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|Title:||Molecular basis of amyloid-β chaperone activity of lipocalin-type prostaglandin D synthase (L-PGDS)||Authors:||Kannaian, Bhuvaneswari||Keywords:||Science::Biological sciences||Issue Date:||2019||Publisher:||Nanyang Technological University||Source:||Kannaian, B. (2019). Molecular basis of amyloid-β chaperone activity of lipocalin-type prostaglandin D synthase (L-PGDS). Doctoral thesis, Nanyang Technological University, Singapore.||Project:||AcRF Tier 2, grant number M4020231||Abstract:||Misfolding and aggregation of specific proteins are involved in neurodegenerative proteinopathies like Alzheimer’s disease (AD), Parkinson’s disease (PD) and Amyotrophic lateral sclerosis (ALS), etc. AD is an irreversible, progressive neurodegenerative disease that causes memory loss and detrimental effects in speaking, mood, and cognitive skills. The amyloid β peptides undergo a conformational change from their soluble monomeric forms to insoluble aggregates, which is believed to be the crucial pathogenesis associated with Alzheimer’s disease. Molecular chaperones are the nanomachinery which primarily aids in protein folding and refolding of misfolded or damaged proteins. Lipocalin-type prostaglandin D synthase (L-PGDS) is found to have chaperone activity in addition to the well-established enzymatic and lipophilic ligand carrier activities. Here, we establish the role of L-PGDS as a chaperone by inhibiting the Aβ aggregation and by disaggregating the preformed fibrils. The protective role of L-PGDS as a chaperone was determined for Aβ40 and Aβ (25-35) using Thioflavin T assay and fluorescence microscopy. L-PGDS inhibits the Aβ40 aggregation by targeting the primary and secondary nucleation. C65A mutant also exhibited inhibitory activity against Aβ40 and Aβ (25-35) aggregation indicating that C65 is not the only critical residue for the chaperone activity of L-PGDS. Interaction between L-PGDS and the monomeric Aβ40 was established using Nuclear magnetic resonance (NMR) spectroscopy and Small-angle X-ray scattering (SAXS) techniques. HSQC titration of L-PGDS and monomeric Aβ40 identified the possible binding interface. The results revealed that the C-terminus of Aβ40 is mainly involved in the interaction with L-PGDS. SAXS results confirm that L-PGDS is rigid and remains as a monomer in solution and does not aggregate even at a concentration of 4mg/ml. The L-PGDS-Aβ40 complex showed a ~1Å increase in the radius of gyration (Rg) compared to the Apo form, and the shape shows an additional domain occupied by the N-terminus of Aβ40. A representative model for L-PGDS-Aβ40 complex generated by classical molecular dynamics simulations showed that the N-terminal residues (1-16) of Aβ40 are not in contact with L-PGDS and are extended which is in agreement with the SAXS model. Moreover, the contact map generated from the MD model showed several residues that were identified by the NMR titration to be in the interaction site. Thus, our MD model is in agreement with the experimental NMR data and SAXS model. In addition to the protective role of molecular chaperones, they are also found to be involved in the disaggregation of preformed fibrils. In this context, L-PGDS has also performed well for dismantling the preformed fibrils of Aβ40 and Aβ (25-35) as monitored by Thioflavin T assay and Transmission electron microscopy (TEM). Since L-PGDS showed chaperone activity for dismantling the fibrils, L-PGDS was used to dissolve the protein aggregates collected from the brain tissue of Alzheimer’s disease patients. The insoluble protein aggregates were treated with L-PGDS, Formic acid, and Hexafluoroisopropanol. Proteomic analysis of L-PGDS treated sample identified 187 proteins, which include several crucial proteins that are commonly found in the AD brain samples. L-PGDS is a highly abundant, ubiquitously expressed, small chaperone protein that inhibits the Aβ aggregation and disintegrates the preformed fibrils of Aβ without ATP consumption. On the whole, both the protective role and the disaggregase role of L-PGDS as a chaperone are discussed in this study thereby elucidating the possible mechanism of action.||URI:||https://hdl.handle.net/10356/137482||DOI:||10.32657/10356/137482||Rights:||This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0).||Fulltext Permission:||open||Fulltext Availability:||With Fulltext|
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Updated on May 24, 2022
Updated on May 24, 2022
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