Nitric oxide-induced carbonylation of Bcl-2, GAPDH and ANT precedes apoptotic events in insulin-secreting RINm5F cells.
Generation of high levels of nitric oxide (NO) following induction of NOS2 by interleukin-1 beta (IL-1beta) triggers beta cell apoptosis in insulin-secreting RINm5F cells. Mitochondrial and nuclear events such as downregulation of the antiapoptotic protein Bcl-2, activation of the pore responsible for the permeability transition (PT) and DNA fragmentation are involved in the process. We report in the present paper that exposure of insulin-producing RINm5F cells to NO donors and to IL-1beta leads to oxidative carbonylation of both Bcl-2 and the adenine nucleotide translocator (ANT) component of the mitochondrial PT pore. When the effect of endogenous generation of high concentrations of NO following exposure of cells to IL-1beta was studied, carbonylation of Bcl-2 preceded downregulation of the protein. Overexpression of Mn-SOD decreases substantially the extent of Bcl-2 carbonylation in SIN-1-exposed cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibition, carbonylation and translocation from cytoplasm to nucleus and DNA fragmentation were also induced by DETA/NO exposure. DETA/NO-induced carbonylation of Bcl-2 and ANT proteins takes place 6 h before apoptotic release of histone-associated DNA to cytoplasm. Time course studies also reveal a close parallel between GAPDH translocation to nucleus and carbonylation. Inhibitors of lipooxidation end products formation such as piridoxamine (PM) and aminoguanidine (AG) block NO-triggered carbonylation of Bcl-2, ANT and GAPDH, prevent NO-induced GAPDH enzyme inhibition and nuclear translocation and DNA fragmentation. Our results support the notion that the oxidative carbonylation of proteins plays a role in the control of NO-induced apoptosis.
Cahuana
Gladys M
GM
Laboratory of Biochemistry of the Immune System, Department of Medical Biochemistry and Molecular Biology, University of Sevilla, 41009 Seville, Spain.
Tejedo
Juan R
JR
Jiménez
Juan
J
Ramírez
Remedios
R
Sobrino
Francisco
F
Bedoya
Francisco J
FJ
D000227:Adenine Nucleotides / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D003671:DEET / Q000494:pharmacology; D016166:Free Radical Scavengers; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007375:Interleukin-1 / Q000494:pharmacology; D007700:Kinetics; D015227:Lipid Peroxidation / Q000187:drug effects; D008981:Molsidomine / Q000031:analogs & derivatives* / Q000494:pharmacology; D009569:Nitric Oxide / Q000494:pharmacology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D013482:Superoxide Dismutase / Q000378:metabolism
D000227:Adenine Nucleotides; D016166:Free Radical Scavengers; D007328:Insulin; D007375:Interleukin-1; D020030:Nitric Oxide Donors; D019253:Proto-Oncogene Proteins c-bcl-2; D003671:DEET; D009569:Nitric Oxide; C002385:linsidomine; D008981:Molsidomine; D013482:Superoxide Dismutase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases