Abstract
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.
Highlights
The human NEET proteins mitoNEET and nutrient-deprivation autophagy factor-1 (NAF-1) play major roles in the regulation of apoptosis, autophagy and iron and reactive oxygen species (ROS) homeostasis in cells [1,2,3,4,5,6]
These include NAF-1 (CISD2), a non-lysosomal calcium-activated protease Calpain-1 (CAPN1), that could be related to CAPN2, previously shown to bind NAF-1 [24], and a number of other proteins that are known to be involved in immune response, cytoskeleton and cancer (e.g., FAT tumor suppressor and UXT)
Fe/Fe-S levels are tightly regulated in cells to prevent the accumulation of ROS that could lead to the activation of cell death pathways such as apoptosis and ferroptosis [50]
Summary
The human NEET proteins mitoNEET (mNT) and NAF-1 (encoded by the CISD1 and CISD2 genes, respectively) play major roles in the regulation of apoptosis, autophagy and iron and reactive oxygen species (ROS) homeostasis in cells [1,2,3,4,5,6]. The studies described above suggest that NAF-1 and mNT use their labile 2Fe-2S clusters to mediate different redox or cluster transfer reactions that help cancer cells alleviate some of the toxic effects of iron and ROS over-accumulation during oxidative challenge in the mitochondria This function could be regulated by mNT or NAF-1 in many different cells types [6, 16,17,18], and represent one of the major functions of these proteins in mammalian and even plant cells [2]
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