Intracellular formation of ”undisruptable” dimers of inducible nitric oxide synthase

Abstract

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.

Regulation of inducible nitric oxide synthase by aggresome formation

“…A172 and A549 cells were stimulated by a cytokine mixture similar to that used for RT4 cells. RAW264.7 cells were stimulated by LPS and IFN-␥ (21,23). In all cell types examined, iNOS formed an aggresome at the perinuclear area (Fig.…”

“…Therefore, we sought to examine the subcellular localization of cytokine-induced iNOS. RT4 is a urinary bladder papilloma cell line that expresses iNOS upon cytokine stimulation (21)(22)(23). At varying times after stimulation by a mixture of IFN-␥ (100 units͞ml), IL-1␤ (0.5 ng͞ml), TNF-␣ (10 ng͞ml), and IL-6 (200 units͞ml), RT4 cells were fixed and immunostained by using a monoclonal anti-iNOS antibody.…”

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

“…Both wild-type iNOS and iNOS G456A were transiently transfected in HEK 293T cells. Wild-type iNOS expressed in HEK 293T cells is mostly dimeric by gel permeation chromatography (20). In contrast, iNOS G456A was inactive and could not form dimers (Fig.…”

“…As expected, Western analysis of the same samples showed gradual disappearance of iNOS undisruptable dimers (UD). iNOS UD have been shown to correlate with the ability of iNOS to form dimers (20). Unexpectedly, however, there was a gradual reduction in the amount of iNOS protein detected in the presence of the inhibitor over time.…”

“…2C), confirming the results obtained with cell lines and further indicating that the observed half-life of iNOS is not restricted to transformed cell lines. (18)(19)(20). We aimed to determine whether reducing the intracellular levels of H 4 B would affect iNOS half-life.…”