Nitrite Reductase Activity and Inhibition of H2S Biogenesis by Human Cystathionine ?-Synthase

Nitrite was recognized as a potent vasodilator >130 years and has more recently emerged as an endogenous signaling molecule and modulator of gene expression. Understanding the molecular mechanisms that regulate nitrite metabolism is essential for its use as a potential diagnostic marker as well as therapeutic agent for cardiovascular diseases. In this study, we have identified human cystathionine ß-synthase (CBS) as a new player in nitrite reduction with implications for the nitrite-dependent control of H₂S production. This novel activity of CBS exploits the catalytic property of its unusual heme cofactor to reduce nitrite and generate NO. Evidence for the possible physiological relevance of this reaction is provided by the formation of ferrous-nitrosyl (Fe^II-NO) CBS in the presence of NADPH, the human diflavin methionine synthase reductase (MSR) and nitrite. Formation of Fe^II-NO CBS via its nitrite reductase activity inhibits CBS, providing an avenue for regulating biogenesis of H₂S and cysteine, the limiting reagent for synthesis of glutathione, a major antioxidant. Our results also suggest a possible role for CBS in intracellular NO biogenesis particularly under hypoxic conditions. The participation of a regulatory heme cofactor in CBS in nitrite reduction is unexpected and expands the repertoire of proteins that can liberate NO from the intracellular nitrite pool. Our results reveal a potential molecular mechanism for cross-talk between nitrite, NO and H₂S biology.     

The Regulation of l-Methionine Synthesis and the Properties of Cystathionine γ-Synthase and β-Cystathionase in Corynebacterimn glutamicum

Cystathionine γ-synthase and β-cystathionase activities were found to be present in cell-free extract of Corynebacterium glutamicum. The reactions catalyzed by cystathionine γ-synthase and β-cystathionase were characterized with respect to Michaelis constant, pH optimum, incubation time and optimal enzyme concentration. Cystathionine γ-synthase was sensitive to the inhibition by S-adenosylmethionine. Formation of cystathionine γ-synthase and β-cystathionase was repressed by the addition of methionine to the growth medium although this repression appeared to be non-coordinate.

The regulation of methionine biosynthesis in C. glutamicum was discussed on the basis of these findings.     

S-Adenosyl-l-methionine Modulates CO and NO? Binding to the Human H2S-generating Enzyme Cystathionine β-Synthase

Cystathionine β-synthase (CBS) is a key enzyme in human (patho)physiology with a central role in hydrogen sulfide metabolism. The enzyme is composed of a pyridoxal 5′-phosphate-binding catalytic domain, flanked by the following two domains: a heme-binding N-terminal domain and a regulatory C-terminal domain binding S-adenosyl-l-methionine (AdoMet). CO or NO^• binding at the ferrous heme negatively modulates the enzyme activity. Conversely, AdoMet binding stimulates CBS activity. Here, we provide experimental evidence for a functional communication between the two domains. We report that AdoMet binding significantly enhances CBS inhibition by CO. Consistently, we observed increased affinity (∼5-fold) and faster association (∼10-fold) of CO to the ferrous heme at physiological AdoMet concentrations. NO^• binding to reduced CBS was also enhanced by AdoMet, although to a lesser extent (∼2-fold higher affinity) as compared with CO. Importantly, CO and NO^• binding was unchanged by AdoMet in a truncated form of CBS lacking the C-terminal regulatory domain. These unprecedented observations demonstrate that CBS activation by AdoMet puzzlingly sensitizes the enzyme toward inhibition by exogenous ligands, like CO and NO^•. This further supports the notion that CBS regulation is a complex process, involving the concerted action of multiple physiologically relevant effectors.     

Enhanced Expression of Cystathionine β-Synthase and Cystathionine γ-Lyase During Acute Cholecystitis-Induced Gallbladder Inflammation

Background

Hydrogen sulfide (H₂S) has recently been shown to play an important role in the digestive system, but the role of endogenous H₂S produced locally in the gallbladder is unknown. The aim of this study was to investigate whether gallbladder possesses the enzymatic machinery to synthesize H₂S, and whether H₂S synthesis is changed in gallbladder inflammation during acute acalculous cholecystitis (AC).

