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23 p.-7 fig., The intermediate filament protein vimentin is involved in essential cellular processes, including cell division and stress responses, as well as in the pathophysiology of cancer, pathogen infection, and autoimmunity. The vimentin network undergoes marked reorganizations in response to oxidative stress, in which modifications of vimentin single cysteine residue, Cys328, play an important role, and is modulated by zinc availability. However, the molecular basis for this regulation is not fully understood. Here, we show that Cys328 displays a low pKa, supporting its reactivity, and is readily alkylated and oxidized in vitro. Moreover, combined oxidation and crosslinking assays and molecular dynamics simulations support that zinc ions interact with Cys328 in its thiolate form, whereas Glu329 and Asp331 stabilize zinc coordination. Vimentin oxidation can induce disulfide crosslinking, implying the close proximity of Cys328 from neighboring dimers in certain vimentin conformations, supported by our computational models. Notably, micromolar zinc concentrations prevent Cys328 alkylation, lipoxidation, and disulfide formation. Moreover, zinc selectively protects vimentin from crosslinking using short-spacer cysteine-reactive but not amine-reactive agents. These effects are not mimicked by magnesium, consistent with a lower number of magnesium ions hosted at the cysteine region, according to molecular dynamics simulations. Importantly, the region surrounding Cys328 is involved in interaction with several drugs targeting vimentin and is conserved in type III intermediate filaments, which include glial fibrillary acidic protein and desmin. Altogether, our results identify this region as a hot spot for zinc binding, which modulates Cys328 reactivity. Moreover, they provide a molecular standpoint for vimentin regulation through the interplay between cysteine modifications and zinc availability., This work was supported by the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie Grant Agreement No. 675132 “Masstrplan”, Grants SAF2015-68590-R and RTI2018-097624-B-I00 from Agencia Estatal de Investigación, MICINN/FEDER, Spain, and Instituto de Salud Carlos III/FEDER, RETIC ARADyAL RD16/0006/0021 to DPS; Grant CTQ2017-88353-R from MICINN to SMS; Grant BES-2015-071588 from MICINN to JGC.

16 p.-5 fig., Intermediate filaments (IFs) play key roles in cell mechanics, signaling and homeostasis. Their assembly and dynamics are finely regulated by posttranslational modifications. The type III IFs, vimentin, desmin, peripherin and glial fibrillary acidic protein (GFAP), are targets for diverse modifications by oxidants and electrophiles, for which their conserved cysteine residue emerges as a hot spot. Pathophysiological examples of these modifications include lipoxidation in cell senescence and rheumatoid arthritis, disulfide formation in cataracts and nitrosation in endothelial shear stress, although some oxidative modifications can also be detected under basal conditions. We previously proposed that cysteine residues of vimentin and GFAP act as sensors for oxidative and electrophilic stress, and as hinges influencing filament assembly. Accumulating evidence indicates that the structurally diverse cysteine modifications, either per se or in combination with other posttranslational modifications,elicit specific functional outcomes inducing distinct assemblies or network rearrangements, including filament stabilization, bundling or fragmentation. Cysteine-deficient mutants are protected from these alterations but show compromised cellular performance in network assembly and expansion, organelle positioning and aggresome formation, revealing the importance of this residue. Therefore, the high susceptibility to modification of the conserved cysteine of type III IFs and its cornerstone position in filament architecture sustains their role in redox sensing and integration of cellular responses. This has deep pathophysiological implications and supports the potential of this residue as a drug target., This work was supported by Grants from Agencia Estatal de Investigación, Ministerio de Ciencia e Innovación (MCI, Spain) and European Regional Development Fund, RTI2018-097624-B-I00,Instituto de Salud Carlos III (Spain) RETIC AraDyal RD16/0006/0021,and CSIC PTI Global Health (PIE202020E223/CSIC-COV19-100). AV-P is supported by the FPI program from MCI, reference BES-2016-076965.

143 p.-17 fig., SIGNIFICANCE:Transsulfuration allows conversion of methionine into cysteine using homocysteine as an intermediate. This pathway produces S-adenosylmethionine, a key metabolite for cell function, and provides 50% of the cysteine needed for hepatic glutathione synthesis. The route requires the intake of essential nutrients (e.g. methionine, vitamins) and is regulated by their availability. Transsulfuration presents multiple interconnections with epigenetics, ATP and glutathione synthesis, polyol and pentose phosphate pathways and detoxification that rely mostly in the exchange of substrates or products. Major hepatic diseases, rare diseases and sensorineural disorders, among others that concur with oxidative stress, present impaired transsulfuration. Recent advances. In contrast to the classical view, a nuclear branch of the pathway, potentiated under oxidative stress, is emerging. Several transsulfuration proteins regulate gene expression, suggesting moonlighting activities. Additionally, abnormalities in homocysteine metabolism link nutrition and hearing loss., CRITICAL ISSUES:Knowledge about the cross-regulation between pathways is mostly limited to the hepatic availability/removal of substrates and inhibitors. However, advances regarding protein-protein interactions involving oncogenes, identification of several post-translational modifications and putative moonlighting activities expand the potential impact of transsulfuration beyond methylations and homocysteine., FUTURE DIRECTIONS:Increasing the knowledge on transsulfuration outside the liver, understanding the protein-protein interaction networks involving these enzymes, the functional role of their post-translational modifications or the mechanisms controlling their nucleocytoplasmic shuttling may provide further insights into the pathophysiological implications of this pathway, allowing design of new therapeutic interventions., This work was support by grants of the Ministerio de Economía y Competitividad (SAF2012-36519 and SAF2015-68590R to DPS) and Instituto de Salud Carlos III (RETICs RIRAAF RD12/0013/0008 and ARADyAL RD16/0006/0021).

