Molecular dynamics simulation of Axillaridine–A: A potent natural cholinesterase inhibitor

Molecular Dynamics (MD) simulations were carried out for human acetylcholinesterase (hAChE) and its complex with Axillaridine–A, in order to dynamically explore the active site of the protein and the behaviour of the ligand at the peripheral binding site. Simulation of the enzyme alone showed that the active site of AChE is located at the bottom of a deep and narrow cavity whose surface is lined with rings of aromatic residues while Tyr72 is almost perpendicular to the Trp286, which is responsible for stable π -π interactions. The complexation of AChE with Axillaridine-A, results in the reduction of gorge size due to interaction between the ligand and the active site residues. The gorge size was determined by the distance between the center of mass of Glu81 and Trp286. As far as the geometry of the active site is concerned, the presence of ligand in the active site alters its specific conformation, as revealed by stable hydrogen bondings established between amino acids. With the increasing interaction between ligand and the active amino acids, size of the active site of the complex decreases with respect to time. Axillaridine-A, forms stable π -π interactions with the aromatic ring of Tyr124 that results in inhibition of catalytic activity of the enzyme. This π -π interaction keeps the substrate stable at the edge of the catalytic gorge by inhibiting its catalytic activity. The MD results clearly provide an explanation for the binding pattern of bulky steroidal alkaloids at the active site of AChE.     

Molecular dynamics simulation on the effect of transition metal binding to the N-terminal fragment of amyloid-β

Abstract

We report molecular dynamics simulations of three possible adducts of Fe(II) to the N-terminal 1–16 fragments of the amyloid-β peptide, along with analogous simulations of Cu(II) and Zn(II) adducts. We find that multiple simulations from different starting points reach pseudo-equilibration within 100–300?ns, leading to over 900?ns of equilibrated trajectory data for each system. The specifics of the coordination modes for Fe(II) have only a weak effect on peptide secondary and tertiary structures, and we therefore compare one of these with analogous models of Cu(II) and Zn(II) complexes. All share broadly similar structural features, with mixture of coil, turn and bend in the N-terminal region and helical structure for residues 11–16. Within this overall pattern, subtle effects due to changes in metal are evident: Fe(II) complexes are more compact and are more likely to occupy bridge and ribbon regions of Ramachandran maps, while Cu(II) coordination leads to greater occupancy of the poly-proline region. Analysis of representative clusters in terms of molecular mechanics energy and atoms-in-molecules properties indicates similarity of four-coordinate Cu and Zn complexes, compared to five-coordinate Fe complex that exhibits lower stability and weaker metal–ligand bonding.

Communicated by Ramaswamy H. Sarma     

Inhibiting effect of αs1-casein on Aβ1-40 fibrillogenesis

Background

α_s1-Casein is one of the four types of caseins, the largest protein component of bovine milk. The lack of a compact folded conformation and the capability to form micelles suggest a relationship of α_s1-casein with the class of the intrinsically disordered (or natively unfolded) proteins. These proteins are known to exert a stabilizing activity on biomolecules through specific interaction with hydrophobic surfaces. In the present work we focused on the effect of α_s1-casein on the fibrillogenesis of 1-40 β-amyloid peptide, involved in Alzheimer's disease.

Methods

The aggregation kinetics of β-peptide in presence and absence of α_s1-casein was followed under shear at 37 °C by recording the Thioflavine fluorescence, usually taken as an indicator of fibers formation. Measurements of Static and Dynamic Light Scattering, Circular Dichroism, and AFM imaging were done to reveal the details of α_s1-casein-Aβ_1-40 interaction.

Results and discussions

α_s1-Casein addition sizably increases the lag-time of the nucleation phase and slows down the entire fibrillization process. α_s1-Casein sequesters the amyloid peptide on its surface thus exerting a chaperone-like activity by means a colloidal inhibition mechanism.

General significance

Insights on the working mechanism of natural chaperones in preventing or controlling the amyloid aggregation.     

