Intracellular formation of "undisruptable" dimers of inducible nitric oxide synthase.
Overproduction of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in the pathogenesis of many diseases. iNOS is active only as a homodimer. Dimerization of iNOS represents a potentially critical target for therapeutic intervention. In this study, we show that intracellular iNOS forms dimers that are "undisruptable" by boiling, denaturants, or reducing agents. Undisruptable (UD) dimers are clearly distinguishable from the easily dissociated dimers formed by iNOS in vitro. UD dimers do not form in Escherichia coli-expressed iNOS and could not be assembled in vitro, which suggests that an in vivo cellular process is required for their formation. iNOS UD dimers are not affected by intracellular depletion of H4B. However, the mutation of Cys-115 (critical for zinc binding) greatly affects the formation of UD dimers. This study reveals insight into the mechanisms of in vivo iNOS dimer formation. UD dimers represent a class of iNOS dimers that had not been suspected. This unanticipated finding revises our understanding of the mechanisms of iNOS dimerization and lays the groundwork for future studies aimed at modulating iNOS activity in vivo.
Kolodziejski
Pawel J
PJ
Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA.
Rashid
Mohammad B
MB
Eissa
N Tony
NT
D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D001980:Bronchi / Q000201:enzymology; D002460:Cell Line; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology; D019281:Dimerization; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D006801:Humans; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000201:enzymology; D009154:Mutation; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection; D015032:Zinc / Q000378:metabolism
D016207:Cytokines; D011994:Recombinant Proteins; D001708:Biopterins; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C003402:sapropterin; D015032:Zinc