The vimentin network displays remarkable plasticity to support basic cellular functions. Here, we show that in several cell types vimentin filaments redistribute to the cell periphery during mitosis, forming a robust scaffold interwoven with cortical actin and affecting the mitotic cortex properties. Importantly, the intrinsically disordered tail domain of vimentin is essential for this redistribution, which allows normal mitotic progression. A tailless vimentin mutant forms curly bundles, which remain entangled with dividing chromosomes leading to mitotic catastrophes or asymmetric partitions. Serial deletions of the tail domain induce increasing impairments of cortical association and mitosis progression. Disruption of actin, but not of microtubules, mimics the impact of tail deletion. Pathophysiological stimuli, including HIV-protease and lipoxidation, induce similar alterations. Interestingly, filament integrity is dispensable for cortical association, which also occurs in vimentin particles. These results unveil novel implications of vimentin dynamics in cell division by means of its interplay with the mitotic cortex.

21 p.-5 fig.-2 tab. (Chapter 3), Homeostasis of the main cellular methyl donor, S-adenosylmethionine, depends on the methionine cycle, which is at the crossroads of metabolic pathways involved in the synthesis and regulation of a large variety of compounds and processes. Impairment of the methionine cycle is detected in a vast majority of liver diseases that concur with oxidative stress. The complex regulation of the pathway is exerted from transcriptional to protein oligomerization and activity levels, subcellular distribution being recently added to the play. Oxidative stress performs a role at all of these regulatory levels either directly (e.g., eliciting protein modifications) or indirectly (e.g., decreasing recycling of vitamin B12). Therefore, the interest in deciphering the mechanisms through which medical interventions could revert methionine cycle impairment, contributing to liver disease treatment. In this line, the control of subcellular distribution has emerged as a putative target for new and existent antioxidants, their evaluation requiring a large research effort., Work carried out in the author’s laboratories was supported by grants of the Ministerio de Economía y Competitividad (BFU2009-08977 to MAP; SAF2012-36519 and SAF2015-68590-R to DPS)

16 p.-5 fig.-1 tab., PDRG1 is a small oncogenic protein of 133 residues. In normal human tissues, the p53 and DNA damage-regulated gene 1 (PDRG1) gene exhibits maximal expression in the testis and minimal levels in the liver. Increased expression has been detected in several tumor cells and in response to genotoxic stress. High-throughput studies identified the PDRG1 protein in a variety of macromolecular complexes involved in processes that are altered in cancer cells. For example, this oncogene has been found as part of the RNA polymerase II complex, the splicing machinery and nutrient sensing machinery, although its role in these complexes remains unclear. More recently, the PDRG1 protein was found as an interaction target for the catalytic subunits of methionine adenosyltransferases. These enzymes synthesize S-adenosylmethionine, the methyl donor for, among others, epigenetic methylations that occur on the DNA and histones. In fact, downregulation of S-adenosylmethionine synthesis is the first functional effect directly ascribed to PDRG1. The existence of global DNA hypomethylation, together with increased PDRG1 expression, in many tumor cells highlights the importance of this interaction as one of the putative underlying causes for cell transformation. Here, we will review the accumulated knowledge on this oncogene, emphasizing the numerous aspects that remain to be explored., The author wishes to thank Drs. Juliana Pérez-Miguelsanz and Dolores Pérez-Sala for their thoughtful comments on the manuscript and former members of her group at the Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM) for their contributions through the years. The author also wishes to acknowledge the longstanding support by the Ministerio Educación y Ciencia and Ministerio de Economía y Competitividad of Spain (until June 2013).

15 p.-8 fig., The intermediate filament protein vimentin constitutes a critical sensor for electrophilic and oxidative stress, which induce extensive reorganization of the vimentin cytoskeletal network. Here, we have investigated the mechanisms underlying these effects. In vitro, electrophilic lipids, including 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and 4-hydroxynonenal (HNE), directly bind to vimentin, whereas the oxidant diamide induces disulfide bond formation. Mutation of the single vimentin cysteine residue (Cys328) blunts disulfide formation and reduces lipoxidation by 15d-PGJ2, but not HNE. Preincubation with these agents differentially hinders NaClinduced filament formation by wild-type vimentin, with effects ranging from delayed elongation and increased filament diameter to severe impairment of assembly or aggregation. Conversely, the morphology of vimentin Cys328Ser filaments is mildly or not affected. Interestingly, preformed vimentin filaments are more resistant to electrophile-induced disruption, although chemical modification is not diminished, showing that vimentin (lip) oxidation prior to assembly is more deleterious. In cells, electrophiles, particularly diamide, induce a fast and drastic disruption of existing filaments, which requires the presence of Cys328. As the cellular vimentin network is under continuous remodeling, we hypothesized that vimentin exchange on filaments would be necessary for diamide-induced disruption. We confirmed that strategies reducing vimentin dynamics, as monitored by FRAP, including cysteine crosslinking and ATP synthesis inhibition, prevent diamide effect. In turn, phosphorylation may promote vimentin disassembly. Indeed, treatment with the phosphatase inhibitor calyculin A to prevent dephosphorylation intensifies electrophile-induced wild-type vimentin filament disruption. However, whereas a phosphorylation-deficient vimentin mutant is only partially protected from disorganization, Cys328Ser vimentin is virtually resistant, even in the presence of calyculin A. Together, these results indicate that modification of Cys328 and vimentin exchange are critical for electrophile-induced network disruption., This work was supported by the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie Grant agreement no. 675132 “Masstrplan”, Grant SAF2015-68590-R from MINECO/FEDER, Spain and Instituto de Salud Carlos III/FEDER,RETIC Aradyal RD16/0006/0021. Feedback from COST Action CA15214 “EuroCellNet” is gratefully acknowledged.

