Molecular insights on the coronavirus MERS-CoV interaction with the CD26 receptor.

The Middle East respiratory syndrome (MERS) is a severe respiratory disease with high fatality rates, caused by the Middle East respiratory syndrome coronavirus (MERS-CoV). The virus initiates infection by binding to the CD26 receptor (also known as dipeptidyl peptidase 4 or DPP4) via its spike protein. Although the receptor-binding domain (RBD) of the viral spike protein and the complex between RBD and the extracellular domain of CD26 have been studied using X-ray crystallography, conflicting studies exist regarding the importance of certain amino acids outside the resolved RBD-CD26 complex interaction interface. To gain atomic-level knowledge of the RBD-CD26 complex, we employed computational simulations to study the complex's dynamic behavior as it evolves from its crystal structure to a conformation stable in solution. Our study revealed previously unidentified interaction regions and interacting amino acids within the complex, determined a novel comprehensive RBD-binding domain of CD26, and by that expanded the current understanding of its structure. Additionally, we examined the impact of a single amino acid substitution, E513A, on the complex's stability. We discovered that this substitution disrupts the complex through an allosteric domino-like mechanism that affects other residues. Since MERS-CoV is a zoonotic virus, we evaluated its potential risk of human infection via animals, and suggest a low likelihood for possible infection by cats or dogs. The molecular structural information gleaned from our insights into the RBD-CD26 complex pre-dissociative states may be proved useful not only from a mechanistic view but also in assessing inter-species transmission and in developing anti-MERS-CoV antiviral therapeutics.

Cytotoxicity and reversal effect of sertraline, fluoxetine, and citalopram on MRP1- and MRP7-mediated MDR.

Cancer is one of the leading causes of death worldwide, and the development of resistance to chemotherapy drugs is a major challenge in treating malignancies. In recent years, researchers have focused on understanding the mechanisms of multidrug resistance (MDR) in cancer cells and have identified the overexpression of ATP-binding cassette (ABC) transporters, including ABCC1/MRP1 and ABCC10/MRP7, as a key factor in the development of MDR. In this study, we aimed to investigate whether three drugs (sertraline, fluoxetine, and citalopram) from the selective serotonin reuptake inhibitor (SSRI) family, commonly used as antidepressants, could be repurposed as inhibitors of MRP1 and MRP7 transporters and reverse MDR in cancer cells. Using a combination of in silico predictions and in vitro validations, we analyzed the interaction of MRP1 and MRP7 with the drugs and evaluated their ability to hinder cell resistance. We used computational tools to identify and analyze the binding site of these three molecules and determine their binding energy. Subsequently, we conducted experimental assays to assess cell viability when treated with various standard chemotherapies, both with and without the presence of SSRI inhibitors. Our results show that all three SSRI drugs exhibited inhibitory/reversal effects in the presence of chemotherapies on both MRP1-overexpressed cells and MRP7-overexpressed cells, suggesting that these medications have the potential to be repurposed to target MDR in cancer cells. These findings may open the door to using FDA-approved medications in combination therapy protocols to treat highly resistant malignancies and improve the efficacy of chemotherapy treatment. Our research highlights the importance of investigating and repurposing existing drugs to overcome MDR in cancer treatment.

Extracellular mutation induces an allosteric effect across the membrane and hampers the activity of MRP1 (ABCC1).

Dynamic conformational changes play a major role in the function of proteins, including the ATP-Binding Cassette (ABC) transporters. Multidrug Resistance Protein 1 (MRP1) is an ABC exporter that protects cells from toxic molecules. Overexpression of MRP1 has been shown to confer Multidrug Resistance (MDR), a phenomenon in which cancer cells are capable to defend themselves against a broad variety of drugs. In this study, we used varied computational techniques to explore the unique F583A mutation that is known to essentially lock the transporter in a low-affinity solute binding state. We demonstrate how macro-scale conformational changes affect MRP1's stability and dynamics, and how these changes correspond to micro-scale structural perturbations in helices 10-11 and the nucleotide-binding domains (NBDs) of the protein in regions known to be crucial for its ATPase activity. We demonstrate how a single substitution of an outward-facing aromatic amino acid causes a long-range allosteric effect that propagates across the membrane, ranging from the extracellular ECL5 loop to the cytoplasmic NBD2 over a distance of nearly 75 Å, leaving the protein in a non-functional state, and provide the putative allosteric pathway. The identified allosteric structural pathway is not only in agreement with experimental data but enhances our mechanical understanding of MRP1, thereby facilitating the rational design of chemosensitizers toward the success of chemotherapy treatments.

