Antitumoral Activity of the Universal Methyl Donor S-Adenosylmethionine in Glioblastoma Cells.

Glioblastoma (GBM), the most frequent and lethal brain cancer in adults, is characterized by short survival times and high mortality rates. Due to the resistance of GBM cells to conventional therapeutic treatments, scientific interest is focusing on the search for alternative and efficient adjuvant treatments. S-Adenosylmethionine (AdoMet), the well-studied physiological methyl donor, has emerged as a promising anticancer compound and a modulator of multiple cancer-related signaling pathways. We report here for the first time that AdoMet selectively inhibited the viability and proliferation of U87MG, U343MG, and U251MG GBM cells. In these cell lines, AdoMet induced S and G2/M cell cycle arrest and apoptosis and downregulated the expression and activation of proteins involved in homologous recombination DNA repair, including RAD51, BRCA1, and Chk1. Furthermore, AdoMet was able to maintain DNA in a damaged state, as indicated by the increased γH2AX/H2AX ratio. AdoMet promoted mitotic catastrophe through inhibiting Aurora B kinase expression, phosphorylation, and localization causing GBM cells to undergo mitotic catastrophe-induced death. Finally, AdoMet inhibited DNA repair and induced cell cycle arrest, apoptosis, and mitotic catastrophe in patient-derived GBM cells. In light of these results, AdoMet could be considered a potential adjuvant in GBM therapy.

S-Adenosylmethionine Inhibits Colorectal Cancer Cell Migration through Mirna-Mediated Targeting of Notch Signaling Pathway.

Metastasis is a leading cause of mortality and poor prognosis in colorectal cancer (CRC). Thus, the identification of new compounds targeting cell migration represents a major clinical challenge. Recent findings evidenced a central role for dysregulated Notch in CRC and a correlation between Notch overexpression and tumor metastasis. MicroRNAs (miRNAs) have been reported to cross-talk with Notch for its regulation. Therefore, restoring underexpressed miRNAs targeting Notch could represent an encouraging therapeutic approach against CRC. In this context, S-adenosyl-L-methionine (AdoMet), the universal biological methyl donor, being able to modulate the expression of oncogenic miRNAs could act as a potential antimetastatic agent. Here, we showed that AdoMet upregulated the onco-suppressor miRNAs-34a/-34c/-449a and inhibited HCT-116 and Caco-2 CRC cell migration. This effect was associated with reduced expression of migration-/EMT-related protein markers. We also found that, in colorectal and triple-negative breast cancer cells, AdoMet inhibited the expression of Notch gene, which, by luciferase assay, resulted the direct target of miRNAs-34a/-34c/-449a. Gain- and loss-of-function experiments with miRNAs mimics and inhibitors demonstrated that AdoMet exerted its inhibitory effects by upregulating miRNAs-34a/-34c/-449a. Overall, these data highlighted AdoMet as a novel Notch inhibitor and suggested that the antimetastatic effects of AdoMet involve the miRNA-mediated targeting of Notch signaling pathway.

Polyphenol Extract from "Greco" Grape Canes: Characterization, Antioxidant Capacity, and Antitumor Effects on Cal-33 and JHU-SCC-011 Head and Neck Squamous Cell Carcinoma.

In the current study, we determined the antioxidant properties of "Greco" grape cane extracts, a typical cultivar of southern Italy. We also explored the anticancer activity of the polyphenol-rich fraction of the extract on head and neck squamous carcinoma cells (HNSCC) and investigated the underlying mechanism. Aqueous extracts were prepared at different pHs and extraction times and the total phenolic and reducing sugar contents were estimated. Radical Scavenging Activity (RSA), Ferric Reducing Antioxidant Power (FRAP), and Total Antioxidant Capacity (TAC) of the extracts were measured. A polyphenol-rich fraction, accounting for 6.7% by weight and characterized mainly by procyanidins and stilbenoids, was prepared from the extract obtained at pH 7 for 60 min. We demonstrated that the extract exerted a cytotoxic effect on HNSCC cell lines by inducing cell cycle arrest via cyclin downregulation and p21 upregulation, and by triggering apoptosis through caspase cascade activation, PARP-1 cleavage, and an increase in the Bax/Bcl-2 ratio. We furnished evidence that the polyphenol-rich fraction played the major role in the anticancer activity of the extract. These outcomes highlighted grape canes from the "Greco" cultivar as a valuable source of polyphenols that may represent good candidates for the design of innovative adjuvant therapies in the treatment of HNSCC.

