Membrane regulation of 15LOX-1/PEBP1 complex prompts the generation of ferroptotic signals, oxygenated PEs.

Ferroptosis is a regulated form of cell death, the mechanism of which is still to be understood. 15-lipoxygenase (15LOX) complex with phosphatidylethanolamine (PE)-binding protein 1 (PEBP1) catalyzes the generation of pro-ferroptotic cell death signals, hydroperoxy-polyunsaturated PE. We focused on gaining new insights into the molecular basis of these pro-ferroptotic interactions using computational modeling and liquid chromatography-mass spectrometry experiments. Simulations of 15LOX-1/PEBP1 complex dynamics and interactions with lipids revealed that association with the membrane triggers a conformational change in the complex. This conformational change facilitates the access of stearoyl/arachidonoyl-PE (SAPE) substrates to the catalytic site. Furthermore, the binding of SAPE promotes tight interactions within the complex and induces further conformational changes that facilitate the oxidation reaction. The reaction yields two hydroperoxides as products, 15-HpETE-PE and 12-HpETE-PE, at a ratio of 5:1. A significant effect of PEBP1 is observed only on the predominant product. Moreover, combined experiments and simulations consistently demonstrate the significance of PEBP1 P112E mutation in generating ferroptotic cell death signals.

Toward Overcoming Pyrethroid Resistance in Mosquito Control: The Role of Sodium Channel Blocker Insecticides.

Diseases spread by mosquitoes lead to the death of 700,000 people each year. The main way to reduce transmission is vector control by biting prevention with chemicals. However, the most commonly used insecticides lose efficacy due to the growing resistance. Voltage-gated sodium channels (VGSCs), membrane proteins responsible for the depolarizing phase of an action potential, are targeted by a broad range of neurotoxins, including pyrethroids and sodium channel blocker insecticides (SCBIs). Reduced sensitivity of the target protein due to the point mutations threatened malaria control with pyrethroids. Although SCBIs-indoxacarb (a pre-insecticide bioactivated to DCJW in insects) and metaflumizone-are used in agriculture only, they emerge as promising candidates in mosquito control. Therefore, a thorough understanding of molecular mechanisms of SCBIs action is urgently needed to break the resistance and stop disease transmission. In this study, by performing an extensive combination of equilibrium and enhanced sampling molecular dynamics simulations (3.2 µs in total), we found the DIII-DIV fenestration to be the most probable entry route of DCJW to the central cavity of mosquito VGSC. Our study revealed that F1852 is crucial in limiting SCBI access to their binding site. Our results explain the role of the F1852T mutation found in resistant insects and the increased toxicity of DCJW compared to its bulkier parent compound, indoxacarb. We also delineated residues that contribute to both SCBIs and non-ester pyrethroid etofenprox binding and thus could be involved in the target site cross-resistance.

Purinergic approach to effective glioma treatment with temozolomide reveals enhanced anti-cancer effects mediated by P2X7 receptor.

The purinergic signaling pathway is the oldest evolutionary transmitter system that regulates a wide array of physiological and pathophysiological processes in central nervous system. However, the question of how the purinergic compounds interact with administrated drugs is rarely addressed. We aimed to clarify the interplay between purinergic signaling and chemotherapeutic drug temozolomide (TMZ) in human glioma cell line. We applied an initial retinoic acid-induced differentiation of A172 glioma cells and tested the P2X7 receptor expression in undifferentiated and differentiated gliomas. We compared the P2X7 receptor agonists/antagonists influence and their co-action with TMZ in both cell types through assessment of cell proliferation, viability and migrative properties. Molecular docking allowed to indicate the potential binding site for TMZ in the structure of hP2X7 receptor. Differentiated cells turned out to be more susceptible to ATP and TMZ alone but also to the concerted action of TMZ and ATP. Enhanced effects triggered by ATP and TMZ treatment include the decreased by 70% viability, and reduced migration ability of differentiated A172 glioma cells. Noteworthy, these results can be achieved already at low non-toxic ATP concentration and at reduced to 125 µM effective concentration of TMZ. Therefore, ATP molecules must be present and maintained at appropriate concentration in glioma cells microenvironment to achieve their co-action with TMZ and enhanced anti-cancer activity. All that, in turn, could shorten the therapy, increase its efficacy and limit the side effects for the patient. Our purinergic approach creates a promising perspective for developing novel combined oncological therapies.

