Spontaneous aggregation of the insulin-derived steric zipper peptide VEALYL results in different aggregation forms with common features.

Recently, several short peptides have been shown to self-assemble into amyloid fibrils with generic cross-beta spines, so-called steric zippers, suggesting common underlying structural features and aggregation mechanisms. Understanding these mechanisms is a prerequisite,for designing fibril-binding compounds and inhibitors of fibril formation. The hexapeptide VEALYL, corresponding to the residues B12-17 of full-length insulin, has been identified as one of these short segments. Here, we analyzed the structures of multiple, morphologically different (fibrillar, microcrystal-like, oligomeric) [C-13,N-15]VEALYL samples by solid-state nuclear magnetic resonance complemented with results from molecular dynamics simulations. By performing NHHC/CHHC experiments, we could determine that the beta-strands within a given sheet of the amyloid-like fibrils formed by the insulin hexapeptide VEALYL are stacked in an antiparallel manner, whereas the sheet-to-sheet packing arrangement was found to be parallel. Experimentally observed secondary chemical shifts for all aggregate forms, as well as empty set and Psi backbone torsion angles calculated with TALOS, are indicative of beta-strand conformation, consistent with the published crystal structure (PDB ID: 2OMQ). Thus, we could demonstrate that the structural features of all the observed VEALYL aggregates are in agreement with the previously observed homosteric zipper spine packing in the crystalline state, suggesting that several distinct aggregate morphologies share the same molecular architecture. (C) 2013 The Authors. Published by Elsevier Ltd. All rights reserved

Membrane protein sequestering by ionic protein–lipid interactions.

Neuronal exocytosis is catalyzed by the SNARE protein syntaxin-1A(1). Syntaxin-1A is clustered in the plasma membrane at sites where synaptic vesicles undergo exocytosis(2,3). However, how syntaxin-1A is sequestered is unknown. Here, we show that syntaxin clustering is mediated by electrostatic interactions with the strongly anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2). We found with super-resolution STED microscopy on the plasma membrane of PC12 cells that PIP2 is the dominant inner-leaflet lipid in ~73 nm-sized microdomains. This high accumulation of PIP2 was required for syntaxin-1A sequestering, as destruction of PIP2 by the phosphatase synaptojanin-1 reduced syntaxin-1A clustering. Furthermore, co-reconstitution of PIP2 and the C-terminal part of syntaxin-1A in artificial giant unilamellar vesicles resulted in segregation of PIP2 and syntaxin-1A into distinct domains even when cholesterol was absent. Our results demonstrate that electrostatic protein-lipid interactions can result in the formation of microdomains independent of cholesterol or lipid phases

Use of zinc phosphate cement as a luting agent for Denzir™ copings: an in vitro study

BACKGROUND: The clinical success rate with zinc phosphate cemented Procera crowns is high. The objective with this study was to determine whether CADCAM processed and zinc phosphate cemented Denzir copings would perform as well as zinc phosphate cemented Procera copings when tested in vitro in tension. METHODS: Twelve Procera copings and twenty-four Denzir copings were made. After the copings had been made, twelve of the Denzir copings were sandblasted on their internal surfaces. All copings were then cemented with zinc phosphate cement to carbon steel dies and transferred to water or artificial saliva. Two weeks after cementation, half of the samples were tested. The remaining samples were tested after one year in the storage medium. All tests were done in tension and evaluated with an ANOVA. RESULTS: Sandblasted and un-sandblasted Denzir copings performed as well as Procera copings. Storage in water or artificial saliva up to one year did not decrease the force needed to dislodge any of the coping groups. Three copings fractured during testing and one coping developed a crack during testing. The three complete fractures occurred in Procera copings, while the partly cracked coping was a Denzir coping. CONCLUSION: No significant differences existed between the different material groups, and the retentive force increased rather than decreased with time. Fewer fractures occurred in Denzir copings, explained by the higher fracture toughness of the Denzir material. Based on good clinical results with zinc phosphate cemented Procera crowns, we foresee that zinc phosphate cement luted Denzir copings are likely to perform well clinically

Synthetic beta cells for fusion-mediated dynamic insulin secretion

Generating artificial pancreatic beta cells by using synthetic materials to mimic glucose-responsive insulin secretion in a robust manner holds promise for improving clinical outcomes in people with diabetes. Here, we describe the construction of artificial beta cells (AβCs) with a multicompartmental 'vesicles-in-vesicle' superstructure equipped with a glucose-metabolism system and membrane-fusion machinery. Through a sequential cascade of glucose uptake, enzymatic oxidation and proton efflux, the AβCs can effectively distinguish between high and normal glucose levels. Under hyperglycemic conditions, high glucose uptake and oxidation generate a low pH (<5.6), which then induces steric deshielding of peptides tethered to the insulin-loaded inner small liposomal vesicles. The peptides on the small vesicles then form coiled coils with the complementary peptides anchored on the inner surfaces of large vesicles, thus bringing the membranes of the inner and outer vesicles together and triggering their fusion and insulin 'exocytosis'