ApoE Isoforms Inhibit Amyloid Aggregation of Proinflammatory Protein S100A9.

Increasing evidence suggests that the calcium-binding and proinflammatory protein S100A9 is an important player in neuroinflammation-mediated Alzheimer's disease (AD). The amyloid co-aggregation of S100A9 with amyloid-ß (Aß) is an important hallmark of this pathology. Apolipoprotein E (ApoE) is also known to be one of the important genetic risk factors of AD. ApoE primarily exists in three isoforms, ApoE2 (Cys112/Cys158), ApoE3 (Cys112/Arg158), and ApoE4 (Arg112/Arg158). Even though the difference lies in just two amino acid residues, ApoE isoforms produce differential effects on the neuroinflammation and activation of the microglial state in AD. Here, we aim to understand the effect of the ApoE isoforms on the amyloid aggregation of S100A9. We found that both ApoE3 and ApoE4 suppress the aggregation of S100A9 in a concentration-dependent manner, even at sub-stoichiometric ratios compared to S100A9. These interactions lead to a reduction in the quantity and length of S100A9 fibrils. The inhibitory effect is more pronounced if ApoE isoforms are added in the lipid-free state versus lipidated ApoE. We found that, upon prolonged incubation, S100A9 and ApoE form low molecular weight complexes with stochiometric ratios of 1:1 and 2:1, which remain stable under SDS-gel conditions. These complexes self-assemble also under the native conditions; however, their interactions are transient, as revealed by glutaraldehyde cross-linking experiments and molecular dynamics (MD) simulation. MD simulation demonstrated that the lipid-binding C-terminal domain of ApoE and the second EF-hand calcium-binding motif of S100A9 are involved in these interactions. We found that amyloids of S100A9 are cytotoxic to neuroblastoma cells, and the presence of either ApoE isoforms does not change the level of their cytotoxicity. A significant inhibitory effect produced by both ApoE isoforms on S100A9 amyloid aggregation can modulate the amyloid-neuroinflammatory cascade in AD.

Mechanism of Secondary Nucleation at the Single Fibril Level from Direct Observations of Aß42 Aggregation.

The formation of amyloid fibrils and oligomers is a hallmark of several neurodegenerative disorders, including Alzheimer's disease (AD), and contributes to the disease pathway. To progress our understanding of these diseases at a molecular level, it is crucial to determine the mechanisms and rates of amyloid formation and replication. In the context of AD, the self-replication of aggregates of the Aß42 peptide by secondary nucleation, leading to the formation of new aggregates on the surfaces of existing ones, is a major source of both new fibrils and smaller toxic oligomeric species. However, the core mechanistic determinants, including the presence of intermediates, as well as the role of heterogeneities in the fibril population, are challenging to determine from bulk aggregation measurements. Here, we obtain such information by monitoring directly the time evolution of individual fibrils by TIRF microscopy. Crucially, essentially all aggregates have the ability to self-replicate via secondary nucleation, and the amplification of the aggregate concentration cannot be explained by a small fraction of "superspreader" fibrils. We observe that secondary nucleation is a catalytic multistep process involving the attachment of soluble species to the fibril surface, followed by conversion/detachment to yield a new fibril in solution. Furthermore, we find that fibrils formed by secondary nucleation resemble the parent fibril population. This detailed level of mechanistic insights into aggregate self-replication is key in the rational design of potential inhibitors of this process.

On the hydrogen evolution reaction activity of graphene-hBN van der Waals heterostructures.

Although graphene technology has reached technology readiness level 9 and hydrogen fuel has been identified as a viable futuristic energy resource, pristine atomic layers such as graphene are found to be inactive towards the hydrogen evolution reaction (HER). Enhancing the intrinsic catalytic activity of a material and increasing its number of active sites by nanostructuring are two strategies in novel catalyst development. Here, electrocatalytically inert graphene (G) and hexagonal boron nitride (hBN) are made active for the HER by forming van der Waals (vdW) heterostructures via vertical stacking. The HER studies are conducted using defect free shear exfoliated graphite and hBN modified glassy carbon electrodes via layer by layer sequential stacking. The G/hBN stacking pattern (AA, AB, and AB') and stacking sequence (G/hBN or hBN/G) have been found to play important roles in the HER activity. Enhancement in the intrinsic activity of graphene by the formation of G/hBN vdW stacks has been further confirmed with thermally reduced graphene oxide and hBN based structures. Tunability in the HER performance of the G/hBN vdW stack is also confirmed via a three-dimensional rGO/hBN electrode. HER active sites in the G/hBN vdW structures are then mapped using density functional theory calculations, and an atomistic interpretation has been identified.

A Double-Resonance CEST Experiment To Study Multistate Protein Conformational Exchange: An Application to Protein Folding.

Despite the importance of protein dynamics to function, studying exchange between multiple conformational states remains a challenge because sparsely populated states are invisible to conventional techniques. CEST NMR experiments can detect minor states with lifetimes between 5 and 200 ms populated to a level of just ∼1%. However, CEST often cannot provide the exchange mechanism for processes involving three or more states, leaving the role of the detected minor states unknown. Here a double-resonance CEST experiment to determine the kinetics of multistate exchange is presented. The approach that involves irradiating resonances from two minor states simultaneously is used to study the exchange of T4 lysozyme (T4L) between the dominant native state and two minor states, the unfolded state and a second minor state (B), each populated to only ∼4%. Regular CEST does not provide the folding mechanism, but double-resonance CEST clearly shows that T4L can fold directly without going through B.

High-affinity multivalent interactions between apolipoprotein E and the oligomers of amyloid-ß.

Although the interaction of apoE isoforms with amyloid-ß (Aß) peptides plays a critical role in the progression of Alzheimer's disease, how they interact with each other remains poorly understood. Here, we investigate the molecular mechanism of apoE-Aß interactions by comparing the effects of the different domains of apoE on Aß. The kinetics of aggregation of Aß1-42 are delayed dramatically in the presence of substoichiometric, nanomolar concentrations of N-terminal fragment (NTF), C-terminal fragment (CTF) and full-length apoE both in lipid-free and in lipidated forms. However, interactions between apoE and Aß as measured by intermolecular Förster resonance energy transfer (FRET) analysis were found to be minimal at t = 0 but to increase in a time-dependent manner. Thus, apoE must interact with one or more 'intermediates' rather than the monomers of Aß. Kinetics of FRET between full-length apoE4 labelled with EDANS at position 62 or 139 or 210 or 247 or 276, and tetramethylrhodamine-labelled Aß (TMR-Aß), further support an involvement of all the three domains of apoE in the interactions. However, the above-mentioned residues do not appear to form a single pocket in the 3-dimensional structure of apoE. A competitive binding assay examining the effects of unlabelled fragments or full-length apoE on the FRET between EDANS-apoE and TMR-Aß show that binding affinity of the full-length apoE to Aß is much higher than that of the fragments. Furthermore, apoE4 is found to interact more strongly than apoE3. We hypothesize that high affinity of the apoE-Aß interaction is attained due to multivalent binding mediated by multiple interactions between oligomeric Aß and full-length apoE.