Biology subjects

Garai, K.

Publications and source records attributed to Garai, K..

9 recordsLinked to original sources

bioRxiv2025

ApoE4 accelerates the condensate to amyloid transition of tau

Aberrant liquid to solid transition (LST) of tau is a pathological hallmark of several tauopathies, most notably Alzheimers disease (AD). ApoE4 is a major genetic risk factor for AD and has been shown to exacerbatetau pathology, although the underlying mechanismremains unclear. Herewe investigate whether apoE isoforms can influenceamyloid aggregation of tau occurring via the liquid-liquid phase separation (LLPS) pathway by monitoring individual tau condensates in real time using total internal reflection fluorescence microscopy (TIRFM) and confocal microscopy. We find that interaction of apoE with tau condensates is dependent on the lipidation status of apoE. Lipid-free apoE, even at 1:1000 substoichiometric concentrations, arrests growth but promotes ageing of the liquid condensates of tau. The strong effect of apoE is accompanied by its preferential accumulation inside the condensates, lowering of the surface tension and promoting amyloid nucleation. Lipidation of apoE2 and apoE3 diminished its amyloid promoting effects very strongly. However, apoE4 even in the lipidated forms accelerated the liquid to amyloid conversion of the tau condensates considerably. Furthermore, the amyloidogenic effect of apoE4 is correlated with its higher delipidation tendency compared to apoE2 and apoE3. Together, our study suggests that lipid-free apoE promotes tau aggregation via dual mechanisms by increasing condensate surface to volume ratio and by facilitating amyloid nucleation possibly at the condensate interfaces. We hypothesize that enhancing apoE4 lipidation in vivo presents a potential strategy to counteract its pathogenic effects on tau aggregation. HighlightsO_LIAll three lipid-free apoE isoforms strongly affect LLPS of tau even at a sub-stoichiometric (1:1000) ratio. C_LIO_LIApoE lowers the surface tension, reduces the size and increases the number of the condensates. C_LIO_LIApoE isoforms accelerate LST of the tau condensates. C_LIO_LILipidation reduces the effects of apoE, strongly in apoE2 and apoE3 but less so in apoE4. C_LIO_LIApoE4 undergoes spontaneous delipidation faster than apoE3 and apoE2. C_LI

biophysics↗

bioRxiv2025

Liquid-solid coexistence at single fibril resolution during tau condensate ageing

Aggregated fibrillar tangles of the microtubule-associated protein tau are a hallmark of Alzheimers disease. It is becoming increasingly clear that a key process that can trigger the formation of such tau tangles is the aberrant ageing of biomolecular condensates of tau formed via liquid-liquid phase separation. This ageing process affects the overall mechanical and structural properties of the condensates, but the molecular-level mechanisms by which aggregation takes place in the condensates have remained elusive. Here, by tracking individual tau molecules inside the condensates using single molecule microscopy, we show that the ageing process is characterized by the coexistence of a growing solid phase of tau within a dense liquid phase. The liquid phase is increasingly confined to the pores of the growing fibril gel network, but maintains its initial viscosity. These findings add a spatial dimension to the ageing of condensates and demonstrate that spatial heterogeneity is a key feature of the liquid-to-solid transition of tau.

biophysics↗

bioRxiv2024

The Alzheimer's Aβ peptide forms biomolecular condensates that trigger amyloid aggregation

The onset and development of Alzheimers disease (AD) is linked to the accumulation of pathological aggregates formed from the normally monomeric amyloid-{beta} peptide within the central nervous system. These A{beta} aggregates are increasingly successfully targeted with clinical therapies, but the fundamental molecular steps that trigger the initial nucleation event leading to the conversion of monomeric A{beta} peptide into pathological aggregates remain unknown. Here we show that the A{beta} peptide can form biomolecular condensates on lipid bilayers both in molecular assays and in living cells. Our results reveal that these A{beta} condensates can significantly accelerate the primary nucleation step in the amyloid conversion cascade that leads to the formation of amyloid aggregates and plaque. We show that A{beta} condensates contain phospholipids, are intrinsically heterogenous, and are prone to undergo a liquid-to-solid transition leading to the formation amyloid fibrils. These findings uncover the liquid-liquid phase separation behaviour of the A{beta} peptide, and reveal a new molecular step very early in the amyloid-{beta} aggregation cascade that can form the basis for novel therapeutic intervention strategies. Significance statementThe hallmark of Alzheimers disease is the abnormal buildup of the normally soluble amyloid {beta} protein aggregates in the central nervous system. While the molecular mechanisms at the late stages of the amyloid {beta} aggregation cascade are well understood, the initial steps remained elusive until now. Our current study demonstrates that amyloid {beta} undergoes liquid-liquid phase separation on lipid surfaces, which triggers primary nucleation and initiates the amyloid {beta} aggregation cascade. This newly identified step in the molecular mechanism of Alzheimers disease represents a promising target for the development of alternative innovative therapeutic strategies.

