Aconitase iron-response element-binding protein

Kennedy, M.C., Mende-Mueller, L., Blondin, G.A., and Beinert, H. 1992. Purification and characterization of cytosolic aconitase from beef hver and its relationship to the iron-responsive element binding protein. Proceedings of the National Academy of Sciences of the USA 89 11730-11734. 

Aconitase exists as both mitochondrial and cytosolic isoenzyme forms of similar structure. However, the cytosolic isoenzyme has a second function. In its apoenzyme form, which lacks the iron-sulfur cluster, it acts as the much-studied iron regulatory factor, or iron-responsive element binding protein (IRE-BP). This protein binds to a specific stem-loop structure in the messenger RNA for proteins involved in iron transport and storage (Chapter 28).86/9°... 

Hail DJ, Rouaoult TA, Tang CK, Chin J, Harford JB, Klausner RD (1992) Reciprocal control of RNA-binding and aconitase activity in the regulation of the iron-responsive element binding protein role of the iron-sulfur cluster. Proc Natl Acad Sci USA 89 7536-7540... 

Guo B, Yu Y, Leibold EA (1994) Iron regulates cytoplasmic levels of a novel iron-responsive element-binding protein without aconitase activity. J Biol Chem 269 24252-24260... 

Basilion JP, Rouault TA, Massinople CM, Klausner RD, Burgess WH. The iron-responsive element-binding protein localization of the RNA-binding site to the aconitase active-site cleft. Proc Natl Acad Sci USA 1994 91 574-578. 

When prepared in the presence of oxygen, IRP-1 contains both the [3Fe-4S] and the [4Fe-4S] forms (2000 to 5000 nmol of c/s-aconitate/min x mg protein) (Soum et al. 2000). When exposed to nitric oxide generated by 3-morpholino-sydnonimine (SIN-1) in the presence of superoxide dismutase for 2 h at 37 °C, IRP-1 lost about 80% of its aconitase activity and its iron responsive element-binding capacity was increased 3- to 5-fold when further ex-... 

Iron regulatory proteins (IRPs) regulate the cellular iron level in mammalian cells. IRPs are known as cytosol mRNA binding proteins which control the stability or the translation rate of mRNAs of iron metabolism-related proteins such as TfR, ferritin, and 5-aminolevulinic acid synthetase in response to the availability of cellular iron [19-21] after uptake [5]. The regulatory mechanism involves the interaction between the iron-responsive element (IRE) in the 3 or 5 untranslated regions of the transcripts and cytosolic IRPs (IRP-1 and -2). IRP-1 is an iron-sulfur (Fe-S) protein with aconitase activity containing a cubane 4Fe-4S cluster. When Fe is replete, IRP-1 prevails in a 4Fe-4S form as a holo-form and is an active cytoplasmic aconitase. As shown in Fig. 3, when Fe is deplete, it readily loses one Fe from the fourth labile Fe in the Fe-S cluster to become a 3Fe-4S cluster and in this state has little enzymatic activity [22, 23]. 

Figure 12-4 Differential binding of IRPl and IRP2 to natural IREs (Iron Responsive Elements). P-5 -RNAs (n = 29-30 nucleotides) were melted and annealed before mixing with recombinant IRP proteins (The proteins were kindly provided by E. A. Leibold, University of Utah, and W. E. Walden, University of Illinois). RNA-protein complexes were separated from RNA by electrophoresis in non-denatuiing polyacrylamide gels [20]. Per contains an internal loop/bulge (Figure 12-3), and TfR, eALAS, and m-aconitase IREs have C-bulges. Per mutation AU6 converts the Per internal loop/bulge to a C-bulge. Per ferritin TfR transferrin receptor eALAS erythroid amino-levulinate synthase and m-aconitase. No IRE/IRP complex was detectable.

Outline the role of RNA secondary structure in the regulation of iron metabolism in animals. Describe the roles of transferrin, transferrin receptor, ferritin, the iron-response element, and IRE-binding protein. Relate the IRE-binding protein to aconitase and iron sensing. 

The iron regulatory proteins, IRP-1 and IRP-2, bind at high affinity (Kd 40-100 pM) to highly conserved RNA stemloops, the Iron-responsive Elements (IREs) at the 5 cap sites of the L- and H-ferritin niRNAs [55-57] (Figure 2). The IRE/IRP interaction impedes the access of the small 40S ribosome subunit to the 5 end of the ferritin niRNAs and thereby suppresses L- and H-ferritin mRNA translation [58-60]. The potency of the IRE to regulate translation is position dependent [61]. Iron influx relieves repression of ferritin translation by removing IRP-1 from the ferritin IREs IRP-1 is simultaneously interconverted to a cytoplasmic cis-aconitase with an iron sulfur cluster, and IRP-2 is degraded by... 

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