Biology subjects

Barrera, F. N.

Publications and source records attributed to Barrera, F. N..

15 recordsLinked to original sources

bioRxiv2025

CAR T cell cytotoxic responses are rapidly generated and sensitive to the unligated TCR

Chimeric antigen receptor (CAR) T cells expressing tumor-targeting engineered receptors can robustly eliminate cancer cells through secretion of cytotoxic factors. Durable remission in leukemia and lymphoma treatment has not been matched in solid tumors. Efforts to maximize tumor destruction and minimize toxicities have driven efforts to tune CAR signaling. However, the molecular mechanisms for CAR triggering and thresholds for activation are incompletely understood. Here, we measured the collection of CAR binding interactions that culminate in polarized delivery of lytic granules to the junction with the target. CAR T cells binarized cytotoxic activities in response to a few binding events and population outcomes were dominated by a subset of cells. Activation at the single molecule level matches the sensitivity of the native T cell receptor (TCR) and points to potent downstream signal propagation. Disruption of the unligated TCR with a transmembrane-targeting inhibitory peptide strongly dampened CAR T cell activation, indicating a critical crosstalk between the two receptors. Harnessing CAR T cell efficacy and reduction of toxicity will require new approaches to modify integration of the binding events, collected stochastically, that are rapidly digitized. These sensitive CAR T cell responses provide new insights into driving cytotoxic signaling through surface interaction engineering.

immunology↗

bioRxiv2025

Cholesterol promotes the formation of dimers and oligomers of the receptor tyrosine kinase ROR1

ROR1 is a member of the receptor tyrosine kinase (RTK) family that plays a crucial role during organogenesis of bone and neural systems by regulating non-canonical Wnt signaling. Misregulation of ROR1 is additionally a causative factor for carcinogenesis in solid and liquid tumors. However, we have a poor understanding of how ROR1 activity is regulated. We employed a recently developed single-molecule method termed SiMPull-POP to study the oligomeric state of ROR1. RTK function is typically triggered by ligand binding, which promotes self-assembly of RTKs to form dimers and in some cases oligomers. However, our data indicate that ROR1 does not follow this paradigm. Instead, ROR1 forms dimers and oligomers in a process that is not affected by the presence of the ROR1 ligand Wnt5a. Additional experiments indicate that the transmembrane domain of ROR1 has a strong tendency to self-assemble, suggesting that this domain modulates ROR1 dimerization. Investigation into a regulatory mechanism for ROR1 self-assembly led to evaluation of the role of the lipid cholesterol, which plays pleiotropic roles in Wnt signaling. Cholesterol was found to promote the assembly of ROR1, and our results point to the transmembrane domain as the region where cholesterol exerts the regulatory effect. Taken together, our results indicate that ROR1 self-assembles in human cells; however, unlike other RTKs, this process is not stabilized by ligand binding but is instead facilitated by membrane cholesterol.

biochemistry↗

bioRxiv2025

Bayesian analysis and efficient algorithms for single-molecule fluorescence data and step counting

With the growing adoption of single-molecule fluorescence experiments, there is an increasing demand for efficient statistical methodologies and accurate analysis of the acquired measurements. Existing analysis frameworks, such as those that use kinetic models, often rely on strong assumptions on the dynamics of the molecules and fluorophores under study that render them inappropriate for general purpose step counting applications, especially when the systems of study exhibit uncharacterized dynamics. Here, we propose a novel Bayesian nonparametric framework to analyze singlemolecule fluorescence data that is kinetic model independent. For the evaluation of our methods, we develop four MCMC samplers, ranging from elemental to highly sophisticated, and demonstrate that the added complexity is essential for accurate data analysis. We apply our methods to experimental data obtained from TIRF photobleaching assays of the EphA2 receptor tagged with GFP. In addition, we validate our approach with synthetic data mimicking realistic conditions and demonstrate its ability to recover ground truth under high- and low-signal-to-noise data, establishing it as a versatile tool for fluorescence data analysis. Significance statementProtein complexes are critical for cell function. Advances in fluorescence experiments have facilitated their direct study, achieving single-molecule resolution in the determination of their stoichiometry. However, the analysis of raw fluorescence data remains a challenge. Traditional visual inspection methods are often time-consuming and susceptible to user bias, while conventional statistical approaches are limited by strong assumptions regarding molecular and probe dynamics or exceedingly high computational requirements. In this study, we introduce a novel statistical methodology for robust and efficient analysis of fluorescence data. Our innovative approach not only enhances the accuracy and reliability of data analysis following a fluorescence experiment but also allows high-throughput applications. Our methods present a significant advancement that may extend the scope of current fluorescence techniques.

