Nuclear shuttling of key methionine cycle enzymes in acute liver injury

Artículo para la web de Divulgación Atlas of Science.Important metabolic enzymes have been classically ascribed to certain subcellular locations, where they perform their well-known function. In the last decades, several technological advances have allowed detection of these proteins in new locations, performing either their known catalytic role or a new function.Peer reviewe

The relationships between human MAT1A mutations and disease: A folding and association problem?

Capítulo 8.-- El pdf es la versión post-print.Methionine adenosyltransferases (MATs) are a family of highly conserved oligomers that catalyze the only known reaction for the synthesis of S-adenosylmethionine (AdoMet), the main cellular methyl donor. Their catalytic subunits exhibit a characteristic structure, organized in three domains formed by nonconsecutive stretches of the sequence. The active sites locate at the interface between subunits in the dimer with amino acids of each monomer contributing to catalysis. Changes in activity, oligomerization level and expression have been detected in several hepatic diseases; the knockout mouse for MAT1A spontaneously developing hepatocellular carcinoma (HCC). However, none of the patients with persistent hypermethioninemia caused by mutations in this gene exhibits hepatic problems, instead a few cases showing demyelination have been described. This chapter discusses aspects related to the structural features of these enzymes and the impact that the mutations found in the human MAT1A gene may have in the final protein structure. The influence of the redox environment in MAT folding and association is also analyzed, in light of the effects that drugs and metals that alter the GSH/GSSG ratio produce in the activity and association level. The recent report of the nuclear localization of the MAT I/III isoenzymes, along with their presence in tissues other than liver opened the option to MAT moonlighting. The possibility exists that disease development is related not only to a decrease in AdoMet production, but also to the role of these particular isoenzymes in different subcellular compartments. Therefore, the influence of MAT1A mutations, especially those leading to protein truncations, on folding and subcellular localization is discussed, paying special attention to the Hazelwood’s hetero-oligomerization hypothesis to explain the demyelination process in patients with persistent hypermethioninemia.Ministerio de Ciencia e Innovación (BFU2008-00666, BFU2009-08977).Peer reviewe

Betaine homocysteine S-methyltransferase emerges as a new player of the nuclear methionine cycle

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).Peer reviewe

The Freundlich model of adsorption for calculation of specific surface areaS

The specific surface area of solids and the surface area occupied by the active phase (metal or oxide) on a support are parameters of the utmost importance in adsorption and catalysis. For the determination of the former, the BET equation is universally established. For the evaluation of the latter the works of selective chemisorption, initiated by Emmett and Brunauer (I ), for metals and by Bridges et al. (2) and Weller et al. (3), for oxides have come to represent important contributions. Some of the classical models of adsorption have also been used for evaluation of specific surfaces (Langmuir equation) (4) and the dispersion of supported metals or oxides (Freundlich equation) (5). Both of them are applicable to physisorption as well as chemisorption processes

Long-term dietary folate deficiency accelerates progressive hearing loss on CBA/Ca mice

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY).Dietary folic acid deficiency induced early hearing loss in C57BL/6J mice after two-months, corroborating the epidemiological association previously described between vitamin deficiency and this sensory impairment. However, this strain is prone to early hearing loss, and hence we decided to analyze whether the effects exerted by folate deprivation follow the same pattern in a mouse strain such as CBA/Ca, which is resistant to hearing impairment. Here, we show results of a long-term study on hearing carried out on CBA/Ca mice subjected to dietary folate deprivation. Systemic changes included decreased serum folate levels, hyperhomocysteinemia and signs of anemia in the group fed the folate-deficient diet. Initial signs of hearing loss were detected in this strain after 8-months of vitamin deficiency, and correlated with histological damage in the cochleae. In conclusion, the data presented reinforce the importance of adequate folic acid levels for the auditory system and suggest that the impact of dietary deficiencies may depend on the genetic background.RM was a fellow of the JAE-CSIC predoctoral program. This work was supported by grants of the Ministerio de Economía y Competitividad (SAF2014-53979-R to IV; BFU2009-08977 to MP), the European Union (FP7-AFHELO and TARGEAR to IV).Peer reviewe

The oncogene PDRG1 is an interaction target of methionine adenosyltransferases

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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 V max (MAT III) isoenzymes, whereas the low V max (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.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 Carlos III (RCMN C03/08 and PI05/0563

Detoxifying enzymes at the cross-roads of inflammation, oxidative stress, and drug hypersensitivity: role of glutathione transferase P1-1 and aldose reductase

