Regulation of divalent metal ions to the aggregation and membrane damage of human islet amyloid polypeptide oligomers.

The accumulation of human islet amyloid polypeptide (hIAPP) on the surface of pancreatic ß cells is closely related to the death of the cells. Divalent metal ions play a significant role in the cytotoxicity of hIAPP. In this study, we examined the roles played by the divalent metal ions of zinc, copper and calcium in the aggregation of both hIAPP18-27 fragment and full-length hIAPP and the ability of their oligomers to damage the membrane of POPC/POPG 4 : 1 LUVs using the ThT fluorescence, TEM, AFM, CD, ANS binding fluorescence and dye leakage experiments. We prepared metal-free and metal-associated oligomers that are similar in size and aggregate slowly using the short peptide and confirmed that the ability of the peptide oligomers to damage the lipid membrane is reduced by the binding to the metal ions, which is closely linked to the reducing hydrophobic exposure of the metal-associated oligomers. The study on the full-length hIAPP showed that the observed membrane damage induced by hIAPP oligomers is either mitigated at a peptide-to-metal ratio of 1 : 0.33 or aggravated at a peptide-to-metal ratio of 1 : 1 in the presence of Zn(ii) and Cu(ii), while the surface hydrophobicity of hIAPP oligomers was reduced at both peptide-to-metal ratios. The observed results of the membrane damage were attributed to the counteraction between a decrease in the disruptive ability of metal-associated oligomer species and an increase in the quantity of oligomers promoted by the binding of the metal ions to hIAPP oligomers. The former could play a predominant role in reducing the membrane damage at a peptide-to-metal ratio of 1 : 0.33, while the latter could play a predominant role in enhancing the membrane damage at a peptide-to-metal ratio of 1 : 1. This study shows that an enhanced membrane damage could be caused by the oligomer species with a decreased instead of an increased disruptive ability, given that the abundance of the oligomer species is high enough.

Role of Chain Extension in the Ability of Peptide Oligomers to Damage the Lipid Membrane Studied by the l- to d-Amino Acid Substitutions of hIAPP18-27.

Exploration of the relation between the structural feature of oligomers and the ability of oligomers to damage the membrane has been an important subject in the study of the cytotoxic mechanism of amyloid proteins. In this work, we selected the hIAPP18-27 fragment as a model peptide and modified it by an alternating substitution of a d-amino acid for an l-amino acid in the hydrophilic N-terminal region, the hydrophobic C-terminal region, and the entire sequence. We prepared the oligomers using these peptides and investigated the effects of chain extension in different regions of the peptide on the ability of the oligomers to damage the membrane composed of POPC/POPG 4:1. We examined the morphology, structure, surface hydrophobicity, and packing compactness of the oligomers and monitored the changes in the structure and aggregation of the peptides upon interaction with the membrane. We found that the surface hydrophobicity and the disruptive ability of the oligomers are increased by an alternating l- and d-amino acid arrangement in the hydrophobic region of the peptide, while the packing compactness of the oligomers is increased and the disruptive ability of the oligomers decreased by an alternating l- and d-amino acid arrangement only in the hydrophilic region. The extension of the hydrophobic chain plays a significant role in the disruptive ability of the oligomers. Our results suggest that a positive relation between the surface hydrophobicity and the disruptive ability could be established only for the oligomers in which the peptide chains are flexible and loosely packed.

Study on the structure and membrane disruption of the peptide oligomers constructed by hIAPP18-27 peptide and its d,l-alternating isomer.

