Nitrogen monoxide activates iron regulatory protein 1 RNA-binding activityby two possible mechanisms: Effect on the [4Fe-4S] cluster and iron mobilization from cells
Sl. Wardrop et al., Nitrogen monoxide activates iron regulatory protein 1 RNA-binding activityby two possible mechanisms: Effect on the [4Fe-4S] cluster and iron mobilization from cells, BIOCHEM, 39(10), 2000, pp. 2748-2758
The iron-regulatory protein 1 (IRP1) regulates the expression of several mo
lecules involved in iron (Fe) metabolism by reversibly binding to iron-resp
onsive elements (IREs) in the untranslated regions (UTR) of particular mRNA
transcripts. Several studies have indicated chat nitrogen monoxide (NO) ma
y influence IRP1 RNA-binding activity by a direct effect on the [4Fe-4S] cl
uster of the protein. It has also been suggested that NO may act indirectly
on IRP1 by affecting the intracellular Fe pools that regulate the function
of this protein [Pantopoulous et al. (1996) Mol. Cell. Biol. 16, 3781-3788
]. There is also the possibility that NO may S-nitrosate sulfhydryl groups
that are crucial for mRNA binding and decrease IRP1 activity by this mechan
ism. We have examined the effect of a variety of NO donors [e.g., S-nitroso
-N-acetylpenicillamine (SNAP), spermine-NONOate (SperNO), and S-nitrosoglut
athione (GSNO)] on IRP1 RNA-binding activity in bath LMTK- fibroblast lysat
es and whole cells. In cell lysates, the effects of NO at increasing RNA-bi
nding activity were only observed when cells were made Fe-replete. Under th
ese circumstances, IRP1 contains an [4Fe-4S] cluster that was susceptible t
o NO. In contrast, when lysates were prepared from cells treated with the F
e chelator desferrioxamine (DFO), NO had no effect on the RNA-binding activ
ity of IRP1. The lack of effect of NO under these conditions was probably b
ecause this protein does not have an [4Fe-4S] cluster. In contrast to the N
O generators above, sodium nitroprusside (SNP) decreased IRP1 RNA binding w
hen cells were incubated with this compound. However, SNP had no effect on
IRP1 RNA-binding activity in lysates, suggesting that the decrease after in
cubation of cells with SNP was not due to S-nitrosation of critical sulfhyd
ryl groups. Apart from the direct effect of NO on IRP1 in Fe-replete cells,
we have shown that NO generated by SNAP, SperNO, and GSNO could also mobil
ize Fe from cells. When NO generation was induced in RAW 264.7 macrophages,
an increase in IRP1 RNA-binding activity occurred but there was only a sma
ll increase in Fe release. Our results suggest that NO could activate IRP1
RNA-binding by two possible mechanisms: (I) its direct effect on the [4Fe-4
S] cluster and (2) mobilization of Fe-59 from cells resulting in Fe depleti
on, which then increases IRP1 RNA-binding activity.