Competition For Binding Sites Research Articles

Study on the Binding of Methylphenanthrene Isomers with Different Methylated Positions to Human Serum Albumin Employing Spectroscopic Techniques Combined with Molecular Docking

The interactions of methylphenanthrene (MP) isomers (1-MP, 3-MP, 4-MP, and 9-MP) with human serum albumin (HSA) were studied in simulated physiological conditions. A fluorescence analysis revealed that each MP formed a 1:1 ground-state complex with HSA. The binding constants at 308 K decreased in the order of 3-MP (9.13 × 104 L·mol−1) > 9-MP (3.57 × 104 L·mol−1) > 4-MP (2.37 × 104 L·mol−1) > 1-MP (1.46 × 104 L·mol−1), which was consistent with the docking results. The K b values at 298 K, 308 K, and 318 K revealed that the β-isomer has a stronger binding affinity with HSA than the α-isomer. Thermodynamic and docking parameters showed that MPs combined with HSA through van der Waals forces. The results of the binding site competition experiment and molecular docking indicated that 4-MP bound at subdomain IIA in HSA molecule, while the other MPs bound at a site between subdomains IB and IIA. The circular dichroism (CD) spectra showed that the α-helical content of HSA reduced in the presence of 1-MP, 4-MP and 9-MP, while no significant change was observed in the presence of 3-MP. Further, the fluorescence spectroscopic studies revealed that the Vitamin B2 (VB2)-transporting function of HSA was inhibited by 3-MP and 9-MP, and enhanced by 1-MP and 4-MP. Moreover, the impact degrees of MPs on the VB2-transporting function had a positive correlation with the binding affinities of the MPs to HSA. Highlights Interactions of MP isomers methylated at varying sites with HSA were studied. The α-substituted MP had a weaker affinity to HSA than β-substituted MP. Binding site of 4-MP in HSA was different from that of 1-MP/3-MP/9-MP. MP isomers unfolded the α-helical structure of HSA to varying degrees. Transporting function of HSA was inhibited by 3-MP/9-MP but enhanced by 1-MP/4-MP.

Vi4-miR-185-5p-Igfbp3 Network Protects the Brain From Neonatal Hypoxic Ischemic Injury via Promoting Neuron Survival and Suppressing the Cell Apoptosis.

Neonatal hypoxic ischemic encephalopathy (HIE) due to birth asphyxia is common and causes severe neurological deficits, without any effective therapies currently available. Neuronal death is an important driving factors of neurological disorders after HIE, but the regulatory mechanisms are still uncertain. Long non-coding RNA (lncRNA) or ceRNA network act as a significant regulator in neuroregeneration and neuronal apoptosis, thus owning a great potential as therapeutic targets in HIE. Here, we found a new lncRNA, is the most functional in targeting the Igfbp3 gene in HIE, which enriched in the cell growth and cell apoptosis processes. In addition, luciferase reporter assay showed competitive regulatory binding sites to the target gene Igfbp3 between TCONS00044054 (Vi4) and miR-185-5p. The change in blood miR-185-5p and Igfbp3 expression is further confirmed in patients with brain ischemia. Moreover, Vi4 overexpression and miR-185-5p knock-out promote the neuron survival and neurite growth, and suppress the cell apoptosis, then further improve the motor and cognitive deficits in rats with HIE, while Igfbp3 interfering got the opposite results. Together, Vi4-miR-185-5p-Igfbp3 regulatory network plays an important role in neuron survival and cell apoptosis and further promote the neuro-functional recovery from HIE, therefore is a likely a drug target for HIE therapy.

Insight into the Dual inhibitory Mechanism of verbascoside targeting serine/threonine phosphatase Stp1 against Staphylococcus aureus

The eukaryotic-like serine/threonine phosphatase (Stp1) is an enzyme-dependent protein phosphatase involved in regulating various virulence factors of Staphylococcus aureus. Owing to its role in S. aureus infections, Stp1 has become a potential target for antibiotic development. Unfortunately, there are very few reports describing Stp1 inhibitors. Using virtual screening, we have identified a potent and effective Stp1 inhibitor, verbascoside (VBS). Interestingly, the kinetics of the enzymatic reaction revealed that this natural inhibitor acts via both competitive and allosteric mechanisms. To explore the mechanism of interaction between VBS and Stp1, standard molecular dynamics (MD) simulations were performed for the Stp1-VBS complex. Consistent with the experimental results, competitive and allosteric binding sites for VBS were identified in Stp1. Met39, Gly41, His42, Arg161, and Asn162 residues were involved in the competitive binding of VBS, while Arg122, Ser136, Asp137, Asn142, and Val145 residues were associated with the allosteric binding of VBS. The contributions of these residues were confirmed by amino acid site-directed mutagenesis and fluorescence quenching experiments. This work demonstrates that VBS is a potent anti-virulence compound against S. aureus infection, laying the foundation for the further development of novel anti-virulence agents.

Studies on Structures and Functions of Kinases leading to Prostate Cancer and Their Inhibitors

Background:Cancer is the uncontrolled growth of abnormal cells in any part of the body. These abnormalities in the cells make them cancer cells, malignant cells, or tumour cells. These cells can infiltrate normal body tissues. Prostate Cancer begins when cells in the prostate gland start to grow out of control.Introduction:According to the National Cancer Institute, an estimated 20 percent of men experience Prostate Cancer in their lifetimes. Prostate Cancer can be divided into castration sensitive or hormone- sensitive Prostate Cancer (CSPC or HSPC) and castration-resistant Prostate Cancer (CRPC). Different therapies showed potential for the treatment of Prostate Cancer in that androgen receptor antagonist, cytochrome p17 inhibitors, radiation therapy, brachytherapy, surgical removal of the gland, androgen deprivation therapy and LnRH antagonists are some of the important ones. Despite various available treatment options in our understanding of the biological basis of Prostate Cancer, the management of the disease, especially in the castration-resistant phase, remains a significant challenge. Several Tyrosine kinase inhibitors (TKIs) have been evaluated in the preclinical setting in Advanced Prostate Cancer. TKIs are small drug molecules that work by competitive ATP inhibition at the catalytic binding site of tyrosine kinase. This results in complete inhibition of the catalytic activity of certain enzymes. If chosen correctly, TKIs can target and inhibit critical, mutated pathways important for the development, progression and metastasis of Prostate Cancer. The review focuses on various tyrosine kinase drug targets and their chemical structure to discuss the mechanism and pathways in the treatment of Prostate Cancer.Methods:The method adopted for the study was mainly based on the secondary search through a systematic literature review. Targets discussed in this review include the epidermal growth factor family (EGFR), vascular endothelial growth factor family (VEGF) receptor, c-Src family kinases (Proto-oncogene tyrosine-protein kinase) (c-Src), platelet-derived growth factor (PDGF) and cmesenchymal- epithelial transition factor (c-Met), which showed some promising results in various studies.Results:Even with the strong scientific rationale for many TKIs in the treatment of Prostate Cancer, the clinical trial experience showed some negative results in advanced phases. However, despite various challenges, the validation studies targeting kinases hold great potential in Prostate Cancer. Given the success of kinase inhibitors across multiple other cancer types, it is likely that this drug class will eventually improve outcomes in Prostate Cancer.Conclusion:Modifications in structures and certain other aspects of TKIs may make these agents promising for the treatment of Prostate Cancer.

Effects of Antagonists of Different Types of NMDA Receptor on Evoked Responses of Pyramidal Neurons in Rat Cerebral Cortex Slices

Despite the extensive pharmacology of NMDA receptors, the use of drugs able to decrease their overactivation in CNS pathologies is limited. One reason for this may be lack of knowledge of the effects of NMDA receptor antagonists on the functional properties of neurons, as a significant quantity of data has been obtained in in vitro models, which do not completely reproduce real pathophysiological processes. The present study addressed the effects of NMDA receptor antagonists with different mechanisms of action on the properties of pyramidal neuron responses in the prefrontal cortex of the rat brain. The competitive antagonist APV (50 μM) and the ion channel blocker memantine (100 μM) were inactive against evoked EPSP. In conditions of suppression of inhibitory transmission with picrotoxin (50 μM), extracellular stimulation evoked epileptiform responses with prolonged membrane depolarization and generation of a burst of action potentials. APV limited the duration and amplitude of these responses. Memantine acquired activity only when magnesium was excluded from the extracellular medium. Memantine’s lack of effect can be explained in terms of competition for the magnesium ion binding site in the channel cavity. These studies show that prediction of the systemic effects of blockers on neuron function and excitability requires use of in vitro models in which NMDA receptors are activated in the presence of a physiological magnesium concentration and without clamping of the membrane potential.

An Epitope on EGFR Loading Catastrophic Internalization Serve as a Novel Oncotarget for Hepatocellular Carcinoma Therapy

The precise role of Epidermal Growth Factor Receptor (EGFR) in Hepatocellular carcinoma (HCC) cells is unknown and EGFR inhibitors have not achieved positive clinical results. The rapid and drastic internalization of EGFR has been proved to successfully treat EGFR inhibitor-resistant patients in recent clinical trials. Here, the anti-tumor efficacy of a protein (rLZ-8) from Ganoderma lucidum was evaluated, it was demonstrated that rLZ-8 could bind to EGFR specifically, drastically enter into Hepatoma cells, abrogate endosomal recycling and induce HCC cell death. Surprisingly, we screened a monoclonal antibody which possesses competitive binding site with rLZ-8, it also trigger catastrophic EGFR internalization. This result suggests that it is necessary to investigate the interface of EGFR and rLZ-8 complex. An internalization related epitope (S222/K269) was identified on the dimerization arm of EGFR extracellular domain (ECD). These results suggest vulnerability of HCC cells to catastrophic EGFR internalization that can be targeted by a novel epitope and point to the possible exploitation in the design of anti-EGFR therapeutic biologics for HCC therapy.

Biometals as conformational modulators of α-synuclein photochemical crosslinking.

Metal dyshomeostasis has long been linked to Parkinson's disease (PD), and the amyloidogenic protein α-synuclein (αS) is universally recognized as a key player in PD pathology. Structural consequences upon coordination of copper and iron to αS have gained attention due to significant dyshomeostasis of both metals in the PD brain. Protein-metal association can navigate protein folding in distinctive pathways based on the identity of the bio-metal in question. In this work, we employed photo-chemical crosslinking of unmodified proteins (PICUP) to evaluate these potential metal ion-induced structural alterations in the folding dynamics of N-terminally acetylated αS (NAcαS) following metal coordination. Through fluorescence analysis and immunoblotting analyses following photoirradiation, we discovered that coordination of iron obstructs copper-promoted crosslinking. The absence of intra-molecular crosslinking upon iron association further supports its C-terminal coordination site and suggests a potential role for iron in mitigating nearby post-translational modification of tyrosine residues. Decreased fluorescence emission upon synergistic coordination of both copper and iron highlighted that although copper acts as a conformational promotor of NAcαS crosslinking, iron inhibits analogous conformational changes within the protein. The metal coordination preferences of NAcαS suggest that both competitive binding sites as well as dual metal coordination contribute to the changes in folding dynamics, unveiling unique structural orientations for NAcαS that have a direct and measureable influence on photoinitiated dityrosine crosslinks. Moreover, our findings have physiological implications in that iron overload, as is associated with PD-insulted brain tissue, may serve as a conformational block of copper-promoted protein oxidation.

Multiple Binding Configurations of Fis Protein Pairs on DNA: Facilitated Dissociation versus Cooperative Dissociation.

As a master transcription regulator, the Fis protein influences over two hundred genes of E. coli. The Fis protein's nonspecific binding to DNA is widely acknowledged, and its kinetics of dissociation from DNA is strongly influenced by its surroundings: the dissociation rate increases as the concentration of the Fis protein in the solution phase increases. In this study, we use computational methods to explore the global binding energy landscape of the Fis1:Fis2:DNA ternary complex. The complex contains a binary-Fis molecular dyad whose formation relies on complex structural rearrangements. The simulations allow us to distinguish several different pathways for the dissociation of the protein from DNA with different functional outcomes and involving different protein stoichiometries: (1) simple exchange of proteins and (2) cooperative unbinding of two Fis proteins to yield bare DNA. In the case of exchange, the protein on the DNA is replaced by the solution-phase protein through competition for DNA binding sites. This process seen in fluorescence imaging experiments has been called facilitated dissociation. In the latter case of cooperative unbinding of pairs, two neighboring Fis proteins on DNA form a unique binary-Fis configuration via protein-protein interactions, which in turn leads to the codissociation of both molecules simultaneously, a process akin to the "molecular stripping" seen in the NFκB/IκB genetic broadcasting system. This simulation shows that the existence of multiple binding configurations of transcription factors can have a significant impact on the kinetics and outcome of transcription factor dissociation from DNA, with important implications for the systems biology of gene regulation by Fis.

Spectral and molecular modelling studies of sulfadoxine interaction with bovine serum albumin

The present study is focused on the binding interaction of sulfadoxine with bovine serum albumin under simulated physiological conditions using spectrofluorimetry. Various spectroscopic and other methods like UV–vis, Raman, FTIR, CD, lifetime measurement, cyclic voltammetry were also employed during the course of study. It was noticeable that sulfadoxine did quench the intrinsic fluorescence of BSA via static quenching process. UV–vis study enabled the calculation of association constant, Kass for the interaction of BSA and sulfadoxine. The change in the secondary structure of BSA and binding mechanism was investigated with the spectral and docking studies. The meticulous investigation of competitive binding sites using various probes was studied and it was clear that drug binding was non-specific. Effect of some co-existent drugs and metal ions were examined which may help in the controlled release of drugs.

Probing the Interaction of Newly Synthesized Pt(II) Complex on Human Serum Albumin Using Competitive Binding Site Markers.

Considering the importance of pharmacology and the influence of drugs on biological materials, the effects of a newly designed and synthesized platin complex (2,2'-Bipyridine-3,3'-dicarboxylic acid, oxalato Platinum(II), as an antitumor drug was tested on the structure of blood carrier protein of human serum albumin (HSA) using various spectroscopic techniques including UV-visible, fluorescence, and circular dichroism at 25 and 37°C. Results of the fluorescence measurements revealed that adding the complex caused reduction in intrinsic fluorescence emission of HSA resulted from dynamic quenching of HSA. The number of binding sites and binding constants were calculated at both temperatures of 25 and 37°C. In addition, in order to identify the complex's binding site on HSA employing spectroscopy, the competitive studies were followed using warfarin, digitoxin and ibuprofen as site markers of Sudlow sites I, II and III. Competitive binding test results have shown that Pt(II) complex bind on the warfarin binding site (or Sudlow sites I) on HSA. Besides, a reduction in thermal stability for HSA was observed in the presence of the newly designed Pt(II) complex.

Thermodynamics and Kinetics of the Divalent-Monovalent Cation Competition for Binding Sites at the μ-Opioid Receptor

Monovalent and divalent cations have been known for years to differentially modulate opioid receptor (OR) signaling. While Na+ was shown to reduce agonist binding, Mg2+ had the opposite effect, particularly in the presence of sodium chloride. The observation of Na+ bound in an inactive, but not active, OR crystal structure, further supports the idea that this monovalent ion reduces agonist binding by stabilizing the receptor inactive state. In contrast, little is known about the preferred interaction site(s) of the divalent ion Mg2+ at ORs, as well as the molecular determinants responsible for its effect on drug affinity and potency. Here, we studied the binding of Mg2+ and Na+ to μ-OR in an explicit lipid/water environment. Using machine learning algorithms and Markov state models applied to tens of microseconds all-atom molecular dynamics simulations, we investigated the competition of Na+ and Mg2+ for binding sites at inactive or active conformations of μ-OR. We found that Mg2+ preferentially binds to the orthosteric ligand binding site of the ligand-free active μ-OR conformation, but not to that of the inactive receptor. However, it does so with a significantly weaker binding affinity compared to Na+. While the orthosteric and sodium allosteric sites could be simultaneously occupied by Na+ in the inactive μ-OR, binding of Mg2+ at the orthosteric site of the active receptor was found to drastically reduce the affinity of Na+ for its allosteric site. Notably, we identified a number of metastable states in which these ions are bound to the extracellular region of the inactive or activated receptor. This information, coupled with thermodynamic and kinetic properties derived from our simulations, suggests testable hypotheses of molecular determinants responsible for the modulatory effects of Mg2+.

Hotspot Identification on Protein Surfaces Using Probe-Based MD Simulations: Successes and Challenges.

Molecular Dynamics (MD) based computational co-solvent mapping methods involve the generation of an ensemble of MD-sampled target protein conformations and using selected small molecule fragments to identify and characterize binding sites on the surface of a target protein. This approach incorporates atomic-level solvation effects and protein mobility. It has shown great promise in the identification of conventional competitive and allosteric binding sites. It is also currently emerging as a useful tool in the early stages of drug discovery. This review summarizes efforts as well as discusses some methodological advances and challenges in binding site identification process through these co-solvent mapping methods.

The Probiotic Lactobacillus rhamnosus for Alleviation of Helicobacter pylori-Associated Gastric Pathology in East Africa.

The probiotic Lactobacillus rhamnosus GG (LGG) can play a role in establishing a harmless relationship with Helicobacter pylori and reduce gastric pathology in East African populations. H. pylori has the ability to inhabit the surface of the mucous layer of the human stomach and duodenum. In the developing world, an estimated 51% of the population is carrier of H. pylori, while in some Western countries these numbers dropped below 20%, which is probably associated with improved sanitation and smaller family sizes. Colonization by H. pylori can be followed by inflammation of the gastric mucus layer, and is a risk factor in the development of atrophic gastritis, peptic ulcers and gastric cancer. Notwithstanding the higher prevalence of H. pylori carriers in developing countries, no equal overall increase in gastric pathology is found. This has been attributed to a less pro-inflammatory immune response to H. pylori in African compared to Caucasian populations. In addition, a relatively low exposure to other risk factors in certain African populations may play a role, including the use of non-steroidal anti-inflammatory drugs, smoking, and diets without certain protective factors. A novel approach to the reduction of H. pylori associated gastric pathology is found in the administration of the probiotic bacterium Lactobacillus rhamnosus yoba 2012 (LRY), the generic variant of LGG. This gastro-intestinal isolate inhibits H. pylori by competition for substrate and binding sites as well as production of antimicrobial compounds such as lactic acid. In addition, it attenuates the host’s H. pylori-induced apoptosis and inflammation responses and stimulates angiogenesis in the gastric and duodenal epithelium. The probiotic LRY is not able to eradicate H. pylori completely, but its co-supplementation in antibiotic eradication therapy has been shown to relieve side effects of this therapy. In Uganda, unlike other African countries, gastric pathology is relatively common, presumably resulting from the lack of dietary protective factors in the traditional diet. Supplementation with LRY through local production of probiotic yogurt, could be a solution to establish a harmless relationship with H. pylori and reduce gastric pathology and subsequent eradication therapy treatment.

Enhanced Nucleocytoplasmic Transport due to Competition for Elastic Binding Sites

Nuclear pore complexes (NPCs) control all traffic into and out of the cell nucleus. NPCs are molecular machines that simultaneously achieve high selectivity and high transport rates. The biophysical details of how cargoes rapidly traverse the pore remain unclear but are known to be mediated by interactions between cargo-binding chaperone proteins and natively unstructured nucleoporin proteins containing many phenylalanine-glycine repeats (FG nups) that line the pore’s central channel. Here, we propose a specific and detailed physical mechanism for the high speed of nuclear import based on the elasticity of FG nups and on competition between individual chaperone proteins for FG nup binding. We develop a mathematical model to support our proposed mechanism. We suggest that the recycling of nuclear import factors back to the cytoplasm is important for driving high-speed import and predict the existence of an optimal cytoplasmic concentration of cargo for enhancing the rate of import over a purely diffusive rate.

A Host–Guest Interaction Assisted Approach for Fabrication of Polybutadiene Nanocomposites Reinforced with Well-Dispersed Cellulose Nanocrystals

Fabrication of homogeneous hydrophobic polymer/cellulose nanocrystals (CNCs) nanocomposites with high mechanical reinforcement is a tough issue owing to the poor interfacial interactions between the two components and the aggregation of CNCs in hydrophobic matrices. In this study, the polybutadiene (PB) nanocomposites reinforced with cotton-derived CNCs were prepared through a host–guest inclusion complex assisted approach. In the system the pendent guest adamantane moieties of PB reacted with β-cyclodextrins (βCDs) to form inclusion complexes, which acted as competitive binding sites to the CNCs via hydrogen bonds, allowing the CNCs to disperse homogeneously in the matrix. Dynamic mechanical analysis and tensile testing studies of the nanocomposites revealed that the incorporation of CNCs into the polymer matrix significantly improved the mechanical properties. For the nanocomposites with 10 wt % βCDs and 15 wt % CNCs, the storage modulus, Young’s modulus, and tensile strength were enhanced by about an order of magnitude compared to neat PB. The increase in the βCD and/or CNC contents led to a shifting of the tan δ peak of the nanocomposite to higher temperatures, suggesting the presence of filler–polymer interactions. Moreover, scanning electron and polarized optical microscopy analyses revealed that in the matrix the individual CNCs aligned in parallel and longitudinal directions to form long and entangled assemblies. The reinforcement could be attributed to the filler–filler and filler–matrix interactions which consequently led to the stress transfer between polymer and CNCs in the system.

Effects of Humic Acids in Chronic Lead Poisoning.

Chronic exposure to lead causes disruption to energy production mechanisms and tissue damage, in particular through its binding to thiol groups and competition for zinc binding sites. We investigated the possibility of preventing the consequences of chronic lead poisoning by administration of three different doses of humic acids (HAs) into feed with the aim of establishing an effective HA dose. During the 10-week experiment, a sub-lethal dose of lead acetate was given to rats during the first 5weeks, with continuous administration of HA over 10weeks. Measurements were taken to determine the content of the metals Pb, Mn, Cu, Fe and Zn; the metalloid Se; and selected antioxidant markers in the heart, liver, kidney and plasma after the first, fifth and tenth weeks of experiment. The administration of lead and HAs clearly affects the redistribution of the elements and the activity of the antioxidant enzymes. This fact was particularly highlighted in the lead-only group as, within the experiment, significantly higher Pb concentrations were found only in the plasma of this group. However, in the group with 1% HA administered with lead, we observed a rise in Zn concentrations in the organs and the deposition of Fe into the liver. Decreased glutathione reductase activity in the plasma and balanced reduced glutathione concentrations indicated sufficient efficiency of redox reactions. SOD activities were among those affected most strongly, with only the 1% HA group showing no effect on heavy metal redistribution as a result of HA administration.

The revelation of selective sphingolipid pathway inhibition mechanism on fumonisin toxin binding to ceramide synthases in susceptible organisms and survival mechanism in resistant species

Fumonisin B1 toxin (FB1) is a well-known competitive inhibitor of ceramide synthase (CS) in yeast. However, FB1 is unable to obstruct CS from Trichoderma spp., which are well-known biocontrol agents. To explore the contrasting binding modes, a comparative structural analysis of complexes of FB1 with these two CS proteins was carried out. Formation of activation loop on the binding of substrates with the CS from yeast was observed but when inhibitor interacted with the activation loop, it transformed into helix leading to the potentially inactivated state of the enzyme. In yeast homologue of the enzyme, the inhibitor and substrates compete for the same binding site. Whereas, in the CS protein from Trichoderma guizhouense, no such competition for substrate binding site was observed and the binding pocket of the enzyme could easily accommodate FB1 molecule along with the two interacting native substrates, which may lead to the successful catalysis.

Isothermal Titration Calorimetry of Be2+ and Ca2+ with Phosphatidylserine Models Guides All-Atom Force Field Development for Lipid-Ion Interactions

Beryllium has multiple industrial applications, but its unsafe manufacturing is associated with developing chronic inflammation in lungs known as berylliosis. Although high incidence of berylliosis was linked to a specific MHC allele that causes immune hypersensitivity to Be2+, recent findings also indicate that Be2+ competes with Ca2+ for binding sites in phosphatidylserine (PS) and thus inhibits the recognition and phagocytosis of apoptotic cells, potentially causing necrotic inflammation. This study aims to develop additive force field parameters that would describe Be2+ competition for divalent ion binding sites on anionic lipids critical for many signaling processes. We started with Lennard-Jones parameters for Be2+ for the CHARMM additive force field based on interactions with water, including free energy of hydration and ion-monohydrate interaction energy and separation distance. The results from isothermal titration calorimetry (ITC) for the binding affinities of Be2+ and Ca2+ to dimethylphosphate and acetate revealed that Be2+ has a stronger affinity than Ca2+ for binding with both component molecules. The existing CHARMM parameters for Ca2+ showed significant overbinding to acetate, while the parameters recently developed for Be2+ resulted in overbinding to both component molecules. To adjust these affinities to fit the experimental free energies of binding, configuration probabilities and free energies of association from molecular dynamics simulations were used to develop Lennard-Jones interaction parameters between the ions and component molecules. Using the revised interaction parameters, binding energies of Be2+ and Ca2+ with pure DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine) monolayers were calculated using free energy perturbation and compared with binding affinities determined from Langmuir monolayer experiments, ITC measurements on soluble short-chain PS and DOPS liposomes.

Binding Selectivity of Methanobactin from Methylosinus trichosporium OB3b for Copper(I), Silver(I), Zinc(II), Nickel(II), Cobalt(II), Manganese(II), Lead(II), and Iron(II).

