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Las prostaglandinas ciclopentenonas (cyPG), como PGA1 y 15d-PGJ2, son mediadores lípidos endógenos que proceden de la deshidratación espontánea de otras prostaglandinas (PG). Estos compuestos ejercen efectos antiinflamatorios y antitumorales. Debido a su elevada reactividad, el principal mecanismo de acción de estos compuestos es la unión covalente a proteínas celulares mediante la adición de Michael. Mediante el uso de abordajes proteómicos, en nuestro grupo de investigación se identificaron proteínas de la superfamilia de las AKR como dianas selectivas de la PGA1. Las AKR son enzimas de metabolismo de fase I que catalizan la oxidorreducción de compuestos tanto endógenos como xenobióticos. En este trabajo hemos caracterizado la modificación por PGA1 de dos proteínas AKR con una elevada importancia fisiopatológica, la aldosa reductasa o AKR1B1 y la AKR1B10, y la repercusión biológica de esta modificación. La aldosa reductasa está implicada en las complicaciones diabéticas secundarias, y la AKR1B10 está implicada en el desarrollo de tumores relacionados con el consumo de tabaco. Hemos demostrado que la PGA1 se une a la proteína recombinante AKR1B1 e inhibe su actividad enzimática. Además, hemos comprobado que la PGA1 se une covalentemente a la proteína AKR1B10 a través del residuo de Cys299 presente en el centro activo de la enzima, e inhibe su actividad enzimática en células. También hemos observado que la PGA1 inhibe la migración in vitro y el crecimiento de colonias en agar de las células A549, y potencia los efectos inducidos por la doxorrubicina (DOX) en estas células. Estos efectos podrían estar potencialmente mediados por la inhibición de la proteína AKR1B10. Los resultados obtenidos en este trabajo aportan nuevas evidencias que ayudan a la comprensión de los mecanismos de acción de las cyPG, confirman la importancia de sus propiedades antitumorales y antiproliferativas, y apoyan su uso como potenciales fármacos para el tratamiento de diferentes patologías en

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.

Sprouty and Spred proteins have been widely implicated in the negative regulation of the fibroblast growth factor receptor-extracellular regulated kinase (ERK) pathway. In considering the functional role of these proteins, we explored their effects on ERK activation induced by cyclopentenone prostanoids, which bind to and activate Ras proteins. We therefore found that ectopic overexpression in HeLa cells of human Sprouty2, or human Spred1 or 2, inhibits ERK1/2 and Elk-1 activation triggered by the cyclopentenone prostanoids PGA1 and 15d-PGJ2. Furthermore, we found that in HT cells that do not express Sprouty2 due to hypermethylation of its gene-promoter, PGA1-provoked ERK activation was more intense and sustained compared to other hematopoietic cell lines with unaltered Sprouty2 expression. Cyclopentenone prostanoids did not induce Sprouty2 tyrosine phosphorylation, in agreement with its incapability to activate tyrosine-kinase receptors. However, Sprouty2 Y55F, which acts as a defective mutant upon tyrosine-kinase receptor stimulation, did not inhibit cyclopentenone prostanoids-elicited ERK pathway activation. In addition, Sprouty2 did not affect the Ras-GTP levels promoted by cyclopentenone prostanoids. These results unveil both common and differential features in the activation of Ras-dependent pathways by cyclopentenone prostanoids and growth factors. Moreover, they provide the first evidence that Sprouty and Spred proteins are negative regulators of the ERK/Elk-1 pathway activation induced not only by growth-factors, but also by reactive lipidic mediators. © 2011 García-Domínguez et al.

