cystathionine γ -lyase) and 3MST inhibitors, which should be required for detailed investigation of a wide range of biological functions of H 2 S. To investigate whether the probe can be used to monitor recombinant enzymatic H 2 S generation, we examined its efficacy with recombinant human CSE, which was purchased from Cayman and 3MST produced by Escherichia coli BL21 strain using a plasmid (3MST/pGEX) expressing 3MST with GST tag. H 2 S production by CSE (20 μg/mL) was readily detected in the presence of millimolar concentrations of the thiol substrate (cysteine; 2.5 mM) and pyridoxal phosphate (50 μM) in 30 mM HEPES buffer (pH 7.4). The fluorescence increment was suppressed by pretreatment with PAG (propargylglycine; 1 mM), which is an inhibitor of CSE (Figure B). Moreover, H 2 S production by 3MST (3.8 μg/mL) was also detected in the presence of thiol substrates (50 μM 3MP and 40 μM DTT) in 30 mM HEPES buffer (pH 7.4) (Figure C). We further confirmed that these systems could be applied to 384-well plate assay. By utilizing HSip-1 as an indicator for H 2 S, we then performed 3MST inhibitor screening of a chemical library containing about 150,000 compounds obtained from Open Innovation Center for Drug Discovery, The University of Tokyo. In this conference, we will show the detailed protocols and results in our poster.

Specificity Protein-1 as a Critical Regulator of Human Cystathionine γ-Lyase in Smooth Muscle Cells

Cystathionine γ-lyase (CSE) is the major enzyme in vascular smooth muscle cells (SMCs) that catalyzes the endogenous production of H(2)S. Phenotypic switching of SMCs is affected by endogenous H(2)S level and alterations of this switching may result in vascular disorders. To date, the mechanisms underlying the alteration of CSE expression and H(2)S production in vascular proliferative diseases have been unclear. In the present study, we found that serum deprivation induced SMC differentiation marker gene expressions and increased CSE expression and H(2)S production in cultured human aorta SMCs (HASMCs). Carotid artery ligation in mice resulted in enhanced neointima formation and down-regulation of CSE expression, suggesting an important role of CSE in SMC differentiation. Transient transfection of HASMCs with human CSE (hCSE) promoter/luciferase reporter revealed that the region between -226 to +140 base pair contains the core promoter for the hCSE gene. Deletion and mutation analysis demonstrated that two specificity protein-1 (Sp1) consensus binding sites were present in the core promoter region of the hCSE gene. Incubation of HASMCs with Sp1 binding inhibitor mithramycin inhibited CSE mRNA expression in a dose-dependent manner. Overexpression of Sp1 alone was sufficient to increase the activity of the hCSE core promoter and CSE protein expression. Chromatin immunoprecipitation assay showed that the binding of Sp1 to the hCSE promoter was increased in differentiated HASMCs compared with that in proliferated HASMCs. Exogenously applied H(2)S at 100 μM stimulated SMC differentiation, which was reversed by p38 MAPK inhibitor SB203580. These results suggest that transcript factor Sp1 is a critical regulator of the hCSE expression during SMC differentiation, and CSE/H(2)S system is essential for maintenance of SMC phenotype.

Site-Directed Mutagenesis on Human Cystathionine-γ-Lyase Reveals Insights into the Modulation of H2S Production

In recent years, increased interest has been directed towards hydrogen sulfide (H2S) as the third gasotransmitter and its role in various diseases. Cystathionine-γ-lyase (CSE) is one of the enzymes responsible for the endogenous production of H2S in mammals. With the aid of the crystal structures of human CSE and site-directed mutagenesis studies, we have identified several amino acid residues in CSE that are actively involved in the catalysis of H2S production. Contrary to reports suggesting that Tyr114 is required for substrate binding, our results reveal a significant increase in the production of H2S upon mutation of Tyr114 to phenylalanine. This is attributed to an increased rate of pyridoxal 5′-phosphate (PLP) regeneration due to weakened π-stacking interactions between Phe114 and PLP. Thr189 is also identified as a crucial residue where hydrogen bonding to Asp187 keeps the latter in an optimal position for hydrogen bonding to the pyridoxal nitrogen of PLP. Furthermore, mutation of Glu339 to lysine, alanine or tyrosine reveals the importance of the hydrophobicity of the 339th amino acid in determining the specificity of the enzyme for the catalysis of α,γ-elimination or α,β-elimination reaction. Our study also shows that the rate of H2S production is increased with increasing exogenous PLP concentration, hence supporting our hypothesis that apo-CSE is formed during the catalysis of H2S production. Taken together, these findings suggest novel routes towards the design of activators or inhibitors that modulate the production of H2S; these modulators may also serve as lead compounds in the development of drugs or mechanistic probes in the study of various H2S-related diseases.