The paradigm of a cytoplasmic methionine cycle synthesizing/eliminating metabolites that are transported into/out of the nucleus as required has been challenged by detection of significant nuclear levels of several enzymes of this pathway. Here, we show betaine homocysteine S-methyltransferase (BHMT), an enzyme that exerts a dual function in maintenance of methionine levels and osmoregulation, as a new component of the nuclear branch of the cycle. In most tissues, low expression of Bhmt coincides with a preferential nuclear localization of the protein. Conversely, the liver, with very high Bhmt expression levels, presents a main cytoplasmic localization. Nuclear BHMT is an active homotetramer in normal liver, although the total enzyme activity in this fraction is markedly lower than in the cytosol. N-terminal basic residues play a role in cytoplasmic retention and the ratio of glutathione species regulates nucleocytoplasmic distribution. The oxidative stress associated with D-galactosamine (Gal) or buthionine sulfoximine (BSO) treatments induces BHMT nuclear translocation, an effect that is prevented by administration of N-acetylcysteine (NAC) and glutathione ethyl ester (EGSH), respectively. Unexpectedly, the hepatic nuclear accumulation induced by Gal associates with reduced nuclear BHMT activity and a trend towards increased protein homocysteinylation. Overall, our results support the involvement of BHMT in nuclear homocysteine remethylation, although moonlighting roles unrelated to its enzymatic activity in this compartment cannot be excluded., This work was supported by grants of the Ministerio de Economía y Competitividad (BFU2008-00666 and BFU2009-08977 to MAP; SAF2012-36519 and SAF2015-68590R to DPS) and Instituto de Salud Carlos III (RETIC RIRAAF RD12/0013/0008 and ARADYAL RD16/0006/0021 to DPS).

This is an open access article distributed under the terms of the Creative Commons Attribution License., Methionine adenosyltransferases MAT I and MAT III (encoded by Mat1a) catalyze S-adenosylmethionine synthesis in normal liver. Major hepatic diseases concur with reduced levels of this essential methyl donor, which are primarily due to an expression switch from Mat1a towards Mat2a. Additional changes in the association state and even in subcellular localization of these isoenzymes are also detected. All these alterations result in a reduced content of the moderate (MAT I) and high Vmax (MAT III) isoenzymes, whereas the low Vmax (MAT II) isoenzyme increases and nuclear accumulation of MAT I is observed. These changes derive in a reduced availability of cytoplasmic S-adenosylmethionine, together with an effort to meet its needs in the nucleus of damaged cells, rendering enhanced levels of certain epigenetic modifications. In this context, the putative role of protein-protein interactions in the control of S-adenosylmethionine synthesis has been scarcely studied. Using yeast two hybrid and a rat liver library we identified PDRG1 as an interaction target for MATα1 (catalytic subunit of MAT I and MAT III), further confirmation being obtained by immunoprecipitation and pull-down assays. Nuclear MATα interacts physically and functionally with the PDRG1 oncogene, resulting in reduced DNA methylation levels. Increased Pdrg1 expression is detected in acute liver injury and hepatoma cells, together with decreased Mat1a expression and nuclear accumulation of MATα1. Silencing of Pdrg1 expression in hepatoma cells alters their steady-state expression profile on microarrays, downregulating genes associated with tumor progression according to GO pathway analysis. Altogether, the results unveil the role of PDRG1 in the control of the nuclear methylation status through methionine adenosyltransferase binding and its putative collaboration in the progression of hepatic diseases., CP was a postdoctoral fellow of the UNAM-CSIC program and ER was supported by RCMN C03/08 and PI05/056. This work was supported by grants of the Ministerio de Economía y Competitividad (BFU2005-00050, BFU2008-00666, BFU2009-08977), and the Instituto de Salud Carlos III (RCMN C03/08 and PI05/0563).

The intermediate filament protein vimentin constitutes a critical sensor for electrophiles and oxidative stress in mesenchymal cells. The structure of the vimentin network structure is altered by lipoxidation, although the mechanisms involved are not completely understood. Here, we have studied the response of vimentin to oxidation and lipoxidation, both in vitro and in cells. Electrophilic lipids including 15d-PGJ2 and HNE bind to isolated vimentin in vitro, whereas the oxidant diamide induces disulfide bond formation. Requirement for the presence of the cysteine residue differs among these modifications. Interestingly, pre-incubation of vimentin with these agents disrupts NaCl-induced filament formation, whereas NaCl polymerized-vimentin appears to be more resistant to filament disruption by electrophiles and oxidants. These observations suggest that (lip)oxidative modifications of soluble vimentin subunits would be more deleterious than modification of polymerized vimentin. Therefore, we are exploring the interplay between vimentin lipoxidation and dynamics in cells. These studies will contribute to understand the implications of vimentin in pathophysiological processes associated with oxidative stress.

Methionine adenosyltransferases (MATs) are the family of enzymes that synthesize the main biological methyl donor, S-adenosylmethionine. The high sequence conservation among catalytic subunits from bacteria and eukarya preserves key residues that control activity and oligomerization, which is reflected in the protein structure. However, structural differences among complexes with substrates and products have led to proposals of several reaction mechanisms. In parallel, folding studies begin to explain how the three intertwined domains of the catalytic subunit are produced, and to highlight the importance of certain intermediates in attaining the active final conformation. This review analyzes the available structural data and proposes a consensus interpretation that facilitates an understanding of the pathological problems derived from impairment of MAT function. In addition, new research opportunities directed toward clarification of aspects that remain obscure are also identified.

