Sanchez-Quiles, V. (Virginia)
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- Regulation of stathmin phosphorylation in mouse liver progenitor-29 cells during proteasome inhibition(Wiley-VCH Verlag Berlin, 2009) Mora, M.I. (María I.); Corrales, F.J. (Fernando José); Sanchez-Quiles, V. (Virginia); Fernandez-Irigoyen, J. (Joaquín); Prieto, J. (Jesús); Muñoz, J. (Javier); Santamaria, E. (Enrique)Proteasome inhibitors are potential therapeutic agents in the treatment of hepatocarcinoma and other liver diseases. The analysis of alternative protein phosphorylation states might contribute to elucidate the underlying mechanisms of proteasome inhibitor-induced apoptosis. We have investigated the response of mouse liver progenitor-29 (MLP-29) cells to MG132 using a combination of phosphoprotein affinity chromatography, DIGE, and nano LC-MS/MS. Thirteen unique deregulated phosphoproteins involved in chaperone activity, stress response, mRNA processing and cell cycle control were unambiguously identified. Alterations in NDRG1 and stathmin suggest new mechanisms associated to proteasome inhibitor-induced apoptosis in MLP-29 cells. Particularly, a transient modification of the phosphorylation state of Ser(16), Ser(25) and Ser(38), which are involved in the regulation of stathmin activity, was detected in three distinct isoforms upon proteasome inhibition. The parallel deregulation of calcium/calmodulin-activated protein kinase II, extracellular regulated kinase-1/2 and cyclin-dependent kinase-2, might explain the modified phosphorylation pattern of stathmin. Interestingly, stathmin phosphorylation profile was also modified in response to epoxomicin treatment, a more specific proteasome inhibitor. In summary, we report here data supporting that regulation of NDRG1 and stathmin by phosphorylation at specific Ser/Thr residues may participate in the cellular response induced by proteasome inhibitors.
- Prohibitin deficiency blocks proliferation and induces apoptosis in human hepatoma cells: molecular mechanisms and functional implications(Wiley-VCH Verlag Berlin, 2010) Corrales, F.J. (Fernando José); Sanchez-Quiles, V. (Virginia); Prieto, J. (Jesús); Sesma, L. (Laura); Segura, V. (Víctor); Santamaria, E. (Enrique)Prohibitin is a multifunctional protein participating in a plethora of essential cellular functions, such as cell signaling, apoptosis, survival and proliferation. In the liver, deficient prohibitin activity participates in the progression of non-alcoholic steatohepatitis and obesity, according to mechanisms that still must be elucidated. In this study, we have used a combination of transcriptomics and proteomics technologies to investigate the response of human hepatoma PLC/PRF/5 cells to prohibitin silencing to define in detail the biological function of hepatic Phb1 and to elucidate potential prohibitin-dependent mechanisms participating in the maintenance of the transformed phenotype. Abrogation of prohibitin reduced proliferation and induced apoptosis in human hepatoma cells in a mechanism dependent on NF kappaB signaling. Moreover, down-regulation of ERp29 together with down-regulation of Erlin 2 suggests ER stress. In agreement, increased C/EBP homologous protein levels, poly-ADP ribose polymerase cleavage and activation of caspase 12 and downstream caspase 7 evidenced ER stress-induced apoptosis. Down-regulation of proteasome activator complex subunit 2 and stathmin as well as accumulation of ubiquitinated proteins suggest interplay between ER stress and proteasome malfunction. Taken together, our results provide evidences for prohibitin having a central role in the maintenance of the transformed and invasive phenotype of human hepatoma cells and may further support previous studies suggesting prohibitin as a potential clinical target.
