CISD3/MiNT is required for complex I function, mitochondrial integrity, and skeletal muscle maintenance.

Mitochondria play a central role in muscle metabolism and function. A unique family of iron-sulfur proteins, termed CDGSH Iron Sulfur Domain-containing (CISD/NEET) proteins, support mitochondrial function in skeletal muscles. The abundance of these proteins declines during aging leading to muscle degeneration. Although the function of the outer mitochondrial CISD/NEET proteins, CISD1/mitoNEET and CISD2/NAF-1, has been defined in skeletal muscle cells, the role of the inner mitochondrial CISD protein, CISD3/MiNT, is currently unknown. Here, we show that CISD3 deficiency in mice results in muscle atrophy that shares proteomic features with Duchenne muscular dystrophy. We further reveal that CISD3 deficiency impairs the function and structure of skeletal muscles, as well as their mitochondria, and that CISD3 interacts with, and donates its [2Fe-2S] clusters to, complex I respiratory chain subunit NADH Ubiquinone Oxidoreductase Core Subunit V2 (NDUFV2). Using coevolutionary and structural computational tools, we model a CISD3-NDUFV2 complex with proximal coevolving residue interactions conducive of [2Fe-2S] cluster transfer reactions, placing the clusters of the two proteins 10 to 16 Å apart. Taken together, our findings reveal that CISD3/MiNT is important for supporting the biogenesis and function of complex I, essential for muscle maintenance and function. Interventions that target CISD3 could therefore impact different muscle degeneration syndromes, aging, and related conditions.

Induction of apoptosis by double-stranded RNA was present in the last common ancestor of cnidarian and bilaterian animals.

Apoptosis, a major form of programmed cell death, is an essential component of host defense against invading intracellular pathogens. Viruses encode inhibitors of apoptosis to evade host responses during infection, and to support their own replication and survival. Therefore, hosts and their viruses are entangled in a constant evolutionary arms race to control apoptosis. Until now, apoptosis in the context of the antiviral immune system has been almost exclusively studied in vertebrates. This limited phyletic sampling makes it impossible to determine whether a similar mechanism existed in the last common ancestor of animals. Here, we established assays to probe apoptosis in the sea anemone Nematostella vectensis, a model species of Cnidaria, a phylum that diverged approximately 600 million years ago from the rest of animals. We show that polyinosinic:polycytidylic acid (poly I:C), a synthetic long double-stranded RNA mimicking viral RNA and a primary ligand for the vertebrate RLR melanoma differentiation-associated protein 5 (MDA5), is sufficient to induce apoptosis in N. vectensis. Furthermore, at the transcriptomic level, apoptosis related genes are significantly enriched upon poly(I:C) exposure in N. vectensis as well as bilaterian invertebrates. Our phylogenetic analysis of caspase family genes in N. vectensis reveals conservation of all four caspase genes involved in apoptosis in mammals and revealed a cnidarian-specific caspase gene which was strongly upregulated. Altogether, our findings suggest that apoptosis in response to a viral challenge is a functionally conserved mechanism that can be traced back to the last common ancestor of Bilateria and Cnidaria.

A VDAC1-mediated NEET protein chain transfers [2Fe-2S] clusters between the mitochondria and the cytosol and impacts mitochondrial dynamics.

Mitochondrial inner NEET (MiNT) and the outer mitochondrial membrane (OMM) mitoNEET (mNT) proteins belong to the NEET protein family. This family plays a key role in mitochondrial labile iron and reactive oxygen species (ROS) homeostasis. NEET proteins contain labile [2Fe-2S] clusters which can be transferred to apo-acceptor proteins. In eukaryotes, the biogenesis of [2Fe-2S] clusters occurs within the mitochondria by the iron-sulfur cluster (ISC) system; the clusters are then transferred to [2Fe-2S] proteins within the mitochondria or exported to cytosolic proteins and the cytosolic iron-sulfur cluster assembly (CIA) system. The last step of export of the [2Fe-2S] is not yet fully characterized. Here we show that MiNT interacts with voltage-dependent anion channel 1 (VDAC1), a major OMM protein that connects the intermembrane space with the cytosol and participates in regulating the levels of different ions including mitochondrial labile iron (mLI). We further show that VDAC1 is mediating the interaction between MiNT and mNT, in which MiNT transfers its [2Fe-2S] clusters from inside the mitochondria to mNT that is facing the cytosol. This MiNT-VDAC1-mNT interaction is shown both experimentally and by computational calculations. Additionally, we show that modifying MiNT expression in breast cancer cells affects the dynamics of mitochondrial structure and morphology, mitochondrial function, and breast cancer tumor growth. Our findings reveal a pathway for the transfer of [2Fe-2S] clusters, which are assembled inside the mitochondria, to the cytosol.

