Biology subjects

Kumita, J. R.

Publications and source records attributed to Kumita, J. R..

10 recordsLinked to original sources

bioRxiv2026

A live-cell autophagy reporter reveals reversible vacuolation in naked mole-rat skin fibroblasts under lysosomal stress

Naked mole-rats (NMRs, Heterocephalus glaber) display unusual longevity and resistance to age-related decline, and accumulating evidence suggests that their autophagy-lysosome pathway (ALP) is regulated differently from that of conventional mammalian models. However, most studies in NMR cells have relied on static biochemical or ultrastructural readouts, leaving the dynamic organisation of autophagy in living cells poorly defined. Here, we establish a stable tandem fluorescent autophagy reporter in NMR skin fibroblasts using an mCherry-EGFP-LC3NMR construct to enable live-cell, single-cell resolution analysis of ALP dynamics. Under basal conditions, NMR skin fibroblasts exhibit a greater abundance of LC3-positive structures than HeLa cells, together with a mixed population of autophagosomes and autolysosomes, indicating a distinct steady-state organisation of the ALP. Chloroquine (CQ)-induced lysosomal stress caused the expected accumulation of LC3-positive structures but also triggered the formation of large cytoplasmic vacuoles in NMR skin fibroblasts. Importantly, this vacuolation was not associated with acute cytotoxicity and progressively resolved following CQ removal, accompanied by reorganisation of LC3-positive compartments and recovery of lysosomal acidity. Electron microscopy showed that CQ-induced vacuoles are membrane-bound, containing internal material and co-existing with multiple ALP-related vesicular compartments. Primary NMR skin fibroblasts display a similar vacuolation phenotype, indicating that this response is not an artefact of immortalisation or reporter expression. Together, these findings establish a live-cell platform for analysing autophagy in NMR cells and identify a distinctive, reversible vacuolation response to lysosomal stress, consistent with dynamic remodelling of the lysosomal system within NMR skin fibroblasts.

cell biology↗

bioRxiv2025

Small Molecule Activators of Protein Phosphatase 2A Exert Global Stabilising Effects on the Scaffold PR65

Protein phosphatase 2A (PP2A), an important therapeutic target, comprises a scaffold subunit PR65 composed of 15 HEAT (Huntingtin/elongation/A-subunit/TOR1) repeats, a catalytic subunit, and one of many different regulatory subunits that enable binding to specific substrates. Recently, small molecule activators of PP2A (SMAPs) were identified, although their mechanisms of action have not been fully defined. Here we explore the interaction of PR65 with two SMAPs, ATUX-8385 and the non-functional DBK-776, using single-molecule optical tweezers, ensemble methods, and computational analysis. In the absence of SMAP, PR65 shows multiple unfolding and refolding transitions, and the force-extension profiles are very heterogeneous with evidence of misfolding. Similar heterogeneity has been observed for chemical-induced unfolding of tandem-repeat proteins like PR65, a consequence of the internal symmetry of the repeat architecture. In the presence of ATUX-8385, higher unfolding and refolding forces are observed globally, and there is less misfolding, suggesting that ATUX-8385 acts like a pharmacological chaperone. In contrast, DBK-766-binding induces higher unfolding forces for only a few repeats of PR65, suggestive of a more localised effect; moreover, subsequent stretch-relax cycles show that PR65 is irreversibly locked in the unfolded state. Docking and molecular dynamics simulations provide additional insights how SMAP binding modulates PR65 structure and function.

biophysics↗

bioRxiv2025

Using a stable protein scaffold to display peptides that bind to alpha-synuclein fibrils

Amyloid fibrils are ordered aggregates that are a pathological hallmark of many neurodegenerative disorders including Alzheimers Disease and Parkinsons Disease. The process of amyloid formation involves a complex cascade by which soluble monomeric protein converts to insoluble, ordered aggregates (amyloid fibrils). Although inhibiting the aggregation pathway is a key target for therapeutic development, the heterogeneous collection of aggregation-prone species formed in this process, including oligomers, protofibrils and fibrils, represent other targets for modifying disease pathology. Developing molecules that can bind to amyloid fibrils and potentially disrupt the harmful interactions between the fibrils and the cellular components would be advantageous. Designing peptide modulators for -synuclein aggregation is of great interest, however effective inhibitory peptides are often hydrophobic and hence difficult to handle; therefore, developing strategies to display these peptides in a soluble scaffold would be very beneficial. Here we demonstrate that the ultra-stable consensus-designed tetratricopeptide repeat (CTPR) protein scaffold can be grafted with "KLVFF" derived peptides previously identified to inhibit protein aggregation and interact with amyloid fibrils, to produce proteins that bind along the surface of -synuclein fibrils with micromolar affinity. Given the ability to insert hydrophobic peptides to produce soluble, CTPR-based binders, this method may prove beneficial in screening for peptide modulators of protein aggregation. Short statementConsensus-designed tetratricopeptide repeat (CTPR) proteins can be endowed with additional functionality including specific client recruitment. Here we created stable, soluble CTPR-based binders that recognise -synuclein fibrils with micromolar affinity. The CTPR scaffold provides a facile way to test potential fibril-binding or aggregation-modulating peptides and to probe their mechanisms of action.