Methods

Adult male guinea pigs underwent either a sham operation or common bile duct ligation (CBDL). One, two, or three days after CBDL, the animals were sacrificed separately. Hematoxylin and eosin-stained slides of gallbladder samples were scored for inflammation. H₂S production rate in gallbladder tissue from each group was determined; immunohistochemistry and western blotting were used to determine expression levels of the H₂S-producing enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) in gallbladder.

Results

There was a progressive inflammatory response after CBDL. Immunohistochemistry analysis showed that CBS and CSE were expressed in the gallbladder epithelium, muscular layer, and blood vessels and that the expression increased progressively with increasing inflammation following CBDL. The expression of CBS protein as well as the H₂S-production rate was significantly increased in the animals that underwent CBDL, compared to those that underwent the sham operation.

Conclusions

Both CBS and CSE are expressed in gallbladder tissues. The expression of these enzymes, as well as H₂S synthesis, was up-regulated in the context of inflammation during AC.     

Detection of Reaction Intermediates during Human Cystathionine β-Synthase-monitored Turnover and H2S Production

Human cystathionine β-synthase (CBS), a novel heme-containing pyridoxal 5′-phosphate enzyme, catalyzes the condensation of homocysteine and serine or cysteine to produce cystathionine and H₂O or H₂S, respectively. The presence of heme in CBS has limited spectrophotometric characterization of reaction intermediates by masking the absorption of the pyridoxal 5′-phosphate cofactor. In this study, we employed difference stopped-flow spectroscopy to characterize reaction intermediates formed under catalytic turnover conditions. The reactions of l-serine and l-cysteine with CBS resulted in the formation of a common aminoacrylate intermediate (k_obs = 0.96 ± 0.02 and 0.38 ± 0.01 mm⁻¹ s⁻¹, respectively, at 24 °C) with concomitant loss of H₂O and H₂S and without detectable accumulation of the external aldimine or other intermediates. Homocysteine reacted with the aminoacrylate intermediate with k_obs = 40.6 ± 3.8 s⁻¹ and re-formed the internal aldimine. In the reverse direction, CBS reacted with cystathionine, forming the aminoacrylate intermediate with k_obs = 0.38 ± 0.01 mm⁻¹ s⁻¹. This study provides the first insights into the pre-steady-state kinetic mechanism of human CBS and indicates that the reaction is likely to be limited by a conformational change leading to product release.     

The Human Cystathionine β-Synthase (CBS) Gene: Complete Sequence, Alternative Splicing, and Polymorphisms

Cystathionine β-synthase [CBS; -serine hydro-lyase (adding homocysteine), EC 4.2.1.22] catalyzes the first committed step of transsulfuration and is the enzyme deficient in classical homocystinuria. In this report, we describe the molecular cloning and the complete nucleotide sequence of the human CBS gene. We report a total of 28,046 nucleotides of sequence, which, in addition to the CBS gene, contains 5 kb of the 5′ flanking region. The human CBS gene contains 23 exons ranging from 42 to 209 bp. The 5′ UTR is formed by 1 of 5 alternatively used exons and 1 invariably present exon, while the 3′ UTR is encoded by exons 16 and 17. We also describe the identification of two alternatively used promoter regions that are GC rich (80%) and contain numerous putative binding sites for Sp1, Ap1, Ap2, and c-myb, but lack the classical TATA box. The CBS locus contains an unusually high number ofAlurepeats, which may predispose this gene to deleterious rearrangements. Additionally, we report on a number of DNA sequence repeats that are polymorphic in North American and European Caucasians.     

Modulation of Cystathionine β-Synthase Activity by the Arg-51 and Arg-224 Mutations

Human cystathionine β-synthase (CBS) catalyzes a pyridoxal 5′-phosphate (PLP) dependent β-replacement reaction to synthesize cystathionine from serine and homocysteine. The enzyme is unique in bearing not only a catalytically important PLP but also heme. In order to study a regulatory process mediated by heme, we performed mutagenesis of Arg-51 and Arg-224, which have hydrogen-bonding interactions with propionate side chains of the prosthetic group. It was found that the arginine mutations decrease CBS activity by approximately 50%. The results indicate that structural changes in the heme vicinity are transmitted to PLP existing 20 Å away from heme. A possible explanation of our results is discussed on the basis of CBS structure.     

