γD-crystallin is an abundant structural protein of the lens that is found in native and modified forms in cataractous aggregates. We establish that UV-B irradiation of γD-crystallin leads to structurally specific modifications and precipitation via two mechanisms: amorphous aggregates and amyloid fibers. UV-B radiation causes cleavage of the backbone, in large measure near the interdomain interface, where side chain oxidations are also concentrated. 2D IR spectroscopy and expressed protein ligation localize fiber formation exclusively to the C-terminal domain of γD-crystallin. The native β-sandwich domains are not retained upon precipitation by either mechanism. The similarity between the amyloid forming pathway when induced by either UV-B radiation or low pH suggests that it is the propensity for the C-terminal β-sandwich domain to form amyloid β-sheets that determines the misfolding pathway independent of the mechanism of denaturation.

Background: Amyloids made of apolipoprotein A-I (apoA-I) contribute to the growth of the atherosclerotic plaques. Results: ApoA-I methionine oxidation by physiological levels of myeloperoxidase induces amyloid formation. Conclusion: Myeloperoxidase-mediated oxidation not only impairs the physiological functions of apoA-I but also promotes protein loss in form of amyloids. Significance: Our findings identify the physiological mechanism transforming wild-type apoA-I into an amyloidogenic protein.

UV light and other factors damage crystallin proteins in the eye lens, resulting in cataracts that scatter light and affect vision. Little information exists about protein structures within these disease-causing aggregates. We examined postmortem lens tissue from individuals with and without cataracts using 2D infrared (2DIR) spectroscopy. Amyloid β-sheet secondary structure was detected in cataract lenses along with denatured structures. No amyloid structures were found in lenses from juveniles, but mature lenses with no cataract diagnosis also contained amyloid, indicating that amyloid structures begin forming before diagnosis. Light scatters more strongly in regions with amyloid structure, and UV light induces amyloid β-sheet structures, linking the presence of amyloid structures to disease pathology. Establishing that age-related cataracts involve amyloid structures gives molecular insight into a common human affliction and provides a possible structural target for pharmaceuticals as an alternative to surgery.

Cataracts are formed by the aggregation of crystallin proteins in the eye lens. Many in vitro studies have established that crystallin proteins precipitate into aggregates that contain amyloid fibers when denatured, but there is little evidence that ex vivo cataracts contain amyloid. In this study, we collect two dimensional infrared (2D IR) spectra on tissue slices of porcine eye lenses. As shown in control experiments on in vitro αB- and γD-crystallin, 2D IR spectroscopy can identify amyloidogenic β-sheet secondary structure because this structure has exceptionally strong coupling and a high degree of ordering that creates characteristic diagonal and cross peaks. In ex vivo experiments of acid treated tissues, characteristic 2D IR features are observed and fibers >50 nm in length are resolved by TEM, consistent with amyloid fibers. In UV-irradiated lens tissues, fibers are not observed with TEM, but amyloidogenic β-sheet secondary structure is identified from the 2D IR spectra. The characteristic 2D IR features of amyloid β-sheet secondary structure are created by as few as 4 or 5 strands, and so identify amyloid secondary structure even if the aggregates themselves are too small to be resolved with TEM. We discuss these findings in the context of the chaperone system of the lens, which we hypothesize sequesters small aggregates, thereby preventing long fibers from forming. This study expands the scope of heterodyned 2D IR spectroscopy to tissues. The results provide a link between in vitro and ex vivo studies, and support the hypothesis that cataracts is an amyloid disease.

Two-dimensional sum-frequency generation spectroscopy (2D SFG) is used to study the structures of the pentapeptide FGAIL on hydrogen bond promoting surfaces. FGAIL is the most amyloidogenic portion of the human islet amyloid polypeptide (hIAPP or amylin). In the presence of a pure gold surface, FGAIL does not form ordered structures. When the gold is coated with a self-assembled monolayer of mercaptobenzoic acid (MBA), 2D SFG spectra reveal features associated with β-sheets. Also observed are cross peaks between the FGAIL peptides and the carboxylic acid groups of the MBA monolayer, indicating that the peptides are in close contact with the surface headgroups. In the second set of samples, FGAIL peptides chemically ligated to the MBA monolayer also exhibited β-sheet features but with a much simpler spectrum. From simulations of the experiments, we conclude that the hydrogen bond promoting surface catalyzes the formation of both parallel and antiparallel β-sheet structures with several different orientations. When ligated, parallel sheets with only a single orientation are the primary structure. Thus, this hydrogen bond promoting surface creates a heterogeneous distribution of polymorph structures, consistent with a concentration effect that allows nucleation of many different amyloid seeding structures. A single well-defined seed favors one polymorph over the others, showing that the concentrating influence of a membrane can be counterbalanced by factors that favor directed fiber growth. These experiments lay the foundation for the measurement and interpretation of β-sheet structures with heterodyne-detected 2D SFG spectroscopy. The results of this model system suggest that a heterogeneous distribution of polymorphs found in nature are an indication of nonselective amyloid aggregation whereas a narrow distribution of polymorph structures is consistent with a specific protein or lipid interaction that directs fiber growth.