AdoHcy Hydrolase Research Articles

Homocysteine regulates fatty acid and lipid metabolism in yeast

S-Adenosyl-l-homocysteine hydrolase (AdoHcy hydrolase; Sah1 in yeast/AHCY in mammals) degrades AdoHcy, a by-product and strong product inhibitor of S-adenosyl-l-methionine (AdoMet)-dependent methylation reactions, to adenosine and homocysteine (Hcy). This reaction is reversible, so any elevation of Hcy levels, such as in hyperhomocysteinemia (HHcy), drives the formation of AdoHcy, with detrimental consequences for cellular methylation reactions. HHcy, a pathological condition linked to cardiovascular and neurological disorders, as well as fatty liver among others, is associated with a deregulation of lipid metabolism. Here, we developed a yeast model of HHcy to identify mechanisms that dysregulate lipid metabolism. Hcy supplementation to wildtype cells up-regulated cellular fatty acid and triacylglycerol content and induced a shift in fatty acid composition, similar to changes observed in mutants lacking Sah1. Expression of the irreversible bacterial pathway for AdoHcy degradation in yeast allowed us to dissect the impact of AdoHcy accumulation on lipid metabolism from the impact of elevated Hcy. Expression of this pathway fully suppressed the growth deficit of sah1 mutants as well as the deregulation of lipid metabolism in both the sah1 mutant and Hcy-exposed wildtype, showing that AdoHcy accumulation mediates the deregulation of lipid metabolism in response to elevated Hcy in yeast. Furthermore, Hcy supplementation in yeast led to increased resistance to cerulenin, an inhibitor of fatty acid synthase, as well as to a concomitant decline of condensing enzymes involved in very long-chain fatty acid synthesis, in line with the observed shift in fatty acid content and composition.

Structure-Activity Relationships of Neplanocin A Analogues as S-Adenosylhomocysteine Hydrolase Inhibitors and Their Antiviral and Antitumor Activities.

On the basis of the potent inhibitory activity of neplanocin A (1) against S-adenosylhomocysteine (AdoHcy) hydrolase, we analyzed the comprehensive structure-activity relationships by modifying the adenine and carbasugar moiety of 1 to find the pharmacophore in the active site of the enzyme. The introduction of 7-deazaadenine instead of adenine eliminated the inhibitory activity against the AdoHcy hydrolase, while 3-deazaadenine maintained the inhibitory activity of the enzyme, indicating that N-7 is essential for its role as a hydrogen bonding acceptor. The substitution of hydrogen at the 6'-position with fluorine increased the inhibitory activity of the enzyme. The one-carbon homologation at the 5'-position generally decreased the inhibitory activity of the enzyme, indicating that steric repulsion exists. A molecular docking study also supported these experimental data. In this study, 6'-fluoroneplanocin A (2) was the most potent inhibitor of AdoHcy hydrolase (IC50 = 0.24 μM). It showed a potent anti-VSV activity (EC50 = 0.43 μM) and potent anticancer activity in all the human tumor cell lines tested.

Abstract 2433: Metabolic reprogramming by an epigenetic mechanism in endocrine therapy resistance of breast cancer

Abstract Metabolic reprogramming is a major mechanism of fueling cancer cell growth and proliferation. However, how tumor metabolism is altered to confer therapeutic resistance is poorly understood. Gene expression profiling of tamoxifen sensitive and resistant breast cancer cells revealed that transition to the resistance was associated with a specific expression reprogramming of genes involved in glucose/energy metabolism, which was accompanied by aberration of specific epigenetic regulators such as the histone methyltransferase NSD2/MMSET/WHSC1. In cell and xenograft tumor models, NSD2 overexpression alone was sufficient to confer tamoxifen resistance. Analysis of clinical specimens indicates that NSD2 was a strong predictive factor for early relapse of tamoxifen therapy. NSD2 wild type, but not its methylase-defective mutant form, coordinately stimulated the expression and enzymatic activity of HK2, TIGAR and G6PD and strongly augmented the pentose phosphate pathway (PPP) production of NADPH for ROS reduction and survival of tamoxifen treated cells and tumors. Further mechanistic studies demonstrated that elevated NSD2 stimulates the metabolic gene expression through H3K36 methylation at the target chromatin. Pharmacological targeting aberrant NSD2 by DZNeP, an S-adenosylhomocysteine (AdoHcy) hydrolase inhibitor, effectively blocked the growth of tamoxifen-resistant tumor. The study reveals a novel mechanism of cancer metabolic reprogramming and suggests targeting the key metabolic re-programmer as a viable option for effective treatment of endocrine resistant tumors. Note: This abstract was not presented at the meeting. Citation Format: June X. Zou, Junjian Wang, Zhijian Duan, Hongwu Chen, Hsing-Jien Kung, Xinbin Chen, Leigh C. Murphy, Alexander Borowsky. Metabolic reprogramming by an epigenetic mechanism in endocrine therapy resistance of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2433. doi:10.1158/1538-7445.AM2014-2433

