Biology subjects

Hristova, K.

Publications and source records attributed to Hristova, K..

16 recordsLinked to original sources

bioRxiv2025

Direct cell reprogramming by a designed agonist inducing HER2-FGFR proximity

Growth factor induced receptor dimerization and activation of downstream pathways can modulate cell fate decisions. Here, we investigate the potential of de novo designed synthetic ligands, termed Novokines, to reprogram cell identity by inducing proximity of novel pairs of receptor subunits. We find that a design, H2F, that brings together HER2 (which has no known natural ligand) and the FGF receptor has potent signaling activity. H2F induces robust signaling and reprograms fibroblasts into myogenic cells. Unlike native FGF ligands, H2F selectively activates the MAPK pathway without engaging PLC{gamma}-mediated Ca{superscript 2} signaling. FRET assays confirm H2F-mediated HER2-FGFR proximity, and phosphoproteomic analysis reveals activation of MAPK effectors. H2F-induced ERK phosphorylation is abolished in cells expressing a kinase-dead FGFR1 (K514M) mutant, confirming the requirement for FGFR catalytic activity. H2F treatment significantly increases myofiber formation from adult patient-derived primary myoblasts, demonstrating its capacity to promote myogenic regeneration. Our findings demonstrate that synthetic receptor pairings can rewire signaling outputs to drive regeneration, providing a programmable platform for cell fate engineering.

biochemistry↗

bioRxiv2024

Absence seizures and sleep abnormalities in a rat model of GRIN2B neurodevelopmental disorder

Pathogenic mutations in GRIN2B are an important cause of severe neurodevelopmental disorders resulting in epilepsy, autism and intellectual disability. GRIN2B encodes the GluN2B subunit of N-methyl-D-aspartate receptors (NMDARs), which are ionotropic glutamate receptors critical for normal development of the nervous system and synaptic plasticity. Here, we characterized a novel Grin2b heterozygous knockout rat model with 24-hour EEG recordings. We found rats heterozygous for the deletion (Grin2b+/-) had a higher incidence of spontaneous spike and wave discharges, the electrographic correlate of absence seizures, than wild-type rats (Grin2b+/+). Spike and wave discharges were longer in duration and displayed higher overall spectral power in Grin2b+/- when compared to those in Grin2b+/+ animals. Heterozygous mutant rats also had abnormal sleep-wake brain state dynamics over the circadian cycle. Specifically, we identified a reduction in total rapid eye movement sleep and, altered distributions of non-rapid eye movement sleep and wake epochs, when compared to controls. This was accompanied by an increase in overall spectral power during non-rapid eye movement sleep in Grin2b+/-. The sleep-wake phenotypes were largely uncorrelated to the incidence of spike and wave discharges. We then tested the antiseizure efficacy of ethosuximide, a T-type voltage-gated calcium channel blocker used in the treatment of absence seizures, and memantine, a noncompetitive NMDAR antagonist currently explored as a mono or adjunctive treatment option in NMDAR related neurodevelopmental disorders. Ethosuximide reduced the number and duration of spike and wave discharges, while memantine did not affect the number of spike and wave discharges but reduced their duration. These results highlight two potential therapeutic options for GRIN2B related epilepsy. Our data shows the new rat Grin2b haploinsufficiency model exhibits clinically relevant phenotypes. As such, it could prove crucial in deciphering underlying pathological mechanisms and developing new therapeutically translatable strategies for GRIN2B neurodevelopmental disorders.

neuroscience↗

bioRxiv2023

Utility of FRET in studies of membrane protein oligomerization: the concept of the effective dissociation constant

The activity of many membrane receptors is controlled through their lateral association into dimers or higher order oligomers. While Forster resonance energy transfer (FRET) measurements have been used extensively to characterize the stability of receptor dimers, the utility of FRET in studies of larger oligomers is unclear. Here we show that we can extract an effective equilibrium dissociation constant from FRET measurements for EphA2, a receptor tyrosine kinase (RTK) known to form active oligomers of heterogeneous distributions in response to its ligand ephrinA1-Fc. The newly introduced effective equilibrium dissociation constant has a well-defined physical meaning and biological significance. It denotes the receptor concentration for which half of the receptors are monomeric and inactive, and the other half are associated into oligomers and are active, irrespective of the exact oligomer size. This work illustrates how FRET, along with fluorescence fluctuation techniques which directly measure the oligomer size, can be a very powerful tool in studies of membrane receptor association and signaling in the plasma membrane.