Allosteric Communication between the Pyridoxal 5′-Phosphate (PLP) and Heme Sites in the H2S Generator Human Cystathionine β-Synthase

Human cystathionine β-synthase (CBS) is a unique pyridoxal 5′-phosphate (PLP)-dependent enzyme that has a regulatory heme cofactor. Previous studies have demonstrated the importance of Arg-266, a residue at the heme pocket end of α-helix 8, for communication between the heme and PLP sites. In this study, we have examined the role of the conserved Thr-257 and Thr-260 residues, located at the other end of α-helix 8 on the heme electronic environment and on activity. The mutations at the two positions destabilize PLP binding, leading to lower PLP content and ∼2- to ∼500-fold lower activity compared with the wild-type enzyme. Activity is unresponsive to PLP supplementation, consistent with the pyridoxine-nonresponsive phenotype of the T257M mutation in a homocystinuric patient. The H₂S-producing activities, also impacted by the mutations, show a different pattern of inhibition compared with the canonical transsulfuration reaction. Interestingly, the mutants exhibit contrasting sensitivities to the allosteric effector, S-adenosylmethionine (AdoMet); whereas T257M and T257I are inhibited, the other mutants are hyperactivated by AdoMet. All mutants showed an increased propensity of the ferrous heme to form an inactive species with a 424 nm Soret peak and exhibited significantly reduced enzyme activity in the ferrous and ferrous-CO states. Our results provide the first evidence for bidirectional transmission of information between the cofactor binding sites, suggest the additional involvement of this region in allosteric communication with the regulatory AdoMet-binding domain, and reveal the potential for independent modulation of the canonical transsulfuration versus H₂S-generating reactions catalyzed by CBS.     

Functional Rescue of Mutant Human Cystathionine ��-Synthase by Manipulation of Hsp26 and Hsp70 Levels in Saccharomyces cerevisiae

