An anti-diabetic drug targets NEET (CISD) proteins through destabilization of their [2Fe-2S] clusters.

Elevated levels of mitochondrial iron and reactive oxygen species (ROS) accompany the progression of diabetes, negatively impacting insulin production and secretion from pancreatic cells. In search for a tool to reduce mitochondrial iron and ROS levels, we arrived at a molecule that destabilizes the [2Fe-2S] clusters of NEET proteins (M1). Treatment of db/db diabetic mice with M1 improved hyperglycemia, without the weight gain observed with alternative treatments such as rosiglitazone. The molecular interactions of M1 with the NEET proteins mNT and NAF-1 were determined by X-crystallography. The possibility of controlling diabetes by molecules that destabilize the [2Fe-2S] clusters of NEET proteins, thereby reducing iron-mediated oxidative stress, opens a new route for managing metabolic aberration such as in diabetes.

Photosynthesis Z-Scheme biomimicry: Photosystem I/BiVO4 photo-bioelectrochemical cell for donor-free bias-free electrical power generation.

Photo-bioelectrochemical cells that are based on photosynthetic proteins are drawing increased attention for both fundamental and applied research. While novel photosynthetic based systems have been introduced, further optimization in terms of stability and efficiency is required. Photosystem I has been utilized extensively in bioelectronic devices, often in conjugation with viologen moieties which act as electron acceptors. It has been shown previously that a partial reduction of oxygen to H2O2 can facilitate damage to proteins hence, limits their long-term activation. Here, we show a newly developed bias-free, donor-free photo-bioelectrochemical system that mimics the natural photosynthetic Z-scheme. Polymethylene blue and polybutyl-viologen were tailored to fit the photosystem I donor and acceptor sides, respectively. Furthermore, we show that by coupling the developed biocathode with a BiVO4/CoP photoanode, a power output of 25 µW/cm2 can be achieved. We further show that our configuration can minimize the damaging effect of H2O2 by two different pathways, oxidation at the photoanode or reduction by the polymethylene blue layer at the biocathode.

Activation of apoptosis in NAF-1-deficient human epithelial breast cancer cells.

Maintaining iron (Fe) ion and reactive oxygen species homeostasis is essential for cellular function, mitochondrial integrity and the regulation of cell death pathways, and is recognized as a key process underlying the molecular basis of aging and various diseases, such as diabetes, neurodegenerative diseases and cancer. Nutrient-deprivation autophagy factor 1 (NAF-1; also known as CISD2) belongs to a newly discovered class of Fe-sulfur proteins that are localized to the outer mitochondrial membrane and the endoplasmic reticulum. It has been implicated in regulating homeostasis of Fe ions, as well as the activation of autophagy through interaction with BCL-2. Here we show that small hairpin (sh)RNA-mediated suppression of NAF-1 results in the activation of apoptosis in epithelial breast cancer cells and xenograft tumors. Suppression of NAF-1 resulted in increased uptake of Fe ions into cells, a metabolic shift that rendered cells more susceptible to a glycolysis inhibitor, and the activation of cellular stress pathways that are associated with HIF1α. Our studies suggest that NAF-1 is a major player in the metabolic regulation of breast cancer cells through its effects on cellular Fe ion distribution, mitochondrial metabolism and the induction of apoptosis.

Structure-function analysis of NEET proteins uncovers their role as key regulators of iron and ROS homeostasis in health and disease.

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.

Photosynthetic reaction center-functionalized electrodes for photo-bioelectrochemical cells.

During the last few years, intensive research efforts have been directed toward the application of several highly efficient light-harvesting photosynthetic proteins, including reaction centers (RCs), photosystem I (PSI), and photosystem II (PSII), as key components in the light-triggered generation of fuels or electrical power. This review highlights recent advances for the nano-engineering of photo-bioelectrochemical cells through the assembly of the photosynthetic proteins on electrode surfaces. Various strategies to immobilize the photosynthetic complexes on conductive surfaces and different methodologies to electrically wire them with the electrode supports are presented. The different photoelectrochemical systems exhibit a wide range of photocurrent intensities and power outputs that sharply depend on the nano-engineering strategy and the electroactive components. Such cells are promising candidates for a future production of biologically-driven solar power.

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.

Nutrient-deprivation autophagy factor-1 (NAF-1): biochemical properties of a novel cellular target for anti-diabetic drugs.

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 in vitro 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.

Photosystem I (PSI)/Photosystem II (PSII)-based photo-bioelectrochemical cells revealing directional generation of photocurrents.