biophysics↗

bioRxiv2023

Determining the Statistical Significance of the Difference Between Arbitrary Curves: A Spreadsheet Approach

We present a simple, spreadsheet-based method to determine the statistical significance of the difference between any two arbitrary curves. This modified Chi-squared method addresses two scenarios: A single measurement at each point with known standard deviation, or multiple measurements at each point averaged to produce a mean and standard error. The method includes an essential correction for the deviation from normality in measurements with small sample size, which are typical in biomedical sciences. Statistical significance is determined without regard to the functionality of the curves, or the signs of the differences. Numerical simulations are used to validate the procedure. Example experimental data are used to demonstrate its application. An Excel spreadsheet is provided for performing the calculations for either scenario.

bioinformatics↗

bioRxiv2023

Quantification of ligand and mutation-induced bias in EGFR phosphorylation in direct response to ligand binding

The 58 receptor tyrosine kinases (RTKs) are critically important for human development, and are implicated in many growth disorders and cancers. Here we introduce a methodology to identify and quantify bias in RTK signal transduction across the plasma membrane, and to quantify phosphorylation efficiencies, without contributions from feedback loops and system bias. We show that RTK biased signaling can occur in the first step of signal transduction not only in response to different ligands, but as a consequence of RTK pathogenic mutations as well. Ligand bias and mutation-induced bias are uncoupled here based on a comprehensive data set of dose response curves acquired for three ligands, EGF, TGF and epiregulin, for wild-type EGFR and for the oncogenic L834R EGFR mutant found in patients with non-small-cell lung cancer (NSCLC). Ligand bias has been extensively studied for GPCRs, and has revolutionized the GPCR field. The demonstration of pathogenic mutation-induced bias in RTK signal transduction across the plasma membrane will open new avenues for the exploration of RTK biased inhibitors as highly specific anti-cancer therapies.

pharmacology and toxicology↗

bioRxiv2022

Activity of EGFR transmembrane region variants indicates specific transmembrane dimers are not required for EGFR activity

The Epidermal Growth Factor Receptor (EGFR) is a Receptor Tyrosine Kinase that mediates cell proliferation and differentiation events during development and maintenance of complex organisms. Formation of specific, ligand-dependent EGFR dimers is a key step in stimulating EGFR signaling, and crystal structures of active, dimeric forms of isolated EGFR extracellular regions and kinase domains have revealed much about how dimer interactions regulate EGFR activity. The nature and role of the transmembrane region in regulating EGFR activity remains less clear, however. Proposed roles for the transmembrane region range from nonspecific but energetically favorable interactions to specific transmembrane dimer conformations being associated with active, inactive, or activity-modulated states of EGFR. To investigate the role of specific transmembrane dimers in modulating EGFR activity we generated thirteen EGFR variants with altered transmembrane sequences designed to favor or disfavor specific types of transmembrane region interactions. We show using FRET microscopy that EGFR transmembrane regions have an intrinsic propensity to associate in mammalian cell membranes that is counteracted by the extracellular region. We show using cell-based assays that each of the EGFR transmembrane variants except the Neu variant, which results in constitutive receptor phosphorylation, is able to autophosphorylate and stimulate phosphorylation of downstream effectors Erk and Akt. Our results indicate that many transmembrane sequences, including polyleucine, are compatible with EGFR activity and provide no evidence for specific transmembrane dimers regulating EGFR function.

molecular biology↗

bioRxiv2022

The efficacy of EphA2 tyrosine phosphorylation increases with EphA2 oligomer size