H2S Biogenesis by Human Cystathionine γ-Lyase Leads to the Novel Sulfur Metabolites Lanthionine and Homolanthionine and Is Responsive to the Grade of Hyperhomocysteinemia

Although there is a growing recognition of the significance of hydrogen sulfide (H(2)S) as a biological signaling molecule involved in vascular and nervous system functions, its biogenesis and regulation are poorly understood. It is widely assumed that desulfhydration of cysteine is the major source of H(2)S in mammals and is catalyzed by the transsulfuration pathway enzymes, cystathionine beta-synthase and cystathionine gamma-lyase (CSE). In this study, we demonstrate that the profligacy of human CSE results in a variety of reactions that generate H(2)S from cysteine and homocysteine. The gamma-replacement reaction, which condenses two molecules of homocysteine, yields H(2)S and a novel biomarker, homolanthionine, which has been reported in urine of homocystinuric patients, whereas a beta-replacement reaction, which condenses two molecules of cysteine, generates lanthionine. Kinetic simulations at physiologically relevant concentrations of cysteine and homocysteine, reveal that the alpha,beta-elimination of cysteine accounts for approximately 70% of H(2)S generation. However, the relative importance of homocysteine-derived H(2)S increases progressively with the grade of hyperhomocysteinemia, and under conditions of severely elevated homocysteine (200 microm), the alpha,gamma-elimination and gamma-replacement reactions of homocysteine together are predicted to account for approximately 90% of H(2)S generation by CSE. Excessive H(2)S production in hyperhomocysteinemia may contribute to the associated cardiovascular pathology.

Structural Basis for the Inhibition Mechanism of Human Cystathionine γ-Lyase, an Enzyme Responsible for the Production of H2S

Impairment of the formation or action of hydrogen sulfide (H(2)S), an endogenous gasotransmitter, is associated with various diseases, such as hypertension, diabetes mellitus, septic and hemorrhagic shock, and pancreatitis. Cystathionine beta-synthase and cystathionine gamma-lyase (CSE) are two pyridoxal-5'-phosphate (PLP)-dependent enzymes largely responsible for the production of H(2)S in mammals. Inhibition of CSE by DL-propargylglycine (PAG) has been shown to alleviate disease symptoms. Here we report crystal structures of human CSE (hCSE), in apo form, and in complex with PLP and PLP.PAG. Structural characterization, combined with biophysical and biochemical studies, provides new insights into the inhibition mechanism of hCSE-mediated production of H(2)S. Transition from the open form of apo-hCSE to the closed PLP-bound form reveals large conformational changes hitherto not reported. In addition, PAG binds hCSE via a unique binding mode, not observed in PAG-enzyme complexes previously. The interaction of PAG-hCSE was not predicted based on existing information from known PAG complexes. The structure of hCSE.PLP.PAG complex highlights the particular importance of Tyr(114) in hCSE and the mechanism of PAG-dependent inhibition of hCSE. These results provide significant insights, which will facilitate the structure-based design of novel inhibitors of hCSE to aid in the development of therapies for diseases involving disorders of sulfur metabolism.