biochemistry↗

bioRxiv2025

pH-Responsive Near Infrared Light Triggered Hydroxyapatite Nanoparticles for Targeted Photothermal Cancer Therapy

Photothermal therapy (PTT), which utilizes photothermal agents (PTAs) to induce localized hyperthermia within tumors upon light irradiation, has emerged as a promising cancer treatment strategy. However, low water solubility, poor in vivo circulation stability and a lack of tumor specificity of many common PTAs limit their applicability. To address these issues, we have developed a simple, yet highly potent, tumor-targeted nanotheranostic system that consists of lipid/PEG-coated hydroxyapatite nanoparticles (LHAPNs) encapsulating the near-infrared (NIR) photothermal dye IR106 (LHAPNIRs). The lipid coat serves to retain the encapsulated dye and prevent serum protein adsorption and macrophage recognition, which would otherwise destabilize the nanoparticles and hinder their tumor targeting efficiency. Additionally, the coat is functionalized with the tumor-acidity-triggered rational membrane (ATRAM) peptide for efficient and specific internalization into tumor cells in the mildly acidic microenvironment of tumors. The nanoparticles facilitated real-time fluorescence and thermal imaging of tumors and demonstrated potent NIR-light triggered anticancer activity in vitro and in vivo, without adversely affecting healthy tissue, leading to markedly prolonged survival. Our results demonstrate that the biocompatible and biodegradable ATRAM-functionalized LHAPNIRs (ALHAPNIRs) effectively combine dual-mode diagnostic imaging with targeted cancer PTT.

bioengineering↗

bioRxiv2024

Cholesterol inhibits oncogenic EphA2 activation

The receptor tyrosine kinase EphA2 drives cancer malignancy by facilitating metastasis. EphA2 can be found in different self-assembly states: as a monomer, dimer, and oligomer. However, our understanding remains limited regarding which EphA2 state is responsible for driving pro-metastatic signaling. To address this limitation, we have developed SiMPull-POP, a single-molecule method for accurate quantification of membrane protein self-assembly. Our experiments revealed that a reduction of plasma membrane cholesterol strongly promoted EphA2 self-assembly. Indeed, low cholesterol caused a similar effect to the EphA2 ligand ephrinA1-Fc. These results indicate that cholesterol inhibits EphA2 assembly. Phosphorylation studies in different cell lines revealed that low cholesterol increased phospho-serine levels, the signature of oncogenic signaling. Investigation of the mechanism that cholesterol uses to inhibit the assembly and activity of EphA2 indicate an in-trans effect, where EphA2 is phosphorylated by protein kinase A downstream of beta-adrenergic receptor activity, which cholesterol also inhibits. Our study not only provides new mechanistic insights on EphA2 oncogenic function, but also suggests that cholesterol acts as a molecular safeguard mechanism that prevents uncontrolled self-assembly and activation of EphA2.

biochemistry↗

bioRxiv2024

A genome-scale screen identifies sulfated glycosaminoglycans as pivotal in epithelial cell damage by Candida albicans

