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

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

Abstract 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

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 diseas

The balancing act of NEET proteins: Iron, ROS, calcium and metabolism

NEET proteins belong to a highly conserved group of [2Fe–2S] proteins found across all kingdoms of life. Due to their unique [2Fesingle bond2S] cluster structure, they play a key role in the regulation of many different redox and oxidation processes. In eukaryotes, NEET proteins are localized to the mitochondria, endoplasmic reticulum (ER) and the mitochondrial-associated membranes connecting these organelles (MAM), and are involved in the control of multiple processes, ranging from autophagy and apoptosis to ferroptosis, oxidative stress, cell proliferation, redox control and iron and iron‑sulfur homeostasis. Through their different functions and interactions with key proteins such as VDAC and Bcl-2, NEET proteins coordinate different mitochondrial, MAM, ER and cytosolic processes and functions and regulate major signaling molecules such as calcium and reactive oxygen species. Owing to their central role in cells, NEET proteins are associated with numerous human maladies including cancer, metabolic diseases, diabetes, obesity, and neurodegenerative diseases. In recent years, a new and exciting role for NEET proteins was uncovered, i.e., the regulation of mitochondrial dynamics and morphology. This new role places NEET proteins at the forefront of studies into cancer and different metabolic diseases, both associated with the regulation of mitochondrial dynamics. Here we review recent studies focused on the evolution, biological role, and structure of NEET proteins, as well as discuss different studies conducted on NEET proteins function using transgenic organisms. We further discuss the different strategies used in the development of drugs that target NEET proteins, and link these with the different roles of NEET proteins in cells

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

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