The receptor tyrosine kinase (RTK) EphA2 is expressed in epithelial and endothelial cells and controls the assembly of cell-cell junctions. EphA2 has also been implicated in many diseases, including cancer. Unlike most RTKs, which signal predominantly as dimers, EphA2 readily forms higher order oligomers upon ligand binding. Here we investigated if a correlation exists between EphA2 signaling properties and the size of the EphA2 oligomers induced by multiple ligands, including the widely used ephrinA1-Fc ligand, the soluble monomeric m-ephrinA1, and novel engineered peptide ligands. We used Fluorescence Intensity Fluctuation (FIF) spectrometry to characterize the EphA2 oligomer populations induced by the different ligands. Interestingly, we found that different monomeric and dimeric ligands induce EphA2 oligomers with widely different size distributions. Comparison of FIF brightness distribution parameters and EphA2 signaling parameters reveals that the efficacy of EphA2 phosphorylation on tyrosine 588, which is indicative of receptor activation, correlates with EphA2 mean oligomer size. However, other characteristics, such as the efficacy of AKT inhibition and ligand bias coefficients, appear to be independent of EphA2 oligomer size. This work highlights the utility of FIF in RTK signaling research and demonstrates a quantitative correlation between the architecture of EphA2 signaling complexes and signaling features.

biophysics↗

bioRxiv2022

Plasticity of transmembrane helix interactions in EphA2 dimers and oligomers

Lateral interactions can stabilize different EphA2 receptor assemblies in the plasma membrane in response to different ligands. Here we use two fluorescent techniques, Forster Resonance Energy Transfer (FRET) and Fluorescence Intensity Fluctuations (FIF) spectrometry, to investigate how mutations in the EphA2 transmembrane (TM) helix affect the association between full-length EphA2 molecules in the absence of ligand and in the presence of three ligands: ephrinA1-Fc, m-ephrinA1, and the YSA peptide. The EphA2 mutations we studied have been previously characterized in the context of the isolated EphA2 TM helix. Working with full-length EphA2, we observed modest effects of the mutations on receptor-receptor interaction. Our data do not support the currently accepted model of a switch between two discrete TM helix dimerization motifs corresponding to active or inactive receptor states. Instead, we propose that different dimeric/oligomeric arrangements of the EphA2 extracellular region couple to an ensemble of TM helix dimer interfaces. Plasticity in the arrangements of receptor tyrosine kinase TM helices in active dimers and oligomers may serve to facilitate the cross-phosphorylation of multiple tyrosines in different positions of the intracellular regions.

biophysics↗

bioRxiv2022

Bias in FGFR1 signaling in response to FGF4, FGF8, and FGF9

The mechanism of differential signaling of multiple FGF ligands through a single FGF receptor is poorly understood. Here, we use biophysical tools to quantify multiple aspects of FGFR1 signaling in response to FGF4, FGF8 and FGF9: potency, efficacy, bias, ligand-induced oligomerization and downregulation, and conformation of the active FGFR1 dimers. We find that the three ligands exhibit distinctly different potencies and efficacies for inducing responses in cells. We further discover qualitative differences in the actions of the three FGFs through FGFR1, as FGF8 preferentially activates some of the probed downstream responses (FRS2 phosphorylation and extracellular matrix loss), while FGF4 and FGF9 preferentially activate different probed responses (FGFR1 phosphorylation and cell growth arrest). Thus, FGF8 is a biased ligand, when compared to FGF4 and FGF9. Forster resonance energy transfer experiments reveal a correlation between biased signaling and the conformation of the FGFR1 transmembrane domain dimer. Our findings expand the mechanistic understanding of FGF signaling during development and bring the poorly understood concept of receptor tyrosine kinase ligand bias into the spotlight.

biophysics↗

bioRxiv2021

Effect of reversible osmotic stress on live cell plasma membranes, probed via Laurdan general polarization measurements