- Redox regulation of methylthioadenosine phosphorylase in liver cells: molecular mechanism and functional implications(Portland Press, 2008) Latasa, M.U. (María Ujué); Corrales, F.J. (Fernando José); Sanchez-del-Pino, M.M. (Manuel M.); Avila, M.A. (Matías Antonio); Sanchez-Quiles, V. (Virginia); Fernandez-Irigoyen, J. (Joaquín); Prieto, J. (Jesús); Muñoz, J. (Javier); Santamaria, M. (Mónica); Santamaria, E. (Enrique); Valero, M.L. (María L.)MTAP (5'-methylthioadenosine phosphorylase) catalyses the reversible phosphorolytic cleavage of methylthioadenosine leading to the production of methylthioribose-1-phosphate and adenine. Deficient MTAP activity has been correlated with human diseases including cirrhosis and hepatocellular carcinoma. In the present study we have investigated the regulation of MTAP by ROS (reactive oxygen species). The results of the present study support the inactivation of MTAP in the liver of bacterial LPS (lipopolysaccharide)-challenged mice as well as in HepG2 cells after exposure to t-butyl hydroperoxide. Reversible inactivation of purified MTAP by hydrogen peroxide results from a reduction of V(max) and involves the specific oxidation of Cys(136) and Cys(223) thiols to sulfenic acid that may be further stabilized to sulfenyl amide intermediates. Additionally, we found that Cys(145) and Cys(211) were disulfide bonded upon hydrogen peroxide exposure. However, this modification is not relevant to the mediation of the loss of MTAP activity as assessed by site-directed mutagenesis. Regulation of MTAP by ROS might participate in the redox regulation of the methionine catabolic pathway in the liver. Reduced MTA (5'-deoxy-5'-methylthioadenosine)-degrading activity may compensate for the deficient production of the precursor S-adenosylmethionine, allowing maintenance of intracellular MTA levels that may be critical to ensure cellular adaptation to physiopathological conditions such as inflammation.
- HSV-1 Cgal+ infection promotes quaking RNA binding protein production and induces nuclear-cytoplasmic shuttling of quaking I-5 isoform in human hepatoma cells(American Society for Biochemistry and Molecular Biology, 2011) Greco, A. (Anna); Mora, M.I. (María I.); Corrales, F.J. (Fernando José); Epstein, A.L. (Alberto L.); Dayon, L. (Loïc); Sanchez-Quiles, V. (Virginia); Foschini, M.G. (María Giovanna); Prieto, J. (Jesús); Sánchez, J.C. (Jean-Charles); Segura, V. (Víctor); Santamaria, E. (Enrique)Herpesvirus type 1 (HSV-1) based oncolytic vectors arise as a promising therapeutic alternative for neoplastic diseases including hepatocellular carcinoma. However, the mechanisms mediating the host cell response to such treatments are not completely known. It is well established that HSV-1 infection induces functional and structural alterations in the nucleus of the host cell. In the present work, we have used gel-based and shotgun proteomic strategies to elucidate the signaling pathways impaired in the nucleus of human hepatoma cells (Huh7) upon HSV-1 Cgal(+) infection. Both approaches allowed the identification of differential proteins suggesting impairment of cell functions involved in many aspects of host-virus interaction such as transcription regulation, mRNA processing, and mRNA splicing. Based on our proteomic data and additional functional studies, cellular protein quaking content (QKI) increases 4 hours postinfection (hpi), when viral immediate-early genes such as ICP4 and ICP27 could be also detected. Depletion of QKI expression by small interfering RNA results in reduction of viral immediate-early protein levels, subsequent decrease in early and late viral protein content, and a reduction in the viral yield indicating that QKI directly interferes with viral replication. In particular, HSV-1 Cgal(+) induces a transient increase in quaking I-5 isoform (QKI-5) levels, in parallel with an enhancement of p27(Kip1) protein content. Moreover, immunofluorescence microscopy showed an early nuclear redistribution of QKI-5, shuttling from the nucleus to the cytosol and colocalizing with nectin-1 in cell to cell contact regions at 16-24 hpi. This evidence sheds new light on mechanisms mediating hepatoma cell response to HSV-1 vectors highlighting QKI as a central molecular mediator.