Wilson's disease is characterized by longterm hepatic accumulation of copper leading to liver disease with reduction of S-adenosylmethionine synthesis. However, the initial changes in this pathway remain unknown and constitute the objective of the present study. Using the Long Evans Cinnamon rat model, early alterations were detected in the mRNA and protein levels, as well as in the activities of several enzymes of the methionine cycle. Notably, the main change was a redox-mediated 80% decrease in the mRNA levels of the methionine adenosyltransferase regulatory subunit as compared to the control group. Moreover, changes in S-adenosylmethionine, S-adenosylhomocysteine, methionine and glutathione levels were also observed. In addition, in vitro experiments show that copper affects the activity and folding of methionine adenosyltransferase catalytic subunits. Taken together, these observations indicate that early copper accumulation alters methionine metabolism with a pattern distinct from that described previously for other liver diseases.

Betaine homocysteine methyltransferase (BHMT), a Zn(2+)-dependent thiolmethyltransferase, contributes to the regulation of homocysteine levels, increases in which are considered a risk factor for cardiovascular diseases. Most plasma homocysteine is generated through the liver methionine cycle, in which BHMT metabolizes approximately 25% of this non-protein amino acid. This process allows recovery of one of the three methylation equivalents used in phosphatidylcholine synthesis through transmethylation, a major homocysteine-producing pathway. Although BHMT has been known for over 40 years, the difficulties encountered in its isolation precluded detailed studies until very recently. Thus, the last 10 years, since the sequence became available, have yielded extensive structural and functional data. Moreover, recent findings offer clues for potential new functions for BHMT. The purpose of this review is to provide an integrated view of the knowledge available on BHMT, and to analyze its putative roles in other processes through interactions uncover to date.

11 pages, 7 figures, 4 tables., Equilibrium folding of rat liver BHMT (betaine-homocysteine methyltransferase), a TIM (triosephosphate isomerase)-barrel tetrameric protein, has been studied using urea as denaturant. A combination of activity measurements, tryptophan fluorescence, CD and sedimentation-velocity studies suggested a multiphasic process including two intermediates, a tetramer (I4) and a monomer (J). Analysis of denaturation curves for single- and six-tryptophan mutants indicated that the main changes leading to the tetrameric intermediate are related to alterations in the helix alpha4 of the barrel, as well as in the dimerization arm. Further dissociation to intermediate J included changes in the loop connecting the C-terminal alpha-helix of contact between dimers, disruption of helix alpha4, and initial alterations in helix alpha7 of the barrel, as well as in the dimerization arm. Evolution of the monomeric intermediate continued through additional perturbations in helix alpha7 of the barrel and the C-terminal loop. Our data highlight the essential role of the C-terminal helix in dimer-dimer binding through its contribution to the increased stability shown by BHMT as compared with other TIM barrel proteins. The results are discussed in the light of the high sequence conservation shown by betaine-homocysteine methyltransferases and the knowledge available for other TIM-barrel proteins., This work was supported by grants of the Ministerio de Ciencia y Tecnología (BMC 2002- 00243 and BIO2000-1279-C02-02) and Fondo de Investigación Sanitaria (01/1077 and RCMN C03/08).

8 pages, 5 figures, 3 tables., A site-directed-mutagenesis study of putative active-site residues in rat liver betaine–homocysteine S-methyltransferase has been carried out. Identification of these amino acids was based on data derived from a structural model of the enzyme. No alterations in the CD spectra or the gel-filtration chromatography elution pattern were observed with the mutants, thus suggesting no modification in the secondary structure content or in the association state of the proteins. All the mutants obtained showed a reduction of the enzyme activity, the most dramatic effect being that of Glu159, followed by Tyr77 and Asp26. Changes in affinity for either of the substrates, homocysteine or betaine, were detected when substitutions were performed of Glu 21, Asp26, Phe74 and Cys186. Interestingly, Asp26, postulated to be involved in homocysteine binding, has a strong effect on affinity for betaine. The relevance of these results is discussed in the light of very recent structural data obtained for the human enzyme., This work was supported by the Fondo de Investigación Sanitaria (grant no. 01/1077 to M.A. P.) and the Ministerio de Ciencia y Tecnología (grant no. BIO2000-1664 to M.G. and BIO2000-1279 to J. S.-A.).

Recombinant rat liver methionine adenosyltransferase has been refolded into fully active tetramers (MAT I) and dimers (MAT III), using as a source chaotrope-solubilized aggregates resulting from specific washes of inclusion bodies. The conditions of refolding, dialysis in the presence of 10 mM dithiothreitol or 10 mM GSH with I mM GSSG, allowed the production of both isoforms, the nature of the redox agent determining the capacity of the final product (MAT I/III) to interconvert. Refolding in the presence of 10 mM dithiothreitol yielded mainly MAT III in a concentration-dependent equilibrium with the homotetramer MAT I. However, refolding in the presence of the redox pair GSH/GSSG resulted in a stable MAT I and III mixture. Blockage of dimer-tetramer interconversion has been found related to the production of a single intramolecular disulfide in methionine adenosyltransferase during the GSH/GSSG folding process. The residues involved in this disulfide have been identified by mass spectrometry and using a set of single cysteine mutants as cysteines 35 and 61. In addition, a kinetic intermediate in the MAT I dissociation to MAT III has been detected. The physiological importance of these results is discussed in light of the structural and regulatory data available., This work was supported by Fondo de Investigación Sanitaria Grant 01/1077 (to M. A. P.), Dirección General de Investigación Científica y Técnica Grant PM 97/0064 (to M. A. P.), Ministerio de Ciencia y Tecnología Grant BMC 2002-00243 (to M. A. P.), and Neuropharma Grants SA-CSIC (to M. G. and M. A. P.) and BMC2001-3337 (to J. J. C.).

