Soluble oligomers of amyloid- peptide (A) are emerging as the primary neurotoxic species in Alzheimer disease, however, whether the membrane is among their direct targets that mediate the downstream adverse effects remains elusive. oligomerization are mutually exclusive processes that proceed through distinct motif interplay, both of which require the action of amino acids 37C40/42 to overcome the auto-inhibitory interaction between amino acids 29C36 and the N-terminal portion albeit via different mechanisms. These results indicate that intra- and extra-membrane oligomerization of A are competing processes and emphasize a critical regulation of membrane on the behavior of A monomer and soluble oligomers, which may determine distinct neurotoxic mechanisms. centrifugation to remove large aggregates (11, 20, 21). A fiber was prepared by incubating 500 m A monomer in TBS for 2 weeks at room temperature and pelleted by centrifugation at 14,000 i yields a straight line with negative ITM2B slope, which intersects the axis at the critical membrane insertion pressure (c). c represents the highest surface pressure of a monolayer below which a protein can insert, thereby quantitatively defining the membrane insertion capacity. The surface pressure of physiological lipid bilayer is 30C32 millinewtons (mN)/m (23C24), indicating that a protein can insert into cell membrane only when its c is 30 mN/m. To detect the aggregation state of monolayer inserted A, experiments were conducted with a constant surface pressure, and A insertion would result in surface area expansion. After 5000 s, monolayers were collected into tubes via negative pressure produced by vacuum for immunoblotting. Liposome Experiments Large unilamellar liposomes were prepared using a mini-extruder (Avanti) as described previously (22). After incubation of A with liposomes for the indicated times, a 10-min centrifugation at 14,000 was conducted to pellet large A aggregates. The resulting supernatant was put through additional SDS-PAGE and centrifugation analysis as indicated in Fig. 3is A monomer, which underwent similar treatment in the lack of liposomes. Virtually all the A monomers had been sedimented with liposomes, and acidity treatment was unable to release liposome-associated A. and are A monomer controls without lipid or liposomes. and are pellet and supernatant fractions of reconstituted A-lipid fusion sample, respectively. and are pellet and supernatant fractions of liposome-A incubation sample, respectively. It was evident that the two samples showed comparable self-assembly patterns. is the A monomer or ADDL at the same concentration incubating for 5000 s. The monolayer-inserted A was largely oligomeric while A in the subphase remained monomeric. Electrophoresis and Immunoblotting A samples were separated on 4% to 16.5% gradient of Tris-Tricine SDS-PAGE (1% SDS), transferred to a polyvinylidene difluoride membrane (GE Healthcare) by a semi-dry trans-blot device (Bio-Rad), and probed with mAb 6E10 (Signet) or 4G8 (Millipore, MA) (1:5000, 2 h, room temperature). 5% fat-free milk was used to block the membrane. Antibodies were diluted in TBS, 0.05% Tween 20, 1% BSA. ECL (Pierce) was used to visualize the A signal. In some experiments, before SDS-PAGE samples were cross-linked with a 50-fold molar excess of freshly prepared bis(sulfosuccinimidyl) suberate (Pierce) for 10 min at room temperature followed by quenching with 1 m Tris (pH 7.4) for 15 min. FRET Assay Fluorescein (0.5 m)-labeled A (donor) and 0.5 m tetramethylrhodamine-labeled A (acceptor) were co-incubated with liposomes at the indicated peptide/lipid ratio with continuous stirring under room temperature. An LS-55 fluorometer (PerkinElmer Instruments) was AMD3100 kinase inhibitor used to detect the fluorescence emission at 588 and 540 nm (5 nm slit width) with an excitation wavelength of 470 nm (2.5 nm slit width). The FRET ratio was calculated as monomer, low molecular weight soluble oligomer (ADDL, A-derived diffusible ligand), PF, and mature fiber following the established protocols (11, 18C20). These A species exhibited expected features in SDS-PAGE, EM observation, ThT fluorescence, and cytotoxicity assays (Fig. 1 and its legend) (11, 18C20, 26). Open in a separate window Physique 1. Characterization of A samples in different assembly says. The characteristics of A monomer, ADDL, protofibril (represent 50 nm) ( 3) are given as mean S.E.; *, 0.05; **, 0.005. At low concentrations PF and ADDL are significantly more toxic than monomer sample in cell viability assay. Soluble A Oligomers Exhibit Impaired Membrane Insertion Capability We examined A-membrane interactions by using Langmuir film balance, which measures changes in surface pressure of lipid monolayer as an index of protein insertion. The term of insertion here means part of the tested AMD3100 kinase inhibitor molecule is incorporated into the hydrophobic core of monolayer resulting in the increase in monolayer surface pressure. The injection of A monomer evoked an abrupt rise of the surface pressure of DPPC monolayer (Fig. 2and = 1), and the corresponding parameters were listed in the shown in Fig. 3for the detailed preparation protocol) AMD3100 kinase inhibitor were obtained by ultracentrifugation at 200,000 for 30 min and probed by.