Predicted Loop Regions Promote Aggregation: A Study of Amyloidogenic Domains in the Functional Amyloid FapC.

Protein fibrillation is traditionally associated with misfolding, loss of functional phenotype, and gain of toxicity in neurodegenerative diseases. However, many organisms exploit fibrils in the form of functional amyloids (FA), as seen in bacteria, such as E. coli, Salmonella, Bacillus, and Pseudomonas. Here, we provide structural information and mechanistic data for fibrillation of the smallest amyloidogenic truncation unit along with the full-length version (FL) of the major amyloid protein FapC from Pseudomonas, predicted to consist of three ß-hairpin-forming imperfect repeats separated by disordered regions. Using a series of truncation mutants, we establish that the putative loops (linkers) increase the rate of aggregation. The minimal aggregation unit consisting of a single repeat with flanking disordered regions (R3C) aggregates in a pathway dominated by secondary nucleation, in contrast to the primary nucleation favored by full-length (FL) FapC. SAXS on FapC FL, R3C, and remaining truncation constructs resolves two major coexisting species in the fibrillation process, namely pre-fibrillar loosely aggregated monomers, and cylindrical, elliptical cross-section fibrils. Solid-state NMR spectra identified rigid parts of the FapC fibril. We assigned Cα-Cß chemical shifts, indicative of a predominant ß-sheet topology with some α-helix or loop chemical shifts. Our work emphasizes the complex nature of FapC fibrillation. In addition, we are able to deduce the importance of non-repeat regions (i.e., predicted loops), which enhance the amyloid protein aggregation and their influence on the polymorphism of the fibril architecture.

Reducing the Amyloidogenicity of Functional Amyloid Protein FapC Increases Its Ability To Inhibit α-Synuclein Fibrillation.

Functional amyloid (FA) proteins have evolved to assemble into fibrils with a characteristic cross-ß structure, which stabilizes biofilms and contributes to bacterial virulence. Some of the most studied bacterial FAs are the curli protein CsgA, expressed in a wide range of bacteria, and FapC, produced mainly by members of the Pseudomonas genus. Though unrelated, both CsgA and FapC contain imperfect repeats believed to drive the formation of amyloid fibrils. While much is known about CsgA biogenesis and fibrillation, the mechanism of FapC fibrillation remains less explored. Here, we show that removing the three imperfect repeats of FapC (FapC ΔR1R2R3) slows down the fibrillation but does not prevent it. The increased lag phase seen for FapC ΔR1R2R3 allows for disulfide bond formation, which further delays fibrillation. Remarkably, these disulfide-bonded species of FapC ΔR1R2R3 also significantly delay the fibrillation of human α-synuclein, a key protein in Parkinson's disease pathology. This attenuation of α-synuclein fibrillation was not seen for the reduced form of FapC ΔR1R2R3. The results presented here shed light on the FapC fibrillation mechanism and emphasize how unrelated fibrillation systems may share such common fibril formation mechanisms, allowing inhibitors of one fibrillating protein to affect a completely different protein.

The random co-polymer glatiramer acetate rapidly kills primary human leukocytes through sialic-acid-dependent cell membrane damage.

The formulation glatiramer acetate (GA) is widely used in therapy of multiple sclerosis. GA consists of random copolymers of four amino acids, in ratios that produce a predominantly positive charge and an amphipathic character. With the extraordinary complexity of the drug, several pharmacological modes-of-action were suggested, but so far none, which rationalizes the cationicity and amphipathicity as part of the mode-of-action. Here, we report that GA rapidly kills primary human T lymphocytes and, less actively, monocytes. LL-37 is a cleavage product of human cathelicidin with important roles in innate immunity. It shares the positive charge and amphipathic character of GA, and, as shown here, also the ability to kill human leukocyte. The cytotoxicity of both compounds depends on sialic acid in the cell membrane. The killing was associated with the generation of CD45+ debris, derived from cell membrane deformation. Nanoparticle tracking analysis confirmed the formation of such debris, even at low GA concentrations. Electric cell-substrate impedance sensing measurements also recorded stable alterations in T lymphocytes following such treatment. LL-37 forms oligomers through weak hydrophobic contacts, which is critical for the lytic properties. In our study, SAXS showed that GA also forms this type of contacts. Taken together, our study offers new insight on the immunomodulatory mode-of-action of positively charged co-polymers. The comparison of LL-37 and GA highlights a consistent requirement of certain oligomeric and chemical properties to support cytotoxic effects of cationic polymers targeting human leukocytes.

The role of stable α-synuclein oligomers in the molecular events underlying amyloid formation.

Studies of proteins' formation of amyloid fibrils have revealed that potentially cytotoxic oligomers frequently accumulate during fibril formation. An important question in the context of mechanistic studies of this process is whether or not oligomers are intermediates in the process of amyloid fibril formation, either as precursors of fibrils or as species involved in the fibril elongation process or instead if they are associated with an aggregation process that is distinct from that generating mature fibrils. Here we describe and characterize in detail two well-defined oligomeric species formed by the protein α-synuclein (αSN), whose aggregation is strongly implicated in the development of Parkinson's disease (PD). The two types of oligomers are both formed under conditions where amyloid fibril formation is observed but differ in molecular weight by an order of magnitude. Both possess a degree of ß-sheet structure that is intermediate between that of the disordered monomer and the fully structured amyloid fibrils, and both have the capacity to permeabilize vesicles in vitro. The smaller oligomers, estimated to contain ∼30 monomers, are more numerous under the conditions used here than the larger ones, and small-angle X-ray scattering data suggest that they are ellipsoidal with a high degree of flexibility at the interface with solvent. This oligomer population is unable to elongate fibrils and indeed results in an inhibition of the kinetics of amyloid formation in a concentration-dependent manner.