Structural Insights into ATP-Sensitive Potassium Channel Mechanics: A Role of Intrinsically Disordered Regions.

Commonly used techniques, such as CryoEM or X-ray, are not able to capture the structural reorganizations of disordered regions of proteins (IDR); therefore, it is difficult to assess their functions in proteins based exclusively on experiments. To fill this gap, we used computational molecular dynamics (MD) simulation methods to capture IDR dynamics and trace biological function-related interactions in the Kir6.2/SUR1 potassium channel. This ATP-sensitive octameric complex, one of the critical elements in the insulin secretion process in human pancreatic ß-cells, has four to five large, disordered fragments. Using unique MD simulations of the full Kir6.2/SUR1 channel complex, we present an in-depth analysis of the dynamics of the disordered regions and discuss the possible functions they could have in this system. Our MD results confirmed the crucial role of the N-terminus of the Kir6.2 fragment and the L0-loop of the SUR1 protein in the transfer of mechanical signals between domains that trigger insulin release. Moreover, we show that the presence of IDRs affects natural ligand binding. Our research takes us one step further toward understanding the action of this vital complex.

In Search of Synergistic Insect Repellents: Modeling of Muscarinic GPCR Interactions with Classical and Bitopic Photoactive Ligands.

Insect vector-borne diseases pose serious health problems, so there is a high demand for efficient molecules that could reduce transmission. Using molecular docking and molecular dynamics (MD) simulation, we studied a series of compounds acting on human and insect muscarinic acetylcholine receptors (mAChRs), a novel target of synergistic agents in pest control. We characterized early conformational changes of human M1 and fruit fly type-A mAChR G protein-coupled receptors (GPCRs) in response to DEET, IR3535, and muscarine binding based on the MD analysis of the activation microswitches known to form the signal transduction pathway in class A GPCRs. We indicated groups of microswitches that are the most affected by the presence of a ligand. Moreover, to increase selectivity towards insects, we proposed a new, bitopic, photoswitchable mAChR ligand-BQCA-azo-IR353 and studied its interactions with both receptors. Modeling data showed that using a bitopic ligand may be a promising strategy in the search for better insect control.

Enhancing the Inhomogeneous Photodynamics of Canonical Bacteriophytochrome.

The ability of phytochromes to act as photoswitches in plants and microorganisms depends on interactions between a bilin-like chromophore and a host protein. The interconversion occurs between the spectrally distinct red (Pr) and far-red (Pfr) conformers. This conformational change is triggered by the photoisomerization of the chromophore D-ring pyrrole. In this study, as a representative example of a phytochrome-bilin system, we consider biliverdin IXα (BV) bound to bacteriophytochrome (BphP) from Deinococcus radiodurans. In the absence of light, we use an enhanced sampling molecular dynamics (MD) method to overcome the photoisomerization energy barrier. We find that the calculated free energy (FE) barriers between essential metastable states agree with spectroscopic results. We show that the enhanced dynamics of the BV chromophore in BphP contributes to triggering nanometer-scale conformational movements that propagate by two experimentally determined signal transduction pathways. Most importantly, we describe how the metastable states enable a thermal transition known as the dark reversion between Pfr and Pr, through a previously unknown intermediate state of Pfr. We present the heterogeneity of temperature-dependent Pfr states at the atomistic level. This work paves a way toward understanding the complete mechanism of the photoisomerization of a bilin-like chromophore in phytochromes.

Photo-Switchable Sulfonylureas Binding to ATP-Sensitive Potassium Channel Reveal the Mechanism of Light-Controlled Insulin Release.