RNA interference of a putative S-adenosyl-L-homocysteine hydrolase gene affects larval performance in Leptinotarsa decemlineata (Say)

In Leptinotarsa decemlineata, juvenile hormones (JHs) play primary roles in the regulation of metamorphosis, reproduction and diapause. In JH biosynthetic pathway in insect corpora allata, methylation of farnesoic acid or JH acid using S-adenosyl-L-methionine generates a potent feedback inhibitor S-adenosyl-L-homocysteine (AdoHcy). Rapid removal of AdoHcy is hypothesized to be essential for JH synthesis. AdoHcy hydrolase (SAHase) is the only eukaryotic enzyme catalyzing the removal. In the present paper, we firstly cloned a putative LdSAHase gene from L. decemlineata. The cDNA consists of 1806bp and encodes a 525 amino acid protein. LdSAHase was expressed in all developmental stages. The gene had the highest and the lowest level of transcription respectively in the 3rd- and 4th-instars’ heads that contain corpora allata, which was positively correlated with JH titer in the haemolymph and the mRNA level of a JH early-inducible gene, the Krüppel homolog 1 gene (Kr-h1). Secondly, dietary ingestion of bacterially-expressed LdSAHase-dsRNA significantly decreased LdSAHase and LdKr-h1 mRNA levels, reduced JH titer, and caused the death of the larvae, and the failure of pupation and adult emergence. After continuous exposure for 12days, 42% of the larvae died, 65% of the prepupae failed to pupate and 100% of the pupae failed to emerge. Moreover, RNAi-mediated LdSAHase knockdown also reduced larval developing time, and decreased larval weight. Lastly, application of JH analogue pyriproxyfen to LdSAHase-dsRNA-exposed larvae did not greatly increase LdSAHase expression level and JH content, but up-regulated LdKr-h1 mRNA level. Expectedly, pyriproxyfen application could partially rescue the negative effects on the survival and the development. Thus, our results support the hypothesis that SAHase plays a critical role in JH biosynthesis in insects.

Protein Arginine Methylation Is More Prone to Inhibition by S-Adenosylhomocysteine than DNA Methylation in Vascular Endothelial Cells

Methyltransferases use S-adenosylmethionine (AdoMet) as methyl group donor, forming S-adenosylhomocysteine (AdoHcy) and methylated substrates, including DNA and proteins. AdoHcy inhibits most methyltransferases. Accumulation of intracellular AdoHcy secondary to Hcy elevation elicits global DNA hypomethylation. We aimed at determining the extent at which protein arginine methylation status is affected by accumulation of intracellular AdoHcy. AdoHcy accumulation in human umbilical vein endothelial cells was induced by inhibition of AdoHcy hydrolase by adenosine-2,3-dialdehyde (AdOx). As a measure of protein arginine methylation status, the levels of monomethylarginine (MMA) and asymmetric and symmetric dimethylated arginine residues (ADMA and SDMA, respectively) in cell protein hydrolysates were measured by HPLC. A 10% decrease was observed at a 2.5-fold increase of intracellular AdoHcy. Western blotting revealed that the translational levels of the main enzymes catalyzing protein arginine methylation, protein arginine methyl transferases (PRMTs) 1 and 5, were not affected by AdoHcy accumulation. Global DNA methylation status was evaluated by measuring 5-methylcytosine and total cytosine concentrations in DNA hydrolysates by LC-MS/MS. DNA methylation decreased by 10% only when intracellular AdoHcy concentration accumulated to 6-fold of its basal value. In conclusion, our results indicate that protein arginine methylation is more sensitive to AdoHcy accumulation than DNA methylation, pinpointing a possible new player in methylation-related pathology.