[Aims]: The discovery of methionine metabolism enzymes in the cell nucleus, together with their association with key nuclear processes, suggested a putative relationship between alterations in their subcellular distribution and disease. [Results]: Using the rat model of D-galactosamine intoxication severe changes in hepatic steady-state mRNA levels were found; the largest decreases corresponded to enzymes exhibiting the highest expression in normal tissue. Cytoplasmic protein levels, activities and metabolite concentrations suffered more moderate changes following a similar trend. Interestingly, galactosamine-treatment induced hepatic nuclear accumulation of MATα1 and S-adenosylhomocysteine hydrolase tetramers, their active assemblies. In fact, galactosamine-treated livers showed enhanced nuclear methionine adenosyltransferase activity. Acetaminophen intoxication mimicked most galactosamine effects on hepatic MATα1, including accumulation of nuclear tetramers. H35 cells overexpressing tagged-MATα1 reproduced the subcellular distribution observed in liver, and the changes induced by galactosamine and acetaminophen that were also observed upon glutathione depletion by buthionine sulfoximine. The H35 nuclear accumulation of tagged-MATα1 induced by these agents correlated with decreased GSH/GSSG ratios and was prevented by N-acetylcysteine and glutathione ethyl ester. However, the changes in epigenetic modifications associated with tagged-MATα1 nuclear accumulation were only prevented by N-acetylcysteine in galactosamine-treated cells. [Innovation]: Cytoplasmic and nuclear changes in proteins regulating the methylation index follow opposite trends in acute liver injury, their nuclear accumulation showing potential as disease marker. [Conclusion]: Altogether these results demonstrate galactosamine- and acetaminophen-induced nuclear accumulation of methionine metabolism enzymes as active oligomers and unveil the implication of redox-dependent mechanisms in the control of MATα1 subcellular distribution., This work was supported by grants of the Ministerio de Economía y Competitividad (BFU2005-00050, BFU2008- 00666, and BFU2009-08977 to M.A.P.; SAF2009-11642 and SAF2012-36519 to D.P.-S.; and PS09/01762 to J.P.-M.) and the Instituto de Salud Carlos III (RCMN C03/08 and PI05/0563 to M.A.P.; RD07/0064/0007 and RD12/0013/0008 to D.P.-S.). M.D. was supported by fellowships of RCMN C03/08 and PI05/0563 and M.P. by BFU2009-08977.

S-adenosylmethionine synthesis is a key process for cell function, and needs to be regulated at multiple levels. In recent years, advances in the knowledge of methionine adenosyltransferases have been significant. The discovery of nuclear localization of these enzymes suggests their transport to provide the methyl donor, S-adenosylmethionine, for DNA and histone methyltransferases in epigenetic modifications, opening new regulatory possibilities. Previous hypotheses considered only the cytoplasmic regulation of these enzymes, hence the need of an update to integrate recent findings. Here, we focus mainly on the liver and redox mechanisms, and their putative effects on localization and interactions of methionine adenosyltransferases., Ministerio de Economía y Competitividad (BFU2008-00666 and BFU2009-08977 to M.A.P., and SAF2009-11642 and SAF2012-36519 to D.P.S.) and Instituto de Salud Carlos III (RETICS RD12/0013/0008 to D.P.S. and FIS PI12/01196 to L.A.).

9 pages, 7 figures, 3 tables., The reversible unfolding of rat liver methionine adenosyltransferase dimer by urea under equilibrium conditions has been monitored by fluorescence spectroscopy, CD, size-exclusion chromatography, analytical ultracentrifugation and enzyme activity measurements. The results obtained indicate that unfolding takes place through a three-state mechanism, involving an inactive monomeric intermediate. This intermediate has a 70% native secondary structure, binds less 8-anilinonaphthalene-1-sulphonic acid than the native dimer and has a sedimentation coefficient of 4.24+/-0.15. The variations of free energy in the absence of denaturant [DeltaG(H(2)O)] and its coefficients of urea dependence (m), calculated by the linear extrapolation model, were 36.15+/-2.3 kJ.mol(-1) and 19.87+/-0.71 kJ.mol(-1).M(-1) for the dissociation of the native dimer and 14.77+/-1.63 kJ.mol(-1) and 5.23+/-0.21 kJ.mol(-1).M(-1) for the unfolding of the monomeric intermediate respectively. Thus the global free energy change in the absence of denaturant and the m coefficient were calculated to be 65.69 kJ.mol(-1) and 30.33 kJ.mol(-1).M(-1) respectively. Analysis of the calculated thermodynamical parameters indicate the instability of the dimer in the presence of denaturant, and that the major exposure to the solvent is due to dimer dissociation. Finally, a minimum-folding mechanism for methionine adenosyltransferase III is established.

Rat liver S-adenosyl-L-methionine synthetase is present in two oligomeric forms, tetramers and dimers, with different substrate kinetics and regulation. In vivo the relative amounts of both forms may change in some instances. The basis of this regulatory mechanism is not known. When rat liver cDNA was used to express the protein in Escherichia coli the two oligomeric forms mere found. Gel filtration chromatography of the purified recombinant enzyme suggested that these two isoforms might be in equilibrium. This was confirmed by kinetic experiments which showed that the specific activity of the enzyme was dependent on the protein concentration. From these experiments, apparent equilibrium constants of (5.6 ± 0.4) x 105 M-1 and (3.5 ± 0.9) x 105 M-1 were obtained at 2 mM and 60 μM methionine concentrations, respectively. Using hydrophobic chromatography on phenyl-Sepharose to separate the tetrameric and dimeric forms, an equilibrium constant of (4.9 ± 0.7) x 105 M-1 was calculated. A rate constant for the dissociation of the tetramer of k-1 = (8.1 ± 0.4) x 10-4 s-1 at 4°C was also calculated using the same approach. In summary, we have shown that the rat liver S-adenosyI-L-methionine synthetase produced in bacterial cells is present in two oligomeric forms, tetramers and dimers, which are in equilibrium. This system might be useful for studying the dynamics and the regulation of the distribution of oligomeric forms in the mammalian liver., This work was supported by grants from the Fondo de Investigaciones Sanitarias (94/0231). DGICYT (PB 94/0087). Europharma and the Science Program of the European Community (SCI*-CTY2-0780).