biophysics↗

bioRxiv2023

Apolipoprotein-E transforms intracellular Amyloid-β oligomers to a more toxic state

It is poorly understood why ApoE variants are major genetic risk factors in Alzheimers disease (AD), which is associated with the aggregation of amyloid beta (A{beta}). Here we directly image specific changes in small A{beta} oligomers in rat brain cells that correlate with the cellular ApoE content. An inhibitor of A{beta}-ApoE interaction suppresses this change and concomitantly reduces A{beta} toxicity in a dose-dependent manner. Single-molecule techniques show changes both in the conformation and the stoichiometry of the oligomers. hiPSC-derived neural stem cells from Alzheimers patients also show similar changes. Interaction with ApoE therefore changes the oligomeric state, membrane affinity, and toxicity of A{beta} oligomers, and can be directly read out in live cells. Our findings suggest a rapid and quantitative assay for AD drug discovery. One-sentence summaryApoE causes specific toxicogenic modifications of A{beta} oligomers, and these changes can be directly imaged in live cells.

biochemistry↗

bioRxiv2021

I-M-150847, a novel GLP-1 and GIP receptor dual agonist, reduces body weight gain and improves glycemic control in the rodent model of type 2 diabetes and obesity.

We report the discovery of a novel unimolecular glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptor dual agonist that exhibits potent glycemic control and weight loss in diet-induced obese mice. The dual agonism of GLP-1 and GIP receptor was achieved by replacing the tryptophan cage of exendin-4 with the C-terminal undecapeptide sequence of oxyntomodulin along with a single amino acid substitution from histidine to tyrosine at the amino terminus of the peptide. The structural modification places lysine 30 of the novel incretin agonist in frame with the corresponding lysine residue in the native GIP sequence. The novel incretin receptor dual agonist, named I-M-150847, induces rapid redistribution of GLP-1R at the plasma membrane following activation ensuring the maintenance of the receptor in a sensitized state. I-M-150847 promotes glucose-stimulated insulin exocytosis in cultured pancreatic beta cells and augments insulin-stimulated glucose uptake in mouse adipocytes. Chronic administration of I-M-150847 enhances insulin sensitivity, improves glycemic control, and achieves significant weight loss relative to the control or exendin-4-treated DIO-mice demonstrating the therapeutic efficacy of dual agonist in ameliorating type 2 Diabetes and Obesity. Significance statement.O_LIReplacement of the Trp-cage with the C-terminal oxyntomodulin undecapeptide along with the tyrosine substitution at the amino terminus converts the selective GLP-1R agonist exendin-4 to a novel GLP-1R and GIPR dual agonist I-M-150847. C_LIO_LIThe GLP-1R and GIPR dual agonist I-M-150847 induces the expeditious redistribution of GLP-1R at the plasma membrane following initial activation thereby maintaining the receptor in a sensitized state. C_LIO_LIThe incretin receptor dual agonist I-M-150847 enhances insulin sensitivity and delivers superior glycemic control and weight loss compared to exendin-4 in the rodent model of diabetes and obesity. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/471325v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1e27480org.highwire.dtl.DTLVardef@4e88acorg.highwire.dtl.DTLVardef@1eee9e7org.highwire.dtl.DTLVardef@3e41c9_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2020

Structural Features and Oligomeric Nature of Human Podocin Domain

Podocytes are crucial cells of the glomerular filtration unit and playing a vital role at the interface of the blood-urine barrier. Podocyte slit-diaphragm is a modified tight junction that facilitates size and shape-dependent permselectivity. Several proteins including podocin, nephrin, CD2AP, and TRPC6 form a macromolecular assembly and constitute the slit-diaphragm. Podocin is an integral membrane protein attached to the inner membrane of the podocyte via a short transmembrane region (101-125). The cytosolic N- and C-terminus help podocin to attain a hook-like structure. Podocin shares 44% homology with stomatin family proteins and similar to the stomatin proteins, podocin was shown to associate into higher-order oligomers at the site of slit-diaphragm. However, the stoichiometry of the homo-oligomers and how it partakes in the macromolecular assemblies with other slit-diaphragm proteins remains elusive. Here we investigated the oligomeric propensity of a truncated podocin construct (residues:126-350). We show that the podocin domain majorly homo-oligomerize into a 16mer. Circular dichroism and fluorescence spectroscopy suggest that the 16mer oligomer has considerable secondary structure and moderate tertiary packing.