Identification of Novel S-Adenosyl-l-Homocysteine Hydrolase Inhibitors through Homology-Model-Based Virtual Screening, Synthesis, and Biological Evaluation

The present study describes a successful application of computational approaches to identify novel Leishmania donovani (Ld) AdoHcyase inhibitors utilizing the differences for Ld AdoHcyase NAD(+) binding between human and Ld parasite. The development and validation of the three-dimensional (3D) structures of Ld AdoHcyase using the L. major AdoHcyase as template has been carried out. At the same time, cloning of the Ld AdoHcyase gene from clinical strains, its overexpression and purification have been performed. Further, the model was used in combined docking and molecular dynamics studies to validate the binding site of NAD in Ld. The hierarchical structure based virtual screening followed by the synthesis of five active hits and enzyme inhibition assay has resulted in the identification of novel Ld AdoHcyase inhibitors. The most potent inhibitor, compound 5, may serve as a "lead" for developing more potent Ld AdoHcy hydrolase inhibitors as potential antileishmanial agents.

(-)-Epigallocatechin-3-gallate and DZNep reduce polycomb protein level via a proteasome-dependent mechanism in skin cancer cells.

Polycomb group (PcG) protein-dependent histone methylation and ubiquitination drives chromatin compaction leading to reduced tumor suppressor expression and increased cancer cell survival. Green tea polyphenols and S-adenosylhomocysteine (AdoHcy) hydrolase inhibitors are important candidate chemopreventive agents. Previous studies indicate that (-)-epigallocatechin-3-gallate (EGCG), a potent green tea polyphenol, suppresses PcG protein level and skin cancer cell survival. Inhibition of AdoHcy hydrolase with 3-deazaneplanocin A (DZNep) inhibits methyltransferases by reducing methyl group availability. In the present study, we examine the impact of EGCG and DZNep cotreatment on skin cancer cell function. EGCG and DZNep, independently and in combination, reduce the level of PcG proteins including Ezh2, eed, Suz12, Mel18 and Bmi-1. This is associated with reduced H3K27me3 and H2AK119ub formation, histone modifications associated with closed chromatin. Histone deacetylase 1 level is also reduced and acetylated H3 formation is increased. These changes are associated with increased tumor suppressor expression and reduced cell survival and are partially reversed by vector-mediated maintenance of Bmi-1 level. The reduction in PcG protein level is associated with increased ubiquitination and is reversed by proteasome inhibitors, suggesting proteasome-associated degradation.

Hypermethioninemias of genetic and non‐genetic origin: A review

This review covers briefly the major conditions, genetic and non-genetic, sometimes leading to abnormally elevated methionine, with emphasis on recent developments. A major aim is to assist in the differential diagnosis of hypermethioninemia. The genetic conditions are: (1) Homocystinuria due to cystathionine β-synthase (CBS) deficiency. At least 150 different mutations in the CBS gene have been identified since this deficiency was established in 1964. Hypermethioninemia is due chiefly to remethylation of the accumulated homocysteine. (2) Deficient activity of methionine adenosyltransferases I and III (MAT I/III), the isoenzymes the catalytic subunit of which are encoded by MAT1A. Methionine accumulates because its conversion to S-adenosylmethionine (AdoMet) is impaired. (3) Glycine N-methyltrasferase (GNMT) deficiency. Disruption of a quantitatively major pathway for AdoMet disposal leads to AdoMet accumulation with secondary down-regulation of methionine flux into AdoMet. (4) S-adenosylhomocysteine (AdoHcy) hydrolase (AHCY) deficiency. Not being catabolized normally, AdoHcy accumulates and inhibits many AdoMet-dependent methyltransferases, producing accumulation of AdoMet and, thereby, hypermethioninemia. (5) Citrin deficiency, found chiefly in Asian countries. Lack of this mitochondrial aspartate-glutamate transporter may produce (usually transient) hypermethioninemia, the immediate cause of which remains uncertain. (6) Fumarylacetoacetate hydrolase (FAH) deficiency (tyrosinemia type I) may lead to hypermethioninemia secondary either to liver damage and/or to accumulation of fumarylacetoacetate, an inhibitor of the high K(m) MAT. Additional possible genetic causes of hypermethioninemia accompanied by elevations of plasma AdoMet include mitochondrial disorders (the specificity and frequency of which remain to be elucidated). Non-genetic conditions include: (a) Liver disease, which may cause hypermethioninemia, mild, or severe. (b) Low-birth-weight and/or prematurity which may cause transient hypermethioninemia. (c) Ingestion of relatively large amounts of methionine which, even in full-term, normal-birth-weight babies may cause hypermethioninemia.

