Biphasic effects of insulin on islet amyloid polypeptide membrane disruption.

Type II diabetes, in its late stages, is often associated with the formation of extracellular islet amyloid deposits composed of islet amyloid polypeptide (IAPP or amylin). IAPP is stored before secretion at millimolar concentrations within secretory granules inside the ß-cells. Of interest, at these same concentrations in vitro, IAPP rapidly aggregates and forms fibrils, yet within secretory granules of healthy individuals, IAPP does not fibrillize. Insulin is also stored within the secretory granules before secretion, and has been shown in vitro to inhibit IAPP fibril formation. Because of insulin's inhibitory effect on IAPP fibrillization, it has been suggested that insulin may also inhibit IAPP-mediated permeabilization of the ß-cell plasma membrane in vivo. We show that although insulin is effective at preventing fiber-dependent membrane disruption, it is not effective at stopping the initial phase of membrane disruption before fibrillogenesis, and does not prevent the formation of small IAPP oligomers on the membrane. These results suggest that insulin has a more complicated role in inhibiting IAPP fibrillogenesis, and that other factors, such as the low pH of the secretory granule, may also play a role.

Determination of the oligomer size of amyloidogenic protein beta-amyloid(1-40) by single-molecule spectroscopy.

Amyloid diseases are traditionally characterized by the appearance of inter- and intracellular fibrillar protein deposits, termed amyloid. Historically, these deposits have been thought to be the etiology of the disease. However, recent evidence suggests that small oligomers of the amyloidogenic protein/peptide are the origin of neurotoxicity. Although the importance of identifying the toxic oligomeric species is widely recognized, such identification is challenging because these oligomers are metastable, occur at low concentration, and are characterized by a high degree of heterogeneity. In this work, a fluorescently labeled beta-amyloid(1-40) is used as a model amyloidogenic peptide to test the effectiveness of what we believe is a novel approach based on single-molecule spectroscopy. We find that by directly counting the photobleaching steps in the fluorescence, we can determine the number of subunits in individual beta-amyloid(1-40) oligomers, which allows us to easily distinguish among different species in the mixtures. The results are further analyzed by comparison with Monte Carlo simulations to show that the variability seen in the size of photobleaching steps can be explained by assuming random dipole orientations for the chromophores in a given oligomer. In addition, by accounting for bias in the oligomer size distribution due to the need to subtract background noise, we can make the results more quantitative. Although the oligomer size determined in this work is limited to only small species, our single-molecule results are in good quantitative agreement with high-performance liquid chromatography gel filtration data and demonstrate that single-molecule spectroscopy can provide useful insights into the issues of heterogeneity and ultimately cellular toxicity in the study of amyloid diseases.

Amyloid-beta membrane binding and permeabilization are distinct processes influenced separately by membrane charge and fluidity.

The 40 and 42 residue amyloid-beta (Abeta) peptides are major components of the proteinaceous plaques prevalent in the Alzheimer's disease-afflicted brain and have been shown to have an important role in instigating neuronal degeneration. Whereas it was previously thought that Abeta becomes cytotoxic upon forming large fibrillar aggregates, recent studies suggest that soluble intermediate-sized oligomeric species cause cell death through membrane permeabilization. The present study examines the interactions between Abeta40 and lipid membranes using liposomes as a model system to determine how changes in membrane composition influence the conversion of Abeta into these toxic species. Abeta40 membrane binding was monitored using fluorescence-based assays with a tryptophan-substituted peptide (Abeta40 [Y10W]). We extend previous observations that Abeta40 interacts preferentially with negatively charged membranes, and show that binding of nonfibrillar, low molecular mass oligomers of Abeta40 to anionic, but not neutral, membranes involves insertion of the peptide into the bilayer, as well as sequential conformational changes corresponding to the degree of oligomerization induced. Significantly, while anionic membranes in the gel, liquid crystalline, and liquid ordered phases induce these conformational changes equally, membrane permeabilization is reduced dramatically as the fluidity of the membrane is decreased. These findings demonstrate that binding alone is not sufficient for membrane permeabilization, and that the latter is also highly dependent on the fluidity and phase of the membrane. We conclude that binding and pore formation are two distinct steps. The differences in Abeta behavior induced by membrane composition may have significant implications on the development and progression of AD as neuronal membrane composition is altered with age.

Amyloid fiber formation and membrane disruption are separate processes localized in two distinct regions of IAPP, the type-2-diabetes-related peptide.

Aggregation of Islet Amyloid Polypeptide (IAPP) has been implicated in the development of type II diabetes. Because IAPP is a highly amyloidogenic peptide, it has been suggested that the formation of IAPP amyloid fibers causes disruption of the cellular membrane and is responsible for the death of beta-cells during type II diabetes. Previous studies have shown that the N-terminal 1-19 region, rather than the amyloidogenic 20-29 region, is primarily responsible for the interaction of the IAPP peptide with membranes. Liposome leakage experiments presented in this study confirm that the pathological membrane disrupting activity of the full-length hIAPP is also shared by hIAPP 1-19. The hIAPP 1-19 fragment at a low concentration of peptide induces membrane disruption to a near identical extent as the full-length peptide. At higher peptide concentrations, the hIAPP 1-19 fragment induces a greater extent of membrane disruption than the full-length peptide. Similar to the full-length peptide, hIAPP 1-19 exhibits a random coil conformation in solution and adopts an alpha-helical conformation upon binding to lipid membranes. However, unlike the full-length peptide, the hIAPP 1-19 fragment did not form amyloid fibers when incubated with POPG vesicles. These results indicate that membrane disruption can occur independently from amyloid formation in IAPP, and the sequences responsible for amyloid formation and membrane disruption are located in different regions of the peptide.