Molecular basis of amyloid-β chaperone activity of lipocalin-type prostaglandin D synthase (L-PGDS)

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.Doctor of Philosoph

Structural and dynamical investigation of histone H2B in well-hydrated nucleosome core particles by solid-state NMR

Abstract H2A-H2B dimer is a key component of nucleosomes and an important player in chromatin biology. Here, we characterized the structure and dynamics of H2B in precipitated nucleosome core particles (NCPs) with a physiologically relevant concentration using solid-state NMR. Our recent investigation of H3-H4 tetramer determined its unique dynamic properties and the present work provides a deeper understanding of the previously observed dynamic networks in NCP that is potentially functionally significant. Nearly complete 13C, 15N assignments were obtained for H2B R30-A121, which permit extracting unprecedented detailed structural and amino-acid site-specific dynamics. The derived structure of H2B in the well-hydrated NCP sample agrees well with that of X-ray crystals. Dynamics at different timescales were determined semi-quantitatively for H2B in a site-specific manner. Particularly, higher millisecond-microsecond dynamics are observed for H2B core regions including partial α1, L1, partial α2, and partial L3. The analysis of these regions in the context of the tertiary structure reveals the clustering of dynamical residues. Overall, this work fills a gap to a complete resonance assignment of all four histones in nucleosomes and delineates that the dynamic networks in NCP extend to H2B, which suggests a potential mechanism to couple histone core with distant DNA to modulate the DNA activities

NMR structure and localization of a large fragment of the SARS-CoV fusion protein : implications in viral cell fusion

The lethal Coronaviruses (CoVs), Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV) and most recently Middle East Respiratory Syndrome Coronavirus, (MERS-CoV) are serious human health hazard. A successful viral infection requires fusion between virus and host cells carried out by the surface spike glycoprotein or S protein of CoV. Current models propose that the S2 subunit of S protein assembled into a hexameric helical bundle exposing hydrophobic fusogenic peptides or fusion peptides (FPs) for membrane insertion. The N-terminus of S2 subunit of SARS-CoV reported to be active in cell fusion whereby FPs have been identified. Atomic-resolution structure of FPs derived either in model membranes or in membrane mimic environment would glean insights toward viral cell fusion mechanism. Here, we have solved 3D structure, dynamics and micelle localization of a 64-residue long fusion peptide or LFP in DPC detergent micelles by NMR methods. Micelle bound structure of LFP is elucidated by the presence of discretely folded helical and intervening loops. The C-terminus region, residues F42-Y62, displays a long hydrophobic helix, whereas the N-terminus is defined by a short amphipathic helix, residues R4-Q12. The intervening residues of LFP assume stretches of loops and helical turns. The N-terminal helix is sustained by close aromatic and aliphatic sidechain packing interactions at the non-polar face. 15N{1H}NOE studies indicated dynamical motion, at ps-ns timescale, of the helices of LFP in DPC micelles. PRE NMR showed that insertion of several regions of LFP into DPC micelle core. Together, the current study provides insights toward fusion mechanism of SARS-CoV.MOE (Min. of Education, S’pore)Accepted versio

Adsorption kinetics, equilibrium and thermodynamics of a textile dye V5BN by a natural nanocomplex material: Clinoptilolite

Dyes are considered as a major pollutant released in industrial (leather, textile, and paper) effluents. In this study, the ability of Clinoptilolite in adsorbing an industrial dye (Violet 5BN) was assessed. Clinoptilolite was characterized by Scanning Electron Microscopy, Energy Dispersive analysis using X-ray and Brunauer, Emmett and Teller analysis. Batch studies at varying adsorbent dosage, pH, temperature, and time revealed that 96% of the dye was adsorbed with an adsorbent mass of 1.5 g at 30 °C, pH 5 and reaction time of 90 min. Both Langmuir and Freundlich isotherms were found to be fit, which proves the process to be heterogeneous. The experimental and calculated values of adsorption capacity were almost similar, with correlation coefficients greater than 0.9, thus implying pseudo-second order and intraparticle diffusion as the favorable models. Negative values of ΔG° indicate strong binding energy between the adsorbent and adsorbate, while negative ΔS° values prove less randomness of the process and higher adsorbate concentration on the adsorbent surface due to ion-exchange interaction. The exothermic nature of adsorption is evident from the negative ΔH° recorded. Thermodynamic studies showed the system was a spontaneous and enthalpy driven process, with chemisorption as the predominant mode of adsorption at 30 °C and physisorption at elevated temperatures. The study demonstrates the significance of natural clinoptilolite in environmental protection, as an adsorbent for remediation of dyes

