The effect of Aß on IAPP aggregation in the presence of an isolated ß-cell membrane.

Fibrillar aggregates of the islet amyloid polypeptide (IAPP) and amyloid-ß (Aß) are known to deposit at pancreatic ß-cells and neuronal cells and are associated with the cell degenerative diseases type-2 diabetes mellitus (T2DM) and Alzheimer's disease (AD), respectively. Since IAPP is secreted by ß-cells and a membrane-damaging effect of IAPP has been discussed as a reason for ß-cell dysfunction and the development of T2DM, studies of the interaction of IAPP with the ß-cell membrane are of high relevance for gaining a molecular-level understanding of the underlying mechanism. Recently, it has also been shown that patients suffering from T2DM exhibit an increased risk to develop AD and vice versa, and a molecular link between AD and T2DM has been suggested. In this study, membrane lipids from the rat insulinoma-derived INS-1E ß-cell line were isolated, and their interaction with the amyloidogenic peptides IAPP and Aß and a mixture of both peptides has been studied. To yield insight into the associated peptides' conformational changes and their effect on the membrane integrity during aggregation, we have carried out attenuated total reflection Fourier transform infrared spectroscopy, fluorescence microscopy, and atomic force microscopy experiments. The IAPP-Aß heterocomplexes formed were shown to adsorb, aggregate, and permeabilize the isolated ß-cell membrane significantly slower than pure IAPP, however, at a rate that is much faster than that of pure Aß. In addition, it could be shown that isolated ß-cell membranes cause similar effects on the kinetics of IAPP and IAPP-Aß fibril formation as anionic heterogeneous model membranes.

Temperature-pressure phase diagram of a heterogeneous anionic model biomembrane system: results from a combined calorimetry, spectroscopy and microscopy study.

By using Fourier transform infrared (FT-IR) spectroscopy in combination with differential scanning calorimetry (DSC) coupled with pressure perturbation calorimetry (PPC), ultrasound velocimetry, Laurdan fluorescence spectroscopy, fluorescence microscopy and atomic force microscopy (AFM), the temperature and pressure dependent phase behavior of the five-component anionic model raft lipid mixture DOPC/DOPG/DPPC/DPPG/cholesterol (20:5:45:5:25 mol%) was investigated. A temperature range from 5 to 65 °C and a pressure range up to 16 kbar were covered to establish the temperature-pressure phase diagram of this heterogeneous model biomembrane system. Incorporation of 10-20 mol% PG still leads to liquid-ordered (l(o))-liquid-disordered (l(d)) phase coexistence regions over a wide range of temperatures and pressures. Compared to the corresponding neutral model raft mixture (DOPC/DPPC/Chol 25:50:25 mol%), the p,T-phase diagram is - as expected and in accordance with the Gibbs phase rule - more complex, the phase sequence as a function of temperature and pressure is largely similar, however. This anionic heterogeneous model membrane system will serve as a more realistic model biomembrane system to study protein interactions with anionic lipid bilayers displaying liquid-disordered/liquid-ordered domain coexistence over a wide range of the temperature-pressure plane, thus allowing also studies of biologically relevant systems encountered under extreme environmental conditions.

Membrane-mediated induction and sorting of K-Ras microdomain signaling platforms.

The K-Ras4B GTPase is a major oncoprotein whose signaling activity depends on its correct localization to negatively charged subcellular membranes and nanoclustering in membrane microdomains. Selective localization and clustering are mediated by the polybasic farnesylated C-terminus of K-Ras4B, but the mechanisms and molecular determinants involved are largely unknown. In a combined chemical biological and biophysical approach we investigated the partitioning of semisynthetic fully functional lipidated K-Ras4B proteins into heterogeneous anionic model membranes and membranes composed of viral lipid extracts. Independent of GDP/GTP-loading, K-Ras4B is preferentially localized in liquid-disordered (l(d)) lipid domains and forms new protein-containing fluid domains that are recruiting multivalent acidic lipids by an effective, electrostatic lipid sorting mechanism. In addition, GDP-GTP exchange and, thereby, Ras activation results in a higher concentration of activated K-Ras4B in the nanoscale signaling platforms. Conversely, palmitoylated and farnesylated N-Ras proteins partition into the l(d) phase and concentrate at the l(d)/l(o) phase boundary of heterogeneous membranes. Next to the lipid anchor system, the results reveal an involvement of the G-domain in the membrane interaction process by determining minor but yet significant structural reorientations of the GDP/GTP-K-Ras4B proteins at lipid interfaces. A molecular mechanism for isoform-specific Ras signaling from separate membrane microdomains is postulated from the results of this study.

