Biology subjects

Webb, K. J.

Publications and source records attributed to Webb, K. J..

7 recordsLinked to original sources

bioRxiv2026

CD1a-Mediated Presentation of Canonical Microbial Peptides to T Cells

Langerhans cells express the nonpolymorphic antigen-presenting molecule CD1a, positioning them as contributors to host immunity against Mycobacterium leprae in human leprosy. CD1a was originally shown to present non-canonical lipopeptide antigens such as dideoxymycobactin and chemically diverse hydrophobic ligands. Here, we generated CD4 T cell lines from leprosy lesions that recognized M. leprae in a CD1a-restricted manner. Unexpectedly, antigen recognition was protease-sensitive, prompting biochemical purification that identified two microbial protein antigens: LppX, a 25-kDa lipoglycoprotein, and Ag85A, a 30-kDa secreted protein with no known lipid modification. Recombinant proteins activated the corresponding T cell lines in a CD1a-dependent manner. Epitope mapping identified 12-mer peptides that fully reconstituted antigenicity, were conserved between M. leprae and M. tuberculosis, and elicited robust, dose-dependent IFN-{gamma} production and T cell proliferation, establishing that DNA-encoded, ribosomally translated peptides serve as CD1a-restricted cognate antigens. Biochemical analyses showed peptide binding to CD1a, supported by isoelectric focusing and surface plasmon resonance (KD [~]75 M for Ag85A). CD1a-peptide tetramers specifically stained cognate T cells, soluble CD1a was sufficient to present peptide antigen, and transfer of the LppX-specific TCR into naive T cells restored antigen responsiveness. Using CD1a-peptide tetramers, we identified antigen-specific T cells enriched in patients undergoing reversal reactions compared with patients with lepromatous leprosy and healthy donors. The CD1a-restricted T cell lines secreted IFN-{gamma} and IL-26, cytokines with established antimicrobial activity. Together, these findings demonstrate that CD1a can present canonical microbial peptides as part of a cell-mediated immune response in leprosy, extending the known spectrum of CD1a ligands. Because CD1a is nonpolymorphic and presents antigens to antimicrobial T cells, CD1a-peptide complexes may provide a broadly applicable platform for studying, detecting, and potentially targeting mycobacterial immunity.

immunology↗

bioRxiv2026

FLIM-FRET as a Molecular Filter for Membrane-Induced Aggregation

Membrane binding is thought to trigger early aggregation of alpha-synuclein (aSyn) in neurons. However, live-cell measurements of membrane-proximal aggregation with high specificity remain challenging. We combine three- channel fluorescence lifetime imaging microscopy (FLIM) with Forster resonance energy transfer (FRET) in a model that uses FRET as a proximity filter. A membrane-tethered donor reports membrane-aSyn separation through lifetime changes, while an aSyn-labeled acceptor reports the aggregation state through its lifetime. We estimate per-cell lifetimes and mixture fractions for membrane-bound and membrane-unbound populations using a hierarchical expectation-maximization (EM) algorithm that pools information across pixels. We validate the estimator using Monte Carlo studies. Using experimental neuronal data, this method resolves changes in membrane-proximal aggregation and aggregate-associated lifetimes. This framework provides quantitative per-cell metrics linking membrane proximity and aggregation for comparative live-cell studies. SIGNIFICANCEMany studies map FRET signals pixel by pixel or average signals over whole cells. Neither approach reliably answers key biological questions: how much membrane-proximal aggregation exists in a given cell, and how does it change across conditions? Here, we combine three-channel FLIM-FRET with a hierarchical analysis that estimates per-cell lifetimes and fractions for bound and unbound populations by pooling information across pixels. This method shifts the focus from noisy images to cell-level metrics that support comparisons across treatments, time points, or genotypes. Simulations and neuronal data show improved accuracy under realistic photon budgets and reveal membrane-proximal aggregation effects that were unattainable otherwise. This approach is broadly applicable and extends beyond alpha-synuclein.

biophysics↗

bioRxiv2025

eIF3d and eIF3e mediate selective translational control of hypoxia that can be inhibited by novel small molecules

Exposure to hypoxia is linked to increased cellular plasticity and enhanced metastasis; effects which are primarily attributed to the transcriptional activation of large gene programs downstream of hypoxia inducible factors (HIFs). However, translational effects in hypoxia, that likely precede transcriptional effects, have remained largely unexplored. Using ribosome-profiling, we uncovered a selective translational response in acute hypoxia that is eIF3d/eIF3e-dependent and controls downstream hypoxic responses including HIF1a accumulation and cellular invasion. We further demonstrated that eIF3e copy number and an eIF3e-expression signature are associated with worsened outcomes for breast cancer patients. Finally, we identified a class of novel small molecules that target eIF3e specifically, reducing the translational response to hypoxia and to ER stress, another stressor that is dependent on eIF3d/eIF3e-mediated translation. Our data uncover critical functions for eIF3d/eIF3e in the hypoxic response and identify a potential means to inhibit stress-induced translation, and potentially plasticity and metastasis, mediated by eIF3e.