Methanobactin (Mb) from Methylosinus trichosporium OB3b is a member of a class of metal binding peptides identified in methanotrophic bacteria. Mb will selectively bind and reduce Cu(II) to Cu(I), and is thought to mediate the acquisition of the copper cofactor for the enzyme methane monooxygenase. These copper chelating properties of Mb make it potentially useful as a chelating agent for treatment of diseases where copper plays a role including Wilson's disease, cancers, and neurodegenerative diseases. Utilizing traveling wave ion mobility-mass spectrometry (TWIMS), the competition for the Mb copper binding site from Ag(I), Pb(II), Co(II), Fe(II), Mn(II), Ni(II), and Zn(II) has been determined by a series of metal ion titrations, pH titrations, and metal ion displacement titrations. The TWIMS analyses allowed for the explicit identification and quantification of all the individual Mb species present during the titrations and measured their collision cross-sections and collision-induced dissociation patterns. The results showed Ag(I) and Ni(II) could irreversibly bind to Mb and not be effectively displaced by Cu(I), whereas Ag(I) could also partially displace Cu(I) from the Mb complex. At pH ≈ 6.5, the Mb binding selectivity follows the order Ag(I)≈Cu(I)>Ni(II)≈Zn(II)>Co(II)>>Mn(II)≈Pb(II)>Fe(II), and at pH 7.5 to 10.4 the order is Ag(I)>Cu(I)>Ni(II)>Co(II)>Zn(II)>Mn(II)≈Pb(II)>Fe(II). Breakdown curves of the disulfide reduced Cu(I) and Ag(I) complexes showed a correlation existed between their relative stability and their compact folded structure indicated by their CCS. Fluorescence spectroscopy, which allowed the determination of the binding constant, compared well with the TWIMS analyses, with the exception of the Ni(II) complex. Graphical abstract ᅟ.

Expression patterns of FSHD-causing DUX4 and myogenic transcription factors PAX3 and PAX7 are spatially distinct in differentiating human stem cell cultures

BackgroundFacioscapulohumeral muscular dystrophy (FSHD) is most commonly inherited in an autosomal dominant pattern and caused by the abnormal expression of DUX4 in skeletal muscle. The DUX4 transcription factor has DNA binding domains similar to several paired class homeotic transcription factors, but only myogenic factors PAX3 and PAX7 rescue cell viability when co-expressed with DUX4 in mouse myoblasts. This observation suggests competition for DNA binding sites in satellite cells might limit muscle repair and may be one aspect of DUX4-associated myotoxicity. The competition hypothesis requires that DUX4 and PAX3/7 be expressed in the same cells at some point during development or in adult tissues. We modeled myogenesis using human isogenic iPS and ES cells and examined expression patterns of DUX4, PAX3, and PAX7 to determine if conditions that promote PAX3 and PAX7 expression in cell culture also promote DUX4 expression in the same cells.MethodsIsogenic iPSCs were generated from human fibroblasts of two FSHD-affected individuals with somatic mosaicism. Clones containing the shortened FSHD-causing D4Z4 array or the long non-pathogenic array were isolated from the same individuals. We also examined myogenesis in commercially available hES cell lines derived from FSHD-affected and non-affected embryos. DUX4, PAX3, and PAX7 messenger RNAs (mRNAs) were quantified during a 40-day differentiation protocol, and antibodies were used to identify cell types in different stages of differentiation to determine if DUX4 and PAX3 or PAX7 are present in the same cells.ResultsHuman iPS and ES cells differentiated into skeletal myocytes as evidenced by Titin positive multinucleated fibers appearing toward the end of a 40-day differentiation protocol. PAX3 and PAX7 were expressed at similar times during differentiation, and DUX4 positive nuclei were seen at terminal stages of differentiation in cells containing the short D4Z4 arrays. Nuclei that expressed both DUX4 and PAX3, or DUX4 and PAX7 were not observed after examining immunostained nuclei at five different time points during myogenic differentiation of pluripotent cells.ConclusionsWe conclude that DUX4, PAX3, and PAX7 have distinct expression patterns during myogenic differentiation of stem cells. Our findings are consistent with the hypothesis that muscle damage in FSHD is due to DUX4-mediated toxicity causing destruction of terminally differentiated myofibers. While these studies examine DUX4, PAX3, and PAX7 expression patterns during stem cell myogenesis, they should not be generalized to tissue repair in adult muscle tissue.