The Amyloid-β (Aβ) peptide is an aggregation-prone peptide linked to neurodegeneration in Alzheimer’s disease (AD). Aβ self-assembles spontaneously in aqueous solution to form aggregates of various sizes, with smaller pre-fibrillar oligomeric aggregates being especially neurotoxic. Such small oligomers are however difficult to study as they are transient, low abundant and heterogenous. We here use a combination of native ion mobility-mass spectrometry and molecular dynamics simulations to systematically study the structure and assembly mechanisms of Aβ oligomers in vitro. It is found that oligomers cannot be formed by a peptide variant that does not have propensity to fold into a β-hairpin motif present in the wild type Aβ peptide. This specific structure motif seems to be a more important determinant for aggregation than the overall hydrophobicity of the peptide. Introduction of an intramolecular disulfide bond in the Aβ peptide increases oligomerization, even though the monomeric peptide is not stabilized in the hairpin conformation. This is probably achieved by pre-arranging the peptide in a conformation which is compatible with oligomeric, but not fibrillar structures. As oligomerization is driven by formation of the hairpin motif it was furthermore possible to decrease the oligomer population by truncating one of the β-strands, and thus decreasing the hairpin propensity of the peptide. These studies provide increased understanding of the earliest steps in Aβ aggregation where species related to AD toxicity might be formed. Prevention of Aβ folding into the hairpin conformation, or specific binding to the hairpin motif could be strategies to design AD therapies. 

A human molecular chaperone protein, DnaJ heat shock protein family (Hsp40) member B6 (DNAJB6), efficiently inhibits amyloid aggregation. This inhibition depends on a unique motif with conserved serine and threonine (S/T) residues that have a high capacity for hydrogen bonding. Global analysis of kinetics data has previously shown that DNAJB6 especially inhibits the primary nucleation pathways. These observations indicated that DNAJB6 achieves this remarkably effective and sub-stoichiometric inhibition by interacting not with the monomeric unfolded conformations of the amyloid-? symbol (A?) peptide but with aggregated species. However, these pre-nucleation oligomeric aggregates are transient and difficult to study experimentally. Here, we employed a native MS-based approach to directly detect oligomeric forms of A? formed in solution. We found that WT DNAJB6 considerably reduces the signals from the various forms of A? (1?40) oligomers, whereas a mutational DNAJB6 variant in which the S/T residues have been substituted with alanines does not. We also detected signals that appeared to represent DNAJB6 dimers and trimers to which varying amounts of A? are bound. These data provide direct experimental evidence that it is the oligomeric forms of A? that are captured by DNAJB6 in a manner which depends on the S/T residues. We conclude that, in agreement with the previously observed decrease in primary nucleation rate, strong binding of A? oligomers to DNAJB6 inhibits the formation of amyloid nuclei.

The DNAJB6 chaperone inhibits fibril formation of aggregation-prone client peptides through interaction with aggregated and oligomeric forms of the amyloid peptides. Its C-terminal domain (CTD) is believed to be functionally important, which is here studied using a set of constructs, either comprising the entire CTD or the first two or all four of the β-strands in the CTD grafted onto a scaffold protein. Each construct was expressed as wild-type and as a mutant variant with alanines replacing five highly conserved and functionally important serine and threonine residues in the first β-strand. The stability, oligomerization, anti-amyloid activity, and affinity for Amyloid-β (Aβ42) species was explored for the constructs using optical spectroscopy, native mass spectrometry, chemical crosslinking and surface plasmon resonance technology. While DNAJB6 forms large and polydisperse oligomers, CTD was found to form only monomers, dimers and tetramers. The dimerization and thermal stability of CTD are significantly reduced by the alanine substitutions. Kinetic analyses show a shift in inhibition mechanism. Full-length DNAJB6 efficiently inhibits primary and secondary nucleation, while all CTD constructs inhibited secondary nucleation but not primary nucleation. Moreover, the anti-amyloid activity of the CTD constructs was not dependent on the serine and threonine residues in the first β-strand. Our findings indicate that the inhibition of primary nucleation in full length DNAJB6 may be due to its high chemical potential and thus its propensity to form oligomers and co-oligomers with Aβ. The CTD constructs instead only bind to Aβ42 fibrils, which affects the nucleation events at the fibril surface.