S-Adenosylmethionine Increases the Sensitivity of Human Colorectal Cancer Cells to 5-Fluorouracil by Inhibiting P-Glycoprotein Expression and NF-κB Activation.

Colorectal cancer (CRC) is the second deadliest cancer worldwide despite significant advances in both diagnosis and therapy. The high incidence of CRC and its poor prognosis, partially attributed to multi-drug resistance and antiapoptotic activity of cancer cells, arouse strong interest in the identification and development of new treatments. S-Adenosylmethionine (AdoMet), a natural compound and a nutritional supplement, is well known for its antiproliferative and proapoptotic effects as well as for its potential in overcoming drug resistance in many kinds of human tumors. Here, we report that AdoMet enhanced the antitumor activity of 5-Fluorouracil (5-FU) in HCT 116p53+/+ and in LoVo CRC cells through the inhibition of autophagy, induced by 5-FU as a cell defense mechanism to escape the drug cytotoxicity. Multiple drug resistance is mainly due to the overexpression of drug efflux pumps, such as P-glycoprotein (P-gp). We demonstrate here that AdoMet was able to revert the 5-FU-induced upregulation of P-gp expression and to decrease levels of acetylated NF-κB, the activated form of NF-κB, the major antiapoptotic factor involved in P-gp-related chemoresistance. Overall, our data show that AdoMet, was able to overcome 5-FU chemoresistance in CRC cells by targeting multiple pathways such as autophagy, P-gp expression, and NF-κB signaling activation and provided important implications for the development of new adjuvant therapies to improve CRC treatment and patient outcomes.

Structures of catalytic cycle intermediates of the Pyrococcus furiosus methionine adenosyltransferase demonstrate negative cooperativity in the archaeal orthologues.

Methionine adenosyltransferases catalyse the biosynthesis of S-adenosylmethionine, the primary methyl group donor in biochemical reactions, through the condensation of methionine and ATP. Here, we report the structural analysis of the Pyrococcus furiosus methionine adenosyltransferase (PfMAT) captured in the unliganded, substrate- and product-bound states. The conformational changes taking place during the enzymatic catalytic cycle are allosterically propagated by amino acid residues conserved in the archaeal orthologues to induce an asymmetric dimer structure. The distinct occupancy of the active sites within a PfMAT dimer is consistent with a half-site reactivity that is mediated by a product-induced negative cooperativity. The structures of intermediate states of PfMAT reported here suggest a distinct molecular mechanism for S-adenosylmethionine synthesis in Archaea, likely consequence of the evolutionary pressure to achieve protein stability under extreme conditions.

S-Adenosyl-l-Methionine Overcomes uL3-Mediated Drug Resistance in p53 Deleted Colon Cancer Cells.

PURPOSE: In order to study novel therapeutic approaches taking advantage of natural compounds showing anticancer and anti-proliferative effects, we focused our interest on S-adenosyl-l-methionine, a naturally occurring sulfur-containing nucleoside synthesized from adenosine triphosphate and methionine by methionine adenosyltransferase, and its potential in overcoming drug resistance in colon cancer cells devoid of p53. RESULTS: In the present study, we demonstrated that S-adenosyl-l-methionine overcomes uL3-mediated drug resistance in p53 deleted colon cancer cells. In particular, we demonstrated that S-adenosyl-l-methionine causes cell cycle arrest at the S phase; inhibits autophagy; augments reactive oxygen species; and induces apoptosis in these cancer cells. CONCLUSIONS: Results reported in this paper led us to propose S-adenosyl-l-methionine as a potential promising agent for cancer therapy by examining p53 and uL3 profiles in tumors to yield a better clinical outcomes.