From virus to diabetes therapy: Characterization of a specific insulin-degrading enzyme inhibitor for diabetes treatment.

Inhibition of insulin-degrading enzyme (IDE) is a possible target for treating diabetes. However, it has not yet evolved into a medical intervention, mainly because most developed inhibitors target the zinc in IDE's catalytic site, potentially causing toxicity to other essential metalloproteases. Since IDE is a cellular receptor for the varicella-zoster virus (VZV), we constructed a VZV-based inhibitor. We computationally characterized its interaction site with IDE showing that the peptide specifically binds inside IDE's central cavity, however, not in close proximity to the zinc ion. We confirmed the peptide's effective inhibition on IDE activity in vitro and showed its efficacy in ameliorating insulin-related defects in types 1 and 2 diabetes mouse models. In addition, we suggest that inhibition of IDE may ameliorate the pro-inflammatory profile of CD4+ T-cells toward insulin. Together, we propose a potential role of a designed VZV-derived peptide to serve as a selectively-targeted and as an efficient diabetes therapy.

Overcoming multidrug resistance with nanomedicines.

INTRODUCTION: Cancer remains the leading cause of death worldwide. Numerous therapeutic strategies that include smart biological treatments toward specific cellular pathways are being developed. Yet, inherent and acquired multidrug resistance (MDR) to chemotherapeutic drugs remains the major obstacle in effective cancer treatments. AREAS COVERED: Herein, we focused on an implementation of nanoscale drug delivery strategies (nanomedicines) to treat tumors that resist MDR. Specifically, we briefly discuss the MDR phenomenon and provide structural and functional characterization of key proteins that account for MDR. We next describe the strategies to target tumors using nanoparticles and provide a mechanistic overview of how changes in the influx:efflux ratio result in overcoming MDR. EXPERT OPINION: Various strategies have been applied in preclinical and clinical settings to overcome cancer MDR. Among them are the use of chemosensitizers that aim to sensitize the cancer cells to chemotherapeutic treatment and the use of nanomedicines as delivery vehicles that can increase the influx of drugs into cancer cells. These strategies can enhance the therapeutic response in resistant tumors by bypassing efflux pumps or by increasing the nominal amounts of therapeutic payloads into the cancer cells at a given time point.

Acetylation of lysine 382 and phosphorylation of serine 392 in p53 modulate the interaction between p53 and MDC1 in vitro.

Occurrence of DNA damage in a cell activates the DNA damage response, a survival mechanism that ensures genomics stability. Two key members of the DNA damage response are the tumor suppressor p53, which is the most frequently mutated gene in cancers, and MDC1, which is a central adaptor that recruits many proteins to sites of DNA damage. Here we characterize the in vitro interaction between p53 and MDC1 and demonstrate that p53 and MDC1 directly interact. The p53-MDC1 interaction is mediated by the tandem BRCT domain of MDC1 and the C-terminal domain of p53. We further show that both acetylation of lysine 382 and phosphorylation of serine 392 in p53 enhance the interaction between p53 and MDC1. Additionally, we demonstrate that the p53-MDC1 interaction is augmented upon the induction of DNA damage in human cells. Our data suggests a new role for acetylation of lysine 382 and phosphorylation of serine 392 in p53 in the cellular stress response and offers the first evidence for an interaction involving MDC1 that is modulated by acetylation.

Ubiquitin: molecular modeling and simulations.

The synthesis and destruction of proteins are imperative for maintaining their cellular homeostasis. In the 1970s, Aaron Ciechanover, Avram Hershko, and Irwin Rose discovered that certain proteins are tagged by ubiquitin before degradation, a discovery that awarded them the 2004 Nobel Prize in Chemistry. Compelling data gathered during the last several decades show that ubiquitin plays a vital role not only in protein degradation but also in many cellular functions including DNA repair processes, cell cycle regulation, cell growth, immune system functionality, hormone-mediated signaling in plants, vesicular trafficking pathways, regulation of histone modification and viral budding. Due to the involvement of ubiquitin in such a large number of diverse cellular processes, flaws and impairments in the ubiquitin system were found to be linked to cancer, neurodegenerative diseases, genetic disorders, and immunological disorders. Hence, deciphering the dynamics and complexity of the ubiquitin system is of significant importance. In addition to experimental techniques, computational methodologies have been gaining increasing influence in protein research and are used to uncover the structure, stability, folding, mechanism of action and interactions of proteins. Notably, molecular modeling and molecular dynamics simulations have become powerful tools that bridge the gap between structure and function while providing dynamic insights and illustrating essential mechanistic characteristics. In this study, we present an overview of molecular modeling and simulations of ubiquitin and the ubiquitin system, evaluate the status of the field, and offer our perspective on future progress in this area of research.