Inhibitory effect of α-ketoglutaric acid on α-glucosidase: integrating molecular dynamics simulation and inhibition kinetics

Abstract

The inhibition of α-glucosidase is used as a key clinical approach to treat type 2 diabetes mellitus and thus, we assessed the inhibitory effect of α-ketoglutaric acid (AKG) on α-glucosidase with both an enzyme kinetic assay and computational simulations. AKG bound to the active site and interacted with several key residues, including ASP68, PHE157, PHE177, PHE311, ARG312, TYR313, ASN412, ILE434 and ARG439, as detected by protein–ligand docking and molecular dynamics simulations. Subsequently, we confirmed the action of AKG on α-glucosidase as mixed-type inhibition with reversible and rapid binding. The relevant kinetic parameter IC₅₀ was measured (IC₅₀ = 1.738?±?0.041?mM), and the dissociation constant was determined (K_{i Slope} = 0.46?±?0.04?mM). Regarding the relationship between structure and activity, a high AKG concentration induced the slight modulation of the shape of the active site, as monitored by hydrophobic exposure. This tertiary conformational change was linked to AKG inhibition and mostly involved regional changes in the active site. Our study provides insight into the functional role of AKG due to its structural property of a hydroxyphenyl ring that interacts with the active site. We suggest that similar hydroxyphenyl ring-containing compounds targeting key residues in the active site might be potential α-glucosidase inhibitors. Abbreviations AKG alpha-ketoglutaric acid

pNPG 4-nitrophenyl-α-d-glucopyranoside

ANS 1-anilinonaphthalene-8-sulfonate

MD molecular dynamics.

Communicated by Ramaswamy H. Sarma     

Molecular dynamics simulation studies of the structural response of an isolated Aβ1–42 monomer localized in the vicinity of the hydrophilic TiO2 surface

We have probed the effect of a model hydrophilic surface, rutile TiO₂, on the full-length amyloid beta (Aβ_1–42) monomer using molecular dynamics simulations. The rutile surface brings about sharp changes in the peptide’s intrinsic behavior in a distance-dependent manner. The intrinsic collapse of the peptide is disrupted, while the β-sheet propensity is sharply enhanced with increased proximity to the surface. The results may have implications for Aβ self-assembly and fibrillogenesis on hydrophilic surfaces and should be taken into consideration in the design of novel nanomaterials for perturbing amyloidogenic behavior.     

The effect of phosphorylation on the conformation of oligo-peptides with Ser–Pro motif: a molecular dynamics simulation

Model tetrapeptide system was designed to investigate the cis/trans isomerization of peptidyl-prolyl imide bond of Ser–Pro motif. To establish the side-chain O-phosphorylation effect in regulating the peptides conformations, molecular dynamics (MD) simulations where carried out on the designed tetrapeptides and their corresponding phosphorylated forms by MD Insight II Discovery3 approach. The most stable configurations and the statistic cis/trans concentration distribution demonstrated that the phosphorylation evidently influences the peptidyl-prolyl imide bond isomerization and works as a key effect in regulating the peptide conformations. The charge state and the site provided for the charge of the phosphate moiety might be an important key. The results also demonstrated that phosphorylation changes the cis conformation ratio of the peptide and the maximum cis value is obtained when the phosphate group has no negative charge.     

Protective effects of EphB2 on Aβ1–42 oligomer-induced neurotoxicity and synaptic NMDA receptor signaling in hippocampal neurons

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized pathologically by the abnormal deposition of extracellular amyloid-β (Aβ) oligomers. However, the nature and precise mechanism of the toxicity of Aβ oligomers are not clearly understood. Aβ oligomers have been previously shown to cause a major loss of EphB2, a member of the EphB family of receptor tyrosine kinases. To determine the effect of EphB2 on Aβ oligomer-induced neurotoxicity and the underlying molecular mechanisms, we examined the EphB2 gene in cultured hippocampal neurons. Using a cellular model of AD, Aβ_1–42 oligomers were confirmed to induce neurotoxicity in a time-dependent manner and result in a major decrease of EphB2. EphB2 overexpression could prevent the neurotoxicity of hippocampal neurons from exposure to Aβ_1–42 oligomers for 1 h. Further analysis revealed that EphB2 overexpression increased synaptic NR1 and NR2B expression in Aβ_1–42 oligomer-treated neurons. Moreover, EphB2 overexpression prevented Aβ_1–42 oligomer-induced downregulation of dephosphorylated p38 MAPK and phosphorylated CREB. Together, these results suggest that EphB2 is a factor which protects hippocampal neurons against the toxicity of Aβ_1–42 oligomers, and we infer that the protection of EphB2 is achieved by increasing the synaptic NMDA receptor level and downstream p38 MAPK and CREB signaling in hippocampal neurons. This study provides new molecular insights into the neuroprotective effect of EphB2 and highlights its potential therapeutic role in the management of AD.     