Candidalysin is a cytolytic peptide produced by the opportunistic fungal pathogen Candida albicans. This peptide is a key virulence factor in mouse models of mucosal and hematogenously disseminated candidiasis. Despite intense interest in the role of candidalysin in C. albicans pathogenicity, its host cell targets have remained elusive. To fill this knowledge gap, we performed a genome-wide loss-of-function CRISPR screen in a human oral epithelial cell line to identify specific host factors required for susceptibility to candidalysin-induced cellular damage. Among the top hits were XYLT2, B3GALT6 and B3GAT3, genes that function in glycosaminoglycan (GAG) biosynthesis. Deletion of these genes led to the absence of GAGs such as heparan sulfate on the epithelial cell surface and increased resistance to damage induced by both candidalysin and live C. albicans. Biophysical analyses including surface plasmon resonance and atomic force and electron microscopy indicated that candidalysin physically binds to sulfated GAGs, facilitating its oligomerization or enrichment on the host cell surface. The addition of exogenous sulfated GAGs or the GAG analogue dextran sulfate protected cells against candidalysin-induced damage. Dextran sulfate, but not non-sulfated dextran, also inhibited epithelial cell endocytosis of C. albicans and fungal-induced epithelial cell cytokine and chemokine production. In a murine model of vulvovaginal candidiasis, topical dextran sulfate administration reduced host tissue damage and decreased intravaginal IL-1{beta} and neutrophil levels. Collectively, these data indicate that GAGs are epithelial cell targets of candidalysin and can be used therapeutically to protect cells from candidalysin-induced damage.

microbiology↗

bioRxiv2024

Phosphatidylinositol (4,5)-bisphosphate drives the formation of EGFR and EphA2 complexes

Receptor tyrosine kinases (RTKs) regulate many cellular functions and are important targets in pharmaceutical development, particularly in cancer treatment. EGFR and EphA2 are two key RTKs that are associated with oncogenic phenotypes. Several studies have reported functional interplay between these receptors, but the mechanism of interaction is still unresolved. Here we utilize a time-resolved fluorescence spectroscopy called PIE-FCCS to resolve EGFR and EphA2 interactions in live cells. We tested the role of ligands and found that EGF, but not ephrin A1 (EA1), stimulated hetero-multimerization between the receptors. To determine the effect of anionic lipids, we targeted phospholipase C (PLC) activity to alter the abundance of phosphatidylinositol (4,5)-bisphosphate (PIP2). We found that higher PIP2 levels increased homo-multimerization of both EGFR and EphA2, as well as hetero-multimerization. This study provides a direct characterization of EGFR and EphA2 interactions in live cells and shows that PIP2 can have a substantial effect on the spatial organization of RTKs.

biophysics↗

bioRxiv2023

pH-responsive upconversion mesoporous silica nanospheres for combined multimodal diagnostic imaging and targeted photodynamic and photothermal cancer therapy

Photodynamic therapy (PDT) and photothermal therapy (PTT) have garnered considerable interest as non-invasive cancer treatment modalities. However, these approaches remain limited by low solubility, poor stability and inefficient targeting of many common photosensitizers (PSs) and photothermal agents (PTAs). To overcome these limitations, we have designed biocompatible and biodegradable tumor-targeted upconversion nanospheres with imaging capabilities. The multifunctional nanospheres consist of a sodium yttrium fluoride core doped with lanthanides (ytterbium, erbium and gadolinium) and bismuth selenide (NaYF4:Yb/Er/Gd,Bi2Se3) within a mesoporous silica shell that encapsulates a PS, Chlorin e6 (Ce6), in its pores. NaYF4:Yb/Er converts deeply penetrating near-infrared (NIR) light to visible light, which excites the Ce6 to generate cytotoxic reactive oxygen species (ROS), while the PTA Bi2Se3 efficiently converts absorbed NIR light to heat. Additionally, Gd enables magnetic resonance imaging (MRI) of the nanospheres. The mesoporous silica shell is coated with lipid/polyethylene glycol (DPPC/cholesterol/DSPE-PEG) to ensure retention of the encapsulated Ce6 and minimize interactions with serum proteins and macrophages that impede tumor targeting. Finally, the coat is functionalized with the acidity-triggered rational membrane (ATRAM) peptide, which promotes specific and efficient internalization into cancer cells within the mildly acidic tumor microenvironment. Following uptake by cancer cells in vitro, NIR laser irradiation of the nanospheres caused substantial cytotoxicity due to ROS production and hyperthermia. The nanospheres facilitated tumor MRI and thermal imaging, and exhibited potent NIR laser light-induced antitumor effects in vivo via combined PDT and PTT, with no observable toxicity to healthy tissue, thereby substantially prolonging survival. Our results demonstrate that the ATRAM-functionalized, lipid/PEG-coated upconversion mesoporous silica nanospheres (ALUMSNs) offer multimodal diagnostic imaging and targeted combinatorial cancer therapy.

