NAF-1 and mitoNEET are central to human breast cancer proliferation by maintaining mitochondrial homeostasis and promoting tumor growth

Mitochondria are emerging as important players in the transformation process of cells, maintaining the biosynthetic and energetic capacities of cancer cells and serving as one of the primary sites of apoptosis and autophagy regulation. Although several avenues of cancer therapy have focused on mitochondria, progress in developing mitochondria-targeting anticancer drugs nonetheless has been slow, owing to the limited number of known mitochondrial target proteins that link metabolism with autophagy or cell death. Recent studies have demonstrated that two members of the newly discovered family of NEET proteins, NAF-1 (CISD2) and mitoNEET (mNT; CISD1), could play such a role in cancer cells. NAF-1 was shown to be a key player in regulating autophagy, and mNT was proposed to mediate iron and reactive oxygen homeostasis in mitochondria. Here we show that the protein levels of NAF-1 and mNT are elevated in human epithelial breast cancer cells, and that suppressing the level of these proteins using shRNA results in significantly reduced cell proliferation and tumor growth, decreased mitochondrial performance, uncontrolled accumulation of iron and reactive oxygen in mitochondria, and activation of autophagy. Our findings highlight NEET proteins as promising mitochondrial targets for cancer therapy

Facile transfer of [2Fe-2S] clusters from the diabetes drug target mitoNEET to an apo-acceptor protein

MitoNEET (mNT) is an outer mitochondrial membrane target of the thiazolidinedione diabetes drugs with a unique fold and a labile [2Fe-2S] cluster. The rare 1-His and 3-Cys coordination of mNT’s [2Fe-2S] leads to cluster lability that is strongly dependent on the presence of the single histidine ligand (His87). These properties of mNT are similar to known [2Fe-2S] shuttle proteins. Here we investigated whether mNT is capable of cluster transfer to acceptor protein(s). Facile [2Fe-2S] cluster transfer is observed between oxidized mNT and apo-ferredoxin (a-Fd) using UV-VIS spectroscopy and native-PAGE, as well as with a mitochondrial iron detection assay in cells. The transfer is unidirectional, proceeds to completion, and occurs with a second-order-reaction rate that is comparable to known iron-sulfur transfer proteins. Mutagenesis of His87 with Cys (H87C) inhibits transfer of the [2Fe-2S] clusters to a-Fd. This inhibition is beyond that expected from increased cluster kinetic stability, as the equivalently stable Lys55 to Glu (K55E) mutation did not inhibit transfer. The H87C mutant also failed to transfer its iron to mitochondria in HEK293 cells. The diabetes drug pioglitazone inhibits iron transfer from WT mNT to mitochondria, indicating that pioglitazone affects a specific property, [2Fe-2S] cluster transfer, in the cellular environment. This finding is interesting in light of the role of iron overload in diabetes. Our findings suggest a likely role for mNT in [2Fe-2S] and/or iron transfer to acceptor proteins and support the idea that pioglitazone’s antidiabetic mode of action may, in part, be to inhibit transfer of mNT’s [2Fe-2S] cluster

Nutrient-Deprivation Autophagy Factor-1 (NAF-1): Biochemical Properties of a Novel Cellular Target for Anti-Diabetic Drugs

<div><p>Nutrient-deprivation autophagy factor-1 (NAF-1) (synonyms: Cisd2, Eris, Miner1, and Noxp70) is a [2Fe-2S] cluster protein immune-detected both in endoplasmic reticulum (ER) and mitochondrial outer membrane. It was implicated in human pathology (Wolfram Syndrome 2) and in BCL-2 mediated antagonization of Beclin 1-dependent autophagy and depression of ER calcium stores. To gain insights about NAF-1 functions, we investigated the biochemical properties of its 2Fe-2S cluster and sensitivity of those properties to small molecules. The structure of the soluble domain of NAF-1 shows that it forms a homodimer with each protomer containing a [2Fe-2S] cluster bound by 3 Cys and one His. NAF-1 has shown the unusual abilities to transfer its 2Fe-2S cluster to an apo-acceptor protein (followed <i>in vitro</i> by spectrophotometry and by native PAGE electrophoresis) and to transfer iron to intact mitochondria in cell models (monitored by fluorescence imaging with iron fluorescent sensors targeted to mitochondria). Importantly, the drug pioglitazone abrogates NAF-1's ability to transfer the cluster to acceptor proteins and iron to mitochondria. Similar effects were found for the anti-diabetes and longevity-promoting antioxidant resveratrol. These results reveal NAF-1 as a previously unidentified cell target of anti-diabetes thiazolidinedione drugs like pioglitazone and of the natural product resveratrol, both of which interact with the protein and stabilize its labile [2Fe-2S] cluster.</p></div