Intracellular Formation of "Undisruptable" Dimers of Inducible Nitric Oxide Synthase

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 100; no. 24; pp. 14263 - 14268
• Main Authors: Kolodziejski, Pawel J., Rashid, Mohammad B., Eissa, N. Tony
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 25.11.2003; National Acad Sciences
• Subjects: Animals; Biological Sciences; Biopterins - analogs & derivatives; Biopterins - metabolism; Bronchi - enzymology; Cell Line; Cell lines; Cells, Cultured; Complementary DNA; Cytokines; Cytokines - pharmacology; Dimerization; Dimers; Enzyme Stability; Epithelial cells; Escherichia coli - genetics; HEK293 cells; Hot Temperature; Humans; In Vitro Techniques; Intracellular Fluid - enzymology; Medical research; Mice; Monomers; Mutation; Nitric oxide; Nitric Oxide Synthase - chemistry; Nitric Oxide Synthase - genetics; Nitric Oxide Synthase - metabolism; Nitric Oxide Synthase Type II; Oxidation-Reduction; Pathogens; Protein Denaturation; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transfection; Zinc; Zinc - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2435290100

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.