Structural basis for transthyretin amyloid formation in vitreous body of the eye

Amyloid transthyretin (ATTR) amyloidosis is characterized by the abnormal accumulation of ATTR fibrils in multiple organs. However, the structure of ATTR fibrils from the eye is poorly understood. Here, we used cryo-EM to structurally characterize vitreous body ATTR fibrils. These structures were distinct from previously characterized heart fibrils, even though both have the same mutation and type A pathology. Differences were observed at several structural levels: in both the number and arrangement of protofilaments, and the conformation of the protein fibril in each layer of protofilaments. Thus, our results show that ATTR protein structure and its assembly into protofilaments in the type A fibrils can vary between patients carrying the same mutation. By analyzing and matching the interfaces between the amino acids in the ATTR fibril with those in the natively folded TTR, we are able to propose a mechanism for the structural conversion of TTR into a fibrillar form.

Reversible supramolecular assembly of the anti-microbial peptide plectasin into helical non-amyloid fibrils

Self-assembly and fibril formation play important roles in protein behavior. Amyloid fibrils formation is well-studied due to its role in neurodegenerative diseases and characterized by refolding of the protein into predominant β-sheet form. However, much less is known about the assembly of proteins into other types of supramolecular structures. Using cryo-electron microscopy at a resolution of 1.97 Angstroem, we show that a triple-mutant of the anti-microbial peptide plectasin assembles reversibly into helical non-amyloid fibrils. Plectasin contains a cysteine-stabilized alpha-helix/beta-sheets structure, which remains intact upon fibril formation. Two fibrils form a right-handed superstructure with each fibril consisting of double helical, left-handed structures. The fibril formation is reversible and follows sigmoidal kinetics with a pH-dependent equilibrium between soluble monomer and protein fibril. The anti-microbial activity does not appear compromised by fibril formation. This is the first high-resolution structure of this type of alpha/beta protein fibrils.

Self-Assembly of Protein Fibrils in Microgravity

Deposits of insoluble protein fibrils in human tissue are associated with amyloidosis and neurodegenerative diseases. Different proteins are involved in each disease; all are soluble in their native conformation in vivo, but by molecular self-assembly, they all form insoluble protein fibril deposits with a similar cross β-sheet structure. This paper reports the results of an experiment in molecular self-assembly carried out in microgravity on the International Space Station (ISS). The Self-Assembly in Biology and the Origin of Life (SABOL) experiment was designed to study the growth of lysozyme fibrils in microgravity. Lysozyme is a model protein that has been shown to replicate the aggregation processes of other amyloid proteins. Here the design and performance of the experimental hardware is described in detail. The flight experiment was carried to the ISS in the Dragon capsule of the SpaceX CRS-5 mission and returned to Earth after 32 days. The lysozyme fibrils formed in microgravity aboard the ISS show a distinctly different morphology compared to fibrils formed in the ground-control (G-C) experiment. The fibrils formed in microgravity are shorter, straighter, and thicker than those formed in the laboratory G-C experiment. For two incubation periods, (2) about 8.5 days and (3) about 14.5 days, the average ISS and G-C fibril diameters are respectively: \matrix{{Period\,2} \hfill & {} \hfill & {{D_{ISS}} = 7.5{\rm{nm}} \pm 31\% ,} \hfill \cr {} \hfill & {\rm and} \hfill & {{D_{G - C}} = 3.4{\rm{nm}} \pm 31\%} \hfill \cr {Period\,3} \hfill & {} \hfill & {{D_{ISS}} = 6.2{\rm{nm}} \pm 33\% ,} \hfill \cr {} \hfill & {\rm and} \hfill & {{D_{G - C}} = 3.6{\rm{nm}} \pm 33\% .}}

The cryoEM structure of the fibril-forming low-complexity domain of hnRNPA2 reveals distinct differences from pathogenic amyloid and shows how mutation converts it to the pathogenic form

AbstracthnRNPA2 is one of a group of human ribonucleoproteins (RNPs) involved in RNA metabolism which form fibrils both under cellular stress and in mutated form in neurodegenerative conditions. Previous work established that the C-terminal low-complexity domain (LCD) of hnRNPA2 fibrillizes under stress, and that missense mutations in this domain are found in the disease multisystem proteinopathy (MSP) with symptoms indistinguishable from ALS and FTD. However, little is known at the atomic level about the hnRNPA2 LCD structure that is involved in those processes and how disease mutations cause structural change. Here we present the cryo-electron microscopy (cryoEM) structure of hnRNPA2 LCD fibril core and demonstrate its capability to form a reversible hydrogel in vitro containing amyloid-like fibrils. Whereas these fibrils, like pathogenic amyloid, are formed from protein chains stacked into β-sheets by backbone hydrogen bonds, they display distinct structural differences: the chains are kinked, enabling non-covalent cross-linking of fibrils and disfavoring formation of pathogenic steric zippers. Both their reversibility and energetic calculations suggest these fibrils are less stable than pathogenic amyloid. Moreover, the crystal structure of the disease-mutation-containing segment of hnRNPA2 suggests that the replacement fundamentally alters the fibril structure to a more stable energetic state. These findings illuminate how molecular interactions promote protein fibril networks and how mutation can transform fibril structure from functional to pathogenic form.

Barbituric Acid Based Fluorogens: Synthesis, Aggregation-Induced Emission, and Protein Fibril Detection

Fluorescent dyes, especially those emitting in the long wavelength region, are excellent candidates in the area of bioassay and bioimaging. In this work, we report a series of simple organic fluorescent dyes consisting of electron-donating aniline groups and electron-withdrawing barbituric acid groups. These dyes are very easy to construct while emitting strongly in the red region in their solid state. The photophysical properties of these dyes, such as solvatochromism and aggregation-induced emission, are systematically characterized. Afterward, the structure–property relationships of these barbituric acid based fluorogens are discussed. Finally, we demonstrate their potential applications for protein amyloid fibril detection.

High-Pressure Response of Amyloid Folds

The abnormal protein aggregates in progressive neurodegenerative disorders, such as Alzheimer’s, Parkinson’s and prion diseases, adopt a generic structural form called amyloid fibrils. The precise amyloid fold can differ between patients and these differences are related to distinct neuropathological phenotypes of the diseases. A key focus in current research is the molecular mechanism governing such structural diversity, known as amyloid polymorphism. In this review, we focus on our recent work on recombinant prion protein (recPrP) and the use of pressure as a variable for perturbing protein structure. We suggest that the amyloid polymorphism is based on volumetric features. Accordingly, pressure is the thermodynamic parameter that fits best to exploit volume differences within the states of a chemical reaction, since it shifts the equilibrium constant to the state that has the smaller volume. In this context, there are analogies with the process of correct protein folding, the high pressure-induced effects of which have been studied for more than a century and which provides a valuable source of inspiration. We present a short overview of this background and review our recent results regarding the folding, misfolding, and aggregation-disaggregation of recPrP under pressure. We present preliminary experiments aimed at identifying how prion protein fibril diversity is related to the quaternary structure by using pressure and varying protein sequences. Finally, we consider outstanding questions and testable mechanistic hypotheses regarding the multiplicity of states in the amyloid fold.