ATP-sensitive potassium (KATP) channels are present in numerous organs, including the heart, brain, and pancreas. Physiological opening and closing of KATPs present in pancreatic ß-cells, in response to changes in the ATP/ADP concentration ratio, are correlated with insulin release into the bloodstream. Sulfonylurea drugs, commonly used in type 2 diabetes mellitus treatment, bind to the octamer KATP channels composed of four pore-forming Kir6.2 and four SUR1 subunits and increase the probability of insulin release. Azobenzene-based derivatives of sulfonylureas, such as JB253 inspired by well-established antidiabetic drug glimepiride, allow for control of this process by light. The mechanism of that phenomenon was not known until now. In this paper, we use molecular docking, molecular dynamics, and metadynamics to reveal structural determinants explaining light-controlled insulin release. We show that both trans- and cis-JB253 bind to the same SUR1 cavity as antidiabetic sulfonylurea glibenclamide (GBM). Simulations indicate that, in contrast to trans-JB253, the cis-JB253 structure generated by blue light absorption promotes open structures of SUR1, in close similarity to the GBM effect. We postulate that in the open SUR1 structures, the N-terminal tail from Kir6.2 protruding into the SUR1 pocket is stabilized by flexible enough sulfonylureas. Therefore, the adjacent Kir6.2 pore is more often closed, which in turn facilitates insulin release. Thus, KATP conductance is regulated by peptide linkers between its Kir6.2 and SUR1 subunits, a phenomenon present in other biological signaling pathways. Our data explain the observed light-modulated activity of photoactive sulfonylureas and widen a way to develop new antidiabetic drugs having reduced adverse effects.

Interactions of Sea Anemone Toxins with Insect Sodium Channel-Insights from Electrophysiology and Molecular Docking Studies.

Animal venoms are considered as a promising source of new drugs. Sea anemones release polypeptides that affect electrical activity of neurons of their prey. Voltage dependent sodium (Nav) channels are the common targets of Av1, Av2, and Av3 toxins from Anemonia viridis and CgNa from Condylactis gigantea. The toxins bind to the extracellular side of a channel and slow its fast inactivation, but molecular details of the binding modes are not known. Electrophysiological measurements on Periplaneta americana neuronal preparation revealed differences in potency of these toxins to increase nerve activity. Av1 and CgNa exhibit the strongest effects, while Av2 the weakest effect. Extensive molecular docking using a modern SMINA computer method revealed only partial overlap among the sets of toxins' and channel's amino acid residues responsible for the selectivity and binding modes. Docking positions support earlier supposition that the higher neuronal activity observed in electrophysiology should be attributed to hampering the fast inactivation gate by interactions of an anemone toxin with the voltage driven S4 helix from domain IV of cockroach Nav channel (NavPaS). Our modelling provides new data linking activity of toxins with their mode of binding in site 3 of NavPaS channel.

Structural Determinants of Insulin Release: Disordered N-Terminal Tail of Kir6.2 Affects Potassium Channel Dynamics through Interactions with Sulfonylurea Binding Region in a SUR1 Partner.

Inward rectifying potassium ion channels (KATP), sensitive to the ATP/ADP concentration ratio, play an important, control role in pancreatic ß cells. The channels close upon the increase of this ratio, which, in turn, triggers insulin release to blood. Numerous mutations in KATP lead to severe and widespread medical conditions such as diabetes. The KATP system consists of a pore made of four Kir6.2 subunits and four accompanying large SUR1 proteins belonging to the ABCC transporters group. How SUR1 affects KATP function is not yet known; therefore, we created simplified models of the Kir6.2 tetramer based on recently determined cryo-EM KATP structures. Using all-atom molecular dynamics (MD) with the CHARMM36 force field, targeted MD, and molecular docking, we revealed functionally important rearrangements in the Kir6.2 pore, induced by the presence of the SUR1 protein. The cytoplasmic domain of Kir6.2 (CTD) is brought closer to the membrane due to interactions with SUR1. Each Kir6.2 subunit has a conserved, functionally important, disordered N-terminal tail. Using molecular docking, we found that the Kir6.2 tail easily docks to the sulfonylurea drug binding region located in the adjacent SUR1 protein. We reveal, for the first time, dynamical behavior of the Kir6.2/SUR1 system, confirming a physiological role of the Kir6.2 disordered tail, and we indicate structural determinants of KATP-dependent insulin release from pancreatic ß cells.

