Biology subjects

Toth, P.

Publications and source records attributed to Toth, P..

5 recordsLinked to original sources

bioRxiv2025

EphA2 and Ephrin-A1 Use the Same Interface for Both in cis and in trans Interactions That Regulate Distinct Cell Signaling and Function

The 14 members of Eph receptor tyrosine kinases (RTK) bind to membrane-tethered ligand called ephrins and mediate cell contact signaling where the receptors and ligands engage in trans on adjacent cells. Previous studies reveal that some Eph and ephrin pairs are coexpressed on the same cells, including EphA3-ephrin-A3 and EphA4/ephrin-A5, can also interact with each other in cis. However, significant discrepancies persist as to the molecular basis and functional significance of the cis interactions, owning to the difficulties to directly interrogate the interactions. Here, we utilize time-resolved live cell fluorescence spectroscopy to demonstrate direct in cis interactions between EphA2 and ephrin-A1. Structure-guided mutagenesis mapped interactions to two salt bridges between the ligand- and receptor-binding domains of EphA2 and ephrin-A1. Interestingly, the same interface is shared with in trans interaction. Consequently, EphA2-ephrin-A1 interaction in cis competes with their interaction in trans, which leads to attenuation of EphA2 canonical signaling and inhibition of cell rounding when ligand is presented in trans. EphA2 and ephrin-A1 are widely coexpressed in many epithelial tissues, and dysregulation of their expression is known to contribute to tumor initiation and progression. The detailed molecular characterization of the mutually exclusive cis and trans interactions uncovers a new mechanism underpinning their unique roles in oncogenesis. Significance StatementEphA2 exerts dual functions in tumorigenesis, depending on the binding status of its membrane-tethered ephrin-A ligands. Ligands presented in trans on adjacent cells activate EphA2 canonical signaling associated with tumor suppression, whereas loss of ligand expression promotes oncogenic noncanonical signaling of EphA2 via serine 897 phosphorylation. Combining time-resolved spectroscopy in live cells, structure-guided mutagenesis, we show strong in cis interactions between EphA2 and ephrin-A1, which shares the same interface as interaction in trans. Moreover, the cis interaction interferes with ligand binding in trans, attenuates EphA2 canonical signaling. Our results uncover a new mechanism of EphA2 regulation by its co-expressed ligand ephrin-A1 with important implications in its known roles in oncogenesis as well as other disease processes including development of cataract.

biophysics↗

bioRxiv2024

Deep-Learning Structure Elucidation from Single-Mutant Deep Mutational Scanning

Deep learning has revolutionized the field of protein structure prediction. AlphaFold2, a deep neural network, vastly outperformed previous algorithms to provide near atomic-level accuracy when predicting protein structures. Despite its success, there still are limitations which prevent accurate predictions for numerous protein systems. Here we show that sparse residue burial restraints from deep mutational scanning (DMS) can refine AlphaFold2 to significantly enhance results. Burial information extracted from DMS is used to explicitly guide residue placement during structure generation. DMS-Fold was validated on both simulated and experimental single-mutant DMS, with DMS-Fold outperforming AlphaFold2 for 89% of protein targets and with 253 proteins having an improvement greater than 0.1 in TM-score. DMS-Fold is free and publicly available: https://github.com/LindertLab/DMS-Fold.

biochemistry↗

bioRxiv2023

Phospholipid Scramblase-1 is required for efficient neurotransmission and synaptic vesicle retrieval at cerebellar synapses

Structural phospholipids are asymmetrically distributed at the plasma membrane, with phosphatidylethanolamine and phosphatidylserine (PS) virtually absent from the outer leaflet. This asymmetric lipid distribution is transiently altered during specific biological processes including calcium-regulated exocytosis. However, the impact of this transient remodeling of membrane asymmetry on presynaptic function remains unknown. PhosphoLipid SCRamblase 1 (PLSCR1), a protein that randomizes phospholipid distribution between the two leaflets of the plasma membrane in response to calcium activation is an ideal candidate to alter this asymmetry. We therefore set out to determine the role of PLSCR1 in both neurotransmitter release and synaptic vesicle recycling by combining electron microscopy, optical live cell imaging of pHluorin probes and electrophysiology in cerebellar granule cells (GrC) from Plscr1 knock-out mice (Plscr1-/-). We report that PLSCR1 is expressed in GrCs and that PLSCR1-dependent PS egress occurred at synapses in response to neuron stimulation. Furthermore, synaptic transmission is impaired at GrC Plscr1-/- synapses and both PS egress and synaptic vesicle endocytosis are inhibited in Plscr1-/- cultured neurons, demonstrating that PLSCR1 controls phospholipid asymmetry remodeling and synaptic vesicle retrieval following neurotransmitter release. Altogether, our data reveal a key role for PLSCR1 in synaptic vesicle recycling and provide the first evidence that phospholipid scrambling at the plasma membrane is a prerequisite for optimal presynaptic performance.

cell biology↗

bioRxiv2021

Cell Surface Multimeric Assemblies Regulate Canonical and Noncanonical EphA2 Receptor Tyrosine Kinase Signaling

The EphA2 receptor tyrosine kinase mediates ligand-induced canonical signaling associated with tumor suppression and ligand-independent noncanonical signaling implicated in tumor progression. Using time-resolved fluorescence spectroscopy in live cells, we find that unliganded EphA2 receptors pre-assemble into multimers, which is mediated by two symmetric and one asymmetric interfaces in the ectodomain. Upon ligand binding, EphA2 receptors are further assemble into large clusters that also requires the three interfaces. Functionally, disrupting either the symmetric or asymmetric contacts individually blocks the autorecycling of the EphA2 apo receptor. However, only symmetric contact disruption promotes noncanonical signaling and inhibits ligand-induced catalytic activation and endocytosis, which are associated with increased cell migration in vitro and reduced survival in a syngeneic murine glioblastoma model. Our results reveal the pivotal role of EphA2 assembly in dictating canonical vs. noncanonical signaling, and identify the precise molecular interfaces that mediate the formation of the EphA2 signaling clusters.

biophysics↗

bioRxiv2019

The atypical Rho GTPase RhoU interacts with Intersectin-2 to regulate endosomal recycling pathways

Rho GTPases play a key role in various membrane trafficking processes. RhoU is an atypical small Rho GTPase related to Rac/Cdc42 which possesses unique N- and C-terminal domains that regulate its function and its subcellular localization. RhoU localized at the plasma membrane, on endosomes and in cell adhesion structures where it governs cell signalling, differentiation and migration. However, despite its endomembrane localization, RhoU function in vesicular trafficking has been unexplored. Here, we identified intersectins (ITSNs) as new binding partners for RhoU and showed that the second PxxP motif at the N-terminus of RhoU mediated interactions with SH3 domains of ITSNs. To evaluate the function of RhoU and ITSNs in vesicular trafficking, we used fluorescent transferrin as a cargo for uptake experiments. We showed that silencing of either RhoU or ITSN2, but not ITSN1 increased transferrin accumulation in early endosomes resulting from defect in fast vesicle recycling. Concomitantly, RhoU and ITSN2 colocalized to a subset of Rab4-positive vesicles suggesting that RhoU-ITSN2 interaction may occur on fast recycling endosomes to regulate the fate of vesicular cargos.

cell biology↗