S-Adenosylmethionine Inhibits Cell Growth and Migration of Triple Negative Breast Cancer Cells through Upregulating MiRNA-34c and MiRNA-449a.

Triple-negative breast cancer (TNBC) is one of the most common malignancies worldwide and shows maximum invasiveness and a high risk of metastasis. Recently, many natural compounds have been highlighted as a valuable source of new and less toxic drugs to enhance breast cancer therapy. Among them, S-adenosyl-L-methionine (AdoMet) has emerged as a promising anti-cancer agent. MicroRNA (miRNA or miR)-based gene therapy provides an interesting antitumor approach to integrated cancer therapy. In this study, we evaluated AdoMet-induced modulation of miRNA-34c and miRNA-449a expression in MDA-MB-231 and MDA-MB-468 TNBC cells. We demonstrated that AdoMet upregulates miR-34c and miR-449a expression in both cell lines. We found that the combination of AdoMet with miR-34c or miR-449a mimic strongly potentiated the pro-apoptotic effect of the sulfonium compound by a caspase-dependent mechanism. For the first time, by video time-lapse microscopy, we showed that AdoMet inhibited the in vitro migration of MDA-MB-231 and MDA-MB-468 cells and that the combination with miR-34c or miR-449a mimic strengthened the effect of the sulfonium compound through the modulation of ß-catenin and Small Mother Against Decapentaplegic (SMAD) signaling pathways. Our results furnished the first evidence that AdoMet exerts its antitumor effects in TNBC cells through upregulating the expression of miR-34c and miR-449a.

Therapeutic Potential of the Natural Compound S-Adenosylmethionine as a Chemoprotective Synergistic Agent in Breast, and Head and Neck Cancer Treatment: Current Status of Research.

The present review summarizes the most recent studies focusing on the synergistic antitumor effect of the physiological methyl donor S-adenosylmethionine (AdoMet) in association with the main drugs used against breast cancer and head and neck squamous cell carcinoma (HNSCC), two highly aggressive and metastatic malignancies. In these two tumors the chemotherapy approach is recommended as the first choice despite the numerous side effects and recurrence of metastasis, so better tolerated treatments are needed to overcome this problem. In this regard, combination therapy with natural compounds, such as AdoMet, a molecule with pleiotropic effects on multiple cellular processes, is emerging as a suitable strategy to achieve synergistic anticancer efficacy. In this context, the analysis of studies conducted in the literature highlighted AdoMet as one of the most effective and promising chemosensitizing agents to be taken into consideration for inclusion in emerging antitumor therapeutic modalities such as nanotechnologies.

AdoMet triggers apoptosis in head and neck squamous cancer by inducing ER stress and potentiates cell sensitivity to cisplatin.

S-Adenosyl-l-methionine (AdoMet) is a naturally and widely occurring sulfonium compound that plays a primary role in cell metabolism and acts as the principal methyl donor in many methylation reactions. AdoMet also exhibits antiproliferative and proapoptotic activities in different cancer cells. However, the molecular mechanisms underlying the effects exerted by AdoMet have only been partially studied. In the current study, we evaluated the antiproliferative effect of AdoMet on Cal-33 oral and JHU-SCC-011 laryngeal squamous cancer cells to define the underlying mechanisms. We demonstrated that AdoMet induced apoptosis in Cal-33 and JHU-SCC-011 cells, involving a caspase-dependent mechanism paralleled by an increased Bax/Bcl-2 ratio. Moreover, we showed, for the first time, that AdoMet induced ER-stress in Cal-33 cells and activated the unfolded protein response, which can be responsible for apoptosis induction through the activation of CHOP and JNK. In addition, AdoMet-induced ER-stress was followed by autophagy with a consistent increase in the levels of the autophagic marker LC3B-II, which was indeed potentiated by the autophago-lysosome inhibitor chloroquine. As both escape from apoptosis and decreased activation of JNK are mechanisms of resistance to cisplatin (cDPP), an agent usually used in cancer therapy, we have evaluated the effects of AdoMet in combination with cDPP on Cal-33 cells. Our data showed that the combined treatment resulted in a strong synergism in inhibiting cell proliferation and in enhancing apoptosis via intrinsic mechanism. These results demonstrate that AdoMet has ER-stress-mediated antiproliferative activity and synergizes with cDDP on cell growth inhibition, thus providing the basis for its use in new anticancer strategies.