X-ray Crystal Structure and Binding Mode Analysis of Human S-Adenosylhomocysteine Hydrolase Complexed with Novel Mechanism-Based Inhibitors, Haloneplanocin A Analogues

The X-ray crystal structure of human S-adenosylhomocysteine (AdoHcy) hydrolase was first determined as a tetrameric form bound with the novel mechanism-based inhibitor fluoroneplanocin A (4b). The crystallized enzyme complex showed the closed conformation and turned out to be the intermediate of mechanism-based inhibition. It confirmed that the cofactor depletion by 3'-oxidation of fluoroneplanocin A contributes to the enzyme inhibition along with the irreversible covalent modification of AdoHcy hydrolase. In addition, a series of haloneplanocin A analogues (4b-e and 5b-e) were designed and synthesized to characterize the binding role and reactivity of the halogen substituents and the 4'-CH(2)OH group. The biological evaluation and molecular modeling studies identified the key pharmacophores and structural requirements for the inhibitor binding of AdoHcy hydrolase. The inhibitory activity was decreased as the size of the halogen atom increased and/or if the 4'-CH(2)OH group was absent. These results could be utilized to design new therapeutic agents operating via AdoHcy hydrolase inhibition.

AdoHcy hydrolase of Trichomonas vaginalis: Studies of the effects of 5′-modified adenosine analogues and related 6-N-cyclopropyl derivatives

Trypanosoma brucei and Trichomonas vaginalis are both parasitic protozoans that are known to share many similar biochemical pathways. Aristeromycin, as well as 5'-iodovinyl and 5'-oxime analogues of adenosine, are potent inhibitors of AdoHcy hydrolase in T. brucei, an enzyme that catalyses the hydrolysis of AdoHcy to adenosine and L-homocysteine. To help determine the role of this enzyme in T. vaginalis, we have tested a library of 5'-modified adenosine derivatives, including 5'-deoxy-5'-(iodomethylene)-adenosine and related 6-N-cyclopropyl analogues. Our results indicate that these inhibitors are effective at inhibiting the growth of T. vaginalis, by as much as 95%.

SET7/9 Catalytic Mutants Reveal the Role of Active Site Water Molecules in Lysine Multiple Methylation

SET domain lysine methyltransferases (KMTs) methylate specific lysine residues in histone and non-histone substrates. These enzymes also display product specificity by catalyzing distinct degrees of methylation of the lysine ε-amino group. To elucidate the molecular mechanism underlying this specificity, we have characterized the Y245A and Y305F mutants of the human KMT SET7/9 (also known as KMT7) that alter its product specificity from a monomethyltransferase to a di- and a trimethyltransferase, respectively. Crystal structures of these mutants in complex with peptides bearing unmodified, mono-, di-, and trimethylated lysines illustrate the roles of active site water molecules in aligning the lysine ε-amino group for methyl transfer with S-adenosylmethionine. Displacement or dissociation of these solvent molecules enlarges the diameter of the active site, accommodating the increasing size of the methylated ε-amino group during successive methyl transfer reactions. Together, these results furnish new insights into the roles of active site water molecules in modulating lysine multiple methylation by SET domain KMTs and provide the first molecular snapshots of the mono-, di-, and trimethyl transfer reactions catalyzed by these enzymes.

S-adenosylhomocysteine hydrolase inhibition by 3-deazaneplanocin A analogues induces anti-cancer effects in breast cancer cell lines and synergy with both histone deacetylase and HER2 inhibition