bioengineering↗

bioRxiv2022

Allosteric inhibition of the epidermal growth factor receptor through disruption of transmembrane interactions

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) commonly targeted for inhibition by anti-cancer therapeutics. Current therapeutics target EGFRs kinase domain or extracellular region. However, these types of inhibitors are not specific for tumors over healthy tissue and therefore cause undesirable side effects. Our lab has recently developed a new strategy to regulate RTK activity by designing a peptide that specifically binds to the transmembrane (TM) region of the RTK to allosterically modify kinase activity. These peptides are acidity-responsive, allowing them to preferentially target acidic environments like tumors. We have applied this strategy to EGFR and created the PET1 peptide. We observed that PET1 behaves as a pH-responsive peptide that modulates the configuration of the EGFR TM through a direct interaction. Our data indicated that PET1 inhibits EGFR-mediated cell migration. Finally, we investigated the mechanism of inhibition through molecular dynamics simulations, which showed that PET1 sits between the two EGFR TM helices; this molecular mechanism was additionally supported by AlphaFold-Multimer predictions. We propose that the PET1-induced disruption of native TM interactions disturbs the conformation of the kinase domain in such a way that it inhibits EGFRs ability to send migratory cell signals. This study is a proof-of-concept that acidity-responsive membrane peptide ligands can be generally applied to RTKs. In addition, PET1 constitutes a viable approach to therapeutically target the TM of EGFR.

cell biology↗

bioRxiv2022

Allosteric Inhibition of the T Cell Receptor by a Designed Membrane Ligand

The T cell receptor (TCR) is a complex molecular machine that directs the activation of T cells, allowing the immune system to fight pathogens and cancer cells. Despite decades of investigation, the molecular mechanism of TCR activation is still controversial. One of the leading activation hypotheses is the allosteric model. This model posits that binding of pMHC at the extracellular domain triggers a dynamic change in the transmembrane (TM) domain of the TCR subunits, which leads to signaling at the cytoplasmic side. We sought to test this hypothesis by creating a TM ligand for TCR. Previously we described a method to create a soluble peptide capable of inserting into membranes and bind to the TM domain of the receptor tyrosine kinase EphA2 (Alves et al., eLife 2018). Here we show that the approach is generalizable to complex membrane receptors, by designing a membrane ligand for TCR. We observed that the designed peptide caused a reduction of Lck phosphorylation of TCR at the CD3{zeta} subunit. As a result, in the presence of this Peptide Inhibitor of TCR (PITCR), the proximal signaling cascade downstream of TCR activation was significantly dampened in T cells. Co-localization and co-immunoprecipitation results in DIBMA native nanodiscs confirmed that PITCR was able to bind to the TCR. We propose that PITCR binds into a crevice present between the TM helices of the CD3{zeta} and CD3{varepsilon}({delta}) subunits. Our results additionally indicate that PITCR disrupts the allosteric changes in the compactness of the TM bundle that occur upon TCR activation, lending support to the allosteric TCR activation model. The TCR inhibition achieved by PITCR might be useful to treat inflammatory and autoimmune diseases and to prevent organ transplant rejection, as in these conditions aberrant activation of TCR contributes to disease.

biophysics↗

bioRxiv2021

The C. albicans virulence factor Candidalysin polymerizes in solution to form membrane pores and damage epithelial cells.