Ras proteins are crucial players in differentiation and oncogenesis and constitute important drug targets. The localization and activity of Ras proteins are highly dependent on posttranslational modifications at their C-termini. In addition to an isoprenylated cysteine, H-Ras, but not other Ras proteins, possesses two cysteine residues (C181 and C184) in the C-terminal hypervariable domain that act as palmitoylation sites in cells.Cyclopentenone prostaglandins (cyPG) are reactive lipidic mediators that covalently bind to H-Ras and activate H-Ras dependent pathways. Dienone cyPG, such as 15-deoxy-D12,14-PGJ2 (15d-PGJ2) and D12-PGJ2 selectively bind to the H-Ras hypervariable domain. Here we show that these cyPG bind simultaneously C181 and C184 of H-Ras, thus potentially altering the conformational tendencies of the hypervariable domain. Based on these results, we have explored the capacity of several bifunctional cysteine reactive small molecules to bind to the hypervariable domain of H-Ras proteins. Interestingly, phenylarsine oxide (PAO), a widely used tyrosine phosphatase inhibitor, and dibromobimane, a cross-linking agent used for cysteine mapping, effectively bind H-Ras hypervariable domain. The interaction of PAO with H-Ras takes place in vitro and in cells and blocks modification of H-Ras by 15d-PGJ2. Moreover, PAO treatment selectively alters H-Ras membrane partition and the pattern of H-Ras activation in cells, from the plasma membrane to endomembranes. These results identify H-Ras as a novel target for PAO. More importantly, these observations reveal that small molecules or reactive intermediates interacting with spatially vicinal cysteines induce intramolecular cross-linking of H-Ras C-terminus potentially contributing to the modulation of Rasdependent pathways.

Cyclopentenone prostaglandins (cyPG) are reactive eicosanoids that may display anti-inflammatory and antiproliferative actions, possibly offering therapeutic potential. Here we report the identification of members of the aldo-keto reductase (AKR) family as selective targets of the cyPG prostaglandin A1 (PGA1). AKR enzymes metabolize aldehydes and drugs containing carbonyl groups and are involved in inflammation and tumorigenesis. Thus, these enzymes represent a class of targets to develop small molecule inhibitors with therapeutic activity. Molecular modeling studies pointed to the covalent binding of PGA1 to Cys299, close to the active site of AKR, with His111 and Tyr49, which are highly conserved in the AKR family, playing a role in PGA1 orientation. Among AKR enzymes, AKR1B10 is considered as a tumor marker and contributes to tumor development and chemoresistance. We validated the direct modification of AKR1B10 by biotinylated PGA1 (PGA1-B) in cells, and confirmed that mutation of Cys299 abolishes PGA1-B incorporation, whereas substitution of His111 or Tyr49 reduced the interaction. Modification of AKR1B10 by PGA1 correlated with loss of enzymatic activity and both effects were increased by depletion of cellular glutathione. Moreover, in lung cancer cells PGA1 reduced tumorigenic potential and increased accumulation of the AKR substrate doxorubicin, potentiating cell-cycle arrest induced by this hemotherapeutic agent. Our findings define PGA1 as a new AKR inhibitor and they offer a framework to develop compounds that could counteract cancer chemoresistance

[EN] The invention relates to the use of a series of compounds having a 2-cyclopentenone nucleus substituted by alkyl and alkenyl chains that are proven inhibitors of aldo-keto reductase (AKR) enzymes, specifically AKRl Bl and AKRl BlO. Consequently, the compounds ofthe invention can be used for the treatment ofpathological conditions or diseases involving said enzymes., [ES] La presente invención se refiere al uso de una serie de compuestos que poseen un núcleo de 2-cíclopentenona sustituido por cadenas alquílicas yalquénilicas que han demostrado ser inhibidores de enzimas aldo-keto reductasas (AKR), más particulannente de la AKRl Bl Y AKRl BlO. Por tanto, los compuestos de la presente invención son útiles para el tratamiento de enfennedades o condiciones patológicas en las que estas enzimas están implicadas.

Purpose of review: To discuss the avenues that proteomic techniques are opening for the study of the chemical basis and cellular mechanisms of immunological reactions to drugs. Recent findings: Technical developments in recent years are allowing a detailed characterization of drug–protein interactions. In addition, novel metabolic pathways for drug biotransformation are being uncovered and potential targets for protein haptenation are being proposed that may help in the understanding of these complex processes. Summary: Immunological reactions to drugs pose important clinical problems. Since early works exploring drug–protein interactions, there has been steady progress in this field. However, the mechanisms involved remain incompletely understood. The availability of proteomic techniques with high resolution and sensitivity presents a unique opportunity to tackle this subject from a broad perspective, integrating work in model systems and in patients. Chemical and metabolic characterization of immunological reactions to drugs may also help in the prevention, diagnosis and/or treatment of these processes

La presente invención se refiere al uso de una serie de compuestos que poseen un núcleo de 2-ciclopentenona sustituido por cadenas alquílicas y alquénilicas que han demostrado ser inhibidores de enzimas aldo-keto reductasas (AKR), más particularmente de la AKR181 Y AKR1 81 O. Por tanto, los compuestos de la presente invención son útiles para el tratamiento de enfermedades o condiciones patológicas en las que estas enzimas están implicadas.

