Nanoscale Heterogeneity of the Molecular Structure of Individual hIAPP Amyloid Fibrils Revealed with Tip-Enhanced Raman Spectroscopy.

Type 2 diabetes mellitus is characterized by the pathological deposition of fibrillized protein, known as amyloids. It is thought that oligomers and/or amyloid fibrils formed from human islet amyloid polypeptide (hIAPP or amylin) cause cell death by membrane damage. The molecular structure of hIAPP amyloid fibrils is dominated by ß-sheet structure, as probed with conventional infrared and Raman vibrational spectroscopy. However, with these techniques it is not possible to distinguish between the core and the surface structure of the fibrils. Since the fibril surface crucially affects amyloid toxicity, it is essential to know its structure. Here the surface molecular structure and amino acid residue composition of hIAPP fibrils are specifically probed with nanoscale resolution using tip-enhanced Raman spectroscopy (TERS). The fibril surface mainly contains unordered or α-helical structures, in contrast to the ß-sheet-rich core. This experimentally validates recent models of hIAPP amyloids based on NMR measurements. Spatial mapping of the surface structure reveals a highly heterogeneous surface structure. Finally, TERS can probe fibrils formed on a lipid interface, which is more representative of amyloids in vivo.

Shear stress-dependent downregulation of the adhesion-G protein-coupled receptor CD97 on circulating leukocytes upon contact with its ligand CD55.

Adhesion G protein-coupled receptors (aGPCRs) are two-subunit molecules, consisting of an adhesive extracellular α subunit that couples noncovalently to a seven-transmembrane ß subunit. The cooperation between the two subunits and the effect of endogenous ligands on the functioning of aGPCRs is poorly understood. In this study, we investigated the interaction between the pan-leukocyte aGPCR CD97 and its ligand CD55. We found that leukocytes from CD55-deficient mice express significantly increased levels of cell surface CD97 that normalized after transfer into wild-type mice because of contact with CD55 on both leukocytes and stromal cells. Downregulation of both CD97 subunits occurred within minutes after first contact with CD55 in vivo, which correlated with an increase in plasma levels of soluble CD97. In vitro, downregulation of CD97 on CD55-deficient leukocytes cocultured with wild-type blood cells was strictly dependent on shear stress. In vivo, CD55-mediated downregulation of CD97 required an intact circulation and was not observed on cells that lack contact with the blood stream, such as microglia. Notably, de novo ligation of CD97 did not activate signaling molecules constitutively engaged by CD97 in cancer cells, such as ERK and protein kinase B/Akt. We conclude that CD55 downregulates CD97 surface expression on circulating leukocytes by a process that requires physical forces, but based on current evidence does not induce receptor signaling. This regulation can restrict CD97-CD55-mediated cell adhesion to tissue sites.

The polyphenol EGCG inhibits amyloid formation less efficiently at phospholipid interfaces than in bulk solution.

Age-related diseases, like Alzheimer's disease and type 2 diabetes mellitus, are characterized by protein misfolding and the subsequent pathological deposition of fibrillized protein, also called amyloid. Several classes of amyloid-inhibitors have recently been tested, traditionally under bulk conditions. However, it has become apparent that amyloid fibrils and oligomers assemble and exert their cytotoxic effect at cellular membranes, rather than in bulk solution. Knowledge is therefore required of inhibitor activity specifically at the phospholipid membrane interface. Here we show, using surface-specific sum-frequency generation (SFG) spectroscopy and atomic force microscopy (AFM), that the commonly used (-)-epigallocatechin gallate (EGCG) is a much less efficient amyloid inhibitor at a phospholipid interface than in bulk solution. Moreover, EGCG is not able to disaggregate existing amyloid fibrils at a phospholipid interface, in contrast to its behavior in bulk. Our results show that interfaces significantly affect the efficiency of inhibition by EGCG inhibitors and should therefore be considered during the design and testing of amyloid inhibitors.

Morphology and persistence length of amyloid fibrils are correlated to peptide molecular structure.

The formation of amyloid fibrils is a self-assembly process of peptides or proteins. The superior mechanical properties of these fibrils make them interesting for materials science but constitute a problem in amyloid-related diseases. Amyloid structures tend to be polymorphic, and their structure depends on growth conditions. To understand and control the assembly process, insights into the relation between the mechanical properties and molecular structure are essential. We prepared long, straight as well as short, worm-like ß-lactoglobulin amyloid fibrils and determined their morphology and persistence length by atomic force microscopy (AFM) and the molecular conformation using vibrational sum-frequency generation (VSFG) spectroscopy. We show that long fibrils with near-100% ß-sheet content have a 40-times higher persistence length than short, worm-like fibrils with ß-sheet contents below 80%.

Multimodal Spectroscopic Study of Amyloid Fibril Polymorphism.

Amyloid fibrils are a large class of self-assembled protein aggregates that are formed from unstructured peptides and unfolded proteins. The fibrils are characterized by a universal ß-sheet core stabilized by hydrogen bonds, but the molecular structure of the peptide subunits exposed on the fibril surface is variable. Here we show that multimodal spectroscopy using a range of bulk- and surface-sensitive techniques provides a powerful way to dissect variations in the molecular structure of polymorphic amyloid fibrils. As a model system, we use fibrils formed by the milk protein ß-lactoglobulin, whose morphology can be tuned by varying the protein concentration during formation. We investigate the differences in the molecular structure and composition between long, straight fibrils versus short, wormlike fibrils. We show using mass spectrometry that the peptide composition of the two fibril types is similar. The overall molecular structure of the fibrils probed with various bulk-sensitive spectroscopic techniques shows a dominant contribution of the ß-sheet core but no difference in structure between straight and wormlike fibrils. However, when probing specifically the surface of the fibrils with nanometer resolution using tip-enhanced Raman spectroscopy (TERS), we find that both fibril types exhibit a heterogeneous surface structure with mainly unordered or α-helical structures and that the surface of long, straight fibrils contains markedly more ß-sheet structure than the surface of short, wormlike fibrils. This finding is consistent with previous surface-specific vibrational sum-frequency generation (VSFG) spectroscopic results ( VandenAkker et al. J. Am. Chem. Soc. , 2011 , 133 , 18030 - 18033 , DOI: 10.1021/ja206513r ). In conclusion, only advanced vibrational spectroscopic techniques sensitive to surface structure such as TERS and VSFG are able to reveal the difference in structure that underlies the distinct morphology and rigidity of different amyloid fibril polymorphs that have been observed for a large range of food and disease-related proteins.