Many human diseases are caused by missense substitutions that result in misfolded proteins that lack biological function. Here we express a mutant form of the human cystathionine β-synthase protein, I278T, in Saccharomyces cerevisiae and show that it is possible to dramatically restore protein stability and enzymatic function by manipulation of the cellular chaperone environment. We demonstrate that Hsp70 and Hsp26 bind specifically to I278T but that these chaperones have opposite biological effects. Ethanol treatment induces Hsp70 and causes increased activity and steady-state levels of I278T. Deletion of the SSA2 gene, which encodes a cytoplasmic isoform of Hsp70, eliminates the ability of ethanol to restore function, indicating that Hsp70 plays a positive role in proper I278T folding. In contrast, deletion of HSP26 results in increased I278T protein and activity, whereas overexpression of Hsp26 results in reduced I278T protein. The Hsp26-I278T complex is degraded via a ubiquitin/proteosome-dependent mechanism. Based on these results we propose a novel model in which the ratio of Hsp70 and Hsp26 determines whether misfolded proteins will either be refolded or degraded.Cells have evolved quality control systems for misfolded proteins, consisting of molecular chaperones (heat shock proteins) and proteases. These molecules help prevent misfolding and aggregation by either promoting refolding or by degrading misfolded protein molecules (1). In eukaryotic cells, the Hsp70 system plays a critical role in mediating protein folding. Hsp70 protein interacts with misfolded polypeptides along with co-chaperones and promotes refolding by repeated cycles of binding and release requiring the hydrolysis of ATP (2). Small heat shock proteins (sHsp)² are small molecular weight chaperones that bind non-native proteins in an oligomeric complex and whose function is poorly understood (3). In mammalian cells, the sHsp family includes the α-crystallins, whose orthologue in Saccharomyces cerevisiae is Hsp26. Studies suggest that Hsp26 binding to misfolded protein aggregates is a prerequisite for effective disaggregation and refolding by Hsp70 and Hsp104 (4, 5).Misfolded proteins can result from missense substitutions such as those found in a variety of recessive genetic diseases, including cystathionine β-synthase (CBS) deficiency. CBS is a key enzyme in the trans-sulfuration pathway that converts homocysteine to cysteine (6). Individuals with CBS deficiency have extremely elevated levels of plasma total homocysteine, resulting in a variety of symptoms, including dislocated lenses, osteoporosis, mental retardation, and a greatly increased risk of thrombosis (7). Approximately 80% of the mutations found in CBS-deficient patients are point mutations that are predicted to cause missense substitutions in the CBS protein (8). The most common mutation found in CBS-deficient patients, an isoleucine to threonine substitution at amino acid position 278 (I278T), has been observed in nearly one-quarter of all CBS-deficient patients. Based on the crystal structure of the catalytic core of CBS, this mutation is located in a β-sheet more than 10 Å distant from the catalytic pyridoxal phosphate and does not directly affect the catalytic binding pocket or the dimer interface (9).Previously, our lab has developed a yeast bioassay for human CBS in which yeast expressing functional human CBS can grow in media lacking cysteine, whereas yeast expressing mutant CBS cannot (10). We have used this assay to characterize the functional effects of many different CBS missense alleles, including I278T (7, 11). However, an unexpected finding was that it was possible to restore function to I278T and a number of other CBS missense mutations by either truncation or the addition of a second missense mutation in the C-terminal regulatory domain (12, 13). The ability to restore function by a cis-acting second mutation suggested to us that it might be possible to restore function in trans via either interaction of mutant CBS with a small molecule (i.e. drug) or a mutation in another yeast gene. In a previous study, we found that small osmolyte chemical chaperones could restore function to mutant CBS presumably by directly stabilizing the mutant CBS protein (14).In this study we report on the surprising finding that exposure of yeast to ethanol can restore function of I278T CBS by altering the ratio of the molecular chaperones Hsp26 and Hsp70. We demonstrate Hsp70 binding promotes I278T folding and activity, whereas Hsp26 binding promotes I278T degradation via the proteosome. By manipulating the levels of Hsp26 and Hsp70, we are able to show that I278T CBS protein can have enzymatic activity restored to near wild-type levels of activity. Our findings suggest a novel function for sHsps.     

Cystathionine β-Synthase (CBS) Domains Confer Multiple Forms of Mg2+-dependent Cooperativity to Family II Pyrophosphatases

Regulated family II pyrophosphatases (CBS-PPases) contain a nucleotide-binding insert comprising a pair of cystathionine β-synthase (CBS) domains, termed a Bateman module. By binding with high affinity to the CBS domains, AMP and ADP usually inhibit the enzyme, whereas ATP activates it. Here, we demonstrate that AMP, ADP, and ATP bind in a positively cooperative manner to CBS-PPases from four bacteria: Desulfitobacterium hafniense, Clostridium novyi, Clostridium perfringens, and Eggerthella lenta. Enzyme interaction with substrate as characterized by the Michaelis constant (K_m) also exhibited positive catalytic cooperativity that decreased in magnitude upon nucleotide binding. The degree of both types of cooperativity increased with increasing concentration of the cofactor Mg²⁺ except for the C. novyi PPase where Mg²⁺ produced the opposite effect on kinetic cooperativity. Further exceptions from these general rules were ADP binding to C. novyi PPase and AMP binding to E. lenta PPase, neither of which had any effect on activity. A genetically engineered deletion variant of D. hafniense PPase lacking the regulatory insert was fully active but differed from the wild-type enzyme in that it was insensitive to nucleotides and bound substrate non-cooperatively and with a smaller K_m value. These results indicate that the regulatory insert acts as an internal inhibitor and confers dual positive cooperativity to CBS domain-containing PPases, making them highly sensitive regulators of the PP_i level in response to the changes in cell energy status that control adenine nucleotide distribution. These regulatory features may be common among other CBS domain-containing proteins.     

