Involvement of heme in the degradation of iron-regulatory protein 2.

Iron-regulatory proteins (IRPs) recognize and bind to specific RNA structures called iron-responsive elements. Mediation of these binding interactions by iron and iron-containing compounds regulates several post-transcriptional events relevant to iron metabolism. There are two known IRPs, IRP1 and IRP2, both of which can respond to iron fluxes in the cell. There is ample evidence that IRP1 is converted by iron to cytoplasmic aconitase in vivo. It has also been shown that, under certain conditions, a significant fraction of IRP1 is degraded in cells exposed to iron or heme. Studies have shown that the degradation of IRP1 that is induced by iron can be inhibited by either desferrioxamine mesylate (an iron chelator) or succinyl acetone (an inhibitor of heme synthesis), whereas the degradation induced by heme cannot. This suggests that heme rather than iron is responsible for this degradation. Several laboratories have shown that IRP2 is also degraded in cells treated with iron salts. We now show evidence suggesting that this IRP2 degradation may be mediated by heme. Thus, in experiments analogous to those used previously to study IRP1, we find that IRP2 is degraded in rabbit fibroblast cells exposed to heme or iron salts. However, as shown earlier with IRP1, both desferrioxamine mesylate and succinyl acetone will inhibit the degradation of IRP2 induced by iron but not that induced by heme.

Irreversible steps in the ferritin synthesis induction pathway.

The ability of cells to re-repress ferritin synthesis after removal of an inducing agent (iron or heme) was investigated. Re-repression was found to be a slow process, requiring approximately 4 (after iron removal) to 10 h (after heme removal) for completion. Desferrioxamine mesylate (Desferal) had only a slight effect on the rate of re-repression, whereas cycloheximide was strongly inhibitory, indicating that new protein synthesis is required for re-repression. Re-repression occurred at a slow but significant rate in the presence of both Desferal and cycloheximide. These results indicate that, in the absence of an iron chelator, the induction of ferritin synthesis is essentially irreversible. The kinetics of the previously reported covalent modification of IRE-binding protein (IRE-BP) were then examined, to see whether this phenomenon might account (at least in part) for the irreversibility of induction. It was found that the heme- or iron-dependent disappearance of 98-kDa IRE-BP occurred rapidly (within 1 h), and was equally rapidly reversed upon removal of heme after a 1-h exposure. By contrast, after a 4-h exposure to heme, little 98-kDa IRE-BP could be regenerated after heme removal. These results suggest that the slow, irreversible covalent modification of IRE-BP correlates closely over time with the induction of ferritin synthesis. The covalent modification of IRE-BP depends on cell growth rate, and is most readily detected in rapidly growing cells.

Inducible expression bovine papillomavirus shuttle vectors containing ferritin translational regulatory elements.

The combination of transcriptional and translational control elements in an inducible expression vector suitable for use in stably transformed cell lines was explored. To this end, ferritin translational control elements have been inserted downstream from a mouse metallothionein (mMT-I) transcriptional promoter (PmMT-I), and upstream from various reporter protein-encoding open reading frames (ORFs), all carried on a bovine papillomavirus shuttle vector. Protocols which stimulate transcription (with zinc) and translation (with iron) were developed to optimize the induction of reporter protein synthesis. It was found that insertion of an iron regulatory element between the PmMT-I and a reporter ORF bestowed a sixfold inducibility of reporter protein synthesis with iron and a 90-fold inducibility with iron plus zinc in a classical superinduction protocol. Surprisingly, inclusion of other rabbit ferritin light chain sequences (rFL), including the ORF, enhanced reporter inducibilities to over 15- and 500-fold, respectively. These additional rFL sequences not only increased inducibility but also (i) increased the half-life of the mRNA and (ii) strongly inhibited translation of an ORF located downstream from the 5' proximal ORF. The maximum levels of reporter proteins attained in transformed cells after prolonged induction represented from 1% to 7% of total cellular protein. These inducible expression vectors should prove useful for the production and study of cytotoxic proteins.

Enhanced degradation of the ferritin repressor protein during induction of ferritin messenger RNA translation.

Induction of ferritin synthesis in cultured cells by heme or iron is accompanied by degradation of the ferritin repressor protein (FRP). Intermediates in the degradative pathway apparently include FRP covalently linked in larger aggregates. The effect of iron on FRP degradation is enhanced by porphyrin precursors but is decreased by inhibitors of porphyrin synthesis, which implies that heme is an active agent. These results suggest that translational induction in this system may be caused by enhanced repressor degradation. While unique among translational regulatory systems, this process is common to a variety of other biosynthetic control mechanisms.