Binding of HIV-1 virions to α4β7 expressing cells and impact of antagonizing α4β7 on HIV-1 infection of primary CD4+ T cells

HIV-1 envelope glycoprotein is reported to interact with α4β7, an integrin mediating the homing of lymphocytes to gut-associated lymphoid tissue, but the significance of α4β7 in HIV-1 infection remains controversial. Here, using HIV-1 strain Ba L, the gp120 of which was previously shown to be capable of interacting with α4β7, we demonstrated that α4β7 can mediate the binding of whole HIV-1 virions to α4β7-expressing transfectants. We further constructed a cell line stably expressing α4β7 and confirmed the α4β7-mediated HIV-1 binding. In primary lymphocytes with activated α4β7 expression, we also observed significant virus binding which can be inhibited by an anti-α4β7 antibody. Moreover, we investigated the impact of antagonizing α4β7 on HIV-1 infection of primary CD4+ T cells. In α4β7-activated CD4+ T cells, both anti-α4β7 antibodies and introduction of shorthairpin RNAs specifically targeting α4β7 resulted in a decreased HIV-1 infection. Our findings indicate that α4β7 may serve as an attachment factor at least for some HIV-1 strains. The established approach provides a promising means for the investigation of other viral strains to understand the potential roles of α4β7 in HIV-1 infection.     

The effect of acid mucopolysaccharides and acid mucopolysaccharide–proteins on fibril formation from collagen solutions

1. The effects of acid mucopolysaccharides and acid mucopolysaccharide-proteins on the size and rate of formation of fibril aggregates from collagen solutions in pH7.6 buffers were studied by turbidimetric and light-scattering methods. 2. Serum albumin, orosomucoid, methylated cellulose, chondroitin sulphate A and chondroitin sulphate C of molecular weight less than 20000, and hyaluronate of molecular weight less than 40000 did not influence rates of fibril formation. Chondroitin sulphate A, chondroitin sulphate C and hyaluronate of high molecular weight retarded the rate of fibril formation. This effect of high-molecular-weight chondroitin sulphate C decreased with increasing ionic strength. Heparin, though of low molecular weight (13000), was highly effective, as was also heparitin sulphate. The chondroitin sulphate-proteins of very high molecular weight were highly effective, despite the fact that for some preparations the component chondroitin sulphate chains had molecular weights much less than 20000. 3. Agents that had delayed fibril formation were also effective in producing an increase in degree of aggregation of fibrillar collagen, as indicated by dissymmetry changes observed in light-scattering experiments at low collagen concentrations. Methylated cellulose and heparin at 2.5mug./ml. were unusual in decreasing aggregation, but heparin at 0.25mug./ml. increased aggregation. Electron microscopy of gels showed fibrils and fibril aggregates with ;normal' collagen spacing and dimensions consistent with the light-scattering results. 4. The rates of electrical transport of agents and of solvent (electro-osmosis) through collagen gels indicated a contribution of molecular entanglement that increased with increase in molecular size of the agents. Electrostatic binding of heparin to collagen was noted. Binding to collagen during fibril formation was also found for heparitin sulphate and a chondroitin sulphate with extra sulphate groups. 5. Electrostatic binding of acid mucopolysaccharide-proteins to collagen may be an important factor in the organization and functioning of connective tissues at all stages of growth and development. Excluded-volume (molecular-entanglement) effects may also be important. These factors operate simultaneously and interact mutually so that precise assessment of their relative importance is difficult.     