El metabolismo oxidativo de los ácidos grasos poliinsaturados origina una gran cantidad de especies electrófilas, entre las que se encuentran las prostaglandinas ciclopentenonas (cyPG), como son la 15d-PGJ2 y la PGA1. Estos compuestos se caracterizan por ser mediadores lipídicos implicados en numerosos procesos celulares y se ha descrito que ejercen efectos antiproliferativos, antiinflamatorios y antivirales. Parte de estos efectos biológicos son debidos a su reactividad específica que les permite modificar covalentemente proteínas celulares mediante la adición de Michael. La identificación de las dianas proteicas de las cyPG resulta esencial en el estudio de los mecanismos de acción que expliquen la actividad biológica de estas PG. Los análogos biotinilados de las cyPG han sido ampliamente utilizados como herramientas para la identificación de las proteínas modificadas por cyPG. Este trabajo se ha centrado en la validación del uso del derivado biotinilado de la PGA1 para la identificación de las dianas biológicas de esta cyPG, así como en la confirmación de la modificación de determinadas proteínas celulares por la PGA1-B. Además hemos caracterizado la modificación de proteínas que componen los filamentos intermedios del citoesqueleto, como la vimentina y las laminas nucleares, por cyPG biotiniladas como la 15d-PGJ2-B y PGA1-B.

Lamin A and lamin C (A-type lamins, both encoded by the LMNA gene) are major components of the mammalian nuclear lamina, a complex proteinaceous structure that acts as a scaffold for protein complexes that regulate nuclear structure and function. Abnormal accumulation of farnesylated-progerin, a mutant form of prelamin A, plays a key role in the pathogenesis of the Hutchinson-Gilford progeria syndrome (HGPS), a devastating disorder that causes the death of affected children at an average age of 13.5 years, predominantly from premature atherosclerosis and myocardial infarction or stroke. Remarkably, progerin is also present in normal cells and appears to progressively accumulate during aging of non-HGPS cells. Therefore, understanding how this mutant form of lamin A provokes HGPS may shed significant insight into physiological aging. In this review, we discuss recent advances into the pathogenic mechanisms underlying HGPS, the main murine models of the disease, and the therapeutic strategies developed in cellular and animal models with the aim of reducing the accumulation of farnesylated-progerin, as well as their use in clinical trials of HGPS.

The methyl donor S-adenosylmethionine is synthesized in mammalian cytosol by three isoenzymes. Methionine adenosyltransferase II is ubiquitously expressed, whereas isoenzymes I (homotetramer) and III (homodimer) are considered the hepatic enzymes. In this work, we identified methionine adenosyltransferase I/III in most rat tissues both in the cytoplasm and the nucleus. Nuclear localization was the preferred distribution observed in extrahepatic tissues, where the protein colocalizes with nuclear matrix markers. A battery of mutants used in several cell lines to decipher the determinants involved in methionine adenosyltransferase subcellular localization demonstrated, by confocal microscopy and subcellular fractionation, the presence of two partially overlapping areas at the C-terminal end of the protein involved both in cytoplasmic retention and nuclear localization. Immunoprecipitation of coexpressed FLAG and EGFP fusions and gel filtration chromatography allowed detection of tetramers and monomers in nuclear fractions that also exhibited S-adenosylmethionine synthesis. Neither nuclear localization nor matrix binding required activity, as demonstrated with the inactive F251D mutant. Nuclear accumulation of the active enzyme only correlated with histone H3K27 trimethylation among the epigenetic modifications evaluated, therefore pointing to the necessity of methionine adenosyltransferase I/III to guarantee the supply of S-adenosylmethionine for specific methylations. However, nuclear monomers may exhibit additional roles.

