Biochemical and Biophysical Research Communications
His-87 ligand in mitoNEET is crucial for the transfer of iron sulfur clusters from mitochondria to cytosolic aconitase
Introduction
Iron-sulfur (Fe-S) cluster proteins play important roles in many essential processes, such as enzymatic catalysis, respiration, nitrogen fixation, DNA repair, ribosome biogenesis and iron homeostasis [1], [2], [3]. Fe-S cluster biogenesis disorder has been attributed to many human diseases, such as Friedreich's ataxia [4]. In mammalian cells, iron-sulfur clusters are assembled in mitochondria by the iron sulfur cluster (ISC) assembly machinery. ISC system assembles nascent iron sulfur clusters using iron and l-cysteine, and transfers them to the target proteins [5]. The ISC system is highly conservative from prokaryotes to eukaryotic organisms. Although it has been known that Fe-S clusters are mainly produced in the mitochondria, how these assembled iron-sulfur clusters are transported to target proteins in cytoplasm is not clear. So far, the only known components of the mitochondrial ISC export machinery are ATP-binding cassette (ABC) transporter system including ABC7 and MTABC3 [6], [7], [8].
Human mitoNEET (mNT) is nearly discovered as a target of type II diabetes drugs pioglitazone [9], [10]. The protein is the first drug binding iron sulfur proteins which contains a unique CDGSH-type zinc finger domain [11]. MitoNEET is located to the outer mitochondrial membrane (OMM) via an amino-terminal anchor sequence tethered to the mitochondrial outer membrane. The CDGSH domain of mitoNEET is oriented toward cytoplasm [11]. Biochemical studies have shown that mNT contains a [2Fe-2S] cluster [12]. Protein crystallographic studies revealed that mNT is a homodimer with each monomer containing a [2Fe-2S] center. Distinct from the classic 4-Cys ferredoxin and 2-Cys, 2-His Rieske [2Fe-2S] clusters, mNT represents a novel hybrid [2Fe-2S] cluster [13]. The [2Fe-2S] cluster center in mNT is hosted by three cysteines (Cys-72, Cys-74 and Cys-83) and one histidine (His-87) in the unique CDGSH domain [14], [15], [16], [17]. His-87 has been shown to mostly affect Fe-S cluster redox and stability properties [9], [13], [18], [19], and low pH can increase its instability [20]. It shares the same unique [2Fe-2S] ligation and protein fold with the proteins of Miner1 and Miner2 [11].
Increasing evidence indicated that mNT may be involved in diverse biological processes, including autophagy, apoptosis, aging, diabetes, and reactive oxygen homeostasis [21], [22], [23], [24]. It has been shown that mNT may transfer its [2Fe-2S] clusters to an apo-acceptor protein in vitro [25], [26], [27], and that NADPH inhibits the cluster transportation [28]. In addition, mNT can also transfers [2Fe-2S] clusters to Anamorsin, a protein required for cytosolic Fe-S cluster biogenesis [29]. Furthermore, a recent study [27] revealed that mNT may transfer the [2Fe-2S] clusters only to the iron regulatory protein 1 (IRP1)/cytosolic aconitase in living cells. However, in their studies, transient transfection was used to interfere mNT expression in cells, and only cytosolic iron-sulfur protein GPAT and aconitase were measured.
In this study, we have constructed stable MCF-7 and HepG2 cell lines in which mNT is depleted using shRNA. We confirm that mNT may mediate the transfer of iron sulfur clusters to the cytosolic aconitase. Furthermore, we find that mutations of the His-87 ligand in mNT block the transfer of iron-sulfur clusters to cytosolic aconitase in the cells, suggesting that the His-87 ligand in mNT is crucial for the iron sulfur cluster transfer to the cytosolic aconitase.
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Cell cultures
Human Epithelial Breast Michigan Cancer Foundation-7 (MCF-7) cells and liver hepatocellular carcinoma (HepG2) cells were cultured in DMEM (Sigma, USA) containing 4.5 g/liter glucose and 1 mM stable l-glutamine and 10% fetal bovine serum (FBS) (HyClone, USA) and supplemented with 1% penicillin-streptomycin. Cells were maintained in 100-mm Petri dishes (Corning Incorporated, USA) in an incubator (HERAcell 150i; Thermo Scientific) controlled at 37 °C and 5% CO2.
Vector construction
PGPU6/GFP/Neo-CISD1-homo plasmid
Knockdown of mNT inhibits cell proliferation
We depleted mNT in MCF-7 and HepG2 cells using a specific shRNA. The knockdown efficacy was evaluated by qRT-PCR and western blotting (Fig. 1A, B). We then investigated the effect of mNT downregulation on the proliferation of MCF-7 and HepG2 cells. Fig. 1C shows that at day 5, the cell growth of MCF-7 decreased nearly 50% in the ells with shRNA-mNT. For HepG2 cells, inhibition of cell growth occurred in day 3, and decreased by 70% at day 5 in the cells with shRNA-mNT. These results indicated
Discussion
Fe-S proteins play critical roles in many essential processes [2]. It has been postulated that biogenesis of Fe-S clusters is performed by the mitochondrial iron sulfur cluster assembly machinery, and Fe-S clusters synthesized in the mitochondria are transported to the cytoplasmic Fe-S proteins to complete the maturation process. However, how these newly synthesized Fe-S clusters are transported to the cytoplasm remains poorly understood. MitoNEET is the first drug binding Fe-S protein that
Acknowledgment
Research reported in this publication was supported by the National Natural Science Foundation of China (31200587, 81500440 and 81301744), the Natural Science Foundation of Zhejiang Province Grant (LY12C05003) and the Key Science and Technology Innovation Team of Zhejiang Province Grant (2010R50048-14).
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The authors contributed to this work equally.