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Significance This work demonstrates that the outer mitochondrial-anchored [2Fe-2S] mitoNEET is able to bind within the central cavity of the voltage-dependent anion channel (VDAC) and regulate its gating in a redox-dependent manner. These findings have implications for ferroptosis, apoptosis, and iron metabolism by linking VDAC function, mitoNEET, and the redox environment of the cell. Furthermore, these findings introduce a potential player to the many mechanisms that may alter VDAC’s governance in times of homeostasis or strife., MitoNEET is an outer mitochondrial membrane protein essential for sensing and regulation of iron and reactive oxygen species (ROS) homeostasis. It is a key player in multiple human maladies including diabetes, cancer, neurodegeneration, and Parkinson’s diseases. In healthy cells, mitoNEET receives its clusters from the mitochondrion and transfers them to acceptor proteins in a process that could be altered by drugs or during illness. Here, we report that mitoNEET regulates the outer-mitochondrial membrane (OMM) protein voltage-dependent anion channel 1 (VDAC1). VDAC1 is a crucial player in the cross talk between the mitochondria and the cytosol. VDAC proteins function to regulate metabolites, ions, ROS, and fatty acid transport, as well as function as a “governator” sentry for the transport of metabolites and ions between the cytosol and the mitochondria. We find that the redox-sensitive [2Fe-2S] cluster protein mitoNEET gates VDAC1 when mitoNEET is oxidized. Addition of the VDAC inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS) prevents both mitoNEET binding in vitro and mitoNEET-dependent mitochondrial iron accumulation in situ. We find that the DIDS inhibitor does not alter the redox state of MitoNEET. Taken together, our data indicate that mitoNEET regulates VDAC in a redox-dependent manner in cells, closing the pore and likely disrupting VDAC’s flow of metabolites.

Identification of novel drug targets and chemotherapeutic agents is a high priority in the fight against cancer. Here, we report that MAD-28, a designed cluvenone (CLV) derivative, binds to and destabilizes two members of a unique class of mitochondrial and endoplasmic reticulum (ER) 2Fe-2S proteins, mitoNEET (mNT) and nutrient-deprivation autophagy factor-1 (NAF-1), recently implicated in cancer cell proliferation. Docking analysis of MAD-28 to mNT/NAF-1 revealed that in contrast to CLV, which formed a hydrogen bond network that stabilized the 2Fe-2S clusters of these proteins, MAD-28 broke the coordinative bond between the His ligand and the cluster's Fe of mNT/NAF-1. Analysis of MAD-28 performed with control (Michigan Cancer Foundation; MCF-10A) and malignant (M.D. Anderson-metastatic breast; MDA-MB-231 or MCF-7) human epithelial breast cells revealed that MAD-28 had a high specificity in the selective killing of cancer cells, without any apparent effects on normal breast cells. MAD-28 was found to target the mitochondria of cancer cells and displayed a surprising similarity in its effects to the effects of mNT/NAF-1 shRNA suppression in cancer cells, causing a decrease in respiration and mitochondrial membrane potential, as well as an increase in mitochondrial iron content and glycolysis. As expected, if the NEET proteins are targets of MAD-28, cancer cells with suppressed levels of NAF-1 or mNT were less susceptible to the drug. Taken together, our results suggest that NEET proteins are a novel class of drug targets in the chemotherapeutic treatment of breast cancer, and that MAD-28 can now be used as a template for rational drug design for NEET Fe-S cluster-destabilizing anticancer drugs.

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.

The NEET proteins are a novel family of iron–sulfur proteins characterized by an unusual three cysteine and one histidine coordinated [2Fe–2S] cluster. Aberrant cluster release, facilitated by the breakage of the Fe–N bond, is implicated in a variety of human diseases, including cancer. Here, the molecular dynamics in the multi-microsecond timescale, along with quantum chemical calculations, on two representative members of the family (the human NAF-1 and mitoNEET proteins), show that the loss of the cluster is associated with a dramatic decrease in secondary and tertiary structure. In addition, the calculations provide a mechanism for cluster release and clarify, for the first time, crucial differences existing between the two proteins, which are reflected in the experimentally observed difference in the pH-dependent cluster reactivity. The reliability of our conclusions is established by an extensive comparison with the NMR data of the solution proteins, in part measured in this work.

A novel family of 2Fe–2S proteins, the NEET family, was discovered during the last decade in numerous organisms, including archea, bacteria, algae, plant and human; suggesting an evolutionary-conserved function, potentially mediated by their CDGSH Iron–Sulfur Domain. In human, three NEET members encoded by the CISD1–3 genes were identified. The structures of CISD1 (mitoNEET, mNT), CISD2 (NAF-1), and the plant At-NEET uncovered a homodimer with a unique “NEET fold”, as well as two distinct domains: a beta-cap and a 2Fe–2S cluster-binding domain. The 2Fe–2S clusters of NEET proteins were found to be coordinated by a novel 3Cys:1His structure that is relatively labile compared to other 2Fe–2S proteins and is the reason of the NEETs' clusters could be transferred to apo-acceptor protein(s) or mitochondria. Positioned at the protein surface, the NEET's 2Fe–2S's coordinating His is exposed to protonation upon changes in its environment, potentially suggesting a sensing function for this residue. Studies in different model systems demonstrated a role for NAF-1 and mNT in the regulation of cellular iron, calcium and ROS homeostasis, and uncovered a key role for NEET proteins in critical processes, such as cancer cell proliferation and tumor growth, lipid and glucose homeostasis in obesity and diabetes, control of autophagy, longevity in mice, and senescence in plants. Abnormal regulation of NEET proteins was consequently found to result in multiple health conditions, and aberrant splicing of NAF-1 was found to be a causative of the neurological genetic disorder Wolfram Syndrome 2. Here we review the discovery of NEET proteins, their structural, biochemical and biophysical characterization, and their most recent structure–function analyses. We additionally highlight future avenues of research focused on NEET proteins and propose an essential role for NEETs in health and disease. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

Iron-sulfur cluster biogenesis is executed by distinct protein assembly systems. Mammals have two systems, the mitochondrial Fe-S cluster assembly system (ISC) and the cytosolic assembly system (CIA), that are connected by an unknown mechanism. The human members of the NEET family of 2Fe-2S proteins, nutrient-deprivation autophagy factor-1 (NAF-1) and mitoNEET (mNT), are located at the interface between the mitochondria and the cytosol. These proteins have been implicated in cancer cell proliferation, and they can transfer their 2Fe-2S clusters to a standard apo-acceptor protein. Here we report the first physiological 2Fe-2S cluster acceptor for both NEET proteins as human Anamorsin (also known as cytokine induced apoptosis inhibitor-1; CIAPIN-1). Anamorsin is an electron transfer protein containing two iron-sulfur cluster-binding sites that is required for cytosolic Fe-S cluster assembly. We show, using UV-Vis spectroscopy, that both NAF-1 and mNT can transfer their 2Fe-2S clusters to apo-Anamorsin with second order rate constants similar to those of other known human 2Fe-2S transfer proteins. A direct protein-protein interaction of the NEET proteins with apo-Anamorsin was detected using biolayer interferometry. Furthermore, electrospray mass spectrometry of holo-Anamorsin prepared by cluster transfer shows that it receives both of its 2Fe-2S clusters from the NEETs. We propose that mNT and NAF-1 can provide parallel routes connecting the mitochondrial ISC system and the CIA. 2Fe-2S clusters assembled in the mitochondria are received by NEET proteins and when needed transferred to Anamorsin, activating the CIA.