Gonzalez, R. (Raúl)
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- Altered protein expression and protein nitration pattern during d-galactosamine-induced cell death in human hepatocytes: a proteomic analysis(John Wiley and Sons, 2005) Rodriguez-Ariza, A. (Antonio); Corrales, F.J. (Fernando José); Muntane, J. (Jordi); Lopez-Sanchez, L.M. (Laura M.); Bernardos, A. (Angel); Gonzalez, R. (Raúl); Lopez-Casas, P. (Pedro)BACKGROUND/AIMS: Hepatic injury by d-galactosamine (d-GalN) is a suitable experimental model of hepatocellular injury. The induction of oxidative and nitrosative stress participates during d-GalN-induced cell death in cultured rat hepatocytes. This study aimed to identify protein expression changes during the induction of apoptosis and necrosis by d-GalN in cultured human hepatocytes. METHODS: A proteomic approach was used to identify the proteins involved and those altered by tyrosine nitration. A high dose of d-GalN (40 mM) was used to induce apoptosis and necrosis in primary culture of human hepatocytes. Cellular lysates prepared at different times after addition of d-GalN were separated by two-dimensional electrophoresis. Gel spots with an altered expression and those matching nitrotyrosine-immunopositive proteins were excised and analyzed by mass spectrometry. RESULTS: d-GalN treatment upregulated microsomal cytochrome b5, fatty acid binding protein and manganese superoxide dismutase, and enhanced annexin degradation. d-GalN increased tyrosine nitration of four cytosolic (Hsc70, Hsp70, annexin A4 and carbonyl reductase) and three mitochondrial (glycine amidinotransferase, ATP synthase beta chain, and thiosulfate sulfurtransferase) proteins in human hepatocytes. CONCLUSIONS: The results provide evidences that oxidative stress and nitric oxide-derived reactive oxygen intermediates induce specific alterations in protein expression that may be critical for the induction of apoptosis and necrosis by d-GalN in cultured human hepatocytes.
- Alteration of S-nitrosothiol homeostasis and targets for protein S-nitrosation in human hepatocytes(Wiley-VCH Verlag Berlin, 2008) Briceño, J. (Javier); Rodriguez-Ariza, A. (Antonio); Corrales, F.J. (Fernando José); Ranchal, I. (Isidora); Muntane, J. (Jordi); Ferrin, G. (Gustavo); Lopez-Sanchez, L.M. (Laura M.); Gonzalez, R. (Raúl); Hidalgo, A.B. (Ana B.); Muñoz-Castañeda, J.R. (Juan R.); Gomez, M.A. (Miguel A.); Lopez-Cillero, P. (Pedro); Mata, M. (Manuel) de laThe liver is one organ clearly influenced by nitric oxide (NO), and acute and chronic exposure to this substance has been associated with distinct patterns of liver disease. Disruption or deregulation of S-nitrosothiol (SNO) signalling leads to impairment of cellular function and disease, and this study was aimed to identify potential targets for protein S-nitrosation during alteration of SNO homeostasis in human hepatocytes. Cells were treated with S-nitroso-L-cysteine (CSNO), an effective physiological nitrosothiol for delivering NO bioactivity to cells. Treatment with CSNO augmented the levels of S-nitrosoproteins detected both by chemiluminescence and the biotin switch method. CSNO treatment also increased S-nitrosoglutathione reductase (GSNOR) activity that returned SNO content to basal levels. This increased enzymatic activity was related to augmented levels of ADH-5 mRNA, the gene encoding for GSNOR in humans. In addition, the treatment with the SNO also increased cell death. Twenty S-nitrosoproteins were identified in CSNO-treated hepatocytes, including mitochondrial aldehyde dehydrogenase, protein disulphide isomerase, Hsp60, GRP75 and Raf kinase inhibitor protein. The identification in the S-nitrosatable proteome of proteins involved in metabolism, maintenance of cellular homeostasis and signalling points to the relevance of protein S-nitrosation to the physiology and pathophysiology of human hepatocytes.