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.

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.

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.

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.

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.

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.

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.

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).

Canine Adipose-Derived Stem Cells: Purinergic Characterization and Neurogenic Potential for Therapeutic Applications.

The presented results evidence that canine adipose-derived stem cells (ADSCs) represent the premature population of stem cells with great biological potential and properties. ADCS are easy to obtain and culture, able to differentiate into the neurogenic lineage as well as it is easy to control their proliferation rate with nucleotides and nucleosides or analogues. We report that in vitro cultured canine ADSCs response to adenosine- and ATP-mediated stimulation. Differences in canine ADSCs and human mesenchymal stem cells in ecto-nucleotidase activity have been observed. The ecto-nucleotidase activity changes during ADSCs in vitro transdifferentiation into neurogenic lineage are fast and simple to analyze. Therefore, the simple analysis of ecto-enzymes activity allows for verification of the stem cells quality: their stemness or initiation of the differentiation process. The biological potential of the cells isolated from canine fat, as well as the good quality control of this cell culture, make them a promising tool for both experimental and therapeutic usage. J. Cell. Biochem. 118: 58-65, 2017. © 2016 Wiley Periodicals, Inc.

Inferring pathological states in cortical neuron microcircuits.

The brain activity is to a large extent determined by states of neural cortex microcircuits. Unfortunately, accuracy of results from neural circuits׳ mathematical models is often biased by the presence of uncertainties in underlying experimental data. Moreover, due to problems with uncertainties identification in a multidimensional parameters space, it is almost impossible to classify states of the neural cortex, which correspond to a particular set of the parameters. Here, we develop a complete methodology for determining uncertainties and the novel protocol for classifying all states in any neuroinformatic model. Further, we test this protocol on the mathematical, nonlinear model of such a microcircuit developed by Giugliano et al. (2008) and applied in the experimental data analysis of Huntington׳s disease. Up to now, the link between parameter domains in the mathematical model of Huntington׳s disease and the pathological states in cortical microcircuits has remained unclear. In this paper we precisely identify all the uncertainties, the most crucial input parameters and domains that drive the system into an unhealthy state. The scheme proposed here is general and can be easily applied to other mathematical models of biological phenomena.

Novel KRT14 mutation causing epidermolysis bullosa simplex with variable phenotype.

About 75% of cases of epidermolysis bullosa simplex result from mutations in KRT5 and KRT14 genes. Here, we report a family with a novel heterozygous missense mutation p.Leu418Gln in the KRT14 gene causing EBS of phenotype varying from EBS-loc to EBS-gen intermed. To the best of our knowledge, the family reported by us is the largest one in which two different phenotypes can be distinguished. The molecular dynamics simulations show that p.Leu418Gln mutation results in clear disruption of intermolecular π-stacking between KRT14:Tyr415 and KRT5:Tyr470, which in turn may affect putative phosphorylation site at KRT14:Thr414. This study further supports the importance of the EIATYR/KLLEGE motif in maintaining structural stability of KRT14:KRT5 heterodimer and indicates that physical properties of introduced amino acid can modulate the disease severity. The results obtained indicate further need of genotype-phenotype studies in EBS. In conclusion, genotype-based prognosis should be given to patients with caution.

Immune thrombocytopenia in patients with chronic lymphocytic leukemia treated with cladribine-based regiments or chlorambucil--follow-up of PALG-CLL randomized trials.

OBJECTIVES: The relationship between treatments of chronic lymphocytic leukemia (CLL) with cladribine (2-CdA) or chlorambucil and immune thrombocytopenia (IT) has not been yet determined. METHODS: The records of 777 patients in two randomized Polish Adult Leukemia Group (PALG)-CLL programs treated with these agents were retrospectively analyzed. RESULTS: Immune thrombocytopenia occurred in 55 of 777 (7.1%) patients. No significant differences in IT prevalence were seen between patients on chlorambucil or 2-CdA-based regiments (P = 0.33). IT developed at a median time of 0.499 yr (0.06-4.8) from the start of CLL therapy. This time was significantly longer in patients treated with chlorambucil (2.03 yr, 95% CI: 0.06-4.22) in relation to patients treated with 2-CdA-based regiments (0.52 yr, 95%CI: 0.34-0.69, P = 0.049). Overall survival (OS) of patients with IT and those without IT were similar (2.65 yr vs. 3.2 yr P = 0.23) but the severity of bleeding was more pronounced in the 2-CdA group. The responses to IT therapy were 35%, 54% and 75% for steroids, chemotherapy and splenectomy, respectively. CONCLUSIONS: In this study, an unexpectedly high percentage of IT incidence was demonstrated in patients with CLL requiring chemotherapy. Although no marked differences were seen in IT frequency in patients treated with 2-CdA-based regiments compared to chlorambucil regimen, the clinical course of hemorrhagic diathesis was more severe in 2-CdA group. Also, the time elapsed from study screening to IT diagnosis was significantly shorter in the 2-CdA group than in the chlorambucil group suggesting a causative relationship. The appearance of IT did not influence the median time of OS.

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.

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.

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.

Molecular jamming--the cystine slipknot mechanical clamp in all-atom simulations.

A recent survey of 17 134 proteins has identified a new class of proteins which are expected to yield stretching induced force peaks in the range of 1 nN. Such high force peaks should be due to forcing of a slip-loop through a cystine ring, i.e., by generating a cystine slipknot. The survey has been performed in a simple coarse grained model. Here, we perform all-atom steered molecular dynamics simulations on 15 cystine knot proteins and determine their resistance to stretching. In agreement with previous studies within a coarse grained structure based model, the level of resistance is found to be substantially higher than in proteins in which the mechanical clamp operates through shear. The large stretching forces arise through formation of the cystine slipknot mechanical clamp and the resulting steric jamming. We elucidate the workings of such a clamp in an atomic detail. We also study the behavior of five top strength proteins with the shear-based mechanostability in which no jamming is involved. We show that in the atomic model, the jamming state is relieved by moving one amino acid at a time and there is a choice in the selection of the amino acid that advances the first. In contrast, the coarse grained model also allows for a simultaneous passage of two amino acids.

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.

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.

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.