Rat liver S-adenosylmethionine synthetase, high- and low-M(r) forms, are regulated in vitro by the GSH/GSSG ratio at pH 8. The inhibition and oxidation constants for both forms have been calculated in the presence of thioltransferases. The mechanism of the reaction appeared to involve the formation of intramolecular disulfides. Increases of 3- to 4-fold in the oxidation constants for both S-adenosylmethionine synthetase isoenzymes in the presence of protein disulfide isomerase suggested the possibility of a thiol-disulfide exchange regulatory mechanism for this enzyme in vivo. The significance of these results is discussed on the light of the data available relating glutathione changes and modulation of enzyme activities, either in vivo and in vitro., This work was supported by grants from the Fondo de Investigaciones Sanitarias (94/ 0231) Direcciónn General de Investigación Científica y Técnica (PB 94/0087) the Science Programme of the European Community (SCI*- CT92-0780) and Europharma S.A.

We have examined the functional importance of the cysteine residues of rat liver S-adenosylmethionine synthetase. For this purpose the ten cysteine residues of the molecule were changed to serines by site-directed mutagenesis. Ten recombinant enzyme mutants were obtained by using a bacterial expression system. The same level of expression was obtained for the wild type and mutants, but the ratio of S-adenosylmethionine synthetase between soluble and insoluble fractions differed for some of the mutant forms. The immunoreactivity against an anti-(rat liver S-adenosylmethionine synthetase) antibody was equivalent in all the cases. Effects on S-adenosylmethionine synthetase activities were also measured. Mutants C57S, C69S, C105S and C121S showed decreased relative specific activity of 68, 85, 63 and 29%, respectively, compared with wild-type, whereas C312S resulted in an increase of 1.6-fold. Separation of tetramer and dimer forms for wild type and mutants was carried out by using phenyl-Sepharose columns. The dimer/tetramer ratio was calculated based on the activity and on the protein level estimated by immunoblotting. No monomeric forms of the enzyme were detected in any case. Comparison of dimer/tetramer ratios indicates the importance of cysteine-69 (dimer/tetramer protein ratio of 88 versus 10.2 in the wild type) in maintaining the oligomeric state of rat liver S-adenosylmethionine synthetase. Moreover, all the mutations carried out of cysteine residues between cysteine-35 and cysteine-105 altered the ratio between oligomeric forms.

5 pages, 3 figures., A cDNA containing the complete coding sequence for rat liver S-adenosylmethionine synthetase was cloned into the prokaryotic expression vector pT7-7 and expressed in Escherichia coli BL21(DE3). A major additional band corresponding to a protein of 48 kDa was detected on SDS/PAGE after induction with isopropyl beta-D-thiogalactopyranoside. This protein was distributed in both the soluble and insoluble fractions and accounted for approx. 30% of the total bacterial protein. The soluble enzyme was fully active, as revealed by assays in vitro of S-adenosylmethionine synthetase activity. In addition, transformed bacteria exhibited highly increased levels of intracellular S-adenosylmethionine. Two active forms of the recombinant enzyme, with apparent molecular masses of 210 kDa and 110 kDa, were detected when cytosolic extracts of the transformed cells were fractionated by gel-filtration chromatography. It is concluded that the expressed S-adenosylmethionine synthetase polypeptide assemble as tetramers and dimers., This work has been supported by grants from Fondo de Investigaciones Sanitarias (FIS 91/0220), Europharma and EC Science program (CSIC-CT92-0780).

7 pages, 5 figures, 2 tables., The regulation of rat liver S-adenosylmethionine synthetase (AdoMet synthetase), a key enzyme in methionine metabolism, by protein kinase C (PKC) phosphorylation has been studied. Both enzyme forms, tetramer and dimer, are phosphorylated by this kinase in the same residue, Thr-342, of the sequence. Phosphorylation of the dimer leads to its dissociation, with production of a fully-active monomer. The kinetics of the monomer have been studied, and a KmMet of 931.9 microM, a KmATP of 708 microM and a Vmax of 66.8 nmol/min/mg have been calculated. Alkaline phosphatase treatment of both enzyme forms (tetramer and dimer) produces a reduction in their activity with no change in the oligomeric state. On the other hand, PKC phosphorylation of the alkaline phosphatase-treated AdoMet synthetase forms leads to the dissociation of the dimer to produce a monomer. Rephosphorylation occurs again in the same residue, Thr-342, of the sequence. The significance of AdoMet synthetase regulation by PKC phosphorylation is further discussed., This work was supported by grants from the Fondo de Investigaciones Sanitarias, Europharma and the Science Program of the European Community.

Rat liver S-adenosylmethionine (AdoMet) synthetase appears as high-M(r) (tetramer) and low-M(r) (dimer) forms. Both are inhibited in the presence of GSSG at pH 8. The calculated K(i) values are 2.14 and 4.03 mM for the high- and low-M(r) forms, respectively. No effect on enzyme activity was observed in the presence of GSH, but modulation of inhibition by GSSG can be obtained by addition of GSH. At a total glutathione concentration (GSH + GSSG) of 10 mM, a K(ox) of 1.74 was calculated for the high-M(r) form, whereas this constant was 2.85 for the low-M(r) AdoMet synthetase. No incorporation of [35S]GSSG was observed in either of the enzyme forms, and inhibition of enzyme activity was correlated with dissociation of both AdoMet synthetases to a monomer. The data obtained in the presence of GSSG seem to suggest that oxidation leads to the formation of an intrasubunit disulfide. The possible regulation of AdoMet synthetase activity by the GSH/GSSG ratio is discussed, as well as its in vivo significance., This work was supported in part by grants from the Fondo de Investigaciones Sanitarias and Europharma.