Orthosteric muscarinic receptor activation by the insect repellent IR3535 opens new prospects in insecticide-based vector control.

The insect repellent IR3535 is one of the important alternative in the fight against mosquito-borne disease such as malaria, dengue, chikungunya, yellow fever and Zika. Using a multidisciplinary approach, we propose the development of an innovative insecticide-based vector control strategy using an unexplored property of IR3535. We have demonstrated that in insect neurosecretory cells, very low concentration of IR3535 induces intracellular calcium rise through cellular mechanisms involving orthosteric/allosteric sites of the M1-muscarinic receptor subtype, G protein ßγ subunits, background potassium channel inhibition generating depolarization, which induces voltage-gated calcium channel activation. The resulting internal calcium concentration elevation increases nicotinic receptor sensitivity to the neonicotinoid insecticide thiacloprid. The synergistic interaction between IR3535 and thiacloprid contributes to significantly increase the efficacy of the treatment while reducing concentrations. In this context, IR3535, used as a synergistic agent, seems to promise a new approach in the optimization of the integrated vector management for vector control.

How strong are hydrogen bonds in the peptide model?

Detailed knowledge of intramolecular hydrogen bonds, including their nanomechanics, in a peptide secondary structure is crucial for understanding mechanisms of numerous biochemical processes. Single-molecule force spectroscopy has become a powerful tool to study directly the mechanical properties of single biopolymers and monitoring the hydrogen bonds. However, the interpretation of such experiments, due to their poor temporal resolution relative to the rate of intramolecular dynamics, requires the support of molecular simulations. In this work, we provide a methodology for determining the kinetic and energetic characteristics of hydrogen bonds in a template model of the protein secondary structure. Our approach, based on the steered molecular dynamics method, employs dynamic force spectroscopy calculations and uses two advanced theoretical models of force-induced unbinding. A systematic analysis of the simulated data with these models allowed for quantitative characterization of a single hydrogen bond in the α-helix of the AAKA(AEAAKA)5AC peptide model and detailed explanation of the mechanism of the α-helix unfolding. The methodology proposed here may be extended to other molecular structures stabilized by internal hydrogen bonds.

Dynamics, nanomechanics and signal transduction in reelin repeats.

Reelin is a large glycoprotein controlling brain development and cell adhesion. It regulates the positioning of neurons, as well as neurotransmission and memory formation. Perturbations in reelin signaling are linked to psychiatric disorders. Reelin participates in signal transduction by binding to the lipoprotein receptors VLDLR and ApoER2 through its central region. This part is rich in repeating BNR-EGF-BNR modules. We used standard molecular dynamics, steered molecular dynamics, and perturbation response scanning computational methods to characterize unique dynamical properties of reelin modules involved in signaling. Each module has specific sensors and effectors arranged in a similar topology. In the modules studied, disulfide bridges play a protective role, probably making both selective binding and protease activity of reelin possible. Results of single reelin molecule stretching by atomic force microscopy provide the first data on the mechanical stability of individual reelin domains. The forces required for partial unfolding of the modules studied are below 60 pN.

Prebiotic Soup Components Trapped in Montmorillonite Nanoclay Form New Molecules: Car-Parrinello Ab Initio Simulations.