Epigenetic abnormalities including abnormal histone methyltransferase activity contribute to breast cancer pathogenesis. An example is over expression of the polycomb repressive complex (PRC) 2 member enhancer of zeste homolog 2 (EZH2) which is linked to epigenetic silencing and poor prognosis. Recent evidence shows that S-adenosylhomocysteine (AdoHcy) hydrolase inhibitors (AHI) such as 3-deazaneplanocin A (DZNep) modulate chromatin through indirect inhibition of histone methyltransferases including EZH2. We investigated the biological effects of AdoHcy hydrolase inhibition using DZNep and its structural analogues 3-deazaadenosine (DZA) and neplanocin A (Nep A) in breast cancer cells. EZH2 protein expression was decreased and dose dependent growth inhibition occurred with variable potencies in MCF7, MDA-MB-231 and SKBr3 breast cancer cells. Cellular proliferation was inhibited through G(2)/M cell cycle arrest and apoptosis. In addition breast cancer cells accumulated cytoplasmic lipid droplets in response to AdoHcy hydrolase inhibition consistent with a differentiating effect. Each analogue induced a similar pattern of biological activity against breast cancer cells but with differences in potency (DZA>DZNep>Nep A). Co-administration with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) induced synergistic inhibition of breast cancer cell proliferation. Furthermore, the relatively AHI resistant human epidermal growth factor receptor 2 (HER2) positive cell line SKBr3 underwent synergistic growth inhibition in response to co-treatment with the HER2 directed therapeutic antibody trastuzumab. In conclusion, AHI induce growth inhibition, cell cycle arrest, apoptosis and differentiation in breast cancer cells and synergise with HDAC and HER2 inhibition. Targeting histone methyltransferase activity might be of therapeutic value in breast cancer.

Abstract 4526: Suppression of Alox5 gene is involved in DZNep induced apoptosis in multiple myeloma cells

Abstract The 3-Deazaneplanocin A (DZNep), one of S-adenosylhomocysteine (AdoHcy) hydrolase inhibitors, has shown antitumor activities in a broad range of solid tumors and acute myeloid leukemia. It was found that DZNep effectively depletes EZH2 levels and inhibits trimethylation of lysine 27 on histone H3. Here, we examined its effects on multiple myeloma (MM) cells and found that, at 500 nM, it potently inhibited growth and induced apoptosis in 2 of 8 MM cell lines(8226, H929, KMS11, KMS12BM, KMS18, MM1S, OPM-2 and U266). We then attempted to identify molecular mechanisms of sensitivity to DZNep. RNA from un-treated and DZNep treated cells was profiled by Affymetrix HG-U133 Plus 2.0 microarray and genes with a significant change in gene expression were determined by significance analysis of microarray (SAM) testing. ALOX5 was the most down-regulated gene (5.8-fold) in sensitive cells and was expressed at low level in resistant cells. The results were corroborated by qRT-PCR. Western-blot analysis indicated ALOX5 was highly expressed only in sensitive cell line H929 and greatly decreased upon DZNep treatment. Furthermore, down-regulation of ALOX5 by RNA interference can also induce apoptosis in H929. Since it was reported ALOX5 can regulate IL-6 mRNA level, we also determined IL-6 and IL-6R expression by qRT-PCR. As expected, IL-6 and IL-6R mRNA levels were decreased (>2-fold) in H929 upon DZNep treatment. May-Grunwald-Giemsa staining demonstrated that treatment with DZNep also dose dependently induced nuclear and cytoplasmic morphologic features of apoptosis in H929. These findings in vitro warrants further investigation of DZNep for the treatment of MM in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4526.

Regulation of phospholipid synthesis in yeast

Phospholipid synthesis in the yeast Saccharomyces cerevisiae is a complex process that involves regulation by both genetic and biochemical mechanisms. The activity levels of phospholipid synthesis enzymes are controlled by gene expression (e.g., transcription) and by factors (lipids, water-soluble phospholipid precursors and products, and covalent modification of phosphorylation) that modulate catalysis. Phosphatidic acid, whose levels are controlled by the biochemical regulation of key phospholipid synthesis enzymes, plays a central role in the regulation of phospholipid synthesis gene expression.

Proteomic analysis of 'hybrid necrosis' in wheat (Triticum aestivum) leaves.

Wheat hybrid necrosis has been genetically characterised for many years, but the specific gene(s) and the protein products involved in the processes remains unknown. In this study, protein expression in the base (B), mid (M) and tip (T) segments of the FL-2 leaves of a necrotic hybrid, PZF1 and its parents, Pan555 and Zheng891, was analysed and compared using a high throughput proteomic approach. Twenty-three protein spots, with significant variations in intensity across the necrotic leaf segments, were analysed by MALDI-TOF-MS, of which, 18 were matched to protein accessions in the NCBI database. Several of these proteins are enzymes involved in the methylation cycle, including AdoHcy hydrolase, AdoMet synthase 3 and methionine synthase 1; AdoHcy hydrolase was downregulated sharply in M and T, and AdoMet synthase 3 and methionine synthase 1 were upregulated gradually from M to T. This result suggests that methylation-associated processes, including epigenetic mechanisms, may play a role in the initiation and development of hybrid necrosis. Several energy cycle-associated proteins and cytoprotective proteins were also differentially expressed across the leaf segments, suggesting their direct association with or possible involvement in the necrotic processes. The significant imbalance of a heat-shock protein, a transposon protein and a RNA- and ssDNA-binding protein also makes these proteins potential molecular components in the necrotic processes.

