Molecular Medicine Branch, 'Eunice Kennedy Shriver' National Institute of Child Health and Human Development, Bethesda, MD, USA.
Molecular Medicine Branch, 'Eunice Kennedy Shriver' National Institute of Child Health and Human Development, Bethesda, MD, USA
Haematologica. 2019 Sep;104(9):1756-1767. doi: 10.3324/haematol.2018.214320. Epub 2019 Feb 14.
Loss-of-function mutations in the ATP-binding cassette (ABC) transporter of the inner mitochondrial membrane, ABCB7, cause X-linked sideroblastic anemia with ataxia, a phenotype that remains largely unexplained by the proposed role of ABCB7 in exporting a special sulfur species for use in cytosolic iron-sulfur (Fe-S) cluster biogenesis. Here, we generated inducible ABCB7-knockdown cell lines to examine the time-dependent consequences of loss of ABCB7. We found that knockdown of ABCB7 led to significant loss of mitochondrial Fe-S proteins, which preceded the development of milder defects in cytosolic Fe-S enzymes. In erythroid cells, loss of ABCB7 altered cellular iron distribution and caused mitochondrial iron overload due to activation of iron regulatory proteins 1 and 2 in the cytosol and to upregulation of the mitochondrial iron importer, mitoferrin-1. Despite the exceptionally large amount of iron imported into mitochondria, erythroid cells lacking ABCB7 showed a profound hemoglobinization defect and underwent apoptosis triggered by oxidative stress. In ABCB7-depleted cells, defective heme biosynthesis resulted from translational repression of ALAS2 by iron regulatory proteins and from decreased stability of the terminal enzyme ferrochelatase. By combining chemical crosslinking, tandem mass spectrometry and mutational analyses, we characterized a complex formed of ferrochelatase, ABCB7 and ABCB10, and mapped the interfaces of interactions of its components. A dimeric ferrochelatase physically bridged ABCB7 and ABCB10 homodimers by binding near the nucleotide-binding domains of each ABC transporter. Our studies not only underscore the importance of ABCB7 for mitochondrial Fe-S biogenesis and iron homeostasis, but also provide the biochemical characterization of a multiprotein complex required for heme biosynthesis.
线粒体内膜 ATP 结合盒（ABC）转运蛋白 ABCB7 的功能丧失突变导致 X 连锁铁粒幼细胞性贫血伴共济失调，ABCB7 在外排用于胞质铁硫（Fe-S）簇生物发生的特殊硫物种方面的作用提出后，这种表型在很大程度上仍未得到解释。在这里，我们生成了可诱导的 ABCB7 敲低细胞系，以检查 ABCB7 缺失的时相关后果。我们发现，ABCB7 的敲低导致线粒体 Fe-S 蛋白显著丢失，这先于胞质 Fe-S 酶出现更轻微的缺陷。在红细胞中，ABCB7 的缺失改变了细胞内铁的分布，并由于胞质中铁调节蛋白 1 和 2 的激活以及线粒体铁摄取体 mitoferrin-1 的上调导致线粒体铁过载。尽管异常大量的铁被导入线粒体，但缺乏 ABCB7 的红细胞显示出严重的血红蛋白化缺陷，并在氧化应激触发下发生凋亡。在 ABCB7 耗尽的细胞中，由于铁调节蛋白对 ALAS2 的翻译抑制以及末端酶亚铁螯合酶的稳定性降低，血红素生物合成出现缺陷。通过结合化学交联、串联质谱和突变分析，我们对由 ferrochelatase、ABCB7 和 ABCB10 组成的复合物进行了表征，并绘制了其成分相互作用界面的图谱。二聚体 ferrochelatase 通过结合每个 ABC 转运蛋白的核苷酸结合域附近，将 ferrochelatase、ABCB7 和 ABCB10 同源二聚体物理桥接在一起。我们的研究不仅强调了 ABCB7 对于线粒体 Fe-S 生物发生和铁稳态的重要性，而且还提供了血红素生物发生所需的多蛋白复合物的生化特征。
