RESUMEN
Methylthioadenosine phosphorylase (MTAP), a key enzyme in the adenine and methionine salvage pathways, catalyzes the hydrolysis of methylthioadenosine (MTA), a compound suggested to affect pivotal cellular processes in part through the regulation of protein methylation. MTAP is expressed in a wide range of cell types and tissues, and its deletion is common to cancer cells and in liver injury. The aim of this study was to investigate the proteome and methyl proteome alterations triggered by MTAP deficiency in liver cells to define novel regulatory mechanisms that may explain the pathogenic processes of liver diseases. iTRAQ analysis resulted in the identification of 216 differential proteins (p < 0.05) that suggest deregulation of cellular pathways as those mediated by ERK or NFκB. R-methyl proteome analysis led to the identification of 74 differentially methylated proteins between SK-Hep1 and SK-Hep1+ cells, including 116 new methylation sites. Restoring normal MTA levels in SK-Hep1+ cells parallels the specific methylation of 56 proteins, including KRT8, TGF, and CTF8A, which provides a novel regulatory mechanism of their activity with potential implications in carcinogenesis. Inhibition of RNA-binding proteins methylation is especially relevant upon accumulation of MTA. As an example, methylation of quaking protein in Arg(242) and Arg(256) in SK-Hep1+ cells may play a pivotal role in the regulation of its activity as indicated by the up-regulation of its target protein p27(kip1) The phenotype associated with a MTAP deficiency was further verified in the liver of MTAP± mice. Our data support that MTAP deficiency leads to MTA accumulation and deregulation of central cellular pathways, increasing proliferation and decreasing the susceptibility to chemotherapeutic drugs, which involves differential protein methylation. Data are available via ProteomeXchange with identifier PXD002957 (http://www.ebi.ac.uk/pride/archive/projects/PXD002957).
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Desoxiadenosinas/metabolismo , Hepatocitos/citología , Proteoma/metabolismo , Purina-Nucleósido Fosforilasa/deficiencia , Purina-Nucleósido Fosforilasa/genética , Tionucleósidos/metabolismo , Animales , Proliferación Celular , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/enzimología , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Modelos Animales de Enfermedad , Femenino , Fluorocarburos/efectos adversos , Regulación de la Expresión Génica , Células Hep G2 , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Masculino , Metilación , Ratones , Transducción de SeñalRESUMEN
Liver diseases are the fifth cause of mortality in Western countries, and as opposed to other major causes of mortality, their incidence is increasing. Understanding the molecular background contributing to the progression of liver ailments will surely open new perspectives for the better management of patients. The aim of this study is to elucidate mechanisms underlying the progression of liver injury associated with deficient prohibitin 1, an essential protein to maintain mitochondrial homeostasis and gene expression. PHB1+/- mice developed a more severe steatohepatitis than WT littermates when exposed to a choline and methionine deficient diet. The increased sensitivity was mediated by mitochondrial dysfunction and metabolic impairment in PHB1+/- livers, including inactivation of AMP kinase, measured under a non-restricted diet. Moreover, pro-inflammatory challenges induced higher mortality and liver injury in PHB+/- mice. The increased proliferative capacity of PHB+/- splenocytes, resulting from constitutive defects in central molecular pathways as stated by deregulation of GSK3ß, Erk, Akt or SHP-1, and the concomitant overproduction of pro-inflammatory mediators in Phb1 deficient mice, might account for these effects. In light of these results it might be concluded that Phb1 deficiency is a potential driver of chronic liver diseases by inducing hepatocyte damage and inflammation.
Asunto(s)
Hígado Graso/etiología , Inflamación/complicaciones , Proteínas Represoras/deficiencia , Animales , Deficiencia de Colina/complicaciones , Masculino , Metionina/deficiencia , Ratones , Prohibitinas , TranscriptomaRESUMEN
Methionine adenosyltransferase I/III (MATI/III) synthesizes S-adenosylmethionine (SAM) in quiescent hepatocytes. Its activity is compromised in most liver diseases including liver cancer. Since SAM is a driver of hepatocytes fate we have studied the effect of re-expressing MAT1A in hepatoma Huh7 cells using proteomics. MAT1A expression leads to SAM levels close to those found in quiescent hepatocytes and induced apoptosis. Normalization of intracellular SAM induced alteration of 128 proteins identified by 2D-DIGE and gel-free methods, accounting for deregulation of central cellular functions including apoptosis, cell proliferation and survival. Human Dead-box protein 3 (DDX3X), a RNA helicase regulating RNA splicing, export, transcription and translation was down-regulated upon MAT1A expression. Our data support the regulation of DDX3X levels by SAM in a concentration and time dependent manner. Consistently, DDX3X arises as a primary target of SAM and a principal intermediate of its antitumoral effect. Based on the parallelism between SAM and DDX3X along the progression of liver disorders, and the results reported here, it is tempting to suggest that reduced SAM in the liver may lead to DDX3X up-regulation contributing to the pathogenic process and that replenishment of SAM might prove to have beneficial effects, at least in part by reducing DDX3X levels. This article is part of a Special Issue entitled: Proteomics: The clinical link.