Alterations in S-adenosylhomocysteine metabolism decrease O6-methylguanine DNA methyltransferase gene expression without affecting promoter methylation

The DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) protects cells against the cytotoxic effects of alkylating agents. Therefore, modulation of MGMT expression in tumors is a possible strategy for improving the efficiency of cancer therapy. MGMT expression and activity is lost frequently in association with DNA hypermethylation of the MGMT promoter region. Since DNA and mRNA methylation are controlled by intracellular S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) levels, we hypothesized a role for AdoMet/AdoHcy ratio in the regulation of MGMT promoter methylation and mRNA expression.Our initial studies showed that AdoMet/AdoHcy ratios vary over a wide range (7.0–50) in different glioblastoma and hepatoma cell lines. The studied cell lines exhibit distinct MGMT promoter methylation patterns: MGMT promoter was completely unmethylated in LN-18 and Tu 132 cells, hypermethylated in LN-229, U87-MG, and Tu 113 cells, and partially methylated in HepG2 cells. Furthermore, MGMT promoter methylation patterns and global DNA methylation are not related to intracellular AdoMet/AdoHcy ratio under control conditions. To lower AdoMet/AdoHcy ratio to values <1 we used AdoHcy hydrolase inhibitor adenosine-2′,3′-dialdehyde (30μM) and found that neither short-term (24h) nor long-term changes (7 weeks) in AdoMet/AdoHcy ratio altered global or MGMT promoter methylation. However, experimentally elevated AdoHcy levels significantly decreased MGMT mRNA levels by >50% in all MGMT-expressing cell lines, which is most likely the result of impaired mRNA methylation. Thus, the present study suggests elevation of AdoHcy levels by AdoHcy hydrolase inhibition as a novel pharmacological approach to modulate MGMT expression and to increase the responsiveness to alkylating agents.

Asymmetric Synthesis of Apio Fluoroneplanocin a Analogs as Potential AdoHcy Hydrolase Inhibitor

Apio fluoroneplanocin A (apio F-NPA, 3) and its uracil analogue 4 have been designed and asymmetrically synthesized starting from D-ribose. Introduction of fluoro group into vinylic position of 5 was accomplished successfully over 5 steps employing key reactions such as iodination according to an addition-elimination reaction mechanism, stereo- and regioselective reduction of α,β -unsaturated ketone, and electrophilic fluorination. This methodology can be adapted to the synthesis of fluoro compounds extensively.

Effect of copper on callus growth and gene expression of in vitro-cultured pith explants of Nicotiana glauca

Copper is a vital component of electron transfer reactions mediated by proteins such as superoxide dismutase, cytochrome c oxidase and plastocyanin, but its concentrations in the cells needs to be maintained at low levels. In fact, the same ability of this essential metal ion to transfer electrons can also make it toxic to cells when present in excess. In vitro cultured explants of Nicotiana have been extensively used as a model to analyse metal-DNA interactions. In this report, we examined the effect of copper (1, 10 and 100 μM CuSO4) on callus growth and protein synthesis of in vitro-cultured pith explants of Nicotiana glauca. In addition, a N. glauca cDNA library from Cu-treated (100 μM CuSO4) pith explants cultured in vitro for 24 h was analysed by mRNA differential screening. The copper treatments inhibited callus growth of pith explants. The extent of inhibition was directly correlated to metal concentration. One and 10 μM CuSO4 induced a notable increase of proteins synthesis relative to control explants. By contrast, 100 μM CuSO4 inhibited protein synthesis relative to control extracts. The SDS-PAGE fluorography of pith proteins revealed, in Cu-treated extracts qualitative and/or quantitative differences in the synthesis of some polypeptides compared with control explants. Copper-modulated patterns of gene expression were also analysed by mRNA differential screening. The N. glauca genes isolated from Cu-treated pith explants shared common identities with other genes known to be elicited by diverse stresses, including pathogenesis and abiotic stress. In particular, the cDNAs were homologues to genes encoding cell wall proteins (i.e., extensin, and arabinogalactan-protein) and pathogenesis-related proteins (i.e., osmotin, endochitinase and a member of the Systemic Acquired Resistance gene family). In addition, an MD-2-related lipid-recognition (ML) domain protein and the enzyme S-adenosyl-L-homocysteine (AdoHcy) hydrolase appeared involved in the response to copper stress. In animal cells, AdoHcy hydrolase is a copper binding protein in vivo, which suggests that, also in plant tissues, this enzyme may play an important role in regulating the levels and intracellular distribution of copper.

