Dissecting the structural and functional roles of a putative metal entry site in encapsulated ferritins.

Encapsulated ferritins belong to the universally distributed ferritin superfamily, whose members function as iron detoxification and storage systems. Encapsulated ferritins have a distinct annular structure and must associate with an encapsulin nanocage to form a competent iron store that is capable of holding significantly more iron than classical ferritins. The catalytic mechanism of iron oxidation in the ferritin family is still an open question because of the differences in organization of the ferroxidase catalytic site and neighboring secondary metal-binding sites. We have previously identified a putative metal-binding site on the inner surface of the Rhodospirillum rubrum encapsulated ferritin at the interface between the two-helix subunits and proximal to the ferroxidase center. Here we present a comprehensive structural and functional study to investigate the functional relevance of this putative iron-entry site by means of enzymatic assays, MS, and X-ray crystallography. We show that catalysis occurs in the ferroxidase center and suggest a dual role for the secondary site, which both serves to attract metal ions to the ferroxidase center and acts as a flow-restricting valve to limit the activity of the ferroxidase center. Moreover, confinement of encapsulated ferritins within the encapsulin nanocage, although enhancing the ability of the encapsulated ferritin to undergo catalysis, does not influence the function of the secondary site. Our study demonstrates a novel molecular mechanism by which substrate flux to the ferroxidase center is controlled, potentially to ensure that iron oxidation is productively coupled to mineralization.

Identification of potential aggregation hotspots on Aß42 fibrils blocked by the anti-amyloid chaperone-like BRICHOS domain.

Protein misfolding can generate toxic intermediates, which underlies several devastating diseases, such as Alzheimer's disease (AD). The surface of AD-associated amyloid-ß peptide (Aß) fibrils has been suggested to act as a catalyzer for self-replication and generation of potentially toxic species. Specifically tailored molecular chaperones, such as the BRICHOS protein domain, were shown to bind to amyloid fibrils and break this autocatalytic cycle. Here, we identify a site on the Aß42 fibril surface, consisting of three C-terminal ß-strands and particularly the solvent-exposed ß-strand stretching from residues 26-28, which is efficiently sensed by a designed variant of Bri2 BRICHOS. Remarkably, while only a low amount of BRICHOS binds to Aß42 fibrils, fibril-catalyzed nucleation processes are effectively prevented, suggesting that the identified site acts as a catalytic aggregation hotspot, which can specifically be blocked by BRICHOS. Hence, these findings provide an understanding how toxic nucleation events can be targeted by molecular chaperones.

Specific inhibition of α-synuclein oligomer generation and toxicity by the chaperone domain Bri2 BRICHOS.

Protein misfolding and aggregation are involved in several neurodegenerative disorders, such as α-synuclein (αSyn) implicated in Parkinson's disease, where new therapeutic approaches remain essential to combat these devastating diseases. Elucidating the microscopic nucleation mechanisms has opened new opportunities to develop therapeutics against toxic mechanisms and species. Here, we show that naturally occurring molecular chaperones, represented by the anti-amyloid Bri2 BRICHOS domain, can be used to target αSyn-associated nucleation processes and structural species related to neurotoxicity. Our findings revealed that BRICHOS predominantly suppresses the formation of new nucleation units on the fibrils surface (secondary nucleation), decreasing the oligomer generation rate. Further, BRICHOS directly binds to oligomeric αSyn species and effectively diminishes αSyn fibril-related toxicity. Hence, our studies show that molecular chaperones can be utilized as tools to target molecular processes and structural species related to αSyn neurotoxicity and have the potential as protein-based treatments against neurodegenerative disorders.

Molecular Mechanisms of Amyloid-ß Self-Assembly Seeded by In Vivo-Derived Fibrils and Inhibitory Effects of the BRICHOS Chaperone.

Self-replication of amyloid-ß-peptide (Aß) fibril formation is a hallmark in Alzheimer's disease (AD). Detailed insights have been obtained in Aß self-assembly in vitro, yet whether similar mechanisms are relevant in vivo has remained elusive. Here, we investigated the ability of in vivo-derived Aß fibrils from two different amyloid precursor protein knock-in AD mouse models to seed Aß42 aggregation, where we quantified the microscopic rate constants. We found that the nucleation mechanism of in vivo-derived fibril-seeded Aß42 aggregation can be described with the same kinetic model as that in vitro. Further, we identified the inhibitory mechanism of the anti-amyloid BRICHOS chaperone on seeded Aß42 fibrillization, revealing a suppression of secondary nucleation and fibril elongation, which is strikingly similar as observed in vitro. These findings hence provide a molecular understanding of the Aß42 nucleation process triggered by in vivo-derived Aß42 propagons, providing a framework for the search for new AD therapeutics.