9 p.-2 figPhase I and II enzymes are involved in the metabolism of endogenous reactive compounds as well as xenobiotics, including toxicants and drugs. Genotyping studies have established several drug metabolizing enzymes as markers for risk of drug hypersensitivity. However, other candidates are emerging that are involved in drug metabolism but also in the generation of danger or costimulatory signals. Enzymes such as aldo-keto reductases (AKR) and glutathione transferases (GST) metabolize prostaglandins and reactive aldehydes with proinflammatory activity, as well as drugs and/or their reactive metabolites. In addition, their metabolic activity can have important consequences for the cellular redox status, and impacts the inflammatory response as well as the balance of inflammatory mediators, which can modulate epigenetic factors and cooperate or interfere with drug-adduct formation. These enzymes are, in turn, targets for covalent modification and regulation by oxidative stress, inflammatory mediators, and drugs. Therefore, they constitute a platform for a complex set of interactions involving drug metabolism, protein haptenation, modulation of the inflammatory response, and/or generation of danger signals with implications in drug hypersensitivity reactions. Moreover, increasing evidence supports their involvement in allergic processes. Here, we will focus on GSTP1-1 and aldose reductase (AKR1B1) and provide a perspective for their involvement in drug hypersensitivityThis work has been supported by grants SAF2012-36519 from MINECO and SAF-2015-68590-R from MINECO/FEDER and ISCIII RETIC RIRAAF RD12/0013/0008 to DP,and RD12/0013/0002 to J A.Peer reviewe

Identification of importin (IPO-8) as the most accurate reference gene for the clinicopathological analysis of lung specimens

Abstract Background: The accurate normalization of differentially expressed genes in lung cancer is essential for the identification of novel therapeutic targets and biomarkers by real time RT-PCR and microarrays. Although classical "housekeeping" genes, such as GAPDH, HPRT1, and beta-actin have been widely used in the past, their accuracy as reference genes for lung tissues has not been proven. Results: We have conducted a thorough analysis of a panel of 16 candidate reference genes for lung specimens and lung cell lines. Gene expression was measured by quantitative real time RTPCR and expression stability was analyzed with the softwares GeNorm and NormFinder, mean of |ΔCt| (= |Ct Normal-Ct tumor|) ± SEM, and correlation coefficients among genes. Systematic comparison between candidates led us to the identification of a subset of suitable reference genes for clinical samples: IPO8, ACTB, POLR2A, 18S, and PPIA. Further analysis showed that IPO8 had a very low mean of |ΔCt| (0.70 ± 0.09), with no statistically significant differences between normal and malignant samples and with excellent expression stability. Conclusion: Our data show that IPO8 is the most accurate reference gene for clinical lung specimens. In addition, we demonstrate that the commonly used genes GAPDH and HPRT1 are inappropriate to normalize data derived from lung biopsies, although they are suitable as reference genes for lung cell lines. We thus propose IPO8 as a novel reference gene for lung cancer samples

Expression of Sirtuin 1 and 2 Is Associated with Poor Prognosis in Non-Small Cell Lung Cancer Patients

Sirtuin 1 (SIRT1) and sirtuin 2 (SIRT2) are NAD+-dependent protein deacetylases involved in the regulation of key cancer-associated genes. In this study we evaluated the relevance of these deacetylases in lung cancer biology

Development of a novel splice array platform and its application in the identification of alternative splice variants in lung cancer

Abstract Background Microarrays strategies, which allow for the characterization of thousands of alternative splice forms in a single test, can be applied to identify differential alternative splicing events. In this study, a novel splice array approach was developed, including the design of a high-density oligonucleotide array, a labeling procedure, and an algorithm to identify splice events. Results The array consisted of exon probes and thermodynamically balanced junction probes. Suboptimal probes were tagged and considered in the final analysis. An unbiased labeling protocol was developed using random primers. The algorithm used to distinguish changes in expression from changes in splicing was calibrated using internal non-spliced control sequences. The performance of this splice array was validated with artificial constructs for CDC6, VEGF, and PCBP4 isoforms. The platform was then applied to the analysis of differential splice forms in lung cancer samples compared to matched normal lung tissue. Overexpression of splice isoforms was identified for genes encoding CEACAM1, FHL-1, MLPH, and SUSD2. None of these splicing isoforms had been previously associated with lung cancer. Conclusions This methodology enables the detection of alternative splicing events in complex biological samples, providing a powerful tool to identify novel diagnostic and prognostic biomarkers for cancer and other pathologies