Molecular dynamics simulations of a lipovitellin-derived amphiphilic beta-sheet homologous to apoB-100 beta-sheets at a hydrophobic decane-water interface

Lipovitellin, an egg-yolk lipoprotein, transports lipids in a pocket surrounded by amphiphilic beta-sheets. Its X-ray structure provides possibilities to study interactions between lipophilic beta-sheets and lipids at the atomic level. Here, we studied a 67-residue-long amphiphilic beta-sheet of lipovitellin previously suggested a suitable working model for studies of the lipid-binding behaviour of amphiphilic beta-sheet regions in apolipoprotein B-100 (apoB-100). We performed four molecular dynamics simulations with different starting configurations to define characteristics of the amphiphilic beta-sheet model at a decane-water interface. In each simulation the model beta-sheet bound keenly to the decane layer via its hydrophobic surface. The structural profiles showed unchanged secondary structure of the beta-sheet during the attachment. Also, aromatic side chains, especially tryptophans and tyrosines, mediated the attachment to the hydrophobic layer and influenced the orientation of the decane molecules that are in contact with the beta-sheet. In conclusion, the present simulations reveal high affinity of a lipovitellin-derived amphiphilic beta-sheet to a hydrophobic decane layer. They lay thereby the basis for further studies of the interaction between amphiphilic beta-sheets and lipids in complex molecular systems, like LDL particles, in which the large apoB-100 is the main protein component.     

Mesoscopic simulation study on the efficiency of surfactants adsorbed at the liquid/liquid interface

Surfactant monolayers at the interface between oil and water has been simulated using dissipative particle dynamics (DPD) technique. With a simple coarse-grained model, how variations in structure of surfactants influence their ability to reduce the interfacial tension has been investigated. The result shows that strong hydrophilic head groups are beneficial to make surfactant molecules more stretched and ordered, and help to enhance the efficiency of surfactant at the interface, it is beneficial to decrease interfacial tension if the hydrophobic chains of the surfactant and the oil have similar structure, and phenyl has a positive effect on interfacial efficiency. The results are in agreement with experimental and other theoretical work on surfactants.     

Emilin, a component of elastic fibers preferentially located at the elastin-microfibrils interface

The fine distribution of the extracellular matrix glycoprotein emilin (previously known as glycoprotein gp115) (Bressan, G. M., I. Castellani, A. Colombatti, and D. Volpin. 1983. J. Biol. Chem. 258: 13262-13267) has been studied at the ultrastructural level with specific antibodies. In newborn chick aorta the protein was exclusively found within elastic fibers. In both post- and pre-embedding immunolabeling emilin was mainly associated with regions where elastin and microfibrils are in close contact, such as the periphery of the fibers. This localization of emilin in aorta has been confirmed by quantitative evaluation of the distribution of gold particles within elastic fibers. In other tissues, besides being associated with typical elastic fibers, staining for emilin was found in structures lacking amorphous elastin, but where the presence of tropoelastin has been demonstrated by immunoelectron microscopy. This was particularly evident in the oxitalan fibers of the corneal stroma, in the Descemet's membrane, and in the ciliary zonule. Analysis of embryonic aorta revealed the presence of emilin at early stages of elastogenesis, before the appearance of amorphous elastin. Immunofluorescence studies have shown that emilin produced by chick embryo aorta cells in culture is strictly associated with elastin and that the process of elastin deposition is severely altered by the presence of antiemilin antibodies in the culture medium. The name of the protein was derived from its localization at sites where elastin and microfibrils are in proximity (emilin, elastin microfibril interface located protein).     