Here we seek to gain insight into changes in the plasma membrane of live cells upon the application of osmotic stress using Laurdan, a fluorescent probe that reports on membrane organization, hydration, and dynamics. It is known that the application of osmotic stress to lipid vesicles causes a decrease in Laurdans generalized polarization (GP), which has been interpreted as an indication of membrane stretching. In cells, we see the opposite effects, as GP increases when the osmolarity of the solution is decreased. This increase in GP is associated with the presence of caveolae, which are known to disassemble and flatten in response to osmotic stress, in a process that supplies extra plasma membrane in physiological processes. SignificanceCells can experience multiple stresses in vivo. Furthermore, the application of osmotic stress is used as a biophysical tool to interrogate membrane proocesses in vitro. We sought to investigate the consequences of osmotic stress on the plasma membrane properties using the fluorescent probe Laurdan. Unexpectedly, we find that osmotic stress leads to an increase in GP in live cells. The opposite change in GP has been observed in model lipid bilayers, reminding us there are limitations to the utility of model systems in understanding cell membrane behavior. Despite years of research, the cell membrane still has ways to surprise us.

biophysics↗

bioRxiv2021

Direct quantification of ligand-induced lipid and protein microdomains with distinctive signaling properties

Lipid rafts are known as highly ordered lipid domains that are enriched in saturated lipids such as the ganglioside GM1. While lipid rafts are believed to exist in cells and to serve as signaling platforms through their enrichment in signaling components, they have never been directly observed in the plasma membrane without treatments that artificially cluster GM1 into large lattices. Here we report that microscopic GM1-enriched domains can form, without lipid cross-linking, in the plasma membrane of live mammalian cells expressing the EphA2 receptor tyrosine kinase in response to its ligand ephrinA1-Fc. The GM1-enriched microdomains form concomitantly with EphA2-enriched microdomains, but only partially co-localize with them. To gain insight into how plasma membrane heterogeneity controls signaling, we quantify the degree of EphA2 segregation and study initial EphA2 signaling steps in both EphA2-enriched and EphA2-depleted domains. By measuring dissociation constants, we demonstrate that EphA2 oligomerization is the same in EphA2-enriched and -depleted domains. However, EphA2 interacts preferentially with its downstream effector SRC in EphA2-depleted domains. The ability to induce microscopic GM1-enriched domains in live cells using a ligand for a transmembrane receptor will give us unprecedented opportunities to study the biology of lipid rafts.

biophysics↗

bioRxiv2021

Neurotrophin signaling is modulated by specific transmembrane domain interactions

The neurotrophin receptors p75 and TrkA play an important role in the development and survival of the nervous system. Biochemical data suggest that p75 and TrkA regulate the activities of each other. For instance, p75 is able to regulate the response of TrkA to lower concentrations of NGF and TrkA promotes p75 shedding by -secretases in a ligand-dependent manner. The current model is that p75 and TrkA are regulated by means of a physical direct interaction, however the nature of such interaction has been elusive so far. Here using NMR in micelles, multiscale molecular dynamics (MD), FRET and functional studies we identified and characterized the direct interaction between TrkA and p75 through the transmembrane domains (TMDs). MD of p75-TMD mutants suggests that although the interaction between TrkA and p75 TMDs is maintained, a specific protein interface is required to facilitate TrkA active homodimerization in the presence of NGF. The same mutations in the TMD protein interface of p75 reduced the activation of TrkA by NGF and cell differentiation. In summary we provide a structural model of the p75/TrkA receptor complex stabilized by transmembrane domain interactions.

biochemistry↗

bioRxiv2021

Human herpesvirus 8 molecular mimicry of ephrin ligands facilitates cell entry and triggers EphA2 signaling

Human herpesvirus 8 (HHV-8) is an oncogenic virus that enters cells by fusion of the viral and endosomal cellular membranes in a process mediated by viral surface glycoproteins. One of the cellular receptors hijacked by HHV-8 to gain access to cells is the EphA2 tyrosine kinase receptor, and the mechanistic basis of EphA2-mediated viral entry remains unclear. Using X-ray structure analysis, targeted mutagenesis and binding studies, we here show that the HHV-8 envelope glycoprotein complex gH/gL binds with sub-nanomolar affinity to EphA2 via molecular mimicry of the receptors cellular ligands, ephrins, revealing a pivotal role for the conserved gH residue E52 and the amino-terminal peptide of gL. Using FSI-FRET and cell contraction assays, we further demonstrate that the gH/gL complex also functionally mimics ephrin ligand by inducing EphA2 receptor association via its dimerization interface, thus triggering receptor signaling for cytoskeleton remodeling. These results now provide novel insight into the entry mechanism of HHV-8, opening avenues for the search of therapeutic agents that could interfere with HHV-8 related diseases.