Structural Basis for Aβ1-42 Toxicity Inhibition by Aβ C-Terminal Fragments: Discrete Molecular Dynamics Study

Amyloid β-protein (Aβ) is central to the pathology of Alzheimer's disease. Of the two predominant Aβ alloforms, Aβ_1-40 and Aβ_1-42, the latter forms more toxic oligomers. C-terminal fragments (CTFs) of Aβ were recently shown to inhibit Aβ_1-42 toxicity in vitro. Here, we studied Aβ_1-42 assembly in the presence of three effective CTF inhibitors and an ineffective fragment, Aβ_21-30. Using a discrete molecular dynamics approach that recently was shown to capture key differences between Aβ_1-40 and Aβ_1-42 oligomerization, we compared Aβ_1-42 oligomer formation in the absence and presence of CTFs or Aβ_21-30 and identified structural elements of Aβ_1-42 that correlated with Aβ_1-42 toxicity. CTFs co-assembled with Aβ_1-42 into large heterooligomers containing multiple Aβ_1-42 and inhibitor fragments. In contrast, Aβ_21-30 co-assembled with Aβ_1-42 into heterooligomers containing mostly a single Aβ_1-42 and multiple Aβ_21-30 fragments. The CTFs, but not Aβ_21-30, decreased the β-strand propensity of Aβ_1-42 in a concentration-dependent manner. CTFs and Aβ_21-30 had a high binding propensity to the hydrophobic regions of Aβ_1-42, but only CTFs were found to bind the Aβ_1-42 region A2-F4. Consequently, only CTFs but not Aβ_21-30 reduced the solvent accessibility of Aβ_1-42 in region D1-R5. The reduced solvent accessibility of Aβ_1-42 in the presence of CTFs was comparable to the solvent accessibility of Aβ_1-40 oligomers formed in the absence of Aβ fragments. These findings suggest that region D1-R5, which was more exposed to the solvent in Aβ_1-42 than in Aβ_1-40 oligomers, is involved in mediating Aβ_1-42 oligomer neurotoxicity.     

A molecular dynamics simulation study of the effect of water–graphene interaction on the properties of confined water

The role of water in determining the structure and stability of biomacromolecules has been well studied. In this work, molecular dynamics simulations have been applied to investigate the effect of surface hydrophobicity on the structure and dynamics of water confined between graphene surfaces. In order to evaluate this effect, we apply various attractive/repulsive water–graphene interaction potentials (hydrophobicity). The properties of confined water are studied by applying a purely repulsive interaction potential between water–graphene (modelled as a repulsive r^?12 potential) and repulsive–attractive forces (modelled as an LJ(12-6) potential). Compared to the case of a purely repulsive graphene–water potential, the inclusion of repulsive–attractive forces leads to formation of sharp peaks for density and the number of hydrogen bonds. Also, it was found that repulsive–attractive graphene–water potential caused slower hydrogen bonds dynamics and restricted the diffusion coefficient of water. Consequently, it was found that hydrogen bond breakage and formation rate with the repulsive r^?12 potential model, will increase compared to the corresponding water confined with the LJ(12-6) potential.     

Zinc binding promotes greater hydrophobicity in Alzheimer's Aβ42 peptide than copper binding: Molecular dynamics and solvation thermodynamics studies

The aggregation of Aβ42 peptides is considered as one of the main causes for the development of Alzheimer's disease. In this context, Zn²⁺ and Cu²⁺ play a significant role in regulating the aggregation mechanism, due to changes in the structural and the solvation free energy of Aβ42. In practice, experimental studies are not able to determine the latter properties, since the Aβ42–Zn²⁺ and Aβ42–Cu²⁺ peptide complexes are intrinsically disordered, exhibiting rapid conformational changes in the aqueous environment. Here, we investigate atomic structural variations and the solvation thermodynamics of Aβ42_, Aβ42–Cu²⁺, and Aβ42–Zn²⁺ systems in explicit solvent (water) by using quantum chemical structures as templates for a metal binding site and combining extensive all-atom molecular dynamics (MD) simulations with a thorough solvation thermodynamic analysis. Our results show that the zinc and copper coordination results in a significant decrease of the solvation free energy in the C-terminal region (Met35-Val40), which in turn leads to a higher structural disorder. In contrast, the β-sheet formation at the same C-terminal region indicates a higher solvation free energy in the case of Aβ42_. The solvation free energy of Aβ42 increases upon Zn²⁺ binding, due to the higher tendency of forming the β-sheet structure at the Leu17-Ala42 residues, in contrast to the case of binding with Cu²⁺. Finally, we find the hydrophobicity of Aβ42–Zn²⁺ in water is greater than in the case of Aβ42–Cu²⁺.     

