Redox regulation of neutral sphingomyelinase-1 activity in HEK293 cells through a GSH-dependent mechanism.
Phospholipases are essential enzymes in cellular signalling processes such as cellular differentiation, proliferation and apoptosis. Based on its high degree of homology with sequences of prokaryote SMases, a type of Mg(2+)-dependent PLC (nSMase-1) was recently discovered which displayed strong redox dependence for activity in vitro [F. Rodrigues-Lima, A.C. Fensome, M. Josephs, J. Evans, R.J. Veldman, M. Katan (2000), J. Biol. Chem. 275 (36) 28316-28325]. The aim of this work was to test the hypothesis that glutathione could be a natural regulator of nSMase-1 activity ex vivo. We studied how altering glutathione levels and redox ratio modulate nSMase-1 activity in a HEK293 cell line that ectopically overexpressed the nSMase-1 gene. Diminishing total glutathione with BSO without altering significantly the GSH/GSSG ratio did not affect nSMase-1 activity. Treatment of cells with diamide produced a transient decrease of total glutathione and a sharp, but also transient, decrease of the GSH/GSSG ratio. Under these conditions, nSMase-1 activity was temporarily activated and then returned to normal levels. Simultaneous treatment with BSO and diamide that resulted in permanent decreases of total glutathione and GSH/GSSG redox ratio produced a sustained activation of nSMase-1 activity. Taken together, these data indicate that altering the GSH/GSSG ratio by increasing GSSG or decreasing GSH levels, but not the total concentration of glutathione, modulates nSMase-1 activity. Our findings are the first evidence supporting the ex vivo regulation of nSMase-1 through a redox glutathione-dependent mechanism.
Martín
Sergio F
SF
Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Ciencias, Universidad de Córdoba, 14071 Córdoba, Spain. magosergio@yahoo.com
Sawai
Hirofumi
H
Villalba
José M
JM
Hannun
Yusuf A
YA
D002460:Cell Line; D004789:Enzyme Activation; D005246:Feedback / Q000502:physiology; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D010084:Oxidation-Reduction; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*
D013108:Sphingomyelin Phosphodiesterase; C519633:SMPD2 protein, human; D005978:Glutathione