cancer biology↗

bioRxiv2024

A FRET-Based FLIM Method to Probe Membrane-Induced Alpha-Synuclein Aggregation in Neurons

1Parkinsons disease (PD) involves the aggregation of the protein alpha-synuclein, a process promoted by interactions with intracellular membranes. To study this phenomenon in neurons for the first time, we developed a fluorescence lifetime imaging (FLIM) method using Forster resonance energy transfer and self-quenching reporters, analyzed with a custom-built FLIM microscope. This method offers insights into aggregate formation in PD and can be broadly applied to probe protein-membrane interactions in neurons.

neuroscience↗

bioRxiv2024

Mechanisms of Alpha-Synuclein Seeded Aggregation in Neurons Revealed by Fluorescence Lifetime Imaging

1The brains of Parkinsons disease (PD) patients are characterized by the presence of Lewy body inclusions enriched with fibrillar forms of the presynaptic protein alpha-synuclein (aSyn). Despite related evidence that Lewy pathology spreads across different brain regions as the disease progresses, the underlying mechanism hence the fundamental cause of PD progression is unknown. The propagation of aSyn pathology is thought to potentially occur through the release of aSyn aggregates from diseased neurons, their uptake by neighboring healthy neurons via endocytosis, and subsequent seeding of native aSyn aggregation in the cytosol. A critical aspect of this process is believed to involve the escape of internalized ag-gregates from the endolysosomal compartment, though direct evidence of this mechanism in cultured neuron models remains lacking. In this study, we utilize a custom-built, time-gated fluorescence lifetime imaging microscope (FLIM) to investigate the progression of seeded ag-gregation over time in live cortical neurons. By establishing fluorescence lifetime sensitivity to aSyn aggregation level, we are able to monitor the proteins aggregation state. Through a FLIM analysis of neurons expressing aSyn-mVenus and exposed to aSyn preformed fibrils labeled with the acid-responsive dye pHrodo, we reveal the proteins aggregation state in both the cytosol and the endolysosomal compartment. The results indicate that aSyn seeds undergo partial disassembly prior to escaping the endocytic pathway, and that this escape is closely linked to the aggregation of cytosolic aSyn. In certain neurons, monomeric aSyn is found to translocate from the cytosol into the endolysosomal compartment, where it appar-ently forms aggregates in proximity to retained seeds. Additional analyses reveals zones of neuritic aSyn aggregates that overlaps with regions of microtubule disruption. Collectively, these findings enhance our understanding of aSyn pathology propagation in PD and other synucleinopathies, motivate additional experiments along these lines, and offer a path to guide the development of disease-modifying therapies.

neuroscience↗

bioRxiv2020

Multi-Omic Analysis Reveals Cannabidiol Disruption of Cholesterol Homeostasis in Human Cell Lines

The non-psychoactive cannabinoid, cannabidiol (CBD), is FDA-approved for treatment of two drug-resistant epileptic disorders, and is seeing increased use among the general public, yet the mechanisms that underlie its therapeutic effects and side-effect profiles remain unclear. Here, we report a systems-level analysis of CBD action in human cell lines using temporal multi-omic profiling. FRET-based biosensor screening revealed that CBD treatment resulted in a sharp rise in cytosolic calcium, and activation of AMPK and ERK kinases in human keratinocyte and neuroblastoma cell lines. CBD treatment led to alterations in the abundance of metabolites, mRNA transcripts, and proteins consistent with activation of cholesterol biosynthesis, transport and storage. We found that CBD rapidly incorporated into cellular membranes and altered cholesterol chemical activity, suggesting direct perturbation of cholesterol-dependent membrane properties. CBD treatment induced apoptosis in a dose-dependent manner in multiple human cell lines, which was rescued by inhibition of cholesterol synthesis, and potentiated by compounds that disrupt cholesterol trafficking and storage. Our data point to a pharmacological interaction of CBD with cholesterol homeostasis pathways, with potential implications in its therapeutic use.

systems biology↗

bioRxiv2019

Rapid discovery of drug target engagement by isothermal shift assay

Identifying protein targets directly bound by drug molecules within living systems remains challenging. Here we present the isothermal shift assay, iTSA, for rapid identification of drug targets. Compared with thermal proteome profiling, a prevailing method for target engagement, iTSA offers a simplified workflow, 4-fold higher throughput, and multiplexed experimental designs with higher replication. We demonstrate application of iTSA to identify targets for several kinase inhibitors in lysates and living cells.

pharmacology and toxicology↗