Interaction of hIAPP with model raft membranes and pancreatic beta-cells: cytotoxicity of hIAPP oligomers.

Type II diabetes mellitus (T2DM) is associated with beta-cell failure, which correlates with the formation of pancreatic islet amyloid deposits. The human islet amyloid polypeptide (hIAPP) is the major component of islet amyloid and undergoes structural changes followed by self-association and pathological tissue deposition during aggregation in T2DM. There is clear evidence that the aggregation process is accelerated in the presence of particular lipid membranes. Whereas hIAPP aggregation has been extensively studied in homogeneous model membrane systems, especially negatively charged lipid bilayers, information on the interaction of hIAPP with heterogeneous model raft membranes has been missing until now. In the present study, we focus on the principles of aggregation and amyloid formation of hIAPP in the presence of model raft membranes. Time-lapse tapping mode AFM and confocal fluorescence microscopy experiments followed membrane permeabilization and localization of hIAPP in the raft membrane. Together with the ThT and WST-1 assay, the data revealed elevated cytotoxicity of hIAPP oligomers on INS-1E cells.

Fluorescence microscopy studies on islet amyloid polypeptide fibrillation at heterogeneous and cellular membrane interfaces and its inhibition by resveratrol.

Type II diabetes mellitus (T2DM) is a disease characterized by progressive deposition of amyloid in the extracellular matrix of beta-cells. We investigated the interaction of the islet amyloid polypeptide (IAPP) with lipid model raft mixtures and INS-1E cells using fluorescence microscopy techniques. Following preferential partitioning of IAPP into the fluid lipid phase, the membrane suffers irreversible damage and predominantly circularly-shaped lipid-containing IAPP amyloid is formed. Interaction studies with the pancreatic beta-cell line INS-1E revealed that growing IAPP fibrils also incorporate substantial amounts of cellular membranes in vivo. Additionally, the inhibitory effect of the red wine compound resveratrol on IAPP fibril formation has been studied, alluding to its potential use in developing therapeutic strategies against T2DM.

Biochemical indication for myristoylation-dependent conformational changes in HIV-1 Nef.

The accessory HIV-1 Nef protein is essential for viral replication, high virus load, and progression to AIDS. These functions are mediated by the alteration of signaling and trafficking pathways and require the membrane association of Nef by its N-terminal myristoylation. However, a large portion of Nef is also found in the cytosol, in line with the observation that myristoylation is only a weak lipidation anchor for membrane attachment. We performed biochemical studies to analyze the implications of myristoylation on the conformation of Nef in aqueous solution. To establish an in vivo myristoylation assay, we first optimized the codon usage of Nef for Escherichia coli expression, which resulted in a 15-fold higher protein yield. Myristoylation was achieved by coexpression with the N-myristoyltransferase and confirmed by mass spectrometry. The myristoylated protein was soluble, and proton NMR spectra confirmed proper folding. Size exclusion chromatography revealed that myristoylated Nef appeared of smaller size than the unmodified form but not as small as an N-terminally truncated from of Nef that omits the anchor domain. Western blot stainings and limited proteolysis of both forms showed different recognition profiles and degradation pattern. Analytical ultracentrifugation revealed that myristoylated Nef prevails in a monomeric state while the unmodified form exists in an oligomeric equilibrium of monomer, dimer, and trimer associations. Finally, fluorescence correlation spectroscopy using multiphoton excitation revealed a shorter diffusion time for the lipidated protein compared to the unmodified form. Taken together, our data indicated myristoylation-dependent conformational changes in Nef, suggesting a rather compact and monomeric form for the lipidated protein in solution.