Polymorphisms in drug-related enzymes and receptors are often strongly related to altered drug response and to the risk of developing drug intolerance. Aspirin, usually available as an over-the-counter drug, is one of the most used drugs worldwide and is a common cause of drug intolerance events. Aspirin undergoes polymorphic metabolism. Among the enzymes involved in aspirin biodisposition a major role is played by the enzymes UDP-glucuronosyltransferase UGT1A6, cytochrome P450 CYP2C9 and the xenobiotic/medium chain fatty acid:CoA ligase ACSM2, although other UGTs and ACSMs enzymes may significantly contribute to aspirin metabolism. UGT1A6, CYP2C9 and ACSM2 are polymorphic, as well as PTGS1 and PTGS2, the genes coding for the enzymes cyclo-oxygenases COX1 and COX2, respectively. The present review analyzes the importance of genetic variations in enzymes involved in the metabolism and in the effects of aspirin and common polymorphisms related to aspirin intolerance, and it raises hypotheses on genetic factors related to altered response to aspirin that require further investigation. Major polymorphisms related to aspirin biodisposition are rs2070959, rs1105879 and rs6759892 for the UGT1A6 gene, rs1133607 for the ACSM2 gene, and rs1799853, rs1057910, rs28371686, rs9332131 and rs28371685 for the CYP2C9 gene. Regarding aspirin effects, major PGTS1 targets are rs3842787 and rs5789 for European subjects, and rs3842789 and rs3842792 for African subjects. For the PTGS2 gene nonsynonymous SNPs are likely to be of low relevance because of the influence of transcriptional and posttranscriptional factors. Combined studies for the above mentioned polymorphisms and those corresponding to other genes related to aspirin intolerance will provide excellent tools to identify individuals with a high risk to develop intolerance to aspirin

Background Protein degradation is essential for cell homeostasis. Targeting of proteins for degradation is often achieved by specific protein sequences or posttranslational modifications such as ubiquitination. Methodology/Principal Findings By using biochemical and genetic tools we have monitored the localization and degradation of endogenous and chimeric proteins in live primary cells by confocal microscopy and ultra-structural analysis. Here we identify an eight amino acid sequence from the C-terminus of the short-lived GTPase RhoB that directs the rapid degradation of both RhoB and chimeric proteins bearing this sequence through a lysosomal pathway. Elucidation of the RhoB degradation pathway unveils a mechanism dependent on protein isoprenylation and palmitoylation that involves sorting of the protein into multivesicular bodies, mediated by the ESCRT machinery. Moreover, RhoB sorting is regulated by late endosome specific lipid dynamics and is altered in human genetic lipid traffic disease. Conclusions/Significance Our findings characterize a short-lived cytosolic protein that is degraded through a lysosomal pathway. In addition, we define a novel motif for protein sorting and rapid degradation, which allows controlling protein levels by means of clinically used drugs

The posttranslational modification of proteins by lipids is a key mechanism in the regulation of protein localization and function. Isoprenylation is a process critical for the membrane association of a plethora of signalling proteins with fundamental roles in cell biology, including G proteins and nuclear lamins. Isoprenylation is irreversible but is frequently associated to reversible posttranslational modifications, such as palmitoylation or phosphorylation, that act like switches that modulate the dynamics of protein-membrane or protein-protein interactions and target the modified proteins to specific membrane compartments. The severe phenotype of animals deficient in the enzymes involved in isoprenylation and postprenylation processing highlights the significance of these processes. Moreover, alterations in the genes involved in the maturation of isoprenylated proteins have been found at the basis of some severe human diseases, like choroideremia or the premature ageing progeria syndromes. Given their critical role in the transformation potential of the Ras oncogenes, isoprenylation and postprenylation processing are targets for the development of inhibitors with antitumoral activity. The recent generation of animal models genetically engineered to target the enzymes involved in isoprenylation and associated modifications has unveiled unpredicted aspects of these modifications. Moreover, these models are proving of crucial importance for the elucidation of the mechanisms of disease, and the identification and validation of therapeutic targets. This review attempts to summarize general aspects of the posttranslational modification of proteins by isoprenylation, paying special attention to the evidences obtained from the use of genetically engineered animals and the avenues that these models open.