Characterization of oligomer formation of amyloid-beta peptide using a split-luciferase complementation assay

Amyloid-β peptide (Aβ) is the amyloid component of senile plaques in Alzheimer disease (AD) brains. Recently a soluble oliomeric form of Aβ in Aβ precursor protein transgenic mouse brains and AD brains was identified as a potential causative molecule for memory impairment, suggesting that soluble Aβ oligomers cause neurodegeneration in AD. Further characterization of this species has been hampered, however, because the concentrations are quite small and it is difficult to monitor Aβ oligomers specifically. Here we developed a novel method for monitoring Aβ oligomers using a split-luciferase complementation assay. In this assay, the N- and C-terminal fragments of Gaussia luciferase (Gluc) are fused separately to Aβ. We found that conditioned media from both N- and C-terminal fragments of Gluc-tagged Aβ1-42 doubly transfected HEK293 cells showed strong luminescence. We used gel filtration analyses to analyze the size of oligomers formed by the luciferase complementation assay, and found that it matched closely with oligomers formed by endogenous Aβ in Tg2576 neurons. Large oligomers (24-36-mers), 8-mers, trimers, and dimers predominate. In both systems, Aβ formed oligomers intracellularly, which then appear to be secreted as oligomers. We then evaluated several factors that might impact oligomer formation. The level of oligomerization of Aβ1-40 was similar to that of Aβ1-42. Homodimers formed more readily than heterodimers. The level of oligomerization of murine Aβ1-42 was similar to that of human Aβ1-42. As expected, the familial AD-linked Arctic mutation (E22G) significantly enhanced oligomer formation. These data suggest that Gluc-tagged Aβ enables the analysis of Aβ oligomers.     

Anomalous temperature dependence of peptide films at air-water interface

The tetrapeptide derivative Tyr-Gly-Phe-Ala-OBz (1) forms monolayers as confirmed by compressibility studies carried out at various temperatures. Peptide 1 monolayer exhibits an anomalous structural transition at 40 degrees C as evidenced by pi-A isotherms recorded at different temperatures. The structural transition is also observed in aqueous solution of trifluoroacetate of peptide 1 as evidenced by fluorescence and Raman scattering intensity measurements.     

Model of Alzheimer's disease amyloid-beta peptide based on a RNA binding protein

Although Alzheimer's Abeta peptide has been shown to form beta-sheet structure, a high-resolution molecular structure is still unavailable to date. A search for a sequence neighbor using Abeta(10-42) as the query in the Protein Data-Bank (PDB) revealed that an RNA binding protein, AF-Sm1 from Archaeoglobus fulgidus (PDB entry: 1i4k chain Z), shared 36% identical residues. Using AF-Sm1 as a template, we built a molecular model of Abeta(10-42) by applying comparative modeling methods. The model of Abeta(10-42) contains an antiparallel beta-sheet formed by residues 16-23 and 32-41. Hydrophobic surface constituted by residues 17-20 (LVFF) separates distinctly charged regions. Residues that interact with RNA in the AF-Sm1 crystal structure were found to be conserved in Abeta. Using a native gel we demonstrate for the first time that RNA can interact with Abeta and selectively retard the formation of fibrils or higher-order oligomers. We hypothesize that in a similar fashion to AF-Sm1, RNA interacts with Abeta in the beta-hairpin (beta-turn-beta) structure and prevents fibril formation.     

Growth and morphology of anchorage-dependent animal cells in a liquid/liquid interface system

In general, anchorage-dependent animal cells cultivated on a solid culture substrate, such as polystyrene, are collected by trypsin treatment. This treatment may have detrimental effects such as the proteolysis of the cell membrane proteins. To avoid these effects, cell cultivation using a liquid/liquid interface system has been investigated. In this cultivation method, the cells grow at the interface between a culture medium and a hydrophobic liquid. In this study, various fluorocarbons (FC-40, FC-70, KPF-91, KPF-102, and KPF-142) were used as substrates for the interface, and the cultivation of fibroblast cells (L-929; the mouse-derived cell line) at the interfaces was investigated. Early in the cultivation period, the growth of L-929 cells depended on the substrate type. Although cell cultivation at the interfaces was possible, it was slower than that at the polystyrene surface. Cell spreading at the interfaces was relatively small, which indicates that cell adhesion at the interfaces may be weak. In particular, the cells at the MEM/FC-70 interface anchored with one another and formed multicellular hemispherical aggregations shaped like spheroids. The difference in the adhesions to the interfaces appears to be dependent on the contaminants contained in the fluorocarbons because the physical properties of the fluorocarbon did not affect the cell growth and adhesion. Moreover, subcultivation from the interfaces to the same interface was possible without trypsin treatment. In this case, the delay of the growth at the interfaces did not occur because the cells were not affected by trypsin treatment.     

