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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-β (Aβ) 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. [ABSTRACT FROM AUTHOR]

Pathogenic changes in γ-secretase activity, along with its response to different drugs, can be affected by changes in the saturation of γ-secretase with its substrate. We analyze the saturation of γ-secretase with its substrate using multiscale molecular dynamics studies. We found that an increase in the saturation of γ-secretase with its substrate could result in the parallel binding of different substrate molecules at the docking site and the active site. The C-terminal domain of the substrate bound at the docking site can interact with the most dynamic presenilin sites at the cytosolic end of the active site tunnel. Such interactions can inhibit the ongoing catalytic activity and increase the production of the longer, more hydrophobic, and more toxic Aβ proteins. Similar disruptions in dynamic presenilin structures can be observed with different drugs and disease-causing mutations. Both, C99-βCTF-APP substrate and its different Aβ products, can support the toxic aggregation. The aggregation depends on the substrate N-terminal domain. Thus, the C99-βCTF-APP substrate and β-secretase path can be more toxic than the C83-αCTF-APP substrate and α-secretase path. Nicastrin can control the toxic aggregation in the closed conformation. The binding of the C99-βCTF-APP substrate to γ-secretase can be controlled by substrate channeling between the nicastrin and β-secretase. We conclude that the presented two-substrate mechanism could explain the pathogenic changes in γ-secretase activity and Aβ metabolism in different sporadic and familial cases of Alzheimer's disease. Future drug-development efforts should target different cellular mechanisms that regulate the optimal balance between γ-secretase activity and amyloid metabolism. [ABSTRACT FROM AUTHOR]

The amyloid cascade is central for the neurodegeneration disease pathology, including Alzheimer's and Parkinson's, and remains the focus of much current research. S100A9 protein drives the amyloid-neuroinflammatory cascade in these diseases. DOPA and cyclen-based compounds were used as amyloid modifiers and inhibitors previously, and DOPA is also used as a precursor of dopamine in Parkinson's treatment. Here, by using fluorescence titration experiments we showed that five selected ligands: DOPA-D-H-DOPA, DOPA-H-H-DOPA, DOPA-D-H, DOPA-cyclen, and H-E-cyclen, bind to S100A9 with apparent Kd in the sub-micromolar range. Ligand docking and molecular dynamic simulation showed that all compounds bind to S100A9 in more than one binding site and with different ligand mobility and H-bonds involved in each site, which all together is consistent with the apparent binding determined in fluorescence experiments. By using amyloid kinetic analysis, monitored by thioflavin-T fluorescence, and AFM imaging, we found that S100A9 co-aggregation with these compounds does not hinder amyloid formation but leads to morphological changes in the amyloid fibrils, manifested in fibril thickening. Thicker fibrils were not observed upon fibrillation of S100A9 alone and may influence the amyloid tissue propagation and modulate S100A9 amyloid assembly as part of the amyloid-neuroinflammatory cascade in neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Significance: The majority of the drugs which target membrane-embedded protease γ-secretase show an unusual biphasic activation–inhibition dose-response in cells, model animals, and humans. Semagacestat and avagacestat are two biphasic drugs that can facilitate cognitive decline in patients with Alzheimer's disease. Initial mechanistic studies showed that the biphasic drugs, and pathogenic mutations, can produce the same type of changes in γ-secretase activity. Results: DAPT, semagacestat LY-411,575, and avagacestat are four drugs that show different binding constants, and a biphasic activation–inhibition dose-response for amyloid-β-40 products in SH-SY5 cells. Multiscale molecular dynamics studies have shown that all four drugs bind to the most mobile parts in the presenilin structure, at different ends of the 29 Å long active site tunnel. The biphasic dose-response assays are a result of the modulation of γ-secretase activity by the concurrent binding of multiple drug molecules at each end of the active site tunnel. The drugs activate γ-secretase by facilitating the opening of the active site tunnel, when the rate-limiting step is the tunnel opening, and the formation of the enzyme–substrate complex. The drugs inhibit γ-secretase as uncompetitive inhibitors by binding next to the substrate, to dynamic enzyme structures which regulate processive catalysis. The drugs can modulate the production of different amyloid-β catalytic intermediates by penetration into the active site tunnel, to different depths, with different flexibility and different binding affinity. Conclusions: Biphasic drugs and pathogenic mutations can affect the same dynamic protein structures that control processive catalysis. Successful drug-design strategies must incorporate transient changes in the γ-secretase structure in the development of specific modulators of its catalytic activity. [ABSTRACT FROM AUTHOR]