The pathogenic fungus Candida albicans causes severe invasive candidiasis. C. albicans infection requires the action of the virulence factor Candidalysin (CL), which damages the plasma membrane of the target human cells. However, the molecular mechanism that CL uses to permeabilize membranes is poorly understood. We employed complementary biophysical, modeling, microscopy, and cell biology methods to reveal that CL forms membrane pores using a unique molecular mechanism. Unexpectedly, it was observed that CL readily assembles into linear polymers in solution. The basic structural unit in polymer formation is a CL 8-mer, which is sequentially added into a string configuration. Finally, the linear polymers can close into a loop. Our data indicate that CL loops spontaneously insert into the membrane to become membrane pores. We identified a CL mutation (G4W) that inhibited the formation of polymers in solution and prevented formation of pores in different synthetic lipid membranes systems. Studies in epithelial cells showed that G4W CL failed to activate the danger response signaling pathway, a hallmark of the pathogenic effect of CL. These results indicate that CL polymerization in solution is a necessary step for the damage of cellular membranes. Analysis of thousands of CL pores by atomic force microscopy revealed the co-existence of simple depressions and complex pores decorated with protrusions. Imaging and modeling indicate that the two types of pores are formed by CL molecules assembled into alternate orientations. We propose that this structural rearrangement represents a maturation mechanism that might stabilize pore formation to achieve more robust cellular damage. Taken together, the data show that CL uses a previously unknown mechanism to damage membranes, whereby pre-assembly of CL loops in solution directly leads to formation of membrane pores. Our investigation not only unravels a new paradigm for the formation of membrane pores, but additionally identifies CL polymerization as a novel therapeutic target to treat candidiasis.

biophysics↗

bioRxiv2021

Conformational clamping by a membrane ligand activates the EphA2 receptor

The EphA2 receptor is a promising drug target for cancer treatment, since EphA2 activation can inhibit metastasis and tumor progression. It has been recently described that the TYPE7 peptide activates EphA2 using a novel mechanism that involves binding to the single transmembrane domain of the receptor. TYPE7 is a conditional transmembrane (TM) ligand, which only inserts into membranes at neutral pH in the presence of the TM region of EphA2. However, how membrane interactions can activate EphA2 is not known. We systematically altered the sequence of TYPE7 to identify the binding motif used to activate EphA2. With the resulting six peptides, we performed biophysical and cell migration assays that identified a new potent peptide variant. We also performed a mutational screen that determined the helical interface that mediates dimerization of the TM domain of EphA2 in cells. These results, together with molecular dynamic simulations, allowed to elucidate the molecular mechanism that TYPE7 uses to activate EphA2, where the membrane peptide acts as a molecular clamp that wraps around the TM dimer of the receptor. We propose that this binding mode stabilizes the active conformation of EphA2. Our data, additionally, provide clues into the properties that TM ligands need to have in order to achieve activation of membrane receptors.

biophysics↗

bioRxiv2020

PIP2 promotes conformation-specific dimerization of the EphA2 membrane region

The impact of the EphA2 receptor on cancer malignancy hinges on the two different ways it can be activated. EphA2 induces anti-oncogenic signaling after ligand binding, but ligand-independent activation of EphA2 is pro-oncogenic. It is believed that the transmembrane (TM) domain of EphA2 adopts two alternate conformations in the ligand-dependent and the ligand-independent states. However, it is poorly understood how the difference in TM helical crossing angles found in the two conformations impacts the activity and regulation of EphA2. We devised a method that uses hydrophobic matching to stabilize two conformations of a peptide comprising the EphA2 TM domain and a portion of the intracellular juxtamembrane (JM) segment. The two conformations exhibit different TM crossing angles, resembling the ligand-dependent and ligand-independent states. We developed a single-molecule technique using SMALPs to measure dimerization in membranes. We observed that the signaling lipid PIP2 promotes TM dimerization, but only in the small crossing angle state, which we propose corresponds to the ligand-independent conformation. In this state the two TM are almost parallel, and the positively charged JM segments are expected to be close to each other, causing electrostatic repulsion. The mechanism PIP2 uses to promote dimerization might involve alleviating this repulsion due to its high density of negative charges. Our data reveal a conformational coupling between the TM and JM regions, and suggest that PIP2 might directly exert a regulatory effect on EphA2 activation in cells that is specific to the ligand-independent conformation of the receptor.