Myosin V movement: lessons from molecular dynamics studies of IQ peptides in the lever arm.

Myosin V moves along actin filaments by an arm-over-arm motion, known as the lever mechanism. Each of its arms is composed of six consecutive IQ peptides that bind light chain proteins, such as calmodulin or calmodulin-like proteins. We have employed a multistage approach in order to investigate the mechanochemical structural basis of the movement of myosin V from the budding yeast Saccharomyces cerevisiae. For that purpose, we previously carried out molecular dynamics simulations of the Mlc1p-IQ2 and the Mlc1p-IQ4 protein-peptide complexes, and the present study deals with the structures of the IQ peptides when stripped from the Mlc1p protein. We have found that the crystalline structure of the IQ2 peptide retains a stable rodlike configuration in solution, whereas that of the IQ4 peptide grossly deviates from its X-ray conformation exhibiting an intrinsic tendency to curve and bend. The refolding process of the IQ4 peptide is initially driven by electrostatic interactions followed by nonpolar stabilization. Its bending appears to be affected by the ionic strength, when ionic strength higher than approximately 300 mM suppresses it from flexing. Considering that a poly-IQ sequence is the lever arm of myosin V, we suggest that the arm may harbor a joint, localized within the IQ4 sequence, enabling the elasticity of the neck of myosin V. Given that a poly-IQ sequence is present at the entire class of myosin V and the possibility that the yeast's myosin V molecule can exist either as a nonprocessive monomer or as a processive dimer depending on conditions (Krementsova, E. B., Hodges, A. R., Lu, H., and Trybus, K. M. (2006) J. Biol. Chem. 281, 6079-6086), our observations may account for a general structural feature for the myosins' arm embedded flexibility.

A molecular dynamics study and free energy analysis of complexes between the Mlc1p protein and two IQ motif peptides.

The Mlc1p protein from the budding yeast Saccharomyces cerevisiae is a Calmodulin-like protein, which interacts with IQ-motif peptides located at the yeast's myosin neck. In this study, we report a molecular dynamics study of the Mlc1p-IQ2 protein-peptide complex, starting with its crystal structure, and investigate its dynamics in an aqueous solution. The results are compared with those obtained by a previous study, where we followed the solution structure of the Mlc1p-IQ4 protein-peptide complex by molecular dynamics simulations. After the simulations, we performed an interaction free-energy analysis using the molecular mechanics Poisson-Boltzmann surface area approach. Based on the dynamics of the Mlc1p-IQ protein-peptide complexes, the structure of the light-chain-binding domain of myosin V from the yeast S. cerevisiae is discussed.

Molecular dynamics study of a calmodulin-like protein with an IQ peptide: spontaneous refolding of the protein around the peptide.

The Calmodulin (CaM) is a small (16.7 kDa), highly acidic protein that is crucial to all eukaryotes by serving as a prototypical calcium sensor. In the present study, we investigated, through molecular dynamics simulations, the dynamics of a complex between the Mlc1p protein, which is a CaM-like protein, and the IQ4 peptide. This protein-peptide interaction is of high importance because IQ motifs are widely distributed among different kinds of CaM-binding proteins. The Mlc1p-IQ4 complex, which had been resolved by crystallography to 2.1 A, confers to a Ca(+2)-independent stable structure. During the simulations, the complex undergoes a complicated modulation process, which involves bending of the angles between the alpha-helices of the protein, breaking of the alpha-helical structure of the IQ4 peptide into two sections, and formation of new contact points between the protein and the peptide. The dynamics of the process consist of fast sub picosecond events and much slower ones that take a few nanoseconds to completion. Our study expands the information embedded in the crystal structure of the Mlc1p-IQ4 complex by describing its dynamic behavior as it evolves from the crystal structure to a form stable in solution. The article shows that careful application of molecular dynamics simulations can be used for extending the structural information presented by the crystal structure, thereby revealing the dynamic configuration of the protein in its physiological environment.