No abstract

The islet amyloid polypeptide (IAPP) or amylin is a pancreatic hormone and crucially involved in the pathogenesis of type-II diabetes mellitus (T2DM). Aggregation and amyloid formation of IAPP is considered as the primary culprit for pancreatic beta-cell loss in T2DM patients. In this study, first X-ray reflectivity (XRR) measurements on IAPP at lipid interfaces have been carried out, providing a molecular level characterization of the first steps of the lipid-induced fibrillation process of IAPP, which is initiated by lipid-induced nucleation, oligomerization, followed by detachment of larger IAPP aggregate structures from the lipid membrane, and terminated by the formation of mature fibrils in the bulk solution. The adsorption process of IAPP at lipid interfaces in the absence and presence of negatively charged lipid has also been studied by complementary ATR-FTIR spectroscopic measurements. The morphological properties were followed by atomic force microscopy (AFM). Moreover, we show that the polyphenolic red wine compound resveratrol is able to inhibit IAPP aggregation also in the presence of aggregation-fostering negatively charged lipid interfaces, revealing its potential as a drug candidate for T2DM.

We report x-ray reflectivity and grazing incidence x-ray diffraction measurements of lipopolysaccharide (LPS) monolayers at the water-air interface. Our investigations reveal that the structure and lateral ordering of the LPS molecules is very different from phospholipid systems and can be modulated by the ionic strength of the aqueous subphase in an ion-dependent manner. Our findings also indicate differential effects of monovalent and divalent ions on the two-dimensional ordering of lipid domains. Na(+) ions interact unspecifically with LPS molecules based on their ability to efficiently screen the negative charges of the LPS molecules, whereas Ca(2+) ions interact specifically by cross-linking adjacent molecules in the monolayer. At low lateral pressures, Na(+) ions present in the subphase lead to a LPS monolayer structure ordered over large areas with high compressibility, nearly hexagonal packing of the hydrocarbon chains, and high density in the LPS headgroup region. At higher film pressures, the LPS monolayer becomes more rigid and results in a less perfect, oblique packing of the LPS hydrocarbon chains as well as a smaller lateral size of highly ordered domains on the monolayer. Furthermore, associated with the increased surface pressure, a conformational change of the sugar headgroups occurs, leading to a thickening of the entire LPS monolayer structure. The effect of Ca(2+) ions in the subphase is to increase the rigidity of the LPS monolayer, leading to an oblique packing of the hydrocarbon chains already at low film pressures, an upright orientation of the sugar moieties, and much smaller sizes of ordered domains in the plane of the monolayer. In the presence of both Na(+)- and Ca(2+) ions in the subphase, the screening effect of Na(+) is predominant at low film pressures, whereas, at higher film pressures, the structure and lateral organization of LPS molecules is governed by the influence of Ca(2+) ions. The unspecific charge-screening effect of the Na(+) ions on the conformation of the sugar moiety becomes less dominant at biologically relevant lateral pressures.

The volumetric properties of proteins yield information about the changes in packing and hydration between various states along the folding reaction coordinate and are also intimately linked to the energetics and dynamics of these conformations. These volumetric characteristics can be accessed via pressure perturbation methods. In this work, we report high-pressure unfolding studies of the ankyrin domain of the Notch receptor (Nank1-7) using fluorescence, small-angle x-ray scattering, and Fourier transform infrared spectroscopy. Both equilibrium and pressure-jump kinetic fluorescence experiments were consistent with a simple two-state folding/unfolding transition under pressure, with a rather small volume change for unfolding compared to proteins of similar molecular weight. High-pressure fluorescence, Fourier transform infrared spectroscopy, and small-angle x-ray scattering measurements revealed that increasing urea over a very small range leads to a more expanded pressure unfolded state with a significant decrease in helical content. These observations underscore the conformational diversity of the unfolded-state basin. The temperature dependence of pressure-jump fluorescence relaxation measurements demonstrated that at low temperatures, the folding transition state ensemble (TSE) lies close in volume to the folded state, consistent with significant dehydration at the barrier. In contrast, the thermal expansivity of the TSE was found to be equivalent to that of the unfolded state, indicating that the interactions that constrain the folded-state thermal expansivity have not been established at the folding barrier. This behavior reveals a high degree of plasticity of the TSE of Nank1-7.

Several proteins and peptides are known to form cytotoxic oligomers and amyloid fibrils-mainly consisting of intermolecular cross-b-sheets-upon misfolding and self-association.[1] As these amyloid aggregates deposit in tissues, they are associated with cell degenerative diseases, such as type-2 diabetes mellitus (T2DM) or Alzheimer's disease (AD).[1]The present study is focused on the membrane-mediated aggregation of heteroassemblies of the islet amyloid polypeptide (IAPP) and the b-amyloid (Ab) peptide. The extracellular deposits in the pancreas of patients with T2DM are mainly composed of the 37-residue IAPP, which is produced, stored, and secreted together with insulin by bcells in the pancreatic islets of Langerhans. [2,3] The major component of the extracellular plaques in AD brains is Ab, a 40-or 42-residue fragment of the membrane-associated amyloid precursor protein. [4,5] Recent clinical studies have pointed to a correlation between T2DM and AD, that is, patients with T2DM have a higher risk to suffer from AD and vice versa.[6] Remarkably, the sequences of IAPP and Ab show a 25 % identity and 50 % similarity and it has been shown that fibrillation of IAPP can be cross-seeded by Ab fibrils (Figure 1). [7] Most importantly, recent studies in vitro in the bulk phase have shown that early nonfibrillar and nontoxic Ab and IAPP species bind each other with high affinity forming soluble, nonfibrillar, and nontoxic heterooligomers and that this interaction delays the cytotoxic selfassociation and amyloidogenesis of both Ab and IAPP. [8] In this context, it has also been shown that the IAPP analogue [(N-Me)G24, (N-Me)I26]-IAPP or IAPP-GI, a mimic of nonamyloidogenic IAPP, forms nonfibrillar and nontoxic heteroassemblies with Ab and thus blocks the cytotoxic oligomer and fibril formation by Ab. [8,[12][13][14] As Ab and IAPP are present in blood and cerebrospinal fluid at similar concentrations, an in vivo interaction might be possible, which could be a molecular link between AD and T2DM. [9][10][11] Several studies have shown that lipid-peptide interactions can play a crucial role in amyloid formation of both IAPP and Ab, [1,3,[15][16][17][18][19][20][21][22][23][24][25][26][27][28] and the enhancement of fibrillation in the presence of membranes is believed to be causatively linked to the cellular damage caused by Ab or IAPP assemblies. [25,26,28] Here, the interaction of IAPP and Ab with a complex heterogeneous (raft-like) model biomembrane system comprising 15 % DOPC, 10 % DOPG, 40 % DPPC, 10 % DPPG, and 25 % cholesterol has been studied. This lipid system allows for addressing effects of lateral heterogeneity (i.e., coexisting liquid-disordered and liquid-ordered domains) as well as charge effects upon peptide-membrane interactions. Both effects have been shown to be important for the membrane interaction of these peptides. [16,18,20,27,28] Furthermore, the cross-interaction of IAPP and Ab in the presence of this membrane system was analyzed.To gain a detailed picture of the membrane-mediated aggregation proc...