Long Non-coding RNAs as Important Biomarkers in Laryngeal Cancer and Other Head and Neck Tumours.

Head and neck carcinoma (HNC) is a heterogeneous disease encompassing a variety of tumors according to the origin. Laryngeal cancer (LC) represents one of the most frequent tumors in the head and neck region. Despite clinical studies and advance in treatment, satisfactory curative strategy has not yet been reached. Therefore, there is an urgent need for the identification of specific molecular signatures that better predict the clinical outcomes and markers that serve as suitable therapeutic targets. Long non-coding RNAs (lncRNA) are reported as important regulators of gene expression and represent an innovative pharmacological application as molecular biomarkers in cancer. The purpose of this review is to discuss the most relevant epigenetic and histological prognostic biomarkers in HNC, with particular focus on LC. We summarize the emerging roles of long non-coding RNAs in HNC and LC development and their possible use in early diagnosis.

S-Adenosylmethionine-mediated apoptosis is potentiated by autophagy inhibition induced by chloroquine in human breast cancer cells.

The naturally occurring sulfonium compound S-adenosyl-L-methionine (AdoMet) is an ubiquitous sulfur-nucleoside that represents the main methyl donor in numerous methylation reactions. In recent years, it has been shown that AdoMet possesses antiproliferative properties in various cancer cells, but the molecular mechanisms at the basis of the effect induced by AdoMet have been only in part investigated. In the present study, we found that AdoMet strongly inhibited the proliferation of breast cancer cells MCF-7 by inducing both autophagy and apoptosis. AdoMet consistently enhanced the levels of the autophagy markers beclin-1 and LC3B-II, and caused a significant increase of pro-apoptotic Bax/Bcl-2 ratio paralleled by poly (ADP ribose) polymerase (PARP) and caspase 9, and 6 cleavage. Notably, AdoMet, already at low doses, raised the percentage of cells in G2 /M phase of cell cycle by down-regulating the expression of cell cycle-regulatory proteins cyclin B and cyclin E with a remarkable increase of p53, p27, and p21. We also evaluated the combination of AdoMet and the autophagy inhibitor chloroquine (CLC) showing that autophagy block is synergistic in inducing both growth inhibition and apoptosis. These effects were paralleled by a strong inhibition of the activity of AKT and of the downstream effector mTOR and by an increased cleavage of caspase-6 and PARP. These data suggest, for the first time, that autophagy can act as an escape mechanism from the apoptotic activity of AdoMet, and that AdoMet could be used in combination with CLC or its analogs in the treatment of breast cancer.

S-Adenosylmethionine regulates apoptosis and autophagy in MCF-7 breast cancer cells through the modulation of specific microRNAs.

BACKGROUND: To get insight into the molecular mechanisms underlying the anti-tumor activity of S-adenosyl-l-methionine (AdoMet), we analyzed AdoMet-induced modulation of microRNAs (miRNAs) expression profile in MCF-7 breast cell line and its correlation with cancer-related biological pathways. METHODS: MiRNA expression profiling was performed using a TaqMan MiRNA Array, following 500 µM AdoMet-treatment. The results were confirmed by Quantitative real-time PCR analysis. MCF-7 were transfected with miR-34a, miR-34c and miR-486-5p, mimics and inhibitors in presence or not of 500 µM AdoMet for 72 h. Apoptosis and autophagy were analyzed by flow cytometry and the modulation of the main antiproliferative signaling pathways were evaluated by Western blotting. The potential mRNA targets for each miRNA were identified by the TargetScan miRNA target prediction software. RESULTS: Twenty-eight microRNAs resulted differentially expressed in AdoMet-treated MCF-7 cells compared to control cells. Among them, miRNA-34a and miRNA-34c were up-regulated while miRNA-486-5p was down-regulated. Moreover, we confirmed the ability of AdoMet to regulate these miRNAs in MDA-MB 231 breast cancer cell line. We demonstrate that, in MCF7 cells, the combination of either miR-34a or miR-34c mimic with AdoMet greatly potentiated the pro-apoptotic effect of AdoMet, by a caspase-dependent mechanism and activates p53 acetylation by inhibiting SIRT1 and HDAC1 expression. We also showed that miR-486-5p inhibitor induces autophagy and enhances AdoMet-induced autophagic process by increasing PTEN expression and by inhibiting AKT signaling. CONCLUSIONS: Our findings provide the first evidence that AdoMet can regulate miRNA expression in MCF-7 increasing our knowledge on the molecular basis of the antitumor effect of the sulfonium compound and suggest the use of AdoMet as an attractive miRNA-mediated chemopreventive and therapeutic strategy in breast cancer.

