Abstract
Mammals incorporate a major proportion of absorbed iron as heme, which is catabolized by the heme oxygenase 1 (HO1)-NADPH-cytochrome P450 reductase (CPR) complex into biliverdin, carbon monoxide, and ferrous iron. Moreover, intestinal iron is incorporated as ferrous iron, which is transported via the iron importer, divalent metal transporter 1 (DMT1). Recently, we demonstrated that the iron chaperone poly(rC)-binding protein 2 (PCBP2) can directly receive ferrous iron from DMT1 or transfer iron to the iron exporter, ferroportin 1. To promote intracellular iron flux, an iron chaperone may be essential for receiving iron generated by heme catabolism, but this hypothesis is untested so far. Herein, we demonstrate that HO1 binds to PCBP2, but not to other PCBP family members, namely PCBP1, PCBP3, or PCBP4. Interestingly, HO1 formed a complex with either CPR or PCBP2, and it was demonstrated that PCBP2 competes with CPR for HO1 binding. Using PCBP2-deletion mutants, we demonstrated that the PCBP2 K homology 3 domain is important for the HO1/PCBP2 interaction. In heme-loaded cells, heme prompted HO1-CPR complex formation and decreased the HO1/PCBP2 interaction. Furthermore, in vitro reconstitution experiments with purified recombinant proteins indicated that HO1 could bind to PCBP2 in the presence of heme, whereas loading of PCBP2 with ferrous iron caused PCBP2 to lose its affinity for HO1. These results indicate that ferrous iron released from heme can be bound by PCBP2 and suggest a model for an integrated heme catabolism and iron transport metabolon.
Highlights
Mammals incorporate a major proportion of absorbed iron as heme, which is catabolized by the heme oxygenase 1 (HO1)– NADPH-cytochrome P450 reductase (CPR) complex into biliverdin, carbon monoxide, and ferrous iron
Considering the intricate interactions of poly(rC)-binding protein 2 (PCBP2) with divalent metal transporter 1 (DMT1) and ferroportin 1 (FPN1) as an iron chaperone [37, 39], it was hypothesized that PCBP2 could act to secure the flux of iron from the key enzyme involved in heme catabolism, HO1
Co-immunoprecipitation of these cellular lysates using an anti-green fluorescent protein (GFP) Ab (Fig. 1A, panel ii, left panel) demonstrated that a band for HO1-GFP was identified at ϳ60 kDa, whereas HO2GFP was at ϳ65 kDa, and GFP alone was at ϳ28 kDa, as expected [48]
Summary
Considering the intricate interactions of PCBP2 with DMT1 and FPN1 as an iron chaperone [37, 39], it was hypothesized that PCBP2 could act to secure the flux of iron from the key enzyme involved in heme catabolism, HO1. The relative expression ratio of these proteins in each fraction is demonstrated in Fig. 7B relative to HO1-GFP, CPR, and PCBP2 In these studies, P-cadherin, which is mainly localized at the plasma membrane [52], was strongly detected in fractions 6 –14, with its distribution being almost unchanged after incubation of cells with heme (Fig. 7B). In contrast to PCBP2, PCBP1, PCBP3, and PCBP4 were mainly detected in fractions 3– 6 and did not alter their distribution after incubation with heme (Fig. 7B) These studies are consistent with the fractionation experiments, panel i, where the PCBPs were shown to be cytosolic proteins, with PCBP2 demonstrating greater microsomal enrichment relative to the other PCBP family members. These results demonstrate that the formation of both the HO1–CPR complex and HO1–PCBP2 complex are functioning as a metabolon during heme degradation and that PCBP2 serves as an iron chaperone, binding the iron as it is released by heme catabolism
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