Kinetic Properties of Polymorphic Variants and Pathogenic Mutants in Human Cystathionine γ-Lyase

Human cystathionine-gamma-lyase (CGL) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme, which functions in the transsulfuration pathway that converts homocysteine to cysteine. In addition, CGL is one of two major enzymes that can catalyze the formation of hydrogen sulfide, an important gaseous signaling molecule. Recently, several mutations in CGL have been described in patients with cystathioninuria, a rare but poorly understood genetic disease. Moreover, a common single nucleotide polymorphism in CGL, c.1364G>T that converts serine at position 403 to isoleucine, has been linked to elevated plasma homocysteine levels. In this study, we have characterized the pathogenic T67I and Q240E missense mutations and the polymorphic variants at amino acid residues 403 using kinetic and spectrophotometric methods. We report that the polymorphism does not influence the cofactor content of the enzyme or its steady-state kinetic properties. In contrast, the T67I mutant exhibits a 3.5-fold decrease in V max compared to that of wild-type CGL, while the Q240E mutant exhibits a 70-fold decrease in V max. The K Ms for cystathionine for both pathogenic mutants are comparable to that of wild type CGL. The PLP content of the T67I and Q240E mutants were about 4-fold and 80-fold lower than that of wild-type enzyme, respectively. Preincubation of the T67I mutant with PLP restored activity to wild-type levels while the same treatment resulted in only partial restoration of activity of the Q240E mutant. These results reveal that both mutations weaken the affinity for PLP and suggest that cystathionuric patients with these mutations should be responsive to pyridoxine therapy.

Murine cystathionine γ-lyase: complete cDNA and genomic sequences, promoter activity, tissue distribution and developmental expression

Cystathionine gamma-lyase (CSE) is the last key enzyme in the trans-sulphuration pathway for biosynthesis of cysteine from methionine. Cysteine could be provided through diet; however, CSE has been shown to be important for the adequate supply of cysteine to synthesize glutathione, a major intracellular antioxidant. With a view to determining physiological roles of CSE in mice, we report the sequence of a complete mouse CSE cDNA along with its associated genomic structure, generation of specific polyclonal antibodies, and the tissue distribution and developmental expression patterns of CSE in mice. A 1.8 kb full-length cDNA containing an open reading frame of 1197 bp, which encodes a 43.6 kDa protein, was isolated from adult mouse kidney. A 35 kb mouse genomic fragment was obtained by lambda genomic library screening. It contained promoter regions, 12 exons, ranging in size from 53 to 579 bp, spanning over 30 kb, and exon/intron boundaries that were conserved with rat and human CSE. The GC-rich core promoter contained canonical TATA and CAAT motifs, and several transcription factor-binding consensus sequences. The CSE transcript, protein and enzymic activity were detected in liver, kidney, and, at much lower levels, in small intestine and stomach of both rats and mice. In developing mouse liver and kidney, the expression levels of CSE protein and activity gradually increased with age until reaching their peak value at 3 weeks of age, following which the expression levels in liver remained constant, whereas those in kidney decreased significantly. Immunohistochemical analyses revealed predominant CSE expression in hepatocytes and kidney cortical tubuli. These results suggest important physiological roles for CSE in mice.

Human cystathionine γ-lyase: developmental and in vitro expression of two isoforms

Human cystathionine γ-lyase: developmental and in vitro expression of two isoforms

Human cystathionine γ-lyase: developmental and in vitro expression of two isoforms

Cystathionine gamma-lyase (CGL) is the last enzyme of the trans-sulphuration pathway, which converts methionine into cysteine. To study the possible differences in enzymic activity of the two human cystathionine gamma-lyase isoforms characterized earlier, these were separately expressed in human kidney embryonic 293T cells. Furthermore, developmental changes in the expression of the two mRNA forms as well as the enzymic activity in human liver were studied, as it has been postulated that a change in the relative expression of CGL isoforms causes the postnatal increase in CGL activity. Transfection with the longer isoform increased the CGL activity 1.5-fold, while the activity of the cells transfected with the shorter form did not differ from the basal activity. In human liver samples, CGL activity was only detected in adult tissue (68+/-9 nmol of cysteine/h per mg of protein), whereas activity in fetal, premature and full-term neonatal liver tissue was undetectable. In contrast, strong mRNA expression of both mRNA isoforms was detected from the 19th gestational week onwards and the longer form of CGL appeared to be predominant. The expression of the two mRNA forms varied in parallel. In conclusion, we have shown that only cells overexpressing the longer form of CGL have increased activity, and CGL appears to be regulated at the post-transcriptional level during development.