biochemistry↗

bioRxiv2024

Amyloid forming human lysozyme intermediates are stabilised by non-native amide-π interactions

Mutational variants of human lysozyme cause a rare but fatal hereditary form of systemic amyloidosis by populating an intermediate state that self-assembles into amyloid fibrils. Despite its significance in lysozyme amyloidosis, the intermediate state has been recalcitrant to detailed structural investigation as it is only transiently and sparsely populated. Here, we investigated the intermediate state of a mutational variant of human lysozyme (I59T) using CEST and CPMG RD NMR at low pH. 15N CEST profiles probed the thermal unfolding of the native state into the denatured ensemble and revealed an additional state distinct from the two major states. Global fitting of 15N CEST and CPMG data provided kinetic and thermodynamic parameters for the exchange between all three states, characterising the intermediate state populated at 0.6%. 1H CEST data also confirmed the presence of the intermediate state displaying unusually high or low 1HN chemical shifts. To further investigate the structural details of the intermediate state we used molecular dynamics (MD) simulations, which recapitulated the experimentally observed folding pathway and free energy landscape. A high-energy intermediate state with a locally disordered {beta}-domain and C-helix was observed, revealing non-native hydrogen bonding and amide-{pi} interactions. These interactions account for the anomalous 1H chemical shifts and likely stabilise the transient intermediate state structure. Together, our NMR and MD data provide the first direct structural information on the intermediate state, offering insights into targeting lysozyme amyloidosis.

biophysics↗

bioRxiv2024

Tandem-repeat proteins introduce tuneable properties to engineered biomolecular condensates

The cells ability to rapidly partition biomolecules into biomolecular condensates is linked to a diverse range of cellular functions. Understanding how the structural attributes of biomolecular condensates are linked with their biological roles can be facilitated by the development synthetic condensate systems that can be manipulated in a controllable and predictable way. Here, we design and characterise a tuneable synthetic biomolecular condensate platform fusing modular consensus-designed tetratricopeptide repeat (CTPR) proteins to intrinsically-disordered domains. Trends between the CTPR structural attributes and condensate propensity were recapitulated across different experimental conditions and by in silico modelling, demonstrating that the CTPR domain can systematically affect the condensates in a predictable manner. Moreover, we show that incorporating short binding motifs into the CTPR domain results in specific target-protein recruitment into the condensates. Our model system can be rationally designed in a versatile manner to both tune condensate propensity and endow the condensates with new functions.

biophysics↗

bioRxiv2023

Alpha-synuclein oligomers displace monomeric alpha-synuclein from lipid membranes

Parkinsons disease (PD) is an increasingly prevalent and currently incurable neurodegenerative disorder linked to the accumulation of -synuclein (S) protein aggregates in the nervous system. While S binding to membranes in its monomeric state is correlated to its physiological role, S oligomerisation and subsequent aberrant interactions with lipid bilayers have emerged as key steps in PD-associated neurotoxicity. However, little is known of the mechanisms that govern the interactions of oligomeric S (OS) with lipid membranes and the factors that modulate such interactions. This is in large part due to experimental challenges underlying studies of OS-membrane interactions due to their dynamic and transient nature. Here, we address this challenge by using a suite of microfluidics-based assays that enable in-solution quantification of OS-membrane interactions. We find that OS bind more strongly to highly curved, rather than flat, lipid membranes. By comparing the membrane-binding properties of OS and monomeric S (MS), we further demonstrate that OS bind to membranes with up to 150-fold higher affinity than their monomeric counterparts. Moreover, OS compete with and displace bound MS from the membrane surface, suggesting that disruption to the functional binding of MS to membranes may provide an additional toxicity mechanism in PD. These findings present a unique binding mechanism of oligomers to model membranes, which can potentially be targeted to inhibit the progression of PD. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=62 SRC="FIGDIR/small/533646v2_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@4ab089org.highwire.dtl.DTLVardef@18c0709org.highwire.dtl.DTLVardef@220077org.highwire.dtl.DTLVardef@4c85b9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Phase separation and molecular ordering of the prion-like domain of the thermosensory protein EARLY FLOWERING 3