Electrostatics of a peptide at a membrane/water interface. The pH dependence of melittin association with lipid vesicles

The association of the peptide melittin with small unilamellar DMPC vesicles was studied as a function of pH. The results are discussed quantitatively assuming a water-membrane partition equilibrium. Electrostatic surface charging is taken into account as more and more of the strongly basic peptide accumulates at the bilayer/water interface. The data could be well described in terms of a Gouy-Chapman approach involving an effective interfacial charge well below the actual physical charge carried by the individual peptide molecules. The partition coefficient turned out to be pH invariant, so that one can exclude deprotonation reactions upon insertion of the peptide into the bilayer. The effective interfacial charge per associated melittin molecule decreased over a broad range of pH (pH 7 to pH above 10). Contributions of the free amino terminus and of the arginine residues could be determined by comparing with results obtained using modified melittin (N-terminally formylated and fully acetylated). The data suggest approximately equal fractional contributions of the amino terminus and the three lysines to the effective interfacial charge. The two arginines contribute less. Thus, they may be located farther away from the interface or be closely associated with counter-ions. The analysis is extended to the effect of different ionic strengths.     

High-resolution structure of a self-assembly-competent form of a hydrophobic peptide captured in a soluble beta-sheet scaffold

β-Rich self-assembly is a major structural class of polypeptides, but still little is known about its atomic structures and biophysical properties. Major impediments for structural and biophysical studies of peptide self-assemblies include their insolubility and heterogeneous composition. We have developed a model system, termed peptide self-assembly mimic (PSAM), based on the single-layer β-sheet of Borrelia outer surface protein A. PSAM allows for the capture of a defined number of self-assembly-like peptide repeats within a water-soluble protein, making structural and energetic studies possible. In this work, we extend our PSAM approach to a highly hydrophobic peptide sequence. We show that a penta-Ile peptide (Ile₅), which is insoluble and forms β-rich self-assemblies in aqueous solution, can be captured within the PSAM scaffold in a form capable of self-assembly. The 1.1-Å crystal structure revealed that the Ile₅ stretch forms a highly regular β-strand within this flat β-sheet. Self-assembly models built with multiple copies of the crystal structure of the Ile₅ peptide segment showed no steric conflict, indicating that this conformation represents an assembly-competent form. The PSAM retained high conformational stability, suggesting that the flat β-strand of the Ile₅ stretch primed for self-assembly is a low-energy conformation of the Ile₅ stretch and rationalizing its high propensity for self-assembly. The ability of the PSAM to “solubilize” an otherwise insoluble peptide stretch suggests the potential of the PSAM approach to the characterization of self-assembling peptides.     

Interaction of a peptide nanotube with a water-membrane interface

Inserting peptide nanotubes into lipid bilayers modulates the permeability properties of the cell wall, thus conferring potential bacteriocidal capability. Interaction of a peptide nanotube formed by eight cyclo[RRKWLWLW] subunits with the surface of a hydrated dimyristoylphosphatidylcholine bilayer is investigated using molecular dynamics simulations. The present sequence of alternated D-L-alpha-amino acids has been shown to yield remarkable antibacterial in vitro activity, and the chosen topoisomer corresponds to the optimum amphipathy of the tubular structure, whereby non-polar and charged side chains are segregated by the aqueous interface. The cohesion of the nanotube is ensured by a scaffold of intermolecular hydrogen bonds between adjacent cyclic peptides, supplemented by favorable like-charged contacts of arginine side chains. It is further reinforced by interactions of charged residues with the lipid head groups and of non-polar residues with the lipid acyl chains. The simulation reveals a partial breaking of the synthetic channel accompanying its early insertion into the lipid bilayer. The latter opens new questions about how peptide nanotubes permeate the membrane, in particular whether or not (i) self-assembly precedes partitioning and (ii) translocation occurs with the complete tubular structure.     