Human Polycomb 2 Protein Is a SUMO E3 Ligase and Alleviates Substrate-Induced Inhibition of Cystathionine β-Synthase Sumoylation

Human cystathionine β-synthase (CBS) catalyzes the first irreversible step in the transsulfuration pathway and commits homocysteine to the synthesis of cysteine. Mutations in CBS are the most common cause of severe hereditary hyperhomocysteinemia. A yeast two-hybrid approach to screen for proteins that interact with CBS had previously identified several components of the sumoylation pathway and resulted in the demonstration that CBS is a substrate for sumoylation. In this study, we demonstrate that sumoylation of CBS is enhanced in the presence of human polycomb group protein 2 (hPc2), an interacting partner that was identified in the initial yeast two-hybrid screen. When the substrates for CBS, homocysteine and serine for cystathionine generation and homocysteine and cysteine for H₂S generation, are added to the sumoylation mixture, they inhibit the sumoylation reaction, but only in the absence of hPc2. Similarly, the product of the CBS reaction, cystathionine, inhibits sumoylation in the absence of hPc2. Sumoylation in turn decreases CBS activity by ∼28% in the absence of hPc2 and by 70% in its presence. Based on these results, we conclude that hPc2 serves as a SUMO E3 ligase for CBS, increasing the efficiency of sumoylation. We also demonstrate that γ-cystathionase, the second enzyme in the transsulfuration pathway is a substrate for sumoylation under in vitro conditions. We speculate that the role of this modification may be for nuclear localization of the cysteine-generating pathway under conditions where nuclear glutathione demand is high.     

Effect of the Disease-Causing R266K Mutation on the Heme and PLP Environments of Human Cystathionine β-Synthase

Cystathionine β-synthase (CBS) is an essential pyridoxal 5'-phosphate (PLP)-dependent enzyme of the transsulfuration pathway that condenses serine with homocysteine to form cystathionine; intriguingly, human CBS also contains a heme b cofactor of unknown function. Herein we describe the enzymatic and spectroscopic properties of a disease-associated R266K hCBS variant, which has an altered hydrogen-bonding environment. The R266K hCBS contains a low-spin, six-coordinate Fe(III) heme bearing a His/Cys ligation motif, like that of WT hCBS; however, there is a geometric distortion that exists at the R266K heme. Using rR spectroscopy, we show that the Fe(III)-Cys(thiolate) bond is longer and weaker in R266K, as evidenced by an 8 cm(-1) downshift in the ν(Fe-S) resonance. Presence of this longer and weaker Fe(III)-Cys(thiolate) bond is correlated with alteration of the fluorescence spectrum of the active PLP ketoenamine tautomer. Activity data demonstrate that, relative to WT, the R266K variant is more impaired in the alternative cysteine-synthesis reaction than in the canonical cystathionine-synthesis reaction. This diminished cysteine synthesis activity and a greater sensitivity to exogenous PLP correlate with the change in PLP environment. Fe-S(Cys) bond weakening causes a nearly 300-fold increase in the rate of ligand switching upon reduction of the R266K heme. Combined, these data demonstrate cross talk between the heme and PLP active sites, consistent with previous proposals, revealing that alteration of the Arg(266)-Cys(52) interaction affects PLP-dependent activity and dramatically destabilizes the ferrous thiolate-ligated heme complex, underscoring the importance of this hydrogen-bonding residue pair.     

Contributions of protein kinases and β-arrestin to termination of protease-activated receptor 2 signaling

