Iron-sulfur (Fe-S) proteins are key players in vital processes involving energy homeostasis and metabolism from the simplest to most complex organisms. We report a 1.5 Å x-ray crystal structure of the first identified outer mitochondrial membrane Fe-S protein, mitoNEET. Two protomers intertwine to form a unique dimeric structure that constitutes a new fold to not only the Ϸ650 reported Fe-S protein structures but also to all known proteins. We name this motif the NEET fold. The protomers form a two-domain structure: a ␤-cap domain and a cluster-binding domain that coordinates two acid-labile 2Fe-2S clusters. Binding of pioglitazone, an insulin-sensitizing thiazolidinedione used in the treatment of type 2 diabetes, stabilizes the protein against 2Fe-2S cluster release. The biophysical properties of mitoNEET suggest that it may participate in a redox-sensitive signaling and/or in Fe-S cluster transfer. diabetes ͉ FeS cluster ͉ iron homeostasis ͉ thiazolidinedione ͉ oxidative stress I ron (Fe) is a vital trace element for virtually all organisms. Incorporation of this transition metal into iron-sulfur (Fe-S) clusters forms cofactors integral to diverse biological pathways involved in the capture and metabolism of light and chemical energy (1, 2). Because free iron can be highly toxic, an elaborate array of proteins has evolved to facilitate the transfer of iron through cell compartments, to insert iron into Fe-S clusters, and to incorporate Fe-S clusters into proteins. Fe-S cluster assembly takes place primarily, although not exclusively, within the mitochondrial matrix of eukaryotic cells, and defects in mitochondrial cluster assembly and export have profound consequences for rates of growth, iron accumulation, oxidative stress, and heme biosynthesis (1, 2).Mitochondrial dysfunction is associated with insulin resistance and the development of type 2 diabetes (3). Recent studies suggest that disease pathogenesis involves diminished mitochondrial oxidative capacity in insulin-sensitive tissues. Pharmacologic agents extensively used to treat insulin resistance such as the thiazolidinedione (TZD) pioglitazone are known to enhance oxidative capacity and normalize lipid metabolism (4, 5). Although TZDs are conventionally thought to operate through binding to peroxisome proliferator-activated receptors, a recent study by Colca and colleagues (6) identified an additional binding target within mitochondrial membranes that was named mitoNEET, on the basis of the subcellular localization (mito) and the presence of the amino acid sequence Asn-Glu-Glu-Thr (NEET).MitoNEET was determined to be an integral protein of the outer mitochondrial membrane (OMM) by a series of studies, including immuno-electron microscopy and detailed fractionation studies of highly purified rat liver mitochondria. An amino-terminal signal sequence within the first 32 residues, containing a predicted transmembrane domain, targets mitoNEET to the outer membrane. The orientation of this protein toward the cytoplasm was established by proteolytic digestion...

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.M itochondria play a key role in many human diseases related to their functions in cellular energy production, biosynthesis of essential cellular compounds, and involvement in autophagy and/or apoptosis regulation (1). Contrary to previously held beliefs, recent studies have demonstrated that mitochondria are also key players in the transformation process of cancer cells and may be used as targets for anticancer therapy (2-6). The involvement of mitochondria in cancer cell function could be linked to the enhanced accumulation of iron and reactive oxygen species (ROS) in mitochondria of cancer cells, which is thought to result from the increased metabolic and energetic demands of the transformed phenotype (7). An interesting, recently discovered group of proteins that could link iron and ROS homeostasis with mitochondrial function in cancer cells are NEET proteins (8-15).NEET proteins [mitoNEET (mNT; CISD1), Nutrient-deprivation autophagy factor-1 (NAF-1; CISD2), and CISD3] are a class of iron-sulfur proteins involved in several human pathologies, including diabetes, cystic fibrosis, Wolfram syndrome 2, neurodegeneration, and muscle atrophy (16)(17)(18)(19)(20)(21)(22). mNT and NAF-1 are localized to the outer mitochondrial membrane. NAF-1 is also localized to the endoplasmic reticulum, where it interacts with BCL-2 and Beclin 1 to regulate autophagy and apoptosis (8-13). Deficiency in mNT causes the accumulation of iron and ROS in mitochondria of animal and plant cells, and deficiency in NAF-1 results in decreased mitochondrial function and stability, as well as activation of autophagy in mice and human cells (13-16, 20, 21).Interestingly, levels of mNT and NAF-1 mRNA are increased significantly in many different human cancer c...

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.

Climate change-driven extreme weather events, combined with increasing temperatures, harsh soil conditions, low water availability and quality, and the introduction of many manmade pollutants, pose a unique challenge to plants. Although our knowledge of the response of plants to each of these individual conditions is vast, we know very little about how a combination of many of these factors, occurring simultaneously, that is multifactorial stress combination, impacts plants. Seedlings of wild-type and different mutants of Arabidopsis thaliana plants were subjected to a multifactorial stress combination of six different stresses, each applied at a low level, and their survival, physiological and molecular responses determined. Our findings reveal that, while each of the different stresses, applied individually, had a negligible effect on plant growth and survival, the accumulated impact of multifactorial stress combination on plants was detrimental. We further show that the response of plants to multifactorial stress combination is unique and that specific pathways and processes play a critical role in the acclimation of plants to multifactorial stress combination. Taken together our findings reveal that further polluting our environment could result in higher complexities of multifactorial stress combinations that in turn could drive a critical decline in plant growth and survival.

Photosynthesis is a sustainable process that converts light energy into chemical energy. substantial research efforts are directed towards the application of the photosynthetic reaction centres, photosystems I and II, as active components for the light-induced generation of electrical power or fuel products. nonetheless, no integrated photo-bioelectrochemical device that produces electrical power, upon irradiation of an aqueous solution that includes two inter-connected electrodes is known. Here we report the assembly of photobiofuel cells that generate electricity upon irradiation of biomaterial-functionalized electrodes in aqueous solutions. The cells are composed of electrically contacted photosystem II-functionalized photoanodes and an electrically wired bilirubin oxidase/carbon nanotubes-modified cathode. Illumination of the photoanodes yields the oxidation of water to o 2 and the transfer of electrons through the external circuit to the cathode, where o 2 is re-reduced to water.