Spatially Localized Entropy-Driven Evolution of Nucleic Acid-Based Constitutional Dynamic Networks for Intracellular Imaging and Spatiotemporal Programmable Gene Therapy.

The primer-guided entropy-driven high-throughput evolution of the DNA-based constitutional dynamic network, CDN, is introduced. The entropy gain associated with the process provides a catalytic principle for the amplified emergence of the CDN. The concept is applied to develop a programmable, spatially localized DNA circuit for effective in vitro and in vivo theranostic, gene-regulated treatment of cancer cells. The localized circuit consists of a DNA tetrahedron core modified at its corners with four tethers that include encoded base sequences exhibiting the capacity to emerge and assemble into a [2 × 2] CDN. Two of the tethers are caged by a pair of siRNA subunits, blocking the circuit into a mute, dynamically inactive configuration. In the presence of miRNA-21 as primer, the siRNA subunits are displaced, resulting in amplified release of the siRNAs silencing the HIF-1α mRNA and fast dynamic reconfiguration of the tethers into a CDN. The resulting CDN is, however, engineered to be dynamically reconfigured by miRNA-155 into an equilibrated mixture enriched with a DNAzyme component, catalyzing the cleavage of EGR-1 mRNA. The DNA tetrahedron nanostructure stimulates enhanced permeation into cancer cells. The miRNA-triggered entropy-driven reconfiguration of the spatially localized circuit leads to the programmable, cooperative bis-gene-silencing of HIF-1α and EGR-1 mRNAs, resulting in the effective and selective apoptosis of breast cancer cells and effective inhibition of tumors in tumor bearing mice.

DNA-Tetrahedra Corona-Modified Hydrogel Microcapsules: "Smart" ATP- or microRNA-Responsive Drug Carriers.

The assembly of adenosine triphosphate (ATP)-responsive and miRNA-responsive DNA tetrahedra-functionalized carboxymethyl cellulose hydrogel microcapsules is presented. The microcapsules are loaded with the doxorubicin-dextran drug or with CdSe/ZnS quantum dots as a drug model. Selective unlocking of the respective microcapsules and the release of the loads in the presence of ATP or miRNA-141 are demonstrated. Functionalization of the hydrogel microcapsules a with corona of DNA tetrahedra nanostructures yields microcarriers that revealed superior permeation into cells. This is demonstrated by the effective permeation of the DNA tetrahedra-functionalized microcapsules into MDA-MB-231 breast cancer cells, as compared to epithelial MCF-10A nonmalignant breast cells. The superior permeation of the tetrahedra-functionalized microcapsules into MDA-MB-231 breast cancer cells, as compared to analog control hydrogel microcapsules modified with a corona of nucleic acid duplexes. The effective permeation of the stimuli-responsive, drug-loaded, DNA tetrahedra-modified microcapsules yields drug carriers of superior and selective cytotoxicity toward cancer cells.

Chemical targeting of NEET proteins reveals their function in mitochondrial morphodynamics.

Several human pathologies including neurological, cardiac, infectious, cancerous, and metabolic diseases have been associated with altered mitochondria morphodynamics. Here, we identify a small organic molecule, which we named Mito-C. Mito-C is targeted to mitochondria and rapidly provokes mitochondrial network fragmentation. Biochemical analyses reveal that Mito-C is a member of a new class of heterocyclic compounds that target the NEET protein family, previously reported to regulate mitochondrial iron and ROS homeostasis. One of the NEET proteins, NAF-1, is identified as an important regulator of mitochondria morphodynamics that facilitates recruitment of DRP1 to the ER-mitochondria interface. Consistent with the observation that certain viruses modulate mitochondrial morphogenesis as a necessary part of their replication cycle, Mito-C counteracts dengue virus-induced mitochondrial network hyperfusion and represses viral replication. The newly identified chemical class including Mito-C is of therapeutic relevance for pathologies where altered mitochondria dynamics is part of disease etiology and NEET proteins are highlighted as important therapeutic targets in anti-viral research.