Increasing lines of evidence show that the oligomeric intermediates of amyloid peptides/proteins are toxic to biological membranes. However, the structural features of the oligomers that are closely associated with the ability to damage biological membranes are far from understanding. In this study, we constructed two species of oligomers using hIAPP18-27 peptide and its d,l-alternating isomer, examined the disruptive ability of the oligomers to POPC/POPG 4:1 vesicles by leakage assay and 31P NMR spectroscopy, and characterized the structural features of the oligomers by CD, TEM, 1H NMR and fluorescence quenching experiments. We found that the d,l-alternating peptide oligomers are more disruptive than the all-L peptide oligomers to the lipid membrane. The characterization of the secondary structure revealed that the d,l-alternating peptide adopts an extended polyproline type-II (PPII) conformation, while the all-L peptide adopts a random coil conformation in oligomers. Compared with the all-L peptide oligomers, the d,l-alternating peptide oligomers are less compact and keep more hydrophobic groups water exposed. Both the changes from PPII to α-sheet in the structure of d,l-alternating peptide and from random coil to ß-sheet in the structure of all-L peptide reduce the ability of the peptide oligomers to disrupt the lipid membrane. Our results suggest that an oligomer with extended peptide chains could be more potent in membrane disruption than an oligomer with folded peptide chains and an increase in peptide-peptide interaction could decrease the disruptive ability of oligomer.

Stabilization of Solvent to α-Sheet Structure and Conversion between α-Sheet and ß-Sheet in the Fibrillation Process of Amyloid Peptide.

α-sheet conformation has been proposed to exist as an intermediate conformational component and mediate the fibrillar assemblies of amyloid proteins at certain conditions. However, less attention has been paid to this form of conformation in the studies of the mechanism of amyloidosis because there is insufficient evidence for the existence of the α-sheet intermediate and the transition from the α-sheet intermediate to the ß-sheet fibril. Herein, we characterized the structures of a d,l-alternating amyloidogenic decapeptide under different conditions and studied the structural conversion between the α-sheet and ß-sheet in the fibrillation processes of the peptide. We obtained for the first time the image of α-sheet structured fibrils in aqueous solution and found the essential role of water molecules in the stabilization of the α-sheet structure. We also provided experimental evidence of the structural conversion from the α-sheet to the ß-sheet in the peptide aggregates.

Exploring the relation between the oligomeric structure and membrane damage by a study on rat islet amyloid polypeptide.

The formation of prefibrillar intermediates is a key stage not only for the fibrillation of amyloid peptides but also for their cytotoxicity. The heterogeneous and transient nature of most prefibrillar intermediates make them difficult to be separated, identified, and characterized. Rat islet amyloid polypeptide (rIAPP) has a weak propensity of fibrillation under most solution conditions and is found to be cytotoxic, which enables us to characterize prefibrillar species formed at various oligomeric states and explore the mechanism of membrane damage by these oligomers. In the present study, we prepared rIAPP oligomers under various conditions and characterized their structures, morphologies, sizes, interactions with the membrane composed of POPC/POPG 4 : 1, and disruptive efficiencies to the membrane using CD, TEM, DLS, NMR spectroscopy, and leaking assays. We found that oligomerization of rIAPP below a size limit of ∼50 nm in diameter rendered rIAPP more damaging to the membrane, whereas the formation of assemblies with sizes above this limit dramatically decreased the disruptive potency of rIAPP to the membrane. The oligomer species with smaller sizes and higher membrane-damage efficiencies have a longer time stability of α-helix at the membrane that is associated with a stronger membrane binding. Our findings show that interplay between the oligomeric size and hydrophobic exposure is implicated in the mechanism of membrane damage. The positive correlation between hydrophobic exposure and disruptive potency is valid only for the oligomers with sizes smaller than certain size limit.

Cholesterol-sensing role of phenylalanine in the interaction of human islet amyloid polypeptide with lipid bilayers.

The interactions between hIAPP and the pancreatic ß-cells are associated with ß-cell death in type II diabetes. Cholesterol modulates hIAPP-membrane interaction and hIAPP aggregation. The molecular mechanism underlying this is not well understood. Here we explore the cholesterol-sensing role of F15 in the interactions of hIAPP and hIAPP1-19 with various compositions of lipids, including DOPC, DPPC and DOPC/DPPC using NMR, CD, ThT fluorescence and dye leakage assays. We show that both hIAPP and hIAPP1-19 are more potent in the disruption to the membranes with cholesterol than they are in the disruption to the membranes without cholesterol. A substitution of F15 by leucine affects the binding and disruption of the peptides to the membranes slightly in the absence of cholesterol, but decreases the activities largely in the presence of cholesterol. F15 also plays a role in accelerating fibrillar assembly of hIAPP, but the function is independent of cholesterol in nature. The promotion of cholesterol to the disruptive potency of hIAPP is more effective in the membrane with raft-like domains than in the membrane with a dispersed distribution of cholesterol. Our results suggest that F15 plays a key role in the cholesterol-sensing binding and disruption of hIAPP to the PC membranes and the distribution of cholesterol in the membranes has an influence on the disruptive activity of hIAPP.

