An ABCB10 cell-free system and the exploration of its substrates and regulators
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ABCB10, or ATP binding cassette sub-family B member 10, is a protein localized in the mitochondrial inner membrane. It belongs to the ABC transporter family whose members are proteins that facilitate substrate transport across various biological membranes. It has been found that ABCB10 is required for normal heme biosynthesis during erythroid differentiation and also plays a role in protection against the damage caused by reactive oxygen species (ROS) production. This protective effect exists both in the erythrocyte development and in the heart recovery after the ischemia-reperfusion injury. However, as an ABC transporter, its transported substrates are not known, neither is the mechanism by which ABCB10 plays a role in protection against ROS damage. In this dissertation an 8-azido-ATP photolabeling system is established to study the ATP binding and hydrolysis properties of ABCB10. Using this approach, it is found that the conserved amino acid residues Gly497 and Lys498 in the Walker A motif of the nucleotide binding domain of ABCB10 are required for ATP binding. On the other hand, Gly602 in the C-loop motif and Glu624 in the end of the Walker B motif are necessary for ATP hydrolysis. In addition, most ABC transporters increase ATP hydrolysis in the presence of their substrates. Therefore, the 8-azido-ATP photolabeling system can be utilized to test potential substrates of ABCB10. Substances related to the heme biosynthesis such as δ-aminolevulinic acid (dALA) and the mitochondrial redox state such as oxidized glutathione (GSSG) and reduced glutathione (GSH) are tested for this purpose. The 8-azido-ATP photolabeling system shows that GSSG stimulates ATP hydrolysis without affecting ATP binding, whereas GSH decreases ATP binding. Further study shows that the nucleotide binding domain of ABCB10 is glutathionylated at the cysteine residue on the position 547 (Cys547), suggesting that GSH may modulate ABCB10 activity via the glutathionylation-regulated ATP binding. This is a first insight into the molecular mechanism by which the mitochondrial redox state, through the regulation by GSH and GSSG, can modulate ABCB10 activity.