Structure of the human monomeric NEET protein MiNT and its role in regulating iron and reactive oxygen species in cancer cells.

The NEET family is a relatively new class of three related [2Fe-2S] proteins (CISD1-3), important in human health and disease. While there has been growing interest in the homodimeric gene products of CISD1 (mitoNEET) and CISD2 (NAF-1), the importance of the inner mitochondrial CISD3 protein has only recently been recognized in cancer. The CISD3 gene encodes for a monomeric protein that contains two [2Fe-2S] CDGSH motifs, which we term mitochondrial inner NEET protein (MiNT). It folds with a pseudosymmetrical fold that provides a hydrophobic motif on one side and a relatively hydrophilic surface on the diametrically opposed surface. Interestingly, as shown by molecular dynamics simulation, the protein displays distinct asymmetrical backbone motions, unlike its homodimeric counterparts that face the cytosolic side of the outer mitochondrial membrane/endoplasmic reticulum (ER). However, like its counterparts, our biological studies indicate that knockdown of MiNT leads to increased accumulation of mitochondrial labile iron, as well as increased mitochondrial reactive oxygen production. Taken together, our study suggests that the MiNT protein functions in the same pathway as its homodimeric counterparts (mitoNEET and NAF-1), and could be a key player in this pathway within the mitochondria. As such, it represents a target for anticancer or antidiabetic drug development.

Breast cancer tumorigenicity is dependent on high expression levels of NAF-1 and the lability of its Fe-S clusters.

Iron-sulfur (Fe-S) proteins are thought to play an important role in cancer cells mediating redox reactions, DNA replication, and telomere maintenance. Nutrient-deprivation autophagy factor-1 (NAF-1) is a 2Fe-2S protein associated with the progression of multiple cancer types. It is unique among Fe-S proteins because of its 3Cys-1His cluster coordination structure that allows it to be relatively stable, as well as to transfer its clusters to apo-acceptor proteins. Here, we report that overexpression of NAF-1 in xenograft breast cancer tumors results in a dramatic augmentation in tumor size and aggressiveness and that NAF-1 overexpression enhances the tolerance of cancer cells to oxidative stress. Remarkably, overexpression of a NAF-1 mutant with a single point mutation that stabilizes the NAF-1 cluster, NAF-1(H114C), in xenograft breast cancer tumors results in a dramatic decrease in tumor size that is accompanied by enhanced mitochondrial iron and reactive oxygen accumulation and reduced cellular tolerance to oxidative stress. Furthermore, treating breast cancer cells with pioglitazone that stabilizes the 3Cys-1His cluster of NAF-1 results in a similar effect on mitochondrial iron and reactive oxygen species accumulation. Taken together, our findings point to a key role for the unique 3Cys-1His cluster of NAF-1 in promoting rapid tumor growth through cellular resistance to oxidative stress. Cluster transfer reactions mediated by the overexpressed NAF-1 protein are therefore critical for inducing oxidative stress tolerance in cancer cells, leading to rapid tumor growth, and drugs that stabilize the NAF-1 cluster could be used as part of a treatment strategy for cancers that display high NAF-1 expression.

Thrombin Aptamer-Modified Metal-Organic Framework Nanoparticles: Functional Nanostructures for Sensing Thrombin and the Triggered Controlled Release of Anti-Blood Clotting Drugs.

