Multinuclear NMR and UV–Vis spectroscopy of site directed mutants of the diabetes drug target protein mitoNEET suggest that folding is intimately coupled to iron–sulfur cluster formation

Benson, Susan Kushner; Boyce, Kristen; Bunker, Ryan; Collins, Natalie B.; Daily, K.J.; Esway, A.S.; Gilmore, Grant T.; Hartzler, C.W.; Howard, G.P.; Kasmar, N.A.; Kennedy, Kevin J.; King, B.L.; Kordahi, T.N.; Mattioli, Tony A.; Pugh, Darryl; Ray, Lucille A.; Ross, Suzanne; Torcasio, Morgan H.; Webber, D.P.; Morris, Daniel L.; Leeper, Thomas C.

doi:10.1016/j.inoche.2015.11.022

Cited by 5 publications

(1 citation statement)

References 24 publications

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“…Qualitatively, the structures appear to be complimentary; the grooves and ridges of mNT fit smoothly with those of NAF-1 (see S3 Fig ). Because Fe-S proteins could exhibit some degree of plasticity when transferring clusters [ 45 , 46 ], mNT and NAF-1 could undergo slight conformational changes during the formation of their complex, which was not accounted for by our model. Our findings that mNT and NAF-1 interact in cells and could mediate cluster transfer reactions ( Table 1 , Figs 1 – 3 ), coupled with our previous analysis of cells with suppressed expression of mNT or NAF-1 (4), support a model in which mNT and NAF-1 function in the same cellular pathway to attenuate iron and ROS levels in the mitochondria.…”

Section: Resultsmentioning

confidence: 99%

Interactions between mitoNEET and NAF-1 in cells

et al. 2017

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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, in vivo 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 in vitro 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.

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Section: Resultsmentioning

confidence: 99%

Interactions between mitoNEET and NAF-1 in cells

et al. 2017

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The unique fold and lability of the [2Fe-2S] clusters of NEET proteins mediate their key functions in health and disease

et al. 2018

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NEET proteins comprise a new class of [2Fe-2S] cluster proteins. In human, three genes encode for NEET proteins: cisd1 encodes mitoNEET (mNT), cisd2 encodes the Nutrient-deprivation autophagy factor-1 (NAF-1) and cisd3 encodes MiNT (Miner2). These recently discovered proteins play key roles in many processes related to normal metabolism and disease. Indeed, NEET proteins are involved in iron, Fe-S, and reactive oxygen homeostasis in cells and play an important role in regulating apoptosis and autophagy. mNT and NAF-1 are homodimeric and reside on the outer mitochondrial membrane. NAF-1 also resides in the membranes of the ER associated mitochondrial membranes (MAM) and the ER. MiNT is a monomer with distinct asymmetry in the molecular surfaces surrounding the clusters. Unlike its paralogs mNT and NAF-1, it resides within the mitochondria. NAF-1 and mNT share similar backbone folds to the plant homodimeric NEET protein (At-NEET), while MiNT’s backbone fold resembles a bacterial MiNT protein. Despite the variation of amino acid composition among these proteins, all NEET proteins retained their unique CDGSH domain harboring their unique 3Cys:1His [2Fe-2S] cluster coordination through evolution. The coordinating exposed His was shown to convey the lability to the NEET proteins’ [2Fe-2S] clusters. In this minireview, we discuss the NEET fold and its structural elements. Special attention is given to the unique lability of the NEETs’ [2Fe-2S] cluster and the implication of the latter to the NEET proteins’ cellular and systemic function in health and disease.Graphical abstract

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