microbiology↗

bioRxiv2021

Reversible blood-brain barrier opening utilizing the membrane active peptide melittin in vitro and in vivo

The blood-brain barrier (BBB) tightly controls entry of molecules and cells into the brain, restricting the delivery of therapeutics. Blood-brain barrier opening (BBBO) utilizes reversible disruption of cell-cell junctions between brain microvascular endothelial cells to enable transient entry into the brain. Development of BBBO techniques has been hindered by a lack of physiological models for in vitro study. Here, we utilize an in vitro tissue-engineered microvessel model to demonstrate that melittin, a membrane active peptide present in bee venom, supports BBBO. From endothelial and neuronal viability studies, we identify the accessible concentration range for BBBO. We then use a tissue-engineered model of the human BBB to optimize dosing and elucidate the mechanism of opening. Melittin and other membrane active variants transiently increase paracellular permeability via disruption of cell-cell junctions. In mice, we demonstrate a minimum clinically effective intra-arterial dose of 3 M{middle dot}min melittin, which is reversible within one day and neurologically safe. Melittin-induced BBBO represents a novel platform for delivery of therapeutics into the brain.

bioengineering↗

bioRxiv2021

Allosteric Regulation of the EphA2 Receptor Intracellular Region by Serine/Threonine Kinases

Eph receptor tyrosine kinases play a key role in cell-cell communication. However, lack of structural information on the entire multi-domain intracellular region of any Eph receptor has hindered detailed understanding of their signaling mechanisms. Here, we use an integrative structural biology approach combining X-ray crystallography, small-angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry, to gain the first insights into the structure and dynamics of the entire EphA2 intracellular region. EphA2 promotes cancer malignancy through a poorly understood non-canonical form of signaling that depends on serine/threonine phosphorylation of the linker connecting the EphA2 kinase and SAM domains. We uncovered two distinct molecular mechanisms that may function in concert to mediate the effects of linker phosphorylation through an orchestrated allosteric regulatory network. The first involves a shift in the equilibrium between a "closed" configuration of the EphA2 intracellular region and an "open" more extended configuration induced by the accumulation of phosphorylation sites in the linker. This implies that cooperation of multiple serine/threonine kinase signaling networks is necessary to promote robust EphA2 non-canonical signaling. The second involves allosteric rearrangements in the kinase domain and juxtamembrane segment induced by phosphorylation of some linker residues, suggesting a link between EphA2 non-canonical signaling and canonical signaling through tyrosine phosphorylation. Given the key role of EphA2 in cancer malignancy, this new knowledge can inform therapeutic strategies.

biophysics↗

bioRxiv2020

Ligand-independent EGFR oligomers do not rely on the active state asymmetric kinase dimer

The human Epidermal Growth Factor Receptor (EGFR/ERBB1) is a Receptor Tyrosine Kinase (RTK) that forms active oligomers in response to ligand. Much evidence indicates that EGFR/ERBB1 forms oligomers in the absence of ligand, but the structure and physiological role of these ligand-independent dimers remain unclear. We use fluorescence microscopy to measure the oligomer stability and FRET efficiency for homo- and hetero-oligomers of fluorescent-protein labeled forms of EGFR and its paralog, Human Epidermal Growth Factor Receptor 2 (HER2/ERBB2) in vesicles derived from native cell membranes. Both receptors form ligand-independent oligomers at physiological plasma membrane concentrations. Mutations introduced in the EGFR kinase region at a key interface within the active state dimer alter the FRET efficiency within ligand-independent EGFR oligomers but do not affect their stability. These results indicate that ligand-independent EGFR oligomers do not require this interface and that the inactive state ensemble is distinct from the EGFR active state ensemble.

biophysics↗