Amyloid β chaperone — lipocalin-type prostaglandin D synthase acts as a peroxidase in the presence of heme

The extracellular transporter, lipocalin-type prostaglandin D synthase (L-PGDS) binds to heme and heme metabolites with high affinity. It has been reported that L-PGDS protects neuronal cells against apoptosis induced by exposure to hydrogen peroxide. Our study demonstrates that when human WT L-PGDS is in complex with heme, it exhibits a strong peroxidase activity thus behaving as a pseudo-peroxidase. Electron paramagnetic resonance studies confirm that heme in the L-PGDS-heme complex is hexacoordinated with high-spin Fe(III). NMR titration of heme in L-PGDS points to hydrophobic interaction between heme and several residues within the beta-barrel cavity of L-PGDS. In addition to the transporter function, L-PGDS is a key amyloid beta chaperone in human cerebrospinal fluid. The presence of high levels of bilirubin and its derivatives, implicated in Alzheimer's disease, by binding to L-PGDS may reduce its chaperone activity. Nevertheless, our ThT binding assay establishes that heme and heme metabolites do not significantly alter the neuroprotective chaperone function of L-PGDS. Guided by NMR data we reconstructed the heme L-PGDS complex using extensive molecular dynamics simulations providing a platform for mechanistic interpretation of the catalytic and transporting functions and their modulation by secondary ligands like A beta peptides and heme metabolites

In vivo liquid–liquid phase separation protects amyloidogenic and aggregation-prone peptides during overexpression in Escherichia coli

Funding Information: This research was funded by the Singapore Ministry of Education (MOE) through an Academic Research Fund (AcRF) Tier 3 grant (grant # MOE 2019‐T3‐1‐012) and the Academy of Finland project 315140. Publisher Copyright: © 2022 The Protein Society.Studying pathogenic effects of amyloids requires homogeneous amyloidogenic peptide samples. Recombinant production of these peptides is challenging due to their susceptibility to aggregation and chemical modifications. Thus, chemical synthesis is primarily used to produce amyloidogenic peptides suitable for high-resolution structural studies. Here, we exploited the shielded environment of protein condensates formed via liquid–liquid phase separation (LLPS) as a protective mechanism against premature aggregation. We designed a fusion protein tag undergoing LLPS in Escherichia coli and linked it to highly amyloidogenic peptides, including β amyloids. We find that the fusion proteins form membraneless organelles during overexpression and remain fluidic-like. We also developed a facile purification method of functional Aβ peptides free of chromatography steps. The strategy exploiting LLPS can be applied to other amyloidogenic, hydrophobic, and repetitive peptides that are otherwise difficult to produce.Peer reviewe

Abundant neuroprotective chaperone Lipocalin-type prostaglandin D synthase (L-PGDS) disassembles the Amyloid-β fbrils

Misfolding of Amyloid β (Aβ) peptides leads to the formation of extracellular amyloid plaques. Molecular chaperones can facilitate the refolding or degradation of such misfolded proteins. Here, for the first time, we report the unique ability of Lipocalin-type Prostaglandin D synthase (L-PGDS) protein to act as a disaggregase on the pre-formed fibrils of Aβ(1–40), abbreviated as Aβ40, and Aβ(25–35) peptides, in addition to inhibiting the aggregation of Aβ monomers. Furthermore, our proteomics results indicate that L-PGDS can facilitate extraction of several other proteins from the insoluble aggregates extracted from the brain of an Alzheimer’s disease patient. In this study, we have established the mode of binding of L-PGDS with monomeric and fibrillar Aβ using Nuclear Magnetic Resonance (NMR) Spectroscopy, Small Angle X-ray Scattering (SAXS), and Transmission Electron Microscopy (TEM). Our results confirm a direct interaction between L-PGDS and monomeric Aβ40 and Aβ(25–35), thereby inhibiting their spontaneous aggregation. The monomeric unstructured Aβ40 binds to L-PGDS via its C-terminus, while the N-terminus remains free which is observed as a new domain in the L-PGDS-Aβ40 complex model.MOE (Min. of Education, S’pore)Published versio