Multiple disulfide bridges modulate conformational stability and flexibility in hyperthermophilic archaeal purine nucleoside phosphorylase.

5'-Deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus is a hexameric hyperthermophilic protein containing in each subunit two pairs of disulfide bridges, a CXC motif, and one free cysteine. The contribution of each disulfide bridge to the protein conformational stability and flexibility has been assessed by comparing the thermal unfolding and the limited proteolysis of the wild-type enzyme and its variants obtained by site-directed mutagenesis of the seven cysteine residues. All variants catalyzed efficiently MTA cleavage with specific activity similar to the wild-type enzyme. The elimination of all cysteine residues caused a substantial decrease of ΔHcal (850 kcal/mol) and Tmax (39°C) with respect to the wild-type indicating that all cysteine pairs and especially the CXC motif significantly contribute to the enzyme thermal stability. Disulfide bond Cys200-Cys262 and the CXC motif weakly affected protein flexibility while the elimination of the disulfide bond Cys138-Cys205 lead to an increased protease susceptibility. Experimental evidence from limited proteolysis, differential scanning calorimetry, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions also allowed to propose a stabilizing role for the free Cys164.

S-Adenosylmethionine Affects ERK1/2 and Stat3 Pathways and Induces Apotosis in Osteosarcoma Cells.

Osteosarcoma is a very aggressive bone tumor. Its clinical outcome remains discouraging despite intensive surgery, radiotherapy, and chemotherapy. Thus, novel therapeutic approaches are demanded. S-Adenosylmethionine (AdoMet) is a naturally occurring molecule that is synthesized in our body by methionine adenosyltransferase isoenzymes and is also available as a nutritional supplement. AdoMet is the principal methyl donor in numerous methylation reactions and is involved in many biological functions. Interestingly, AdoMet has been shown to exert antiproliferative action in various cancer cells. However, the underlying molecular mechanisms are just starting to be studied. Here, we investigated the effects of AdoMet on the proliferation of osteosarcoma U2OS cells and the underlying mechanisms. We carried out direct cell number counting, MTT and flow cytometry-based assays, and immunoblotting experiments in response to AdoMet treatment. We found that AdoMet strongly inhibits proliferation of U2OS cells by slowing-down cell cycle progression and by inducing apoptosis. We also report that AdoMet consistently causes an increase of p53 and p21 cell-cycle inhibitor, a decrease of cyclin A and cyclin E protein levels, and a marked increase of pro-apoptotic Bax/Bcl-2 ratio, with caspase-3 activation and PARP cleavage. Moreover, the AdoMet-induced antiproliferative effects were dynamically accompanied by profound changes in ERK1/2 and STAT3 protein and phosphorylation levels. Altogether, our data enforce the evidence of AdoMet acting as a biomolecule with antiproliferative action in osteosarcoma cells, capable of down-regulating ERK1/2 and STAT3 pathways leading to cell cycle inhibition and apoptosis, and provide a rationale for the possible use of AdoMet in osteosarcoma therapy.

Efficient Fludarabine-Activating PNP From Archaea as a Guidance for Redesign the Active Site of E. Coli PNP.

