Formation of amyloid-beta (Aβ) oligomer pores in the membrane of neurons has been proposed to explain neurotoxicity in Alzheimerʼs disease (AD). Here, we present the threedimensional structure of an Aβ oligomer formed in a membrane mimicking environment, namely an Aβ(1-42) tetramer, which comprises a six stranded β-sheet core. The two faces of the β-sheet core are hydrophobic and surrounded by the membrane-mimicking environment while the edges are hydrophilic and solvent-exposed. By increasing the concentration of Aβ(1-42) in the sample, Aβ(1-42) octamers are also formed, made by two Aβ(1-42) tetramers facing each other forming a β-sandwich structure. Notably, Aβ(1-42) tetramers and octamers inserted into lipid bilayers as well-defined pores. To establish oligomer structure-membrane activity relationships, molecular dynamics simulations were carried out. These studies revealed a mechanism of membrane disruption in which water permeation occurred through lipid-stabilized pores mediated by the hydrophilic residues located on the core β-sheets edges of the oligomers.

Alzheimer’s Disease (AD) is the most common neurodegenerative disease, and efficient therapeutic and early diagnostic agents for AD are still lacking. Herein, we report the development of a novel amphiphilic compound, LS-4, generated by linking a hydrophobic amyloid-binding distyrylbenzene fragment with a hydrophilic triazamacrocycle, which dramatically increases the binding affinity toward various amyloid β (Aβ) peptide aggregates, especially for soluble Aβ oligomers. Moreover, upon the administration of LS-4 to 5xFAD mice, fluorescence imaging of LS-4-treated brain sections reveals that LS-4 can penetrate the blood-brain barrier and bind to the Aβ oligomers in vivo. In addition, the treatment of 5xFAD mice with LS-4 reduces the amount of both amyloid plaques and associated phosphorylated tau aggregates vs the vehicle-treated 5xFAD mice, while microglia activation is also reduced. Molecular dynamics simulations corroborate the observation that introducing a hydrophilic moiety into the molecular structure of LS-4 can enhance the electrostatic interactions with the polar residues of the Aβ species. Finally, exploiting the Cu2+-chelating property of the triazamacrocycle, we performed a series of imaging and biodistribution studies that show the 64Cu-LS-4 complex binds to the amyloid plaques and can accumulate to a significantly larger extent in the 5xFAD mouse brains vs the wild-type controls. Overall, these results illustrate that the novel strategy, to employ an amphiphilic molecule containing a hydrophilic moiety attached to a hydrophobic amyloid-binding fragment, can increase the binding affinity for both soluble and insoluble Aβ aggregates and can thus be used to detect and regulate various Aβ species in AD.

The formation of amyloid-beta (Aβ) oligomer pores in the membrane of neurons has been proposed as the means to explain neurotoxicity in Alzheimer's disease (AD). It is therefore critical to characterize Aβ oligomer samples in membrane-mimicking environments. Here we present the first three-dimensional structure of an Aβ oligomer formed in dodecyl phosphocholine (DPC) micelles, namely an Aβ (1-42) tetramer. It comprises a β -sheet core made of six β -strands, connected by only two β turns. The two faces of the β -sheet core are hydrophobic and surrounded by the membrane-mimicking environment. In contrast, the edges of the core are hydrophilic and are solvent-exposed. By increasing the concentration of Aβ(1-42), we prepared a sample enriched in Aβ(1-42) octamers, formed by two Aβ(1-42) tetramers facing each other forming a β -sandwich structure. Notably, samples enriched in Aβ (1-42) tetramers and octamers are both active in lipid bilayers and exhibit the same types of pore-like behaviour, but they show different occurrence rates. Remarkably, molecular dynamics simulations showed a new mechanism of membrane disruption in which water and ion permeation occurred through lipid-stabilized pores mediated by the hydrophilic residues located on the core β -sheets edges of the Aβ(1-42) tetramers and octamers.

Eukaryotic cell homeostasis requires transfer of cellular components among organelles and relies on membrane fusion catalyzed by SNARE proteins. Inactive SNARE bundles are reactivated by hexameric N-ethylmaleimide-sensitive factor, vesicle-fusing ATPase (Sec18/NSF)-driven disassembly that enables a new round of membrane fusion. We previously found that phosphatidic acid (PA) binds Sec18 and thereby sequesters it from SNAREs and that PA dephosphorylation dissociates Sec18 from the membrane, allowing it to engage SNARE complexes. We now report that PA also induces conformational changes in Sec18 protomers and that hexameric Sec18 cannot bind PA membranes. Molecular dynamics (MD) analyses revealed that the D1 and D2 domains of Sec18 contain PA-binding sites and that the residues needed for PA binding are masked in hexameric Sec18. Importantly, these simulations also disclosed that a major conformational change occurs in the linker region between the D1 and D2 domains, which is distinct from the conformational changes that occur in hexameric Sec18 during SNARE priming. Together, these findings indicate that PA regulates Sec18 function by altering its architecture and stabilizing membrane-bound Sec18 protomers.Membrane fusion is necessary for all eukaryotes to effectively transport cellular components between organelles. The trafficking and fusion of vesicles is carried out through a series of events that are highly conserved across eukarya (1). Although many proteins that drive the process may differ between eukaryotic species, they all perform similar roles allowing compartment contact, bilayer fusion, and luminal content mixing (2). The final stage of membrane fusion, and luminal content mixing, is catalyzed by SNARE 3 proteins. Each participating membrane contributes either an R-SNARE or three Q-SNARE coils that wrap around each other to form a parallel four-helical trans-SNARE complex that brings membranes into close apposition. The formation of such complexes releases free energy that is transmitted to the membranes to trigger fusion. Once fusion occurs and membranes are merged, the four-helical SNARE bundle, now a cis-SNARE complex, is inactive and requires disassembly to undergo a new round of fusion.The disassembly of cis-SNAREs, also known as Priming, is carried out by the AAA ϩ protein Sec18/NSF and its adaptor protein Sec17/␣-SNAP (3) (Fig. 1A). Current models suggest that NSF primes cis-SNAREs through a "loaded spring" mechanism triggered by cis-SNARE recognition and ATP hydrolysis (4). NSF binds to cis-SNAREs with the help of ␣-SNAP to form what is known as the 20S complex (5-8). Although NSF was originally isolated as a trimer or tetramer, it can only prime SNAREs as a homohexamer that surrounds the cis-SNAREs and ␣-SNAP proteins to form the 20S particle (9 -11). Association with cis-SNARE-␣-SNAP complexes triggers ATP hydrolysis, which leads to a large conformational change in the protein, with the major change occurring at the N terminus where it folds back over the D1-D2 rings (8). This generates eno...

Cyclooxygenase‐2 (COX‐2) overexpression is prominent in inflammatory diseases, neurodegenerative disorders, and cancer. Directly monitoring COX‐2 activity within its native environment poses an exciting approach to account for and illuminate the effect of the local environments on protein activity. Herein, we report the development of CoxFluor, the first activity‐based sensing approach for monitoring COX‐2 within live cells with confocal microscopy and flow cytometry. CoxFluor strategically links a natural substrate with a dye precursor to engage both the cyclooxygenase and peroxidase activities of COX‐2. This catalyzes the release of resorufin and the natural product, as supported by molecular dynamics and ensemble docking. CoxFluor enabled the detection of oxygen‐dependent changes in COX‐2 activity that are independent of protein expression within live macrophage cells.