Smallest Secondary Nucleation Competent Aβ Aggregates Probed by an ATP-Independent Molecular Chaperone Domain

Abstract

Protein oligomerization is a commonly encountered strategy by which the functional repertoire of proteins is increased. This, however, is a double-edged sword strategy because protein oligomerization is notoriously difficult to control. Living organisms have therefore developed a number of chaperones that prevent protein aggregation. The small ATP-independent molecular chaperone domain proSP-C BRICHOS, which is mainly trimeric, specifically inhibits fibril surface-catalyzed nucleation reactions that give rise to toxic oligomers during the aggregation of the Alzheimer’s disease-related amyloid-β peptide (Aβ42). Here, we have created a stable proSP-C BRICHOS monomer mutant and show that it does not bind to monomeric Aβ42 but has a high affinity for Aβ42 fibrils, using surface plasmon resonance. Kinetic analysis of Aβ42 aggregation profiles, measured by thioflavin T fluorescence, reveals that the proSP-C BRICHOS monomer mutant strongly inhibits secondary nucleation reactions and thereby reduces the level of catalytic formation of toxic Aβ42 oligomers. To study binding between the proSP-C BRICHOS monomer mutant and small soluble Aβ42 aggregates, we analyzed fluorescence cross-correlation spectroscopy measurements with the maximum entropy method for fluorescence correlation spectroscopy. We found that the proSP-C BRICHOS monomer mutant binds to the smallest emerging Aβ42 aggregates that are comprised of eight or fewer Aβ42 molecules, which are already secondary nucleation competent. Our approach can be used to provide molecular-level insights into the mechanisms of action of substances that interfere with protein aggregation.

Remarked Suppression of Aβ42 Protomer-Protomer Dissociation Reaction Elucidated by Molecular Dynamics Simulation

“…We then observed kinetic inactivation of protomer-protomer dissociation reactions even for Apentamer dimer. It is noted that A42 pentamer is found in the range of size of experimentally observed soluble A42 oligomers (15,23), and such small oligomeric aggregates are supposed to promote amyloid fibril formation by functioning as secondary nucleation competent aggregates (15). Our observation then suggests that stable formation of this protomer species is involved in a turning point in A42 amyloid fibril formation processes, then giving an important clue toward comprehensive understanding of microscopic mechanisms for shift from the lag phase to the growth phase.…”

“…The recent experimental studies reported that the number of subunits in soluble A42 aggregates ranges from 4 to 8 (15,23). Besides, such smaller oligomer species are supposed to be secondary nucleation competent fibril-like aggregates which can convert natively folded monomers into growth-competent monomers.…”

“…We calculated potential of mean forces for monomer dissociations from A42(4:4) and A42(10:10) (Figure 6A and 6B), the smallest and largest protomer dimers examined in this study, respectively. Besides, recalling that the smallest A42 fibril species is composed of 8 monomers or fewer (15), we suppose that A42(10:10) is sufficient in size as a model of A42 fibril species Each of the PMFs has one activation barrier toward the dissociation direction and the height of activation barrier appears to increase with the size of protomer. However, the C atoms of the m th , m+1 th … n th residues for l monomers in the protomer, and the case of A42(4:4) is illustrated here as an example.…”

“…In this context, earlier studies on amyloid- (1-42) (A42) have discussed the functional roles of oligomeric aggregates as growth nuclei in amyloid fibril growth, for example. (15)(16)(17) Nontheless, the minimum size of growth nuclei species remains to be elucidated so far even for the paradigmatic amyloid protein. Solving the problem appears to be difficult due to molecular diversity of A42 aggregates found in lag phase (18)(19)(20)(21).…”

“…It is still challenging for experimental approaches to separately examine physicochemical characters of individual oligomeric aggregate species. Under these circumstances, an experimental study reported that A42 oligomers take fibril forms in the aqueous solution (15), suggesting that such oligomeric A42 protomers contribute to the formation of growth nuclei species.…”