The in vivo regulation of S-adenosylmethionine synthetase, a key enzyme in methionine metabolism, is so far unknown. The enzyme activity has been shown to be modulated by glutathione and the oxidation state of its sulfhydryl groups. Analysis of the protein sequence has revealed the presence of putative phosphorylation sites. A mixed regulatory mechanism combining phosphorylation and the oxido/reduction of sulfhydryl groups is proposed. The role of glutathione in this mechanism is also discussed., L.A. and F.C. are Fellows from Ministerio de Educación y Ciencia. C.D. is a Fellow from Fondo de Investigaciones Sanitarias. Work from the author's laboratory has been supported by grants from Fondo de Investigaciones Sanitarias (FIS 91/0220) and Europharma.

5 pages, 3 figures, 3 tables., The present paper reports the tryptic digestion of N-ethylmaleimide-treated S-adenosyl-L-methionine synthetase (high- and low-Mr forms) and the isolation of the modified peptides by h.p.l.c. There is only one site modified after 5 min incubation, and the modification at this site correlates with the main activity decrease. The amino acid composition of this peptide was determined, and its localization in the sequence shows the modified residue as cysteine-150, which is located close to the putative ATP-binding site. Modification of the enzyme for 20 min led to the appearance of a second labelled peptide, which seems to be responsible for about a further 10% of the activity loss. The modification by N-ethylmaleimide of the enzyme was partially prevented in the presence of adenosine 5'-[beta gamma-imido]triphosphate and methionine, further supporting the hypothesis that the modified residues lie within the active site. Urea treatment of the enzyme, followed by modification with N-ethylmaleimide, produces the modification of 7 of the 10 cysteine residues present in the sequence. The results obtained were the same for either of the isoforms., This work was supported by grants from Fondo de Investigaciones Sanitarias and Europharma.

A 3 kb cDNA coding for rat liver S-adenosylmethionine (AdoMet) synthetase has been isolated. The M(r) of the protein has been unequivocally determined by cDNA sequencing and enzyme purification of a thiopropyl-Sepharose column. The length of the mRNA 5' non-coding region has been defined by primer-extension analysis. The rat liver cloned cDNA has been also used to detect S-adenosylmethionine synthetase mRNA in human liver., This work was supported by grants from Fondo de lnvestigaciones Sanitarias (FIS 91/0220) and Europharma. L.A. and M.A.P. are fellows from Ministerio de Educación y Ciencia. M.A. and F,C. are fellows from Europharma and Fundación Conchita Rábago respectively.

Phospholipid methyltransferase, the enzyme that converts phosphatidylethanolamine into phosphatidylcholine with S-adenosyl-L-methionine as the methyl donor, was purified to apparent homogeneity from rat liver microsomal fraction. When analysed by SDS/polyacrylamide-gel electrophoresis only one protein, with molecular mass about 50 kDa, is detected. This protein could be phosphorylated at a single site by incubation with [alpha-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. A less-purified preparation of the enzyme is mainly composed of two proteins, with molecular masses about 50 kDa and 25 kDa, the 50 kDa form being phosphorylated at the same site as the homogeneous enzyme. After purification of both proteins by electro-elution, the 25 kDa protein forms a dimer and migrates on SDS/polyacrylamide-gel electrophoresis with molecular mass about 50 kDa. Peptide maps of purified 25 kDa and 50 kDa proteins are identical, indicating that both proteins are formed by the same polypeptide chain(s). It is concluded that rat liver phospholipid methyltransferase can exist in two forms, as a monomer of 25 kDa and as a dimer of 50 kDa. The dimer can be phosphorylated by cyclic AMP-dependent protein kinase.

Calmodulin, an acidic protein that binds calcium with high affinity, has multiple roles in the activation of many enzymes involved in cellular regulation of eukaryotes. In this study we show that calmodulin binding to hen egg-white lysozyme, in a Ca2+-dependent way, was observed using electroblots incubated with biotinylated calmodulin and detected with avidin-alkaline phosphatase or for affinity chromatography on a gel calmodulin column. Antimicrobial activity of lysozyme was not modified in the presence of Ca2+-calmodulin.

The 11-cis-retinal binding site of rhodopsin is of great interest because it is buried in the membrane but yet must provide an environment for charged amino acids. In addition, the active-site lysine residue must be able to engage in rapid Schiff base formation with 11-cis-retinal at neutral and lower pH values. This requires that this lysine be unprotonated. We have begun to study the environment of the active-site lysine using a reporter group adducted to it. Non-active-site permethylated opsin was reacted with 5-nitrosalicylaldehyde, and the resulting Schiff base was permanently fixed by borohydride reduction. The stoichiometry of incorporation was one. This chromophoric and pH-sensitive reporter group affords information on the active-site environment of rhodopsin by determining the ionization constants of its ionizable groups at different pH values. The pH titration of the modified protein showed a single pKa = 7.8 +/- 0.19 ascribable to the ionization of the phenol. The ionization of the modified lysine residue was not observed at all pH values studied. These studies are interpreted to mean that a negatively charged amino acid is propinquous to the active-site lysine residue and that this latter residue does not have an unusually low pKa., This work was supported by United States Public Health Service Research Grant EY 03624 from the National Institutes of Health. Supported by the Fundacion Juan March (Spain).

