Parkinson's disease (PD) is an age-related movement disorder characterized by a progressive degeneration of dopaminergic neurons in the midbrain. Although the presence of amyloid deposits of α-synuclein (α-syn) is the main pathological feature, PD brains also present a severe permanent inflammation, which largely contributes to neuropathology. Although α-syn has recently been implicated in this process, the molecular mechanisms underlying neuroinflammation remain unknown. In the present study, we investigated the ability of different α-syn aggregates to trigger inflammatory responses. We showed that α-syn induced inflammation through activation of Toll-like receptor 2 (TLR2) and the nucleotide oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome only when folded as amyloid fibrils. Oligomeric species, thought to be the primary species responsible for the disease, were surprisingly unable to trigger the same cascades. As neuroinflammation is a key player in PD pathology, these results put fibrils back to the fore and rekindles discussions about the primary toxic species contributing to the disease. Our data also suggest that the inflammatory properties of α-syn fibrils are linked to their intrinsic structure, most probably to their cross-β structure. Since fibrils of other amyloids induce similar immunological responses, we propose that the canonical fibril-specific cross-β structure represents a new generic motif recognized by the innate immune system.
Nowadays, the emerging role of amyloid-β peptide (Aβ) oligomers in Alzheimer's disease (AD) is widely accepted, putting aside the old idea that fibrils are the primary entities responsible for the onset of the disease. Besides, carrying the E4 isoform of apolipoprotein E (apoE) represents the highest risk of developing AD. Nevertheless, the involvement of apoE4 in AD remains confusing. The goal of this study was to bring new insights into the role of apoE4 in Aβ aggregation. We used infrared spectroscopy, thioflavin T fluorescence, and Western blots to evaluate the influence of apoE isoforms on Aβ aggregation in vitro. Comparing Aβ controls with Aβ incubated either with the apoE3 or apoE4 isoform, we report a 30% reduction of the Aβ fibrillar content, whereas the oligomeric content is 2 times higher on incubation with the pathological isoform apoE4. ApoE4 would bind and block Aβ in its oligomeric conformation, inhibiting further formation of less toxic fibrillar forms of Aβ. While previous studies mostly correlated E4 with fibrils, our report underlines a link between apoE4 and Aβ oligomers and therefore reconciles apoE4 with the new amyloid cascade hypothesis. Our observations suggest that apoE4 strongly stabilizes Aβ oligomers, the pathological species responsible for Alzheimer's disease.
Human multidrug resistance protein 1 (MRP1) is a member of the ATP-binding cassette transporter family and transports chemotherapeutic drugs as well as diverse organic anions such as leukotriene LTC 4 . The transport of chemotherapeutic drugs requires the presence of reduced GSH. By using hydrogen/deuterium exchange kinetics and limited trypsin digestion, the structural changes associated with each step of the drug transport process are analyzed. Purified MRP1 is reconstituted into lipid vesicles with an inside-out orientation, exposing its cytoplasmic region to the external medium. The resulting proteoliposomes have been shown previously to exhibit both ATP-dependent drug transport and drug-stimulated ATPase activity. Our results show that during GSH-dependent drug transport, MRP1 does not undergo secondary structure changes but only modifications in its accessibility toward the external environment. Drug binding induces a restructuring of MRP1 membrane-embedded domains that does not affect the cytosolic domains, including the nucleotide binding domains, responsible for ATP hydrolysis. This demonstrates that drug binding to MRP1 is not sufficient to propagate an allosteric signal between the membrane and the cytosolic domains. On the other hand, GSH binding induces a conformational change that affects the structural organization of the cytosolic domains and enhances ATP binding and/or hydrolysis suggesting that GSH-mediated conformational changes are required for the coupling between drug transport and ATP hydrolysis. Following ATP binding, the protein adopts a conformation characterized by a decreased stability and/or an increased accessibility toward the aqueous medium. No additional change in the accessibility toward the solvent and/or the stability of this specific conformational state and no change of the transmembrane helices orientation are observed upon ATP hydrolysis. Binding of a non-transported drug affects the dynamic changes occurring during ATP binding and hydrolysis and restricts the movement of the drug and its release.Multidrug resistance-associated protein 1 (MRP1), 1 a member of the ATP-binding cassette transporter family, confers resistance to a wide range of chemotherapeutic drugs including anthracyclines, vinca alkaloids, and epipodophyllotoxins by exporting them out of cells (1-6). ATP binding and hydrolysis as well as the presence of GSH is essential for transport to occur (2,3,(7)(8)(9)(10)(11)(12)(13)(14). In addition, MRP1 transports anionic compounds such as LTC 4 (8, 15), bilirubin glucuronide (16), glucuronide, and sulfate-conjugated estrogens (17, 18) and bile salts (19), glutathione disulfide (20), and arsenical and antimonial oxyanions (2).Its predicted topology is characteristic of a typical ABC transporter: a core structure containing two membrane-spanning domains (MSD1 and MSD2), each of them composed of six transmembrane (TM) segments and two nucleotide-binding domains (NBD1 and NBD2) that are located at the cytoplasmic face of the membrane (6, 21-23). In addition to this co...
ATP-binding cassette (ABC) transporters constitute a large class of molecular pumps whose central role in chemotherapy resistance has highlighted their clinical relevance. We investigated whether the lipid composition of the membrane affects the function and structure of HorA, a bacterial ABC multidrug transporter. When the transporter was reconstituted in a bilayer where phosphatidylethanolamine (PE), the main lipid of the bacterial membrane, was replaced with phosphatidylcholine (PC), ATP hydrolysis and substrate transport became uncoupled. Although ATPase activity was maintained, HorA lost its ability to extrude the prototypical substrate Hoechst33342. Attenuated Total Reflection-Fourier Transform Infrared spectroscopy (ATR-FTIR) revealed that, although the secondary structure of the protein was unaffected, the orientation of the transmembrane helices (TM) was modified by the change in lipid composition. The orientation of the backbone carbonyls indicated that the helices opened wider in PE versus PC-containing liposomes, with 10 degrees difference. This was supported by hydrogen/deuterium exchange studies showing increased protection of the backbone from the solvent in PC-containing liposomes. Electron Paramagnetic Resonance was used to further probe the structural change. In the PC-containing liposomes we observed increased mobility of the spin label in TM4, along with increased exposure to molecular oxygen, used as a hydrophobic quencher. This indicates that the lipid change induced modification of the orientation of TM4, exposing Cys-180 to the lipid phase. The lipid composition of the bilayer thus modulates the structure of HorA, and in turn its ability to extrude its substrates.Over the last thirty years, our vision of the biological membrane has evolved from the fluid mosaic model (1) to that of a remarkably complex system where a myriad of molecules are tightly organized along the membrane plane to properly interact and achieve numerous biological functions.Precise interplay between lipids and proteins must occur to achieve biological function, and membrane proteins have evolved specific sequence motifs to adapt to their environment. Studies have begun to evidence how proteins and lipids interact. It emerges notably that membrane proteins are surrounded by a shell of slow-moving lipids (annular lipids) distinguishable from the bulk phase. Other lipids (non-annular lipids) can achieve tight and specific interactions with a protein and act as cofactors essential to protein function (2, 3). The lipid composition of the membrane can have significant effects on, and even regulate, membrane protein function. In a number of cases, protein function is proposed to be influenced by the bulk physico-chemical properties of the membrane, which in turn are strictly determined by the exact lipid composition of the bilayer (2-4). Such properties include hydrophobic thickness (4), phase transition (5), curvature (6), and lateral pressure (3). It has also been proposed that a specific lipid might act as a molecular cha...
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