The combination of the gene of purine nucleoside phosphorylase (PNP) from Escherichia coli and fludarabine represents one of the most promising systems in the gene therapy of solid tumors. The use of fludarabine in gene therapy is limited by the lack of an enzyme that is able to efficiently activate this prodrug which, consequently, has to be administered in high doses that cause serious side effects. In an attempt to identify enzymes with a better catalytic efficiency than E. coli PNP towards fludarabine to be used as a guidance on how to improve the activity of the bacterial enzyme, we have selected 5'-deoxy-5'-methylthioadenosine phosphorylase (SsMTAP) and 5'-deoxy-5'-methylthioadenosine phosphorylase II (SsMTAPII), two PNPs isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. Substrate specificity and catalytic efficiency of SsMTAP and SsMTAPII for fludarabine were analyzed by kinetic studies and compared with E. coli PNP. SsMTAP and SsMTAPII share with E. coli PNP a comparable low affinity for the arabinonucleoside but are better catalysts of fludarabine cleavage with k(cat)/K(m) values that are 12.8-fold and 6-fold higher, respectively, than those reported for the bacterial enzyme. A computational analysis of the interactions of fludarabine in the active sites of E. coli PNP, SsMTAP, and SsMTAPII allowed to identify the crucial residues involved in the binding with this substrate, and provided structural information to improve the catalytic efficiency of E. coli PNP by enzyme redesign.

Protective Effect of Tyrosol and S-Adenosylmethionine against Ethanol-Induced Oxidative Stress of Hepg2 Cells Involves Sirtuin 1, P53 and Erk1/2 Signaling.

Oxidative stress plays a major role in ethanol-induced liver damage, and agents with antioxidant properties are promising as therapeutic opportunities in alcoholic liver disease. In the present work, we investigated the effect of S-adenosylmethionine (AdoMet), Tyrosol (Tyr), and their combination on HepG2 cells exposed to ethanol exploring the potential molecular mechanisms. We exposed HepG2 cells to 1 M ethanol for 4 and 48 h; thereafter, we recorded a decreased cell viability, increase of intracellular reactive oxygen species (ROS) and lipid accumulation, and the release into culture medium of markers of liver disease such as triacylglycerol, cholesterol, transaminases, albumin, ferritin, and homocysteine. On the other hand, AdoMet and Tyrosol were able to attenuate or antagonize these adverse changes induced by acute exposure to ethanol. The protective effects were paralleled by increased Sirtuin 1 protein expression and nuclear translocation and increased ERK1/2 phosphorylation that were both responsible for the protection of cells from apoptosis. Moreover, AdoMet increased p53 and p21 expression, while Tyrosol reduced p21 expression and enhanced the expression of uncleaved caspase 3 and 9, suggesting that its protective effect may be related to the inhibition of the apoptotic machinery. Altogether, our data show that AdoMet and Tyrosol exert beneficial effects in ethanol-induced oxidative stress in HepG2 cells and provide a rationale for their potential use in combination in the prevention of ethanol-induced liver damage.

Neddylation inhibition prevents acetaminophen-induced liver damage by enhancing the anabolic cardiolipin pathway.

Drug-induced liver injury (DILI) is a significant cause of acute liver failure (ALF) and liver transplantation in the Western world. Acetaminophen (APAP) overdose is a main contributor of DILI, leading to hepatocyte cell death through necrosis. Here, we identified that neddylation, an essential post-translational modification involved in the mitochondria function, was upregulated in liver biopsies from patients with APAP-induced liver injury (AILI) and in mice treated with an APAP overdose. MLN4924, an inhibitor of the neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8)-activating enzyme (NAE-1), ameliorated necrosis and boosted liver regeneration in AILI. To understand how neddylation interferes in AILI, whole-body biotinylated NEDD8 (bioNEDD8) and ubiquitin (bioUB) transgenic mice were investigated under APAP overdose with and without MLN4924. The cytidine diphosphate diacylglycerol (CDP-DAG) synthase TAM41, responsible for producing cardiolipin essential for mitochondrial activity, was found modulated under AILI and restored its levels by inhibiting neddylation. Understanding this ubiquitin-like crosstalk in AILI is essential for developing promising targeted inhibitors for DILI treatment.

Role of disulfide bonds in conformational stability and folding of 5'-deoxy-5'-methylthioadenosine phosphorylase II from the hyperthermophilic archaeon Sulfolobus solfataricus.