The assembly of proteins and peptides into amyloid fibrils is causally linked to serious disorders such as Alzheimer’s disease. Multiple proteins have been shown to prevent amyloid formation in vitro and in vivo, ranging from highly specific chaperone–client pairs to completely nonspecific binding of aggregation-prone peptides. The underlying interactions remain elusive. Here, we turn to the machine learning–based structure prediction algorithm AlphaFold2 to obtain models for the nonspecific interactions of β-lactoglobulin, transthyretin, or thioredoxin 80 with the model amyloid peptide amyloid β and the highly specific complex between the BRICHOS chaperone domain of C-terminal region of lung surfactant protein C and its polyvaline target. Using a combination of native mass spectrometry (MS) and ion mobility MS, we show that nonspecific chaperoning is driven predominantly by hydrophobic interactions of amyloid β with hydrophobic surfaces in β-lactoglobulin, transthyretin, and thioredoxin 80, and in part regulated by oligomer stability. For C-terminal region of lung surfactant protein C, native MS and hydrogen–deuterium exchange MS reveal that a disordered region recognizes the polyvaline target by forming a complementary β-strand. Hence, we show that AlphaFold2 and MS can yield atomistic models of hard-to-capture protein interactions that reveal different chaperoning mechanisms based on separate ligand properties and may provide possible clues for specific therapeutic intervention.

The amphiphilic nature of the amyloid-beta (A beta) peptide associated with Alzheimer's disease facilitates various interactions with biomolecules such as lipids and proteins, with effects on both structure and toxicity of the peptide. Here, we investigate these peptide-amphiphile interactions by experimental and computational studies of A beta(1-40) in the presence of surfactants with varying physicochemical properties. Our findings indicate that electrostatic peptide-surfactant interactions are required for coclustering and structure induction in the peptide and that the strength of the interaction depends on the surfactant net charge. Both aggregation-prone peptide-rich coclusters and stable surfactant-rich coclusters can form. Only A beta(1-40) monomers, but not oligomers, are inserted into surfactant micelles in this surfactant-rich state. Surfactant headgroup charge is suggested to be important as electrostatic peptide-surfactant interactions on the micellar surface seems to be an initiating step toward insertion. Thus, no peptide insertion or change in peptide secondary structure is observed using a nonionic surfactant. The hydrophobic peptide-surfactant interactions instead stabilize the A beta monomer, possibly by preventing self-interaction between the peptide core and C terminus, thereby effectively inhibiting the peptide aggregation process. These findings give increased understanding regarding the molecular driving forces for A beta aggregation and the peptide interaction with amphiphilic biomolecules.

The mechanisms behind the Amyloid-beta (A beta) peptide neurotoxicity in Alzheimer's disease are intensely studied and under debate. One suggested mechanism is that the peptides assemble in biological membranes to form beta-barrel shaped oligomeric pores that induce cell leakage. Direct detection of such putative assemblies and their exact oligomeric states is however complicated by a high level of heterogeneity. The theory consequently remains controversial, and the actual formation of pore structures is disputed. We herein overcome the heterogeneity problem by employing a native mass spectrometry approach and demonstrate that A beta(1-42) peptides form coclusters with membrane mimetic detergent micelles. The coclusters are gently ionized using nanoelectrospray and transferred into the mass spectrometer where the detergent molecules are stripped away using collisional activation. We show that A beta(1-42) indeed oligomerizes over time in the micellar environment, forming hexamers with collision cross sections in agreement with a general beta-barrel structure. We also show that such oligomers are maintained and even stabilized by addition of lipids. A beta(1-40) on the other hand form significantly lower amounts of oligomers, which are also of lower oligomeric state compared to A beta(1-42) oligomers. Our results thus support the oligomeric pore hypothesis as one important cell toxicity mechanism in Alzheimer's disease. The presented native mass spectrometry approach is a promising way to study such potentially very neurotoxic species and how they could be stabilized or destabilized by molecules of cellular or therapeutic relevance.

Substantial research efforts have gone into elucidating the role of protein misfolding and self-assembly in the onset and progression of Alzheimer’s disease (AD). Aggregation of the Amyloid-β (Aβ) peptide into insoluble fibrils is closely associated with AD. Here, we use biophysical techniques to study a peptide-based approach to target Aβ amyloid aggregation. A peptide construct, NCAM-PrP, consists of a largely hydrophobic signal sequence linked to a positively charged hexapeptide. The NCAM-PrP peptide inhibits Aβ amyloid formation by forming aggregates which are unavailable for further amyloid aggregation. In a membrane-mimetic environment, Aβ and NCAM-PrP form specific heterooligomeric complexes, which are of lower aggregation states compared to Aβ homooligomers. The Aβ:NCAM-PrP interaction appears to take place on different aggregation states depending on the absence or presence of a membrane-mimicking environment. These insights can be useful for the development of potential future therapeutic strategies targeting Aβ at several aggregation states.