Layered assemblies of photosystem I, PSI, and/or photosystem II, PSII, on ITO electrodes are constructed using a layer-by-layer deposition process, where poly N,N'-dibenzyl-4,4'-bipyridinium (poly-benzyl viologen, PBV(2+) ) is used as an inter-protein "glue". While the layered assembly of PSI generates an anodic photocurrent only in the presence of a sacrificial electron donor system, such as dichlorophenol indophenol (DCPIP)/ascorbate, the PSII-modified electrode leads, upon irradiation, to the formation of an anodic photocurrent (while evolving oxygen), in the absence of any sacrificial component. The photocurrent is generated by transferring the electrons from the PSII units to the PBV(2+) redox polymer. The charge-separated species allow, then, the injection of the electrons to the electrode, with the concomitant evolution of O2 . A layered assembly, consisting of a PSI layer attached to a layer of PSII by the redox polymer PBV(2+) , leads to an anodic photocurrent that is 2-fold higher, as compared to the anodic photocurrent generated by a PSII-modified electrode. This observation is attributed to an enhanced charge separation in the two-photosystem assembly. By the further nano-engineering of the two photosystems on the electrode using two different redox polymers, vectorial electron transfer to the electrode is demonstrated, resulting in a ca. 6-fold enhancement in the photocurrent. The reversed bi-layer assembly, consisting of a PSII layer linked to a layer of PSI by the PBV(2+) redox polymer, yields, upon irradiation, an inefficient cathodic current. This observation is attributed to a mixture of photoinduced electron transfer reactions of opposing effects on the photocurrent directions in the two-photosystem assembly.

Electrochemical switching of photoelectrochemical processes at CdS QDs and photosystem I-modified electrodes.

Photoactive inorganic CdS quantum dots (QDs) or the native photosystem I (PSI) is immobilized onto a pyrroloquinoline quinone (PQQ) monolayer linked to Au electrodes to yield hybrid relay/QDs (or photosystem) assemblies. By the electrochemical biasing of the electrode potential, the relay units are retained in their oxidized PQQ or reduced PQQH(2) states. The oxidized or reduced states of the relay units dictate the direction of the photocurrent (anodic or cathodic). By the cyclic biasing of the electrode potential between the values E ≥ -0.05 V and E ≤ -0.3 V vs Ag quasi-reference electrode (Ag QRE), retaining the relay units in the oxidized PQQ or reduced PQQH(2) states, the photocurrents are respectively switched between anodic and cathodic values. Different configurations of electrically switchable photoelectrochemical systems are described: (i) the PQQ/CdS QDs/(triethanolamine, TEOA) or PQQ/PSI/(ascorbic acid/dichlorophenolindophenol, DCPIP) systems, leading to anodic photocurrents; (ii) the PQQ/CdS QDs (or PSI)/(flavin adenine dinucleotide) systems, leading to cathodic photocurrents; (iii) the PQQ/CdS QDs (or PSI)/(O(2)) switchable systems, leading to cyclic anodic/cathodic switching of the photocurrents.

Characterization of Arabidopsis NEET reveals an ancient role for NEET proteins in iron metabolism.

The NEET family is a newly discovered group of proteins involved in a diverse array of biological processes, including autophagy, apoptosis, aging, diabetes, and reactive oxygen homeostasis. They form a novel structure, the NEET fold, in which two protomers intertwine to form a two-domain motif, a cap, and a unique redox-active labile 2Fe-2S cluster binding domain. To accelerate the functional study of NEET proteins, as well as to examine whether they have an evolutionarily conserved role, we identified and characterized a plant NEET protein. Here, we show that the Arabidopsis thaliana At5g51720 protein (At-NEET) displays biochemical, structural, and biophysical characteristics of a NEET protein. Phenotypic characterization of At-NEET revealed a key role for this protein in plant development, senescence, reactive oxygen homeostasis, and Fe metabolism. A role in Fe metabolism was further supported by biochemical and cell biology studies of At-NEET in plant and mammalian cells, as well as mutational analysis of its cluster binding domain. Our findings support the hypothesis that NEET proteins have an ancient role in cells associated with Fe metabolism.

Integrated photosystem II-based photo-bioelectrochemical cells.

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.

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.

Allostery in the ferredoxin protein motif does not involve a conformational switch.

Regulation of protein function via cracking, or local unfolding and refolding of substructures, is becoming a widely recognized mechanism of functional control. Oftentimes, cracking events are localized to secondary and tertiary structure interactions between domains that control the optimal position for catalysis and/or the formation of protein complexes. Small changes in free energy associated with ligand binding, phosphorylation, etc., can tip the balance and provide a regulatory functional switch. However, understanding the factors controlling function in single-domain proteins is still a significant challenge to structural biologists. We investigated the functional landscape of a single-domain plant-type ferredoxin protein and the effect of a distal loop on the electron-transfer center. We find the global stability and structure are minimally perturbed with mutation, whereas the functional properties are altered. Specifically, truncating the L1,2 loop does not lead to large-scale changes in the structure, determined via X-ray crystallography. Further, the overall thermal stability of the protein is only marginally perturbed by the mutation. However, even though the mutation is distal to the iron-sulfur cluster (∼20 Å), it leads to a significant change in the redox potential of the iron-sulfur cluster (57 mV). Structure-based all-atom simulations indicate correlated dynamical changes between the surface-exposed loop and the iron-sulfur cluster-binding region. Our results suggest intrinsic communication channels within the ferredoxin fold, composed of many short-range interactions, lead to the propagation of long-range signals. Accordingly, protein interface interactions that involve L1,2 could potentially signal functional changes in distal regions, similar to what is observed in other allosteric systems.