Role of S-adenosylhomocysteine hydrolase in adenosine-induced apoptosis in HepG2 cells

Adenosine has been shown to initiate apoptosis through different mechanisms: (i) activation of adenosine receptors, (ii) intracellular conversion to AMP and stimulation of AMP-activated kinase, (iii) conversion to S-adenosylhomocysteine (AdoHcy), which is an inhibitor of S-adenosylmethionine (AdoMet)-dependent methyltransferases. Since the pathways involved are still not completely understood, we further investigated the role of AdoHcy hydrolase in adenosine-induced apoptosis. In HepG2 cells, adenosine induced caspase-like activity and DNA fragmentation, a marker of apoptosis. These effects were potentiated by co-incubation with homocysteine or adenosine deaminase inhibitor, pentostatin, and were mimicked by inhibition of AdoHcy hydrolase by adenosine-2′,3′-dialdehyde (Adox). Adenosine-induced effects were significantly inhibited by dipyridamole, an inhibitor of adenosine transporter, whereas inhibitors of adenosine kinase did not affect adenosine-induced changes. Various adenosine receptor agonists and AICAR, an activator of AMP-activated kinase, did not mimic the effect of adenosine. Thus, adenosine-induced apoptosis is likely due to intracellular action of AdoHcy and independent of AMP-activated kinase and adenosine receptors. Because elevated AdoHcy levels are associated with reduced mRNA methylation, we studied mRNA expression in Adox-treated cells by microarray analysis. Since several p53-target genes and other apoptosis-related genes were up-regulated by Adox, we conclude that AdoHcy is involved in adenosine-induced apoptosis by altering gene expression.

Effects of Ligand Binding and Oxidation on Hinge-Bending Motions in S-Adenosyl-l-homocysteine Hydrolase

Domain motions of S-adenosyl-l-homocysteine (AdoHcy) hydrolase have been detected by time-resolved fluorescence anisotropy measurements. Time constants for reorientational motions in the native enzyme were compared with those for enzymes where key residues were altered by site-directed mutation. Mutations M351P, H353A, and P354A were selected in a hinge region for motion between the open and closed forms of the enzyme, as identified in a previous normal-mode study [Wang et al. (2005) Domain motions and the open-to-closed conformational transition of an enzyme: A normal-mode analysis of S-adenosyl-l-homocysteine hydrolase, Biochemistry 44, 7228-7239]. In wild-type, substrate-free AdoHcy hydrolase (NAD(+) cofactor in each subunit), reorientational motions were detected on time scales of 10-20 and 80-90 ns. The faster motion is attributed to the domain motion, and the slower motion is attributed to the tumbling of the enzyme. The domain motion was also detected for the enzyme complexes E(NADH/3'-keto-adenosine) and E(NAD(+)/3'-deoxyadenosine) but was absent for the complex E(NADH/3'-keto-neplanocin A). The results indicate that AdoHcy hydrolase exists in equilibrium of open and closed structures, with the equilibrium shifted toward the more mobile open form for the substrate-free enzyme, E(NAD(+)), and for intermediates formed early in the catalytic cycle after substrate binding or formed late prior to product release, E(NAD(+)/ligand). However, the strong inhibitor neplanocin A upon binding undergoes oxidation, forming the complex E(NADH/3'-keto-neplanocin). For this complex, which is analogous to the enzyme complex with the central catalytic intermediate, the equilibrium was shifted toward the more rigid closed form. A similar pattern was observed for M351P and P354A mutants. In contrast, the domain motion could not be detected, either in the absence or presence of ligands or with the cofactor in either the oxidized or reduced state, for the H353A protein, suggesting that this mutation changes the hinge-bending dynamics of the enzyme.