This paper features the synthesis of thrombin-responsive, nucleic acid-gated, UiO-68 metal-organic framework nanoparticles (NMOFs) loaded with the drug Apixaban or rhodamine 6G as a drug model. Apixaban acts as an inhibitor of blood clots formation. The loads in the NMOFs are locked by duplex nucleic acids that are composed of anchor nucleic acids linked to the NMOFs that are hybridized with the anti-thrombin aptamer. In the presence of thrombin, the duplex gating units are separated through the formation of thrombin-aptamer complexes. The unlocking of the NMOFs releases the drug (or the drug model). The release of the drug is controlled by the concentration of thrombin. The Apixaban-loaded NMOFs revealed improved inhibition, as compared to free Apixaban, toward blood clot formation. This is reflected by their longer time intervals for inducing clot formation and the decreased doses of the drug required to affect clots formation. The beneficial effects of the Apixaban-loaded NMOFs are attributed to the slow-release mechanism induced by the NMOFs carriers, where the inhibition of factor Xa in the blood clotting cycle retards the formation of thrombin, which slows down the release of the drug.

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.

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

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.

CISD3 is required for Complex I function, mitochondrial integrity, and skeletal muscle maintenance.

Mitochondria play a central role in muscle metabolism and function. In skeletal muscles, a unique family of iron-sulfur proteins, termed CISD proteins, support mitochondrial function. The abundance of these proteins declines with aging leading to muscle degeneration. Although the function of the outer mitochondrial proteins CISD1 and CISD2 has been defined, the role of the inner mitochondrial protein CISD3, is currently unknown. Here we show that CISD3 deficiency in mice results in muscle atrophy that shares proteomic features with Duchenne Muscular Dystrophy. We further reveal that CISD3 deficiency impairs the function and structure of skeletal muscle mitochondria, and that CISD3 interacts with, and donates its clusters to, Complex I respiratory chain subunit NDUFV2. These findings reveal that CISD3 is important for supporting the biogenesis and function of Complex I, essential for muscle maintenance and function. Interventions that target CISD3 could therefore impact muscle degeneration syndromes, aging, and related conditions.

A combination of a cell penetrating peptide and a protein translation inhibitor kills metastatic breast cancer cells.

Cell Penetrating Peptides (CPPs) are promising anticancer and antimicrobial drugs. We recently reported that a peptide derived from the human mitochondrial/ER membrane-anchored NEET protein, Nutrient Autophagy Factor 1 (NAF-1; NAF-144-67), selectively permeates and kills human metastatic epithelial breast cancer cells (MDA-MB-231), but not control epithelial cells. As cancer cells alter their phenotype during growth and metastasis, we tested whether NAF-144-67 would also be efficient in killing other human epithelial breast cancer cells that may have a different phenotype. Here we report that NAF-144-67 is efficient in killing BT-549, Hs 578T, MDA-MB-436, and MDA-MB-453 breast cancer cells, but that MDA-MB-157 cells are resistant to it. Upon closer examination, we found that MDA-MB-157 cells display a high content of intracellular vesicles and cellular protrusions, compared to MDA-MB-231 cells, that could protect them from NAF-144-67. Inhibiting the formation of intracellular vesicles and dynamics of cellular protrusions of MDA-MB-157 cells, using a protein translation inhibitor (the antibiotic Cycloheximide), rendered these cells highly susceptible to NAF-144-67, suggesting that under certain conditions, the killing effect of CPPs could be augmented when they are applied in combination with an antibiotic or chemotherapy agent. These findings could prove important for the treatment of metastatic cancers with CPPs and/or treatment combinations that include CPPs.

Biocatalytic cascades and intercommunicated biocatalytic cascades in microcapsule systems.

