Abstract
Heme biosynthesis occurs through a series of reactions that take place within the cytoplasm and mitochondria, so intermediates need to move across these cellular compartments. However, the specific membrane transport mechanisms involved in the process are not yet identified. The ATP-binding cassette protein ABCB10 is essential for normal heme production, as knocking down this transporter in mice is embryonically lethal and accompanied by severe anemia plus oxidative damage. The role of ABCB10 is unknown, but given its location in the inner mitochondrial membrane, it has been proposed as a candidate to export either an early heme precursor or heme. Alternatively, ABCB10 might transport a molecule important for protection against oxidative damage. To help discern between these possibilities, we decided to study the effect of heme analogs, precursors, and antioxidant peptides on purified human ABCB10. Since substrate binding increases the ATP hydrolysis rate of ABC transporters, we have determined the ability of these molecules to activate purified ABCB10 reconstituted in lipid nanodiscs using ATPase measurements. Under our experimental conditions, we found that the only heme analog increasing ABCB10 ATPase activity was Zinc-mesoporphyrin. This activation of almost seventy percent was specific for ABCB10, as the ATPase activity of a negative control bacterial ABC transporter was not affected. The activation was also observed in cysteine-less ABCB10, suggesting that Zinc-mesoporphyrin's effect did not require binding to typical heme regulatory motifs. Furthermore, our data indicate that ABCB10 was not directly activated by neither the early heme precursor delta-aminolevulinic acid nor glutathione, downsizing their relevance as putative substrates for this transporter. Although additional studies are needed to determine the physiological substrate of ABCB10, our findings reveal Zinc-mesoporphyrin as the first tool compound to directly modulate ABCB10 activity and raise the possibility that some actions of Zinc-mesoporphyrin in cellular and animal studies could be mediated by ABCB10.
Highlights
Given that the basal ATPase activity of ATP-binding cassette (ABC) transporters is increased by the presence of substrate, we decided to explore the effect of putative substrates on the activity of ABCB10
This ZnMP-induced activation occurred in the low micromolar range (Km = 0.9 μM; Fig 2C) and was specific for ABCB10 since under the same experimental conditions the activity of the negative control bacterial ABC transporter MsbA was unaltered by the presence of ZnMP (Fig 2C, inset)
On another hand, when ferric Fe3+ ion was coordinated to the mesoporphyrin IX dihydrochloride (MP) ring (FeMP), the activity of ABCB10 was not affected, suggesting that the presence of a coordinated divalent cation is needed for activation
Summary
The ATP-binding cassette (ABC) transporter ABCB10 was first described 20 years ago as a protein whose expression was induced by GATA-1, a transcription factor essential for normal. Heme biosynthesis (Fig 1A) occurs through eight sequential reactions [8] that start in the mitochondrial matrix with the production of delta-aminolevulinic acid (ALA), which is transported to the cytoplasm and converted to Coproporphyrinogen. Synthetic metal-porphyrin complexes cannot be enzymatically degraded to bile pigments by heme oxygenase and in vivo they might have dual actions both inducing expression and inhibiting activity of this enzyme, which can lead to different biological actions depending on the nature of the metalloporphyrin [25] In addition to their possible therapeutic roles [26,27,28], some metalloporphyrins can be naturally available in certain conditions, such as occurs with the ZnPP formed by ferrochelatase when iron levels are low [29, 30]. Future studies are needed to determine the structural bases that make ZnMP an activator of ABCB10
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