biophysics↗

bioRxiv2019

The effect of phosphatidylserine on the membrane insertion of the cancer-targeting ATRAM peptide is defined by its non-inserting end

The acidity-triggered rational membrane (ATRAM) peptide was designed to target acidic diseases such as cancer. An acidic extracellular medium, such as that found in aggressive tumors, drives the protonation of the glutamic acids in ATRAM, leading to the membrane translocation of its C-terminus and the formation of a transmembrane helix. Compared to healthy cells, cancerous cells often increase exposure of the negatively charged phosphatidylserine (PS) on the outer leaflet of the plasma membrane. Here we use a reconstituted vesicle system to explore how phosphatidylserine influences the interaction of ATRAM with membranes. To explore this, we used two new variants of ATRAM, termed K2-ATRAM and Y-ATRAM, with small modifications at the non-inserting N-terminus. We observed that the effect of PS on the membrane insertion pK and lipid partitioning hinged on the sequence of the non-inserting end. Our data additionally indicate that the effect of PS on the insertion pK does not merely depend on electrostatics, but it is multifactorial. Here we show how small sequence changes can impact the interaction of a peptide with membranes of mixed lipid composition. These data illustrate how model studies using neutral bilayers, which do not mimic the negative charge found in the plasma membrane of cancer cells, may fail to capture important aspects of the interaction of anticancer peptides with tumor cells. This information can guide the design of therapeutic peptides that target the acidic environments of different diseased tissues.\n\nStatement of SignificanceCurrent targeted therapies for cancer have limited success due to drug resistance. Resistance often arises after mutation of the receptor being targeted. A more general target is needed to prevent drug resistance. Most aggressive solid tumors have an extracellular medium. We propose that extracellular acidity is promising for improved targeted therapies. The acidity-triggered rational membrane (ATRAM) inserts in membranes only under acidic conditions. However, it is now known how the lipid changes that occur in the plasma membrane of cancer cells impact the membrane insertion of ATRAM. Here we perform biophysical experiments that show that PS, a lipid exposed in the cancer cell, can impact the membrane insertion of ATRAM. We also uncovered a region of the peptide important for insertion.

biophysics↗

bioRxiv2018

PS membrane asymmetry influences the folding and insertion of a transmembrane helix

The plasma membrane (PM) contains an asymmetric distribution of lipids between the inner and outer leaflets of its bilayer. A lipid of special interest in eukaryotic cells is the negatively charged phosphatidylserine (PS). In healthy cells, PS is actively sequestered to the inner leaflet of the PM but can redistribute to the outer leaflet when the cell is damaged or at the onset of apoptosis. The influence of PS asymmetry and its loss on membrane protein structure and organization have not been widely addressed. Marginally hydrophobic membrane proteins contain acidic residues in their transmembrane sequence, which can enable topological transitions after membrane association. The pH low insertion peptide (pHLIP), which undergoes a topological reorientation and inserts into the membrane at acidic pH - as its name implies, is a useful and well-characterized model for studying these transitions. Although it is known that the inclusion of PS in symmetric vesicles affects the membrane insertion process of pHLIP by lowering the pH midpoint of insertion, it is unclear how PS asymmetry influences these topological transitions. Here, we studied pHLIPs topology using freely-floating asymmetric phosphatidylcholine (PC)/PS vesicles with PS enriched in the inner leaflet. We developed a modified protocol to create asymmetric vesicles containing PS and employed Annexin V labeled with an Alexa 568 fluorophore as a new probe to quantifying PS asymmetry. For pHLIP, membrane insertion was affected by the surface charge difference between bilayer leaflets caused by the asymmetric distribution of charged lipids between the leaflets. We thus conclude that lipid asymmetry can have consequences for the behavior of membrane-associated proteins. A corollary is that model studies using symmetric bilayers to mimic the PM may fail to capture important aspects of protein-membrane interactions.

biophysics↗