Structure of the human monomeric NEET protein MiNT and its role in regulating iron and reactive oxygen species in cancer cells

Abstract

The NEET family is a relatively new class of three related [2Fe-2S] proteins (CISD1-3), important in human health and disease. While there has been growing interest in the homodimeric gene products of CISD1 (mitoNEET) and CISD2 (NAF-1), the importance of the inner mitochondrial CISD3 protein has only recently been recognized in cancer. The CISD3 gene encodes for a monomeric protein that contains two [2Fe-2S] CDGSH motifs, which we term mitochondrial inner NEET protein (MiNT). It folds with a pseudosymmetrical fold that provides a hydrophobic motif on one side and a relatively hydrophilic surface on the diametrically opposed surface. Interestingly, as shown by molecular dynamics simulation, the protein displays distinct asymmetrical backbone motions, unlike its homodimeric counterparts that face the cytosolic side of the outer mitochondrial membrane/endoplasmic reticulum (ER). However, like its counterparts, our biological studies indicate that knockdown of MiNT leads to increased accumulation of mitochondrial labile iron, as well as increased mitochondrial reactive oxygen production. Taken together, our study suggests that the MiNT protein functions in the same pathway as its homodimeric counterparts (mitoNEET and NAF-1), and could be a key player in this pathway within the mitochondria. As such, it represents a target for anticancer or antidiabetic drug development.

Keywords: NEET proteins; cancer; iron homeostasis; iron-sulfur proteins; mitochondria.

Fig. 1.

Domain organization of the NEET proteins and mutation of the cluster-coordinating His ligand of MiNT. (A) Domain organization. All members of the NEET family contain at least one CDGSH domain that binds a [2Fe-2S] cluster. MiNT has two CDGSH cluster-binding domains in a single polypeptide chain, while mNT and NAF-1 each have one domain per chain. MiNT lacks a transmembrane domain, but contains a classic cleavable mitochondrial targeting sequence. (B and C) Mutation of the cluster-coordinating His ligand to Cys stabilizes the [2Fe-2S] cluster. (B) UV-Vis absorption spectra of MiNT wild-type and H75C/H113C mutant proteins. (C) Relative stabilities of the [2Fe-2S] clusters of wild-type and H75C/H114C mutant MiNTs were measured by monitoring the decrease in absorbance of the 458-nm peak at pH 8.0 and 37 °C over time. Stability of the H75C/H113C mutant is greatly increased relative to wild-type MiNT under these conditions.

Fig. 2.

Structural organization of monomeric MiNT highlights the C2 pseudosymmetry and the asymmetrical surface. (A) MiNT contains a four-stranded beta-cap domain composed of two antiparallel hydrogen-bonded stranded sheets that dock to form the four-stranded cap domain. Each contains a structured loop and a turn of helix that contains the [2Fe-2S] cluster-coordinating ligands. However, CBD2 has additional helix and long N- and C-terminal loop extensions. (B) Close-up of the cluster-coordinating ligands as well as the hydrogen-bond network that connects the two clusters across the pseudosymmetrical axis. Residue numbers are indicated. (C) Ribbon diagram of MiNT with surface aromatics highlighted. The clear asymmetry in the surface of MiNT is evident in this representation. (D) Displacements around the average structure for the first principal component of simulated native-state dynamics, scaled 10-fold.

Fig. 3.

MiNT donates its [2Fe-2S] clusters to human mitochondrial ferredoxins. The [2Fe-2S] cluster transfer from holo-MiNT to human apo-FDX1 and to apo-FDX2 was monitored by UV-Vis absorption spectroscopy. Spectra from select time points of the transfer to apo-FDX1 (A) and to apo-FDX2 (C) are shown. Transfer progress as determined by the ratio of absorbance at 420 nm to 458 nm vs. time is shown for the transfer to apo-FDX1 (B) and to apo-FDX2 (D). The traces shown were obtained with 15 μM MiNT (30 μM [2Fe-2S] clusters) and 60 μM apo-FDX1 or apo-FDX2 at 25 °C. AU, absorbance units.

Fig. 4.

Impaired mitochondrial function and enhanced mitochondrial iron and ROS accumulation in cancer cells with suppressed expression of MiNT. (A) Decreased MMP in MDA-MB-231 cancer cells with suppressed MiNT expression. (Top) Confocal microscopy images of control cells (transformed with scrambled vector), and two different cell lines with suppressed expression of MiNT [CISD3(−)A and CISD3(−)D], stained with the MMP indicator TMRE (0.1 μM). (Bottom) Bar graph showing quantification of TMRE fluorescence in the different cell lines calculated for n = 3 independent experiments, each with 90 cells per group. ***P < 0.001. Pretreatment with DFP (50 μM) for 16 h prevents the effect of CISD3 suppression on MMP. r.u., relative units. (B) Enhanced accumulation of mLI in cancer cells with suppressed CISD3 expression. (Left) Bar graph showing a decrease (quenching) in RPA fluorescence (indicative of high mLI levels) in two different cell lines with suppressed expression of MiNT [CISD3(−)A and CISD3(−)D], compared with control. Pretreatment with DFP (100 μM) for 60 min prevents the effect of CISD3 suppression on mLI. (Right) Bar graph showing the relative RPA fluorescence change upon addition of DFP, indicative of the relative change in mLI induced by the iron chelator. Results are calculated for n = 3 independent experiments each with 90 cells per group. ***P < 0.001. Δr.u., change in relative units. (C) Enhanced accumulation of mROS in cancer cells with suppressed MiNT expression compared with control. mROS was measured following 60 min of incubation with mitoSOX (5 μM). Pretreatment with DFP (100 μM) for 60 min prevents the effect of CISD3 suppression.

Fig. 5.

MiNT provides a link between FDXR and ferredoxins (FDX1 and FDX2) in the mitochondrial matrix. MiNT provides [2Fe-2S] clusters produced inside the mitochondria by the iron-sulfur cluster assembly system (ISC) to FDX1 and FDX2. MiNT can provide parallel routes linking the ISC to mitochondrial matrix processes. Matrix-produced clusters are important for cell metabolism, maintenance, and proliferation.