Posttranslational glutathiolation of aldose reductase (AKR1B1): a possible mechanism of protein recovery from S-nitrosylation.
Nitric oxide (NO) is an important regulator of the catalytic activity of aldose reductase (AR). It reacts with the active site cysteines of AR and this reaction results in the formation of several kinetically distinct forms of the protein. The catalytic activity of AR is increased in the ischemic heart and this increase in activity is associated with NO-dependent modification of AR. During reperfusion, the enzyme reverts back to its un-activated form. Although, AR activation has been linked to thiol oxidation, the mechanisms of de-activation remain unclear. Here we report that treatment of recombinant human AR (AKR1B1) by a non-thiol-based NO-donor (DEANO) results in activation and S-nitrosylation of the protein. The nitrosylated (ARSNO), but not the reduced (ARSH), protein reacted with reduced glutathione (GSH) and this reaction resulted in the formation of glutathiolated AR (ARSSG). The modification of AR by NO was site-specific at Cys-298 and was not affected by selective mutation of the neighboring residue, Cys-303 to an alanine. Incubation of the glutathiolated AR (ARSSG) with GSH resulted in the regeneration of the reduced form of the protein (ARSH). Treatment of nitrosylated AR (ARSNO) with ascorbic acid also led to the conversion of the protein to its reduced form. These observations suggest that intracellular reductants such as GSH and ascorbate could convert the nitrosylated form of AR to its basal or reduced state. In general, such reductive reactions might represent a common mechanism for denitrosylating proteins or an "off" switch in NO-mediated signaling pathways involving protein S-nitrosylation reactions.
Baba
Shahid Pervez
SP
Institute of Molecular Cardiology, University of Louisville, Louisville, KY 40202, United States.
Wetzelberger
Karin
K
Hoetker
Joseph David
JD
Bhatnagar
Aruni
A
D000449:Aldehyde Reductase / Q000737:chemistry / Q000378:metabolism*; D001205:Ascorbic Acid / Q000378:metabolism; D015153:Blotting, Western; D003545:Cysteine / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009603:Nitroso Compounds / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021241:Spectrometry, Mass, Electrospray Ionization
D020030:Nitric Oxide Donors; D009603:Nitroso Compounds; D000449:Aldehyde Reductase; D005978:Glutathione; D003545:Cysteine; D001205:Ascorbic Acid