Activated G_q protein–coupled receptors (G_qPCRs) can be desensitized by phosphorylation and β-arrestin binding. The kinetics and individual contributions of these two mechanisms to receptor desensitization have not been fully distinguished. Here, we describe the shut off of protease-activated receptor 2 (PAR2). PAR2 activates G_q and phospholipase C (PLC) to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP₂) into diacylglycerol and inositol trisphosphate (IP₃). We used fluorescent protein–tagged optical probes to monitor several consequences of PAR2 signaling, including PIP₂ depletion and β-arrestin translocation in real time. During continuous activation of PAR2, PIP₂ was depleted transiently and then restored within a few minutes, indicating fast receptor activation followed by desensitization. Knockdown of β-arrestin 1 and 2 using siRNA diminished the desensitization, slowing PIP₂ restoration significantly and even adding a delayed secondary phase of further PIP₂ depletion. These effects of β-arrestin knockdown on PIP₂ recovery were prevented when serine/threonine phosphatases that dephosphorylate GPCRs were inhibited. Thus, PAR2 may continuously regain its activity via dephosphorylation when there is insufficient β-arrestin to trap phosphorylated receptors. Similarly, blockers of protein kinase C (PKC) and G protein–coupled receptor kinase potentiated the PIP₂ depletion. In contrast, an activator of PKC inhibited receptor activation, presumably by augmenting phosphorylation of PAR2. Our interpretations were strengthened by modeling. Simulations supported the conclusions that phosphorylation of PAR2 by protein kinases initiates receptor desensitization and that recruited β-arrestin traps the phosphorylated state of the receptor, protecting it from phosphatases. Speculative thinking suggested a sequestration of phosphatidylinositol 4-phosphate 5 kinase (PIP5K) to the plasma membrane by β-arrestin to explain why knockdown of β-arrestin led to secondary depletion of PIP₂. Indeed, artificial recruitment of PIP5K removed the secondary loss of PIP₂ completely. Altogether, our experimental and theoretical approaches demonstrate roles and dynamics of the protein kinases, β-arrestin, and PIP5K in the desensitization of PAR2.     

Characterization of Cystathionine β-Synthase TtCbs1 and Cysteine Synthase TtCsa1 Involved in Cysteine Biosynthesis in Tetrahymena thermophila

Cysteine is implicated in important biological processes. It is synthesized through two different pathways. Cystathionine β-synthase and cystathionine γ-lyase participate in the reverse transsulfuration pathway, while serine acetyltransferase and cysteine synthase function in the de novo pathway. Two evolutionarily related pyridoxal 5′-phosphate-dependent enzymes, cystathionine β-synthase TtCBS1 (TTHERM_00558300) and cysteine synthase TtCSA1 (TTHERM_00239430), were identified from a freshwater protozoan Tetrahymena thermophila. TtCbs1 contained the N-terminal heme binding domain, catalytic domain, and C-terminal regulatory domain, whereas TtCsa1 consisted of two α/β domains. The catalytic core of the two enzymes is similar. TtCBS1 and TtCSA1 showed high expression levels in the vegetative growth stage and decreased during the sexual developmental stage. TtCbs1 and TtCsa1 were localized in the cytoplasm throughout different developmental stages. His-TtCbs1 and His-TtCsa1 were expressed and purified in vitro. TtCbs1 catalyzed the canonical reaction with the highest velocity and possessed serine sulfhydrylase activity. TtCsa1 showed cysteine synthase activity with high K_m for O-acetylserine and low K_m for sulfide and also had serine sulfhydrylase activity toward serine. Both TtCbs1 and TtCsa1 catalyzed hydrogen sulfide producing. TtCBS1 knockdown and TtCSA1 knockout mutants affected cysteine and glutathione synthesis. TtCbs1 and TtCsa1 are involved in cysteine synthesis through two different pathways in T. thermophila.     

The AP-3 �� Adaptin Mediates the Biogenesis and Function of Lytic Vacuoles in Arabidopsis

Plant vacuoles are essential multifunctional organelles largely distinct from similar organelles in other eukaryotes. Embryo protein storage vacuoles and the lytic vacuoles that perform a general degradation function are the best characterized, but little is known about the biogenesis and transition between these vacuolar types. Here, we designed a fluorescent marker–based forward genetic screen in Arabidopsis thaliana and identified a protein affected trafficking2 (pat2) mutant, whose lytic vacuoles display altered morphology and accumulation of proteins. Unlike other mutants affecting the vacuole, pat2 is specifically defective in the biogenesis, identity, and function of lytic vacuoles but shows normal sorting of proteins to storage vacuoles. PAT2 encodes a putative β-subunit of adaptor protein complex 3 (AP-3) that can partially complement the corresponding yeast mutant. Manipulations of the putative AP-3 β adaptin functions suggest a plant-specific role for the evolutionarily conserved AP-3 β in mediating lytic vacuole performance and transition of storage into the lytic vacuoles independently of the main prevacuolar compartment-based trafficking route.