Amyloids are filamentous polymers of aberrantly folded proteins distinguished by cross-beta structure. Accumulation of amyloid is associated with approximately 20 human diseases, including Alzheimer's, type-2 diabetes, and rheumatoid arthritis. Amyloids are distinguished into two broad categories: infectious and non-infectious. Infectious amyloids are called prions. We started studying yeast prion structures in 1998, focusing initially on Ure2p, a negative regulator of nitrogen catabolism. We showed that its N-terminal domain is responsible for prionogenesis, while the C-terminal domain which performs its regulatory function remains folded in filaments but is inactivated by a steric mechanism. In our amyloid backbone concept, the prion domains form the filament backbone and are surrounded by the C-terminal domains. In 2005, we published the parallel superpleated beta-structure model for the amyloid backbone. With this model, it envisages arrays of parallel beta-sheets generated by stacking monomers with planar beta-serpentine folds. Topologically similar structures are good candidates for other amyloid fibrils, including amylin, and growing support for models of this kind is appearing in the scientific literature. Ongoing work is aimed at testing and refining this model;investigating fibril polymorphism;and relating amyloids to native conformations. In FY 13 we focused on two projects. (1) Polyglutamine-rich amyloids. Many neurodegenerative disorders such as Huntingtons disease exhibit intracerebral deposition of aggregated polypeptides with abnormally extended tracts of tandem glutamine residues (polyQ). These proteins are highly insoluble and form amyloid fibril-containing aggregates in vivo and in vitro. High-resolution structures for these fibrils would be of great value for investigating the molecular basis and the mechanism of progression of these diseases. However, analysis of polyQ fibrils has remained difficult. A major challenge has been their extreme insolubility whereby these polypeptides rapidly aggregate into higher-order complexes in which individual fibrils are barely distinguishable.
Aimi ng to produce fibrils suitable for structural analysis, we designed polyQ-containing peptides of suitable length ( and RT;30 amino acids) in which some L-lysine residues were included to enhance solubility and two D-lysines to interrupt the putative beta-strand by inserting reverse turns. Two polypeptides were synthesized: polyQKd33 (33 residues) and polyQKd32 (32 residues), which has one fewer glut amines in the central part. Both peptides were found to be soluble at neutral pH and to assemble into well dispersed fibrils at pH 11-12 suitable for EM analysis. These fibrils were imaged by negative staining EM, cryo-EM, and electron diffraction from unstained specimens and by dark-field STEM of unstained freeze-dried specimens at the Brookhaven STEM laboratory. When analyzed by electron diffraction, both fibrils showed a sharp ring at a spacing of (0. 47nm)-1 (Fig. 1A, B), indicative of cross-beta structure, the hallmark of amyloid. However, the polyQKd32 fibrils were slightly but consistently thicker than polyQKd33 by both negative staining EM and cryo-EM. Furthermore, dark-field STEM imaging revealed the same trend in fibril diameters. Mass-per-length analyses revealed an accompanying increase in subunit packing density from 1.0 to 1.5 molecules per axial repeat (0.47 nm) of the cross-beta structure. Underlying these changes in overall fibril architecture is the insertion of a single additional glutamine residue in the central part of the polypeptide. These observations - which are being prepared for publication - suggest models for the three-dimensional structures of these and other polyQ-containing fibrils. 2) Alfa-synuclein-containing amyloids. Parkinsons disease (PD) is a chronic and progressive neurodegenerative disease affecting motor function. PD is characterized by dopaminergic neuronal cell death and by the presence of Lewy bodies. Alfa-synuclein (aS) fibrils are the main component of Lewy bodies, and previous research suggests that its fibrillation is part of the disease pathology. Normally, the 140 aa long protein has a membrane remodeling function which we are also researching, as reported in project AR027015-18. aS is alfa-helical when associated with lipid and a random coil in solution. In fibril formation, the protein polymerizes into a cross-beta structure. Despite their high clinical relevance, structural information on aS-containing amyloid fibrils has been elusive and such information is the goal of this project. Recombinant aS was expressed in E. coli, purified and assembled into fibrils, which were observed by cryo-EM in our laboratory and by dark-field STEM at the Brookhaven STEM facility. The resulting cryo-EM images revealed that aS fibrils are 5 nm in diameter and more or less straight, and they have an axial repeat of 75 nm. Mass-per-length measurements made from the STEM data gave a unimodal distribution with a mean density equivalent to two subunits per 0.47 nm axial rise. This suggests that the fibril is in fact a pair of protofibrils - each with a parallel superpleated structure - wrapping around a common axis. Reconstruction of the cross-section indicates that each protofibril has two components. These observations, which afford a basis for modeling studies, are currently being prepared for publication.
|Huang, Rick K; Baxa, Ulrich; Aldrian, Gudrun et al. (2014) Conformational switching in PolyGln amyloid fibrils resulting from a single amino acid insertion. Biophys J 106:2134-42|
|Varkey, Jobin; Mizuno, Naoko; Hegde, Balachandra G et al. (2013) ýý-Synuclein oligomers with broken helical conformation form lipoprotein nanoparticles. J Biol Chem 288:17620-30|
|Baxa, Ulrich (2008) Structural basis of infectious and non-infectious amyloids. Curr Alzheimer Res 5:308-18|