6 pages, 5 figures., When a partially purified rat liver phospholipid methyltransferase is incubated with [gamma-32P]ATP and rat brain protein kinase C, phospholipid methyltransferase (Mr 50,000, pI 4.75) becomes phosphorylated. Phosphorylation of the enzyme showed Ca2+/lipid-dependency. Protein kinase C-dependent phosphorylation of phospholipid methyltransferase was accompanied by an approx. 2-fold activation of the enzyme activity. Activity changes and enzyme phosphorylation showed the same time course. Activation of the enzyme also showed Ca2+/lipid-dependency. Protein kinase C mediates phosphorylation of predominantly serine residues of the methyltransferase. One major peak of phosphorylation was identified by analysis of tryptic phosphopeptides by isoelectrofocusing. This peak (pI 5.2) differs from that phosphorylated by the cyclic AMP-dependent protein kinase (pI 7.2), demonstrating the specificity of phosphorylation of protein kinase C. Tryptic-peptide mapping by h.p.l.c. of the methyltransferase phosphorylated by protein kinase C revealed one major peak of radioactivity, which could be resolved into two labelled phosphopeptides by t.l.c. The significance of protein kinase C-mediated phosphorylation of phospholipid methyltransferase is discussed., This work was supported by grants from CAICYT, FISS and Europharma.

An enzyme that catalyses the three-step methylation of phosphatidylethanolamine to phosphatidylcholine as well as the methylation of fatty acids and that uses S-adenosylmethionine as the methyl donor has been purified about 200-fold from rat liver. Irradiation of the purified enzyme with a short-wavelength u.v. light in the presence of [methyl-3H]8-azido-S-adenosylmethionine followed by electrophoresis results in the incorporation of radioactivity into a single protein band of about 25 kDa. It is concluded that a single catalytic subunit catalyses the conversion of phosphatidylethanolamine into phosphatidylcholine and fatty acid methylation.

The present results show that the catalytic subunit of cyclic AMP-dependent protein kinase phosphorylates the 50 kDa protein of rat liver phospholipid methyltransferase at one single site on a serine residue. Phosphorylation of this site is stimulated 2- to 3-fold by S-adenosylmethionine. S-adenosylmethionine-dependent protein phosphorylation is time- and dose-dependent and occurs at physiological concentrations. S-adenosylhomocysteine has no effect on protein phosphorylation but inhibits S-adenosylmethionine-dependent protein phosphorylation. S-Adenosylmethionine/S-adenosylhomocysteine ratios varying from 0 to 5 produce a dose-dependent stimulation of the phosphorylation of the 50 kDa protein. In conclusion, these results show, for the first time, that the ratio S-adenosylmethionine/S-adenosylhomocysteine can modulate phosphorylation of a specific protein.

Incubation of partially purified rat liver lipid methyltransferase with MgATP and the catalytic subunit of the cyclic AMP dependent protein kinase results in up to 4-fold activation of the methylation reaction. When (γ- 32 P) MgATP is included in the assay mixture, the analysis of the phosphoprotein products by electrophoresis shows the incorporation of 32 P into a single protein band of about 50K and pI 4.75. It is concluded that rat liver lipid methyltransferase can be converted from a low activity dephosphorylated form to a high activity phosphorylated form.

The active site of rat liver S-adenosylmethionine synthetase was studied using 8-azido ATP, a photolabile analogue of ATP. Both forms of the enzyme, tetramer and dimer, could be labelled by using concentrations of the analogue similar to the KmATP values for each form, 350 microM and 1 mM respectively. Labelling of both S-adenosylmethionine synthetase forms with 8-azido [alpha-32P]ATP, followed by tryptic digestion and purification by HPLC, afforded one specifically labelled peptide in each case. Identification of the labelled peptide by amino acid analysis and peptide sequencing, and comparison with the enzyme sequence, indicated that the same peptide (267-286) was modified in both enzyme forms. The results are discussed on the basis of the high degree of similarity that this peptide shows in all the known S-adenosylmethionine synthetase sequences.

Chinese hamster ovary cells were stably transfected with rat liver S-adenosylmethionine synthetase cDNA. As a result, S-adenosylmethionine synthetase activity increased 2.3-fold, an effect that was accompanied by increased S-adenosylmethionine, a depletion of ATP and NAD levels, elevation of the S-adenosylmethionine/S-adenosylhomocysteine ratio (the methylation ratio), increased DNA methylation and polyamine levels (spermidine and spermine), and normal GSH levels. By contrast, the transfected cells showed normal growth curves and morphology. Exposure to an oxidative stress by the addition of H2O2 resulted in a greater consumption of ATP and NAD in the transfected cells than in the wild-type cells. In turn, cell killing by H2O2 was greater in the transfected cells than in the wild-type cells. This killing of Chinese hamster ovary cells by H2O2 involved the activation of poly(ADP-ribose) polymerase with the resultant loss of NAD and ATP. 3-Aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase, but not the antioxidant N,N´-diphenylphenylenediamine, prevented the killing of Chinese hamster ovary cells by H2O2 and maintained the contents of NAD and ATP. The results of this study indicate that a moderate activation of the synthesis of S-adenosylmethionine leads to ATP and NAD depletion and to a greater sensitivity to cell killing by oxidative stress.

The conversion of phosphatidylethanolamine to phosphatidylcholine occurs by three successive N -methylations of the amino moiety of phosphatidylethanolamine. It has been proposed that two enzymes are needed for the conversion of phosphatidylethanolamine to phosphatidylcholine. However, several reports question this hypothesis in mammalian cells. Present evidence indicates that, in the liver, only one enzyme controls the flow from phosphatidylethanolamine to phosphatidylcholine.