Biomolecule-loaded nucleic acid-functionalized carboxymethyl cellulose hydrogel-stabilized microcapsules (diameter ca. 2 µm) are introduced as cell-like containments. The microcapsules are loaded with two DNA tetrahedra, T1 and T2, functionalized with guanosine-rich G-quadruplex subunits, and/or with native enzymes (glucose oxidase, GOx, and/or ß-galactosidase, ß-gal). In the presence of K+-ions and hemin, the T1/T2 tetrahedra constituents, loaded in the microcapsules, assemble into a hemin/G-quadruplex bridged tetrahedra dimer DNAzyme catalyzing the oxidation of Amplex Red to Resorufin by generating H2O2. In the presence of co-loaded GOx or GOx/ß-gal, the GOx//T1/T2 hemin/G-quadruplex cascade catalyzing the glucose-mediated oxidation of Amplex Red to Resorufin, and the three-biocatalysts cascade consisting of ß-gal//GOx//hemin/G-quadruplex bridged T1/T2 catalyzing the lactose-driven oxidation of Amplex Red to Resorufin proceed in the microcapsules. Enhanced biocatalytic transformations in the microcapsules, as compared to the performance of the reactions in a homogeneous phase, are observed, due to the proximity of the biocatalysts in a confined volume. As the synthetic methodology to prepare the microcapsules yields boundaries functionalized with complementary nucleic acid tethers, the dynamic association of different microcapsules, loaded selectively with biomolecular catalysts, proceeds. The dynamic dimerization of GOx-loaded microcapsules and hemin/G-quadruplex bridged T1/T2 DNAzyme-loaded microcapsules yields effective intercommunicated microcapsules driving the GOx//hemin/G-quadruplex bridged T1/T2 DNAzyme cascade. In addition, the dynamic dimerization of GOx-loaded microcapsules with ß-gal//hemin/G-quadruplex bridged T1/T2-loaded microcapsules enables the bi-directional intercommunicated operation of the lactose-stimulated three catalysts ß-gal//GOx//hemin/G-quadruplex bridged T1/T2 DNAzyme cascade. The guided separation and formation of dynamic supramolecular dimer microcapsular containments, and the dictated switchable operation of intercommunicated biocatalytic cascades are demonstrated.

miRNA-Guided Imaging and Photodynamic Therapy Treatment of Cancer Cells Using Zn(II)-Protoporphyrin IX-Loaded Metal-Organic Framework Nanoparticles.

An analytical platform for the selective miRNA-21-guided imaging of breast cancer cells and miRNA-221-guided imaging of ovarian cancer cells and the selective photodynamic therapy (PDT) of these cancer cells is introduced. The method is based on Zn(II)-protoporphyrin IX, Zn(II)-PPIX-loaded UiO-66 metal-organic framework nanoparticles, NMOFs, gated by two hairpins Hi/Hj through ligation of their phosphate residues to the vacant Zr4+-ions associated with the NMOFs. The hairpins are engineered to include the miRNA recognition sequence in the stem domain of Hi, and in the Hi and Hj, partial locked stem regions of G-quadruplex subunits. Intracellular phosphate-ions displace the hairpins, resulting in the release of the Zn(II)-PPIX and intracellular miRNAs open Hi, and this triggers the autonomous cross-opening of Hi and Hj. This activates the interhairpin hybridization chain reaction and leads to the assembly of highly fluorescent Zn(II)-PPIX-loaded G-quadruplex chains. The miRNA-guided fluorescent chains allow selective imaging of cancer cells. Moreover, PDT with visible light selectively kills cancer cells and tumor cells through the formation of toxic reactive oxygen species.

The [2Fe-2S] protein CISD2 plays a key role in preventing iron accumulation in cardiomyocytes.

Considered a key aging gene, CISD2, encoding CDGSH iron-sulfur domain-containing protein 2, plays a central role in regulating calcium homeostasis, preventing mitochondrial dysfunction, and the activation of autophagy and apoptosis in different cells. Here, we show that cardiomyocytes from CISD2-null mice accumulate high levels of iron and contain high levels of transferrin receptor and ferritin. Using proteomics and transmission electron microscopy, we further show that the lack of CISD2 induces several features of the aging process in young mice, but other features are not induced. Taken together, our findings suggest that CISD2 protects cardiomyocytes from overaccumulation of iron, which is common in aging hearts and can contribute to the pathogenesis of heart failure.

Aptamer-Functionalized Ce4+-Ion-Modified C-Dots: Peroxidase Mimicking Aptananozymes for the Oxidation of Dopamine and Cytotoxic Effects toward Cancer Cells.