An application of ellipsometry: Assessment of polysaccharide and glycoprotein interaction with lectin at a liquid/solid interface

Lectins were specifically adsorbed from solution onto metallized glass slides coated with polysacchride, glycopeptide and glycoprotein films. The degree of interaction was determined by measuring the thickness of the bound lectin layer with an ellipsometer after washing and drying the slide. The binding of concanavalin A (tetrameric) and succinyl concanavalin A (dimeric) to a yeast mannan film was studied as a function of lectin concentration, temperature, rinsing time and the extent of stirring of the slide. The maximum thickness of bound concanavalin A and succinyl concanavalin A was 11 and 3.8 nm, respectively. The method permitted the measurement of the association constants for both lectins (1.0 · 10⁷ M^?1 for concanavalin A, 2 · 10⁶ M^?1 for succinyl concanavalin A) and the detection of 0.6 pmol concanavalin A. The same sensitivity was observed with anti-mannan antibodies. The binding of both lectins was shown to be specific using sugar haptens. When compared with methyl α-D-mannoside, the affinity of concanavalin A for D-mannose and D-glucose was 14 and 3%, respectively. A film of mucin glycopeptide (universal adsorbent) interacted similarly with concanavalin A, Ricinus communis I, soya bean and wheat germ lectins. However, films of glycoproteins such as fetuin, ceruloplasmin and Aspergillus niger β-D-galactosidase interacted to different degrees with these lectins. The relative affinity of wheat germ agglutinin for $\text{N-}\text{acetyl}\text{-}\text{D}\text{-}\text{glucosamine}$ and for chitin-derived oligosaccharides was also determined. When films of sialoglyproteins were treated with neuraminidase, the thickness of the bound peanut agglutinin layer increased. Although this method cannot determine quantitatively the sugar composition of the film, it permits rapid estimation of the interaction of lectins with polysaccharides and glycoproteins, usingg little material.     

Neuritic deposits of amyloid-beta peptide in a subpopulation of central nervous system-derived neuronal cells

Our goal is to understand the pathogenesis of amyloid-beta (Abeta) deposition in the Alzheimer's disease (AD) brain. We established a cell culture system where central nervous system-derived neuronal cells (CAD cells) produce and accumulate within their processes large amounts of Abeta peptide, similar to what is believed to occur in brain neurons, in the initial phases of AD. Using this system, we show that accumulation of Abeta begins within neurites, prior to any detectable signs of neurodegeneration or abnormal vesicular transport. Neuritic accumulation of Abeta is restricted to a small population of neighboring cells that express normal levels of amyloid-beta precursor protein (APP) but show redistribution of BACE1 to the processes, where it colocalizes with Abeta and markers of late endosomes. Consistently, cells that accumulate Abeta appear in isolated islets, suggesting their clonal origin from a few cells that show a propensity to accumulate Abeta. These results suggest that Abeta accumulation is initiated in a small number of neurons by intracellular determinants that alter APP metabolism and lead to Abeta deposition and neurodegeneration. CAD cells appear to recapitulate the biochemical processes leading to Abeta deposition, thus providing an experimental in vitro system for studying the molecular pathobiology of AD.     

Rapid synthesis of photoreceptor membrane and assembly of new microvilli in a crab at dusk

Summary Large areas of photoreceptor membrane are synthesized in the retinula cells of the crab Leptograpsus variegatus at dusk. Initially, new membrane differentiates from rough endoplasmic reticulum (ER) as large tubules of smooth ER. These tubules transform to concentric ellipsoids of closely apposed pairs of membranes (doublet ER), sometimes passing through an intervening crenate form. The new membrane is transported through bridges of cytoplasm that cross the palisade to the rhabdom region, from which the remains of the rhabdomeres that were built during the previous dusk have been dissolved. The degradation of the old microvilli of one rhabdomere is accomplished without affecting neighbouring rhabdomeres of other cells. New microvilli are assembled in situ from sheets of doublet ER, which are converted to tubules oriented in the same direction as the future microvilli. The cytoplasmic face of the ER remains the cytoplasmic face of the tubules, which become progressively narrower, partly by further longitudinal division, until the final diameter of the microvillus is reached. A central core is often seen in transverse sections of mature microvilli. It may be involved in the final consolidation, but rhabdomeric microvilli are not formed in the same manner as those of intestinal brush border cells. There is no evidence that new membrane passes through the Golgi compartment before incorporation into the rhabdom, as is the case for rod outer segment membrane in vertebrate photoreceptors.     

