title: Investigation of the role of the mitochondrial ABC transporter ABCB7 in heme and iron-sulfur protein biosynthesis
title: Untersuchung der Rolle des mitochondrialen ABC-Transporters ABCB7 bei der Biosynthese von Häm- und Eisen-Schwefel-Proteinen
titleType: AlternativeTitle
Creators: name: Mathea, Nadine Arlett
Publisher: name: Philipps-Universität Marburg
Year: 2024
Subjects: ABCB7
Fe/S cluster
Eisen-Schwefel-Cluster
Medical sciences Medicine
Medizin
Iron-sulfur cluster

description: Mutations in the gene encoding the human mitochondrial transporter ABCB7 have been associated with X-linked sideroblastic anemia and cerebellar ataxia and refractory anemia with ring sideroblasts. ABCB7 is located in the mitochondrial inner membrane and considered to serve as an exporter of sulfur-containing compounds crucial for cytosolic and nuclear iron-sulfur protein biogenesis. While a few previous studies have also demonstrated a deficiency in heme-containing proteins in ABCB7-depleted cells, the precise molecular mechanisms behind these observations remain ill-defined. Various cytosolic-nuclear iron-sulfur proteins play pivotal roles in cellular iron and heme metabolism, and consequently could explain the X-linked sideroblastic anemia and cerebellar ataxia or refractory anemia with ring sideroblasts-related patient phenotypes. Other studies have suggested physical as well as functional interactions between ABCB7 and ferrochelatase, an enzyme associated with the matrix side of the mitochondrial inner membrane and mediating the final step in heme biosynthesis by inserting iron into protoporphyrin IX. These observations raised the question of a direct involvement of ABCB7 in both heme synthesis and mitochondrial heme export with possible consequences for erythropoiesis. This study aimed to assess the role of ABCB7 in hemoprotein maturation in human cells. To examine a potential mitochondrial heme export function of ABCB7, a HeLa tissue culture system overexpressing the peroxisomal heme-containing enzyme catalase was established. Using this system, RNA interference was applied to deplete ABCB7, and the consequences for both heme formation and mitochondrial export were compared to those of a ferrochelatase deficiency by using a combination of cell fractionation, enzymatic activity and immunoblotting assays. In addition, the maturation of selected mitochondrial and cytosolic-nuclear iron-sulfur proteins were assessed to study the requirement of ABCB7 in iron-sulfur protein biosynthesis. Upon depletion of either ABCB7 or ferrochelatase, the HeLa cell yield declined markedly, highlighting the indispensable nature of both ABCB7 and ferrochelatase for cell viability. Knockdown of ferrochelatase specifically affected the stability and function of hemoproteins both within and outside mitochondria, including respiratory chain complex II, cytochrome c1 of respiratory chain complex III, respiratory chain complex IV and catalase. In contrast, deficiency of ABCB7 did not significantly impair catalase activity, clearly indicating that ABCB7 does not play a role in heme export from mitochondria and the subsequent maturation of extramitochondrial hemoproteins. Moreover, the present study did not identify a general requirement of ABCB7 for mitochondrial heme proteins, arguing against a functional influence of the transporter on heme formation by ferrochelatase. Rather, loss of ABCB7 led to a compromised maturation of cytosolic and nuclear Fe/S proteins including GPAT, DPYD and NTHL1, three enzymes involved in nucleotide metabolism and DNA repair, and iron-regulatory protein 1. Plasmid-based expression of RNA interference-resistant ABCB7 reverted these effects. Together, the results corroborated earlier findings which had indicated the requirement of ABCB7 for cytosolic and nuclear iron-sulfur cluster biosynthesis. In agreement with an ABCB7 function in maturation of iron-regulatory protein 1, ABCB7 deficiency also influenced the levels of two iron regulatory protein-regulated key proteins of cellular Fe homeostasis, the iron storage protein ferritin and the iron uptake component transferrin receptor, resulting in a cellular iron deficiency phenotype. Consistently, ABCB7 deficiency triggered an increase in iron-regulatory protein 2 levels, an effect indicating decreased iron levels in the cytosolic compartment. Moreover, RNA interference-mediated depletion of ABCB7 elicited defects in mitochondrial iron-sulfur proteins, likely caused by the altered iron metabolism. Taken together, the current investigation clearly refutes critical roles of human ABCB7 in both mitochondrial heme synthesis and heme export to the cytosol and thus contradicts a direct involvement of ABCB7 in erythropoiesis. In accordance with previous studies on ABCB7 orthologs from multiple model organisms, this transporter was found to be required for the biogenesis of cytosolic-nuclear iron-sulfur proteins. The exact substrate of the transporter remains unclear and thus will be an exciting subject of future cell biological research. The concomitant, yet weaker effects on mitochondrial iron-sulfur proteins were likely caused by the oxidative stress resulting from the iron accumulation observed in ABCB7-deficient cells. The results of the present study may explain the mitochondrial phenotypes of X-linked sideroblastic anemia and cerebellar ataxia and refractory anemia with ring sideroblasts. Similar pathological mechanisms may be effective in other iron-overload diseases such as Friedreich's ataxia (caused by frataxin deficiency) and sideroblastic anemia 3 (caused by GLRX5 deficiency). Other cellular processes involving cytosolic-nuclear iron-sulfur proteins, such as iron metabolism, genome integrity and protein biosynthesis, are also affected in the case of ABCB7 deficiency, and contribute to the complex phenotype of patients who suffer from X-linked sideroblastic anemia and cerebellar ataxia or refractory anemia with ring sideroblasts.