The catalytic effects of complex minerals or meteorites are often mentioned as important factors for the origins of life. To assess the possible role of nanoconfinement within a catalyst consisting of montmorillonite (MMT) and the impact of local electric field on the formation efficiency of the simple hypothetical precursors of nucleic acid bases or amino acids, we performed ab initio Car-Parrinello molecular dynamics simulations. We prepared four condensed-phase systems corresponding to previously suggested prototypes of a primordial soup. We monitored possible chemical reactions occurring within gas-like bulk and MMT-confined four simulation boxes on a 20-ps time scale at 1 atm and 300 K, 400 K, and 600 K. Elevated temperatures did not affect the reactivity of the elementary components of the gas-like boxes considerably; however, the presence of the MMT nanoclay substantially increased the formation probability of new molecules. Approximately 20 different new compounds were found in boxes containing carbon monoxide or formaldehyde molecules. This observation and an analysis of the atom-atom radial distribution functions indicated that the presence of Ca2+ ions at the surface of the internal MMT cavities may be an important factor in the initial steps of the formation of complex molecules at the early stages of the Earth's history.

Towards A New Approach for the Description of Cyclo⁻2,4-Dihydroxybenzoate, A Substance Which Effectively Mimics Zearalenone in Imprinted Polymers Designed for Analyzing Selected Mycotoxins in Urine.

A method of purifying cyclododecyl 2,4-dihydroxybenzoate as a potential replacement template molecule for preparation of molecularly-imprinted polymers for isolation of zearalenone in urine was developed. Full physicochemical characteristics of cyclododecyl 2,4-dihydroxybenzoate for the first time included crystallographic analysis and molecular modelling, which made possible the determination of the similarity between the cyclododecyl 2,4-dihydroxybenzoate and zearalenone molecules. The obtained molecularly-imprinted polymers show very high in vitro selectivity towards zearalenone due to specific interactions (e.g., hydrogen bonding, molecular recognition interaction). The achieved extraction recovery exceeds 94% at the tested concentration levels (20⁻500 ng·mL-1) with a relative standard deviation below 2%. Immunosorbents were found to have lower recoveries (below 92.5%) and RSD value between 2 and 4% for higher concentrations of the studied substance (400 ng·mL-1).

A novel de novo mutation p.Ala428Asp in KRT5 gene as a cause of localized epidermolysis bullosa simplex.

Epidermolysis bullosa is a group of inherited blistering skin diseases resulting in most cases from missense mutations in KRT5 and KRT14 genes encoding the basal epidermal keratins 5 and 14. Here, we present a patient diagnosed with a localized subtype of epidermolysis bullosa simplex caused by a heterozygous mutation p.Ala428Asp in the KRT5 gene, that has not been previously identified. Moreover, a bioinformatic analysis of the novel mutation was performed, showing changes in the interaction network between the proteins. Identification of novel mutations and genotype-phenotype correlations allow to better understanding of underlying pathophysiologic bases and is important for genetic counselling, patients' management, and disease course prediction.

Spacial models of malfunctioned protein complexes help to elucidate signal transduction critical for insulin release.

Mutations in gene KCNJ11 encoding the Kir6.2 subunit of the ATP-sensitive potassium channel (KATP), a representative of a quite complex biosystem, may affect insulin release from pancreatic beta-cells. Both gain and loss of channel activity are observed, which lead to varied clinical phenotypes ranging from neonatal diabetes to congenital hyperinsulinism. In order to understand the mechanisms of the channel function better we mapped, based on the literature review, known medically relevant Kir6.2/SUR1 mutations into recently (2017) determined CryoEM 3D structures of this complex. We used a clustering algorithm to find hots spots in the 3D structure, thus we may hypothesize about their nano-mechanical role in the channel gating and the insulin level control. We also adapted a simple model of the channel gating to cover all currently known factors that can influence the KATP biosystem functions.

Dominant ELOVL1 mutation causes neurological disorder with ichthyotic keratoderma, spasticity, hypomyelination and dysmorphic features.