Solid-state NMR investigations of peptide-lipid interaction and orientation of a beta-sheet antimicrobial peptide,protegrin

Protegrin-1 (PG-1) is a broad-spectrum beta-sheet antimicrobial peptide found in porcine leukocytes. The mechanism of action and the orientation of PG-1 in lipid bilayers are here investigated using (2)H, (31)P, (13)C, and (15)N solid-state NMR spectroscopy. (2)H spectra of mechanically aligned and chain-perdeuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) bilayers indicate that PG-1 at high concentrations destroys the orientational order of the aligned lamellar bilayer. The conformation of the lipid headgroups in the unoriented region is significantly altered, as seen from the (31)P spectra of POPC and the (2)H spectra of headgroup-deuterated 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine. These observations indicate that PG-1 disrupts microbial membranes by breaking the extended bilayer into smaller disks, where a significant fraction of lipids is located in the edges of the disks with a distribution of orientations. These edges allow the lipid bilayer to bend back on itself as in toroidal pores. Interestingly, this loss of bilayer orientation occurs only in long-chain lipids such as POPC and not in shorter chain lipids such as 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine (DLPC). To understand the mode of binding of PG-1 to the lipid bilayer, we determined the orientation of PG-1 in DLPC bilayers. The (13)CO and (15)N chemical shifts of Val-16 labeled PG-1 indicate that the beta-strand axis is tilted by 55 degrees +/- 5 degrees from the bilayer normal while the normal of the beta-sheet plane is 48 degrees +/- 5 degrees from the bilayer normal. This orientation favors interaction of the hydrophobic backbone of the peptide with the hydrophobic core of the bilayer and positions the cationic Arg side chains to interact with the anionic phosphate groups. This is the first time that the orientation of a disulfide-stabilized beta-sheet membrane peptide has been determined by solid-state NMR.     

Neuropathology of human Alzheimer disease after immunization with amyloid-beta peptide: a case report

Amyloid-beta peptide (Abeta) has a key role in the pathogenesis of Alzheimer disease (AD). Immunization with Abeta in a transgenic mouse model of AD reduces both age-related accumulation of Abeta in the brain and associated cognitive impairment. Here we present the first analysis of human neuropathology after immunization with Abeta (AN-1792). Comparison with unimmunized cases of AD (n = 7) revealed the following unusual features in the immunized case, despite diagnostic neuropathological features of AD: (i) there were extensive areas of neocortex with very few Abeta plaques; (ii) those areas of cortex that were devoid of Abeta plaques contained densities of tangles, neuropil threads and cerebral amyloid angiopathy (CAA) similar to unimmunized AD, but lacked plaque-associated dystrophic neurites and astrocyte clusters; (iii) in some regions devoid of plaques, Abeta-immunoreactivity was associated with microglia; (iv) T-lymphocyte meningoencephalitis was present; and (v) cerebral white matter showed infiltration by macrophages. Findings (i)-(iii) strongly resemble the changes seen after Abeta immunotherapy in mouse models of AD and suggest that the immune response generated against the peptide elicited clearance of Abeta plaques in this patient. The T-lymphocyte meningoencephalitis is likely to correspond to the side effect seen in some other patients who received AN-1792 (refs. 7-9).     

Rapid assembly of a multimeric membrane protein pore

We have observed the assembly of the staphylococcal pore-forming toxin α-hemolysin using single-molecule fluorescence imaging. Surprisingly, assembly from the monomer to the complete heptamer is extremely rapid, occurring in <5 ms. No lower order oligomeric intermediates are detected. Monte Carlo simulation of our experiment shows that assembly is diffusion limited, and pore formation is dependent on the stability of intermediate species. There are close similarities between bacterial pore-forming toxins, such as staphylococcal α-hemolysin, the anthrax protective antigen, and the cholesterol-dependent cytolysins, and their eukaryotic analogs, such as the complement pore membrane attack complex and perforin domain. The assembly mechanism we have observed for α-hemolysin provides a simple model that aids our understanding of these important pore formers.