Formation of lamellar micelle-like oligomers and membrane disruption revealed by the study of short peptide hIAPP18-27.

Prefibrillar amyloid aggregates of proteins are known as cytotoxic species and a common pathogenic factor for many degenerative diseases. The mechanism underlying the formation and cytotoxicity of prefibrillar aggregates is believed to be independent of the actual nature of the amyloid protein. In this study, we monitored the formation of the peptide oligomers and examined the disruptive effects of the oligomers on liposomes using the human islet amyloid polypeptide fragment hIAPP18-27 as a model peptide. We observed various morphologies of oligomers formed at different aggregation stages that precede the formation of mature amyloid fibrils. These oligomer species were sufficiently stable to maintain their structures and properties under acidic conditions. We presented the first evidence that an oligomer species with a lamellar crystalline structure and a size of about 20-60 nm in length, 8 nm in width and 1.5 nm in thickness was the most disruptive to the membrane containing the anionic component and toxic to the INS-1 cells. Our results showed that short peptides, in light of their slower fibrillation, could be used as a model system in the study of the toxic mechanism of misfolding oligomers of amyloid peptides.

Nonenzymatic biosensor based on Cu(x)O nanoparticles deposited on polypyrrole nanowires for improving detection range.

Cu(x)O (CuO and Cu2O composite) nanoparticles modified polypyrrole (PPy) nanowires were fabricated and used as a biosensor for detecting glucose (GLC). PPy nanowires were prepared through electrodeposition, while Cu(x)O nanoparticles were deposited on PPy nanowires by electrodeposition and electrochemical oxidation in situ. The scanning electron microscopy images showed the Cu(x)O nanoparticles aligned along the PPy nanowires uniformly and the average size of Cu(x)O nanoparticles is about 90 nm. The electrocatalytic activity of Cu(x)O/PPy/Au towards GLC was investigated under alkaline conditions using cyclic voltammetry and chronoamperometry. The sensor exhibited a linear range up to 8 mM of GLC, which is more than two times of most of the existing non-enzymatic GLC sensors based on CuO or Cu2O. The sensitivity of the sensor is 232.22 µAmM⁻¹ cm⁻² and detection limit is 6.2 µM (at signal/noise=3). Moreover, the sensor showed excellent selectivity, reproducibility and stability properties. These excellent performances make Cu(x)O/PPy/Au a good nonenzymatic GLC sensor.

Synthesis of thiolated chitosan and preparation nanoparticles with sodium alginate for ocular drug delivery.

PURPOSE: The goal of the present study was to synthesize mucoadhesive polymer - thiolated chitosan (TCS) from chitosan (CS), then prepared CS/TCS-sodium alginate nanoparticles (CS/TCS-SA NPs), determined which was more potential for ocular drug delivery. METHODS: A new method for preparing TCS was developed, and the characteristics were determined using Fourier transform infrared spectroscopy and the degree of thiol immobilized was measured by Ellman's reagent. Human corneal epithelium (HCE) cells were incubated with different concentrations of TCS for 48 h to determine the cell viabilities. CS/TCS-SA NPs were prepared and optimized by a modified ionic gelation method. The particle sizes, zeta potentials, Scanning electron microscopy images, mucoadhesion, in vitro cell uptake and in vivo studies of the two types of NP were compared. RESULTS: The new method enabled a high degree of thiol substitution of TCS, up to 1,411.01±4.02 µmol/g. In vitro cytocompatibility results suggest that TCS is nontoxic. Compared to CS-SA NPs, TCS-SA NPs were more stable, with higher mucoadhesive properties and could deliver greater amounts of drugs into HCE cells in vitro and cornea in vivo. CONCLUSIONS: TCS-SA NPs have better delivery capability, suggesting they have good potential for ocular drug delivery applications.