Characterisation of α3β1 and αvβ3 integrin N-oligosaccharides in metastatic melanoma WM9 and WM239 cell lines

It is well documented that glycan synthesis is altered in some pathological processes, including cancer. The most frequently observed alterations during tumourigenesis are extensive expression of β1,6-branched complex type N-glycans, the presence of poly-N-acetyllactosamine structures, and high sialylation of cell surface glycoproteins. This study investigated two integrins, α₃β₁ and α_vβ₃, whose expression is closely related to cancer progression. Their oligosaccharide structures in two metastatic melanoma cell lines (WM9, WM239) were analysed with the use of matrix-assisted laser desorption ionisation mass spectrometry. Both examined integrins possessed heavily sialylated and fucosylated glycans, with β1,6-branches and short polylactosamine chains. In WM9 cells, α₃β₁ integrin was more variously glycosylated than α_vβ₃; in WM239 cells the situation was the reverse. Functional studies (wound healing and ELISA integrin binding assays) revealed that the N-oligosaccharide component of the tested integrins influenced melanoma cell migration on vitronectin and α₃β₁ integrin binding to laminin-5. Additionally, more variously glycosylated integrins exerted a stronger influence on these parameters. To the best of our knowledge, this is the first report concerning structural characterisation of α_vβ₃ integrin glycans in melanoma or in any cancer cells.     

A modified protocol to prepare seed-free starting solutions of amyloid-β (Aβ)1–40 and Aβ1–42 from the corresponding depsipeptides

Preparing reliable, seed-free stock solutions of the highly amyloidogenic peptides amyloid-β (Aβ) is difficult. Besides the formation of aggregates during synthesis and storage, dissolution of the peptide is a critical step because vortexing can induce aggregation. To overcome this, synthesis of the more water-soluble depsi-Aβ_1–42 peptide, from which the native sequence is easily obtained, has been suggested. We further refined this technique, including a cutoff filtration step and switching the depsipeptide in basic conditions, to stabilize the formed native peptide. The obtained solutions of native Aβ_1–40 and Aβ_1–42 peptides were homogeneous and aggregate free, as indicated by thioflavin T and circular dichroism analysis.     

The mitogenic effect of testosterone and 17β-estradiol on airway smooth muscle cells

Airway disease distribution and/or severity exhibit sex differences suggesting that sex hormones are involved in the respiratory system physiology and pathophysiology. The implication of airway smooth muscle cells (ASMCs) in the physiology of the airways and the pathogenetic mechanism of airway remodeling is of great interest. Therefore, we studied the effect of testosterone and 17β-estradiol on ASMC proliferation and the mechanisms involved.Cell proliferation was estimated using the methyl-[³H]thymidine incorporation and Cell Titer 96^® AQueous One Solution Assay methods. ASMC isolated from adult male or female rabbit trachea were incubated with testosterone (1 pM-1 μM) or 17β-estradiol (1 pM-1 μM), in the presence or absence of the androgen receptor antagonist flutamide (10 nM) or estrogen receptor antagonist ICI182780 (10 nM), as well as of the PI3K inhibitors LY294002 (20 μM) or wortmannin (1 μM), or the MAPK inhibitors PD98059 (100 μM) or U0126 (1 μM).After 24 h of incubation, testosterone and 17β-estradiol increased methyl-[³H]thymidine incorporation and cell number, in ASMC isolated from male or female animals. The induction of ASMC proliferation by testosterone or 17β-estradiol was inhibited by flutamide or ICI182780 respectively, as well as by LY294002, wortmannin, PD98059 or U0126.In conclusion, testosterone and 17β-estradiol have a mitogenic effect on ASMC, which is receptor-mediated and involves the MAPK and PI3K signaling pathways. Moreover, their effect is the same for ASMC from male and female animals. It is possible that gender-related differences in ASMC remodeling, may be influenced by the different patterns of sex steroid hormone secretion in males and females.     