BACKGROUND: Ichthyosis and neurological involvement occur in relatively few known Mendelian disorders caused by mutations in genes relevant both for epidermis and neural function. OBJECTIVES: To identify the cause of a similar phenotype of ichthyotic keratoderma, spasticity, mild hypomyelination (on MRI) and dysmorphic features (IKSHD) observed in two unrelated paediatric probands without family history of disease. METHODS: Whole exome sequencing was performed in both patients. The functional effect of prioritised variant in ELOVL1 (very-long-chain fatty acids (VLCFAs) elongase) was analysed by VLCFA profiling by gas chromatography-mass spectrometry in stably transfected HEK2932 cells and in cultured patient's fibroblasts. RESULTS: Probands shared novel heterozygous ELOVL1 p.Ser165Phe mutation (de novo in one family, while in the other family, father could not be tested). In transfected cells p.Ser165Phe: (1) reduced levels of FAs C24:0-C28:0 and C26:1 with the most pronounced effect for C26:0 (P=7.8×10-6 vs HEK293 cells with wild type (wt) construct, no difference vs naïve HEK293) and (2) increased levels of C20:0 and C22:0 (P=6.3×10-7, P=1.2×10-5, for C20:0 and C22:0, respectively, comparison vs HEK293 cells with wt construct; P=2.2×10-7, P=1.9×10-4, respectively, comparison vs naïve HEK293). In skin fibroblasts, there was decrease of C26:1 (P=0.014), C28:0 (P=0.001) and increase of C20:0 (P=0.033) in the patient versus controls. There was a strong correlation (r=0.92, P=0.008) between the FAs profile of patient's fibroblasts and that of p.Ser165Phe transfected HEK293 cells. Serum levels of C20:0-C26:0 FAs were normal, but the C24:0/C22:0 ratio was decreased. CONCLUSION: The ELOVL1 p.Ser165Phe mutation is a likely cause of IKSHD.

Author Correction: Nanomechanics of multidomain neuronal cell adhesion protein contactin revealed by single molecule AFM and SMD.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Conformational Sampling of a Biomolecular Rugged Energy Landscape.

The protein structure refinement using conformational sampling is important in hitherto protein studies. In this paper, we examined the protein structure refinement by means of potential energy minimization using immune computing as a method of sampling conformations. The method was tested on the x-ray structure and 30 decoys of the mutant of [Leu]Enkephalin, a paradigmatic example of the biomolecular multiple-minima problem. In order to score the refined conformations, we used a standard potential energy function with the OPLSAA force field. The effectiveness of the search was assessed using a variety of methods. The robustness of sampling was checked by the energy yield function which measures quantitatively the number of the peptide decoys residing in an energetic funnel. Furthermore, the potential energy-dependent Pareto fronts were calculated to elucidate dissimilarities between peptide conformations and the native state as observed by x-ray crystallography. Our results showed that the probed potential energy landscape of [Leu]Enkephalin is self-similar on different metric scales and that the local potential energy minima of the peptide decoys are metastable, thus they can be refined to conformations whose potential energy is decreased by approximately 250 kJ/mol.

Nanomechanics of multidomain neuronal cell adhesion protein contactin revealed by single molecule AFM and SMD.

Contactin-4 (CNTN4) is a complex cell adhesion molecule (CAM) localized at neuronal membranes, playing a key role in maintaining the mechanical integrity and signaling properties of the synapse. CNTN4 consists of six immunoglobulin C2 type (IgC2) domains and four fibronectin type III (FnIII) domains that are shared with many other CAMs. Mutations in CNTN4 gene have been linked to various psychiatric disorders. Toward elucidating the response of this modular protein to mechanical stress, we studied its force-induced unfolding using single molecule atomic force microscopy (smAFM) and steered molecular dynamics (SMD) simulations. Extensive smAFM and SMD data both indicate the distinctive mechanical behavior of the two types of modules distinguished by unique force-extension signatures. The data also reveal the heterogeneity of the response of the individual FNIII and IgC2 modules, which presumably plays a role in the adaptability of CNTN4 to maintaining cell-cell communication and adhesion properties under different conditions. Results show that extensive sampling of force spectra, facilitated by robot-enhanced AFM, can help reveal the existence of weak stabilizing interactions between the domains of multidomain proteins, and provide insights into the nanomechanics of such multidomain or heteromeric proteins.