biophysics↗

bioRxiv2020

Hsp70 inhibits aggregation of Islet amyloid polypeptide by binding to the heterogeneous prenucleation oligomers

Molecular chaperone Hsp70 plays important roles in the pathology of amyloid diseases by inhibiting aberrant aggregation of proteins. However, mechanism of the interactions of Hsp70 with the amyloidogenic intrinsically disordered proteins (IDPs) is not clear. Here, we use Hsp70 from different organisms to show that it inhibits aggregation of Islet amyloid polypeptide (IAPP) at substoichiometric concentrations even in absence of ATP. The effect is found to be the strongest if Hsp70 is added in the beginning of aggregation but progressively less if added later, indicating role of Hsp70 in preventing primary nucleation possibly via interactions with the prefibrillar oligomers of IAPP. Fluorescence Correlation Spectroscopy (FCS) measurements of the solutions containing fluorescently labelled Hsp70 and IAPP exhibit fluorescence bursts suggesting formation of heterogeneous complexes of oligomeric IAPP binding to multiple molecules of Hsp70. Size exclusion chromatography and field flow fractionation are then used to fractionate the smaller complexes. Multiangle light scattering and FCS measurements suggest that these complexes comprise of monomers of Hsp70 and small oligomers of IAPP. However, concentration of the complexes is measured to be a few nanomolar amidst several molar of free Hsp70 and IAPP. Hence, our results indicate that Hsp70 interacts poorly with the monomers but strongly with oligomers of IAPP. This is likely a common feature of the interactions between the chaperones and the amyloidogenic IDPs. While strong interactions with the oligomers prevent aberrant aggregation, poor interaction with the monomers avert interference with the functions of the IDPs.

biophysics↗

bioRxiv2018

Apolipoprotein E interacts with amyloid-β oligomers via positively cooperative multivalent binding

Interaction of apolipoprotein E (apoE) isoforms with amyloid-{beta} (A{beta}) peptides is considered a critical determinant of the progression of Alzheimers disease. However, molecular mechanism of the apoE-A{beta} interaction is poorly understood. Here we characterize the nature of the apoE-A{beta} complexes and identify the region of apoE that interacts with A{beta}. We have prepared three distinct fragments of apoE4, viz., the N-terminal fragment (NTF), hinge domain fragment (HDF) and C-terminal fragment (CTF) to compare its interactions with A{beta}. Kinetics of aggregation of A{beta} is delayed dramatically in presence of low, substoichiometric concentrations of both NTF and CTF in lipid-free, as well as, in lipidated forms. Effect of HDF is found to be small. Strong inhibition by NTF and CTF at substoichiometric concentrations indicate interactions with the intermediates or the oligomers of A{beta}. Kinetics of Forster Resonance Energy Transfer (FRET) between full-length apoE4 labeled with EDANS at positions 62, 139, 210, 247, and 276 and tetramethylrhodamine (TMR)-labeled A{beta} further support involvement of multiple regions of apoE in the interactions. Since the interactions involve intermediates of A{beta} quantitative evaluation of the binding affinities are not feasible. Hence we employed a competitive binding assay to examine whether the N- and C-terminal domains interact cooperatively. Addition of unlabeled full-length apoE eliminates the FRET between EDANS-NTF + EDANS-CTF and TMR-A{beta} almost completely but not vice versa. Furthermore, full-length apoE but not the equimolar mixture of the fragments could displace the already bound EDANS-apoE molecules from the complexes. Therefore, binding affinity of the A{beta} oligomers to the intact full-length apoE is much higher than the affinity to the domains when mixed together as fragments. Thus, our results indicate that apoE-A{beta} complex formation is mediated by positively cooperative multivalent binding between the multiple sites on apoE and the oligomeric forms of A{beta}.

biochemistry↗

bioRxiv2018

A fluorescence correlation spectrometer for measurements in cuvettes

We have developed a fluorescence correlation spectroscopy (FCS) setup for performing single molecule measurements on samples inside regular cuvettes. We built this by using an Extra Long Working Distance (ELWD), 0.7 NA, air objective with working distance > 1.8 mm. We have achieved counts per molecule > 44 kHz, diffusion time < 64 s for rhodamine B in aqueous buffer and a confocal volume < 2 fl. The cuvette-FCS can be used for measurements over a wide range of temperature that is beyond the range permitted in the microscope-based FCS. Finally, we demonstrate that cuvette-FCS can be coupled to automatic titrators to study urea dependent unfolding of proteins with unprecedented accuracy. The ease of use and compatibility with various accessories will enable applications of cuvette-FCS in the experiments that are regularly performed in fluorimeters but are generally avoided in microscope-based FCS.

biophysics↗