The role of copper(II) in the aggregation of human amylin

Amylin is the 37-residue peptide hormone produced by the islet β-cells in the pancreas and the formation of amylin aggregates is strongly associated with β-cells degeneration in type 2 diabetes, as demonstrated by more than 95% of patients exhibiting amylin amyloid upon autopsy. It is widely recognized that metal ions such as copper(II) have been implicated in the aggregation process of amyloidogenic peptides such as Aβ and α-synuclein and there is evidence that also amylin self-assembly is largely affected by copper(II). For this reason, in this work, the role of copper(II) in the aggregation of amylin has been investigated by several different experimental approaches. Mass spectrometric investigations show that copper(II) induces significant changes in the amylin structure which decrease the protein fibrillogenesis as observed by ThT measurements. Accordingly, solid-state NMR experiments together with computational analysis carried out on a model amylin fragment confirmed the non fibrillogenic nature of the copper(II) induced aggregated structure. Finally, the presence of copper(II) is also shown to have a major influence on amylin proneness to be degraded by proteases and cytotoxicity studies on different cell cultures are reported

Inhibitory Mechanism of Pancreatic Amyloid Fibril Formation: Formation of the Complex between Tea Catechins and the Fragment of Residues 22–27

Islet amyloid polypeptide (IAPP) is a major component of pancreatic amyloid deposits associated with type 2 diabetes. Polyphenols contained in plant foods have been found to inhibit amyloid fibril formation of proteins and/or peptides. However, the inhibition mechanism is not clear for a variety of systems. Here the inhibition mechanism of green tea polyphenols, catechins, on amyloid fibril formation of the IAPP fragment (IAPP22–27), which is of sufficient length for formation of β-sheet-containing amyloid fibrils, was investigated by means of kinetic analysis. A quartz crystal microbalance (QCM) determined that the association constants of gallate-type catechins [epicatechin 3-gallate (ECg) and epigallocatechin 3-gallate] for binding to IAPP22–27 immobilized on the gold plate in QCM were 1 order of magnitude larger than those of the free IAPP22–27 peptide, and also those of epicatechin and epigallocatechin. Kinetic analysis using a two-step autocatalytic reaction mechanism revealed that ECg significantly reduced the rate constants of the first nucleation step of amyloid fibril formation, while the rate of autocatalytic growth was less retarded. ¹H nuclear magnetic resonance studies clarified that a IAPP22–27/ECg complex clearly forms as viewed from the ¹H chemical shift changes and line broadening. Our study suggests that tea catechins specifically inhibit the early stages of amyloid fibril formation to form amyloid nuclei by interacting with the unstructured peptide and that this inhibition mechanism is of great therapeutic value because stabilization of the native state could delay the pathogenesis of amyloid diseases and also the toxicity of the small oligomer (protofibril) is reported to be greater than that of the mature fibril

Interaction of epicatechin gallate with phospholipid membranes as revealed by solid-state NMR spectroscopy

AbstractEpicatechin gallate (ECg), a green tea polyphenol, has various physiological effects. Our previous nuclear Overhauser effect spectroscopy (NOESY) study using solution NMR spectroscopy demonstrated that ECg strongly interacts with the surface of phospholipid bilayers. However, the dynamic behavior of ECg in the phospholipid bilayers has not been clarified, especially the dynamics and molecular arrangement of the galloyl moiety, which supposedly has an important interactive role. In this study, we synthesized [13C]-ECg, in which the carbonyl carbon of the galloyl moiety was labeled by 13C isotope, and analyzed it by solid-state NMR spectroscopy. Solid-state 31P NMR analysis indicated that ECg changes the gel-to-liquid-crystalline phase transition temperature of DMPC bilayers as well as the dynamics and mobility of the phospholipids. In the solid-state 13C NMR analysis under static conditions, the carbonyl carbon signal of the [13C]-ECg exhibited an axially symmetric powder pattern. This indicates that the ECg molecules rotate about an axis tilting at a constant angle to the bilayer normal. The accurate intermolecular–interatomic distance between the labeled carbonyl carbon of [13C]-ECg and the phosphorus of the phospholipid was determined to be 5.3±0.1Å by 13C–31P rotational echo double resonance (REDOR) measurements. These results suggest that the galloyl moiety contributes to increasing the hydrophobicity of catechin molecules, and consequently to high affinity of galloyl-type catechins for phospholipid membranes, as well as to stabilization of catechin molecules in the phospholipid membranes by cation–π interaction between the galloyl ring and quaternary amine of the phospholipid head-group

Helix Conformations in 7TM Membrane Proteins Determined Using Oriented-Sample Solid-State NMR with Multiple Residue-Specific 15N Labeling

Oriented solid-state NMR in combination with multiple-residue-specific 15N labeling and extensive numerical spectral analysis is proposed to determine helix conformations of large membrane proteins in native membranes. The method is demonstrated on uniaxially oriented samples of 15N-methionine, -valine, and -glycine-labeled bacteriorhopsin in native purple membranes. Experimental two-dimensional 1H-15N dipole-dipole coupling versus 15N chemical shift spectra for all samples are analyzed numerically to establish combined constraints on the orientation of the seven transmembrane helices relative to the membrane bilayer normal. Since the method does not depend on specific resonance assignments and proves robust toward nonidealities in the sample alignment, it may be generally feasible for the study of conformational arrangement and function-induced conformation changes of large integral membrane proteins