Sulfolobus solfataricus 5'-deoxy-5'-melthylthioadenosine phosphorylase II (SsMTAPII), is a hyperthermophilic hexameric protein with two intrasubunit disulfide bonds (C138-C205 and C200-C262) and a CXC motif (C259-C261). To get information on the role played by these covalent links in stability and folding, the conformational stability of SsMTAPII and C262S and C259S/C261S mutants was studied by thermal and guanidinium chloride (GdmCl)-induced unfolding and analyzed by fluorescence spectroscopy, circular dichroism, and SDS-PAGE. No thermal unfolding transition of SsMTAPII can be obtained under nonreducing conditions, while in the presence of the reducing agent Tris-(2-carboxyethyl) phosphine (TCEP), a Tm of 100°C can be measured demonstrating the involvement of disulfide bridges in enzyme thermostability. Different from the wild-type, C262S and C259S/C261S show complete thermal denaturation curves with sigmoidal transitions centered at 102°C and 99°C respectively. Under reducing conditions these values decrease by 4°C and 8°C respectively, highlighting the important role exerted by the CXC disulfide on enzyme thermostability. The contribution of disulfide bonds to the conformational stability of SsMTAPII was further assessed by GdmCl-induced unfolding experiments carried out under reducing and nonreducing conditions. Thermal unfolding was found to be reversible if the protein was heated in the presence of TCEP up to 90°C but irreversible above this temperature because of aggregation. In analogy, only chemical unfolding carried out in the presence of reducing agents resulted in a reversible process suggesting that disulfide bonds play a role in enzyme denaturation. Thermal and chemical unfolding of SsMTAPII occur with dissociation of the native hexameric state into denatured monomers, as indicated by SDS-PAGE.

Hepatic levels of S-adenosylmethionine regulate the adaptive response to fasting.

There has been an intense focus to uncover the molecular mechanisms by which fasting triggers the adaptive cellular responses in the major organs of the body. Here, we show that in mice, hepatic S-adenosylmethionine (SAMe)-the principal methyl donor-acts as a metabolic sensor of nutrition to fine-tune the catabolic-fasting response by modulating phosphatidylethanolamine N-methyltransferase (PEMT) activity, endoplasmic reticulum-mitochondria contacts, ß-oxidation, and ATP production in the liver, together with FGF21-mediated lipolysis and thermogenesis in adipose tissues. Notably, we show that glucagon induces the expression of the hepatic SAMe-synthesizing enzyme methionine adenosyltransferase α1 (MAT1A), which translocates to mitochondria-associated membranes. This leads to the production of this metabolite at these sites, which acts as a brake to prevent excessive ß-oxidation and mitochondrial ATP synthesis and thereby endoplasmic reticulum stress and liver injury. This work provides important insights into the previously undescribed function of SAMe as a new arm of the metabolic adaptation to fasting.

New determinants in the catalytic mechanism of nucleoside hydrolases from the structures of two isozymes from Sulfolobus solfataricus.

The purine- and pyrimidine-specific nucleoside hydrolases (NHs) from the archaeon Sulfolobus solfataricus participate in the fundamental pathway of nucleotide catabolism and function to maintain adequate levels of free nitrogenous bases for cellular function. The two highly homologous isozymes display distinct specificities toward nucleoside substrates, and both lack the amino acids employed for activation of the leaving group in the hydrolytic reaction by the NHs characterized thus far. We determined the high-resolution crystal structures of the purine- and pyrimidine-specific NHs from S. solfataricus to reveal that both enzymes belong to NH structural homology group I, despite the different substrate specificities. A Na(+) ion is bound at the active site of the pyrimidine-specific NH instead of the prototypical Ca(2+), delineating a role of the metals in the catalytic mechanism of NHs in the substrate binding rather than nucleophile activation. A conserved His residue, which regulates product release in other homologous NHs, provides crucial interactions for leaving group activation in the archaeal isozymes. Modeling of the enzyme-substrate interactions suggests that steric exclusion and catalytic selection underlie the orthogonal base specificity of the two isozymes.