Aptamer-functionalized Ce4+-ion-modified C-dots act as catalytic hybrid systems, aptananozymes, catalyzing the H2O2 oxidation of dopamine. A series of aptananozymes functionalized with different configurations of the dopamine binding aptamer, DBA, are introduced. All aptananozymes reveal substantially enhanced catalytic activities as compared to the separated Ce4+-ion-modified C-dots and aptamer constituents, and structure-catalytic functions between the structure and binding modes of the aptamers linked to the C-dots are demonstrated. The enhanced catalytic functions of the aptananozymes are attributed to the aptamer-induced concentration of the reaction substrates in spatial proximity to the Ce4+-ion-modified C-dots catalytic sites. The oxidation processes driven by the Ce4+-ion-modified C-dots involve the formation of reactive oxygen species (•OH radicals). Accordingly, Ce4+-ion-modified C-dots with the AS1411 aptamer or MUC1 aptamer, recognizing specific biomarkers associated with cancer cells, are employed as targeted catalytic agents for chemodynamic treatment of cancer cells. Treatment of MDA-MB-231 breast cancer cells and MCF-10A epithelial breast cells, as control, with the AS1411 aptamer- or MUC1 aptamer-modified Ce4+-ion-modified C-dots reveals selective cytotoxicity toward the cancer cells. In vivo experiments reveal that the aptamer-functionalized nanoparticles inhibit MDA-MB-231 tumor growth.

Aptamer-Modified Au Nanoparticles: Functional Nanozyme Bioreactors for Cascaded Catalysis and Catalysts for Chemodynamic Treatment of Cancer Cells.

Polyadenine-stabilized Au nanoparticles (pA-AuNPs) reveal dual nanozyme catalytic activities toward the H2O2-mediated oxidation of dopamine to aminochrome and toward the aerobic oxidation of glucose to gluconic acid and H2O2. The conjugation of a dopamine-binding aptamer (DBA) to the pA-AuNPs yields aptananozyme structures catalyzing simultaneously the H2O2-mediated oxidation of dopamine to aminochrome through the aerobic oxidation of glucose. A set of aptananozymes consisting of DBA conjugated through the 5'- or 3'-end directly or spacer bridges to pA-AuNPs were synthesized. The set of aptananozymes revealed enhanced catalytic activities toward the H2O2-catalyzed oxidation of dopamine to dopachrome, as compared to the separated pA-AuNPs and DBA constituents, and structure-function relationships within the series of aptananozymes were demonstrated. The enhanced catalytic function of the aptananozymes was attributed to the concentration of the dopamine at the catalytic interfaces by means of aptamer-dopamine complexes. The dual catalytic activities of aptananozymes were further applied to design bioreactors catalyzing the effective aerobic oxidation of dopamine in the presence of glucose. Mechanistic studies demonstrated that the aptananozymes generate reactive oxygen species. Accordingly, the AS1411 aptamer, recognizing the nucleolin receptor associated with cancer cells, was conjugated to the pA-AuNPs, yielding a nanozyme for the chemodynamic treatment of cancer cells. The AS1411 aptamer targets the aptananozyme to the cancer cells and facilitates the selective permeation of the nanozyme into the cells. Selective cytotoxicity toward MDA-MB-231 breast cancer cells (ca. 70% cell death) as compared to MCF-10A epithelial cells (ca. 2% cell death) is demonstrated.

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.

A peptide-derived strategy for specifically targeting the mitochondria and ER of cancer cells: a new approach in fighting cancer.

An effective anti-cancer therapy should exclusively target cancer cells and trigger in them a broad spectrum of cell death pathways that will prevent avoidance. Here, we present a new approach in cancer therapy that specifically targets the mitochondria and ER of cancer cells. We developed a peptide derived from the flexible and transmembrane domains of the human protein NAF-1/CISD2. This peptide (NAF-144-67) specifically permeates through the plasma membranes of human epithelial breast cancer cells, abolishes their mitochondria and ER, and triggers cell death with characteristics of apoptosis, ferroptosis and necroptosis. In vivo analysis revealed that the peptide significantly decreases tumor growth in mice carrying xenograft human tumors. Computational simulations of cancer vs. normal cell membranes reveal that the specificity of the peptide to cancer cells is due to its selective recognition of their membrane composition. NAF-144-67 represents a promising anti-cancer lead compound that acts via a unique mechanism.