Pawel Kolodziejski

Regulation of inducible nitric oxide synthase by rapid cellular turnover and cotranslational down-regulation by dimerization inhibitors

Overproduction of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of many disorders. iNOS is notably distinguished from constitutive NOSs by its production of large amounts of NO for a prolonged period; hence, it was termed the high-output NOS. Understanding how cells regulate iNOS is a prerequisite for strategies aimed at modulating NO synthesis. iNOS is thought to be regulated primarily at the transcriptional level in response to cytokines and inflammatory mediators. In this study, we report a posttranslational regulatory mechanism for control of iNOS expression through a rapid cellular rate of turnover. Unexpectedly, iNOS cellular half-life was found to be relatively short. In primary bronchial epithelial cells, iNOS half-life was 1.6 ± 0.3 h. A similar half-life was found for iNOS in several cell lines. This fast rate of turnover is in sharp contrast to that reported for the constitutive NOS isoforms. iNOS half-life was not affected by intracellular depletion of tetrahydrobiopterin, a critical cofactor required for iNOS activity. Further, iNOS monomers and dimers had a similar half-life. Importantly, we discovered a previously unrecognized cotranslational down-regulation mechanism by which the newly discovered pyrimidineimidazole-based allosteric dimerization inhibitors of iNOS lead to reduced iNOS expression. This study provides insights into the cellular posttranslational mechanisms of iNOS and has important implications for design of selective iNOS inhibitors and their use in therapeutic strategies.

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.