Interactions between mitoNEET and NAF-1 in cells

<div><p>The NEET proteins mitoNEET (mNT) and nutrient-deprivation autophagy factor-1 (NAF-1) are required for cancer cell proliferation and resistance to oxidative stress. NAF-1 and mNT are also implicated in a number of other human pathologies including diabetes, neurodegeneration and cardiovascular disease, as well as in development, differentiation and aging. Previous studies suggested that mNT and NAF-1 could function in the same pathway in mammalian cells, preventing the over-accumulation of iron and reactive oxygen species (ROS) in mitochondria. Nevertheless, it is unknown whether these two proteins directly interact in cells, and how they mediate their function. Here we demonstrate, using yeast two-hybrid, <i>in vivo</i> bimolecular fluorescence complementation (BiFC), direct coupling analysis (DCA), RNA-sequencing, ROS and iron imaging, and single and double shRNA lines with suppressed mNT, NAF-1 and mNT/NAF-1 expression, that mNT and NAF-1 directly interact in mammalian cells and could function in the same cellular pathway. We further show using an <i>in vitro</i> cluster transfer assay that mNT can transfer its clusters to NAF-1. Our study highlights the possibility that mNT and NAF-1 function as part of an iron-sulfur (2Fe-2S) cluster relay to maintain the levels of iron and Fe-S clusters under control in the mitochondria of mammalian cells, thereby preventing the activation of apoptosis and/or autophagy and supporting cellular proliferation.</p></div

DCA analysis and complex model of NAF-1-mNT interaction.

<p><b>A</b>. A cartoon representation of the mNT-NAF-1 complex, with mNT on the left (blue) and NAF-1 on the right (pink). This figure was generated by aligning the PDB crystal structures of mNT (PDB 2QH7) and NAF-1 (PDB 3FNV) with the result of the simulation. The distance between the iron-sulfur clusters is shown to be about 12.6 Å (highlighted in red). <b>B</b>. Surface representation of the mNT-NAF-1 complex demonstrating the close fit between the two proteins. A closer view of this lock-and-key part of the interface is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175796#pone.0175796.s003" target="_blank">S3 Fig</a>. The DCA couplings are depicted as green lines. A full list of the couplings, along with their approximate distances is included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175796#pone.0175796.s005" target="_blank">S1 Table</a>.</p

Mitochondrial membrane potential (MMP), labile iron and ROS measurements in cancer cell lines with suppressed mNT and/or NAF-1 expression.

<p>Compared to single suppression of mNT [mNT(-)] or NAF-1 [NAF-1(-)], double shRNA suppression of mNT and NAF-1 [mNT(-)/NAF-1(-)] stable lines do not result in a significantly larger impairment in MMP <b>(A)</b>, mitochondrial labile iron <b>(B)</b> and mitochondrial ROS <b>(C)</b> accumulation. The iron chelator DFP (100 μM) is shown to recover the effect of mNT and/or NAF-1 suppression on MMP, mitochondrial labile iron and ROS in all lines in a similar manner <b>(A-C)</b>. All measurements were compared to control MDA-MB-231 cell lines transfected with the same shRNA vector containing a scrambled RNA. The expression levels of the mNT and NAF-1 proteins in all lines are shown <b>(D)</b>, and Coomassie blue loading controls in <b>(E)</b>. ***P <0.001.</p

A hypothetical model for the interaction of mNT with NAF-1 in cells.

<p>mNT is shown to accept Fe-S clusters from the mitochondria and transfer them to NAF-1. The flow of clusters from mNT to NAF-1 is shown to be used by NAF-1 to regulate different processes such as apoptosis/autophagy activation, as well as cellular proliferation. The cluster relay between mNT and NAF-1 is proposed to link mitochondrial Iron/ROS/Fe-S homeostasis and function with the regulation of cell death/proliferation by the NAF-1/BCL-2/MAPK/PI3K-Akt pathways.</p

2Fe-2S cluster transfer from holo-mNT to apo-NAF-1.

<p>Apo-NAF-1 was incubated at 37°C for 20 min with β-mercaptoethanol and holo-mNT, and chromatographed on a native gel as described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175796#pone.0175796.ref031" target="_blank">31</a>] with the modification described in Materials and Methods. Red-colored bands in the upper native gels are indicative of the [2Fe–2S] cluster presence in the two proteins. Blue-colored bands in the lower duplicate gel are the result of Coomassie Blue staining to confirm the presence and levels of the two proteins.</p

Overlap between transcripts altered in cancer cells with suppressed mNT [mNT(-)] or NAF-1 [NAF-1(-)] expression.

<p><b>A</b>. Venn diagram showing the overlap between transcripts altered (p≤0.05) in cancer cells with suppressed mNT [mNT(-)] or NAF-1 [NAF-1(-)] expression detected with RNA-seq analysis. <b>B</b>. KEGG annotation of transcripts common to cells with suppressed mNT [mNT(-)] or NAF-1 [NAF-1(-)] expression.</p