Liquid-liquid phase separation (LLPS) is an important mechanism enabling the dynamic compartmentalisation of macromolecules, including complex polymers such as proteins and nucleic acids, and occurs as a function of the physicochemical environment. In the model plant, Arabidopsis thaliana, LLPS by the protein EARLY FLOWERING3 (ELF3) occurs in a temperature sensitive manner and controls thermoresponsive growth. ELF3 contains a largely unstructured prion-like domain (PrLD) that acts as a driver of LLPS in vivo and in vitro. The PrLD contains a poly-glutamine (polyQ) tract, whose length varies across natural Arabidopsis accessions. Here, we use a combination of biochemical, biophysical and structural techniques to investigate the dilute and condensed phases of the ELF3 PrLD with varying polyQ lengths. We demonstrate that the dilute phase of the ELF3 PrLD forms a monodisperse higher order oligomer that does not depend on the presence of the polyQ sequence. This species undergoes LLPS in a pH and temperature-sensitive manner and the polyQ region of the protein tunes the initial stages of phase separation. The liquid phase rapidly undergoes aging and forms a hydrogel as shown by fluorescence and atomic force microscopies. Furthermore, we demonstrate that the hydrogel assumes a semi-ordered structure as determined by small angle X-ray scattering, electron microscopy and X-ray diffraction. These experiments demonstrate a rich structural landscape for a PrLD protein and provide a framework to describe the structural and biophysical properties of biomolecular condensates.

biophysics↗

bioRxiv2020

Cholesterol rich, highly phase separated, naked mole-rat brain lipids are exquisitely sensitive to amyloid induced membrane damage

Naked mole-rats are extraordinarily long-lived rodents that do not develop age-related neurodegenerative diseases. Remarkably, they do not accumulate amyloid plaques, even though their brains contain high concentrations of amyloid beta peptide, even from a young age Therefore, these animals offer an opportunity to investigate mechanisms of resistance against the neurotoxicity of amyloid beta aggregation. Working in this direction, here we examine the composition, phase behaviour, and amyloid beta interactions of naked mole-rat brain lipids. Relative to mouse, naked mole-rat brain lipids are rich in cholesterol and contain sphingomyelin in lower amounts and of shorter chain lengths. Proteins associated with metabolism of ceramides, sphingomyelin and ceramide receptor activity were also found to be decreased in naked mole-rat brain lysates. Correspondingly, we find that naked mole-rat brain lipid membranes exhibit a high degree of phase separation, with the liquid ordered phase occupying up to 80% of the supported lipid bilayer. These observations are consistent with the membrane pacemaker hypothesis of ageing, according to which long-living species have lipid membranes particularly resistant to oxidative damage. However, we found that exposure to amyloid beta disrupts the naked mole-rat brain lipid membranes while those formed from mouse brain lipids exhibit small, well-defined footprints, whereby the amyloid beta penetrates deeply into the lipid membranes. These results suggest that in naked mole-rats the lipid composition of cell membranes may offer neuroprotection through resistance to oxidative processes rather than through mechanical effects.

biophysics↗

bioRxiv2020

Rapid Fractionation and Characterisation of Alpha-Synuclein Oligomers in Solution

Oligomeric intermediates are implicated as neurotoxins in the pathogenesis of protein mis-folding diseases. Structural, biophysical and biochemical characterisation of these species is challenging due to their heterogeneous and transient nature, and their typically low abun-dance. Here, we show that microfluidic free-flow electrophoresis is capable of separating heterogeneous oligomer mixtures on a timescale of seconds, at least two orders of magnitude faster than conventional techniques. This enables analysis of oligomer structural heterogeneity, zeta-potential and immunochemistry with minimal sample perturbation under physiologically-relevant conditions.

biophysics↗

bioRxiv2019

Structural characterization of covalently stabilized human cystatin C oligomers

Human cystatin C (HCC), a cysteine-protease inhibitor, exists as a folded monomer under physiological conditions but has the ability to self-assemble via domain swapping into multimeric states, including oligomers with a doughnut-like structure. The structure of the monomeric HCC has been solved by X-ray crystallography, and a covalently linked version of HCC (stab-1 HCC) is able to form stable oligomeric species containing 10-12 monomeric subunits. We have performed molecular modeling, and in conjunction with experimental parameters obtained from AFM, TEM and SAXS measurements, we observe that the structures are essentially flat, with a height of about 2 nm, and the distance between the outer edge of the ring and the edge of the central cavity is ~5.1 nm. These dimensions correspond to the height and diameter of one stab-1 HCC subunit and we present a dodecamer model for stabilized cystatin C oligomers using molecular dynamics simulations and experimentally measured parameters. Given that oligomeric species in protein aggregation reactions are often transient and very highly heterogeneous, the structural information presented here on these isolated stab-1 HCC oligomers may provide useful to further explore the physiological relevance of different structural species of cystatin C in relationship to protein misfolding disease

biophysics↗