Preliminary in vivo efficacy studies of a recombinant rhesus anti-α4β7 monoclonal antibody

Recent findings established that primary targets of HIV/SIV are lymphoid cells within the gastrointestinal (GI) tract. Focus has therefore shifted to T-cells expressing α₄β₇ integrin which facilitates trafficking to the GI tract via binding to MAdCAM-1. Approaches to better understand the role of α₄β₇+ T-cells in HIV/SIV pathogenesis include their depletion or blockade of their synthesis, binding and/or homing capabilities in vivo. Such studies can ideally be conducted in rhesus macaques (RM), the non-human primate model of AIDS. Characterization of α₄β₇ expression on cell lineages in RM blood and GI tissues reveal low densities of expression by NK cells, B-cells, naïve and TEM (effector memory) T-cells. High densities were observed on TCM (central memory) T-cells. Intravenous administration of a single 50 mg/kg dose of recombinant rhesus α₄β₇ antibody resulted in significant initial decline of α₄β₇+ lymphocytes and sustained coating of the α₄β₇ receptor in both the periphery and GI tissues.     

Molecular dynamics simulation of the reciprocal fused LiF–KBr mixture: local structure and self-diffusion coefficients

This paper describes the molecular dynamics simulation of the reciprocal fused LiF–KBr mixture, which is located above the critical mixing point, in the temperature range 1280–1450 K. The first coordination sphere is found to form as follows: a smaller ion is formed around a smaller counter-ion, and a larger ion is formed around a larger counter-ion. The calculated concentration dependence of the self-diffusion coefficients and the radial distribution functions of all ion pairs indicate that the degree of association of the Li–F pair increases as the lithium fluoride fraction in the mixture decreases.     

Phosphatidic acid potentiates Gαq stimulation of phospholipase C-β1 signaling

Phosphatidic acid (PA) is interactive with Gα_q-linked agonists to stimulate GPCR signaling via phospholipase C-β₁ (PLC-β₁). Phorbol 12-myristate 13-acetate (PMA) increases cellular levels of PA and phospholipase D activity (PLD). This study evaluated whether PMA can stimulate PLC-β₁ activity via PA, independent of GPCR input in transfected COS 7 cells. PMA alone had little effect on PLC activity in cells co-transfected with PLC-β₁ and Gα_q. Activated Gα_q, induced by co-transfecting muscarinic cholinergic receptor (m1R), was necessary for stimulation of PLC-β₁ activity by PMA. Stimulation by PMA was dependent on the PA-regulatory motif of PLC-β₁ implicating PA in this mechanism. PLD1 knockdown by antisense decreased responsiveness of PLC-β₁ to both PMA and carbachol. PA alone thus has little effect on PLC-β₁ activity, but PA and PLD1 synergize with activated Gα_q to stimulate PLC-β₁ signaling. Coordinate interaction with activated Gα_q may serve as an important mechanism to fine tune response to ligands while preventing spurious initiation of PLC-β signaling by PA in cells.     

Polymorphism of Alzheimer's Aβ17-42 (p3) Oligomers: The Importance of the Turn Location and Its Conformation

Aβ_17-42 (so-called p3) amyloid is detected in vivo in the brains of individuals with Alzheimer's disease or Down's syndrome. We investigated the polymorphism of Aβ_17-42 oligomers based on experimental data from steady-state NMR measurements, electron microscopy, two-dimensional hydrogen exchange, and mutational studies, using all-atom molecular-dynamics simulation with explicit solvent. We assessed the structural stability and the populations. Our results suggest that conformational differences in the U-turn of Aβ_17-42 lead to polymorphism in β-sheet registration and retention of an ordered β-strand organization at the termini. Further, although the parallel Aβ_17-42 oligomer organization is the most stable of the conformers investigated here, different antiparallel Aβ_17-42 organizations are also stable and compete with the parallel architectures, presenting a polymorphic population. In this study we propose that 1), the U-turn conformation is the primary factor leading to polymorphism in the assembly of Aβ_17-42 oligomers, and is also coupled to oligomer growth; and 2), both parallel Aβ_17-42 oligomers and an assembly of Aβ_17-42 oligomers that includes both parallel and antiparallel organizations contribute to amyloid fibril formation. Finally, since a U-turn motif generally appears in amyloids formed by full proteins or long fragments, and since to date these have been shown to exist only in parallel architectures, our results apply to a broad range of oligomers and fibrils.