Biology subjects

Donahue, C.

Publications and source records attributed to Donahue, C..

4 recordsLinked to original sources

bioRxiv2026

A Yeast Two-Hybrid Protein Domain Screening Approach for Ebola Virus-Human Protein Interactions Identifies PABPC1 as a Host Factor Required for Replication

Ebola virus (EBOV) causes severe hemorrhagic fever and remains a global health threat despite advances in vaccines and antibody-based therapeutics. Viral replication and immune evasion depend on interactions between EBOV proteins and host factors, yet the full spectrum of these interactions is unknown. To systematically identify EBOV-human protein-protein interactions, we performed more than 200 yeast two-hybrid (Y2H) screens using full-length EBOV proteins and domain fragments as baits and cDNA libraries derived from human liver tissue, macrophages, and interferon-stimulated macrophages. This approach revealed 521 unique interactions supported by confidence scoring based on retest assays and library screen data. The interactome includes proteins involved in RNA metabolism, transcriptional regulation, and post-translational modification, with enrichment for RNA-binding proteins among partners of NP, VP30, and L. By employing multiple constructs for each EBOV gene, interaction interfaces could be localized to specific viral domains. This approach identified a likely MYND domain binding site in the intrinsically ordered region of NP. Similarly, overlapping cDNA fragments from Y2H screens revealed potential binding sites within human proteins, including putative VP30 binding sites on NUFIP2 and PABPC1 that lacked canonical PPPPxY motifs. PABPC1 interacted with VP30 in uninfected human cells and accumulated in inclusion bodies in infected cells that resembled respiratory syncytial virus inclusion body associated granules (IBAGs). Inhibiting PABPC1 expression reduced EBOV replication early in infection. These findings expand the EBOV-host interaction network, identify candidate regulators of viral RNA synthesis, and provide a resource for mechanistic studies and antiviral target discovery. ImportanceEbola virus (EBOV) remains a major global health threat due to its high mortality rate and limited treatment options. Viral replication and immune evasion depend on interactions between EBOV proteins and host factors, yet these interactions are incompletely understood. This study uses a large-scale yeast two-hybrid approach to systematically map EBOV-human protein-protein interactions, revealing hundreds of previously unreported partners and identifying cellular pathways that may be exploited by the virus. By integrating domain mapping, confidence scoring, and functional validation, we provide a resource that advances understanding of EBOV biology and may be used to discover candidates for antiviral development.

microbiology↗

bioRxiv2026

Dynamic conformational ensembles of soluble Tau encode neuronal toxicity prior to aggregation

Tau aggregation is a defining feature of Alzheimers disease and related tauopathies, yet the conformational states of Tau in neurons prior to aggregation remain poorly understood. Existing structural models are derived largely from fibrillar assemblies and provide limited insight into the dynamic, soluble Tau species that initiate pathology. Here, we combine hydrogen-deuterium exchange mass spectrometry with super-resolution imaging and neuronal models to define the conformational ensemble of soluble Tau under physiological and disease-relevant conditions. We show that soluble Tau populates distinct, dynamic conformations characterized by regional stabilization and long-range intramolecular interactions that are invisible to fibril-based structures. Disease-associated perturbations selectively remodel these conformational ensembles, exposing aggregation-prone regions and altering Tau subcellular organization in neurons. Notably, these Tau species inhibit axonal transport, which is essential for neuronal health, linking specific ensemble states to neuronal toxicity. These findings establish soluble Tau conformation as a dynamic, regulatable state that precedes aggregation and encodes disease relevance. By defining the structural logic of Tau before fibril formation, this work provides a framework for understanding early tauopathy mechanisms and for targeting Tau pathology at its earliest stages. SUMMARYTau pathology is a hallmark of Alzheimers disease (AD) and related dementias (ADRDs). Although Tau is often described as intrinsically disordered, it is a dynamic protein with distinct but poorly defined conformations. Here we conduct a systematic time-resolved structure-function analysis of normal and pathologic Tau, including hyperphosphorylated, mutant Tau, and posttranslational-modification-mimetic Tau. To characterize dynamic conformational changes of Tau, we combined state-of-the-art hydrogen deuterium exchange mass spectrometry with structured illumination microscopy, demonstrating a novel Tau-MT binding mode: "dynamic oscillation". To correlate Tau structure with neuronal function, we evaluated axonal transport as a sensitive readout of neuronal health. Many toxic Tau forms share a common signature of increased exposure of the N-terminal phosphate activating domain (PAD) in vitro and in vivo. Aberrant exposure of PAD correlates with Tau pathology and axonal transport defects. Tau phosphorylation at S262 alone is sufficient to alter Tau-microtubule interactions beyond R1-R4 motifs, globally changing Tau conformation, disrupting "dynamic oscillation" on MTs, and inhibiting axonal transport. Frontotemporal dementia-associated P301L-Tau remains associated with microtubules but also inhibits axonal transport. Our results reveal a well-defined conformation of soluble WT Tau in neurons and its highly dynamic interaction with microtubules, altered by AD/ADRD-Tau forms. Our multidisciplinary approach comprising biochemical manipulations, innovative MS tools, advanced microscopy, cellular assays, and mouse and human data pair Tau conformations with distinct neuronal functions and pathologies in health and disease.

neuroscience↗

bioRxiv2022

Individual Astrocyte Morphology in the Collagenous Lamina Cribrosa Revealed by Multicolor DiOlistic Labeling

Astrocytes in the lamina region of the optic nerve head play vital roles in supporting retinal ganglion cell axon health. In glaucoma, these astrocytes are implicated as early responders to stressors, undergoing characteristic changes in cell function as well as cell morphology. Much of what is currently known about individual lamina astrocyte morphology has been learned from rodent models which lack a defining feature of the human optic nerve head, the collagenous lamina cribrosa (LC). Current methods available for evaluation of collagenous LC astrocyte morphology have significant shortcomings. We aimed to evaluate Multicolor DiOlistic labeling (MuDi) as an approach to reveal individual astrocyte morphologies across the collagenous LC. Gold microcarriers were coated with all combinations of three fluorescent cell membrane dyes, DiI, DiD, and DiO, for a total of seven dye combinations. Microcarriers were delivered to 150m-thick coronal vibratome slices through the LC of pig, sheep, goat, and monkey eyes via MuDi. Labeled tissues were imaged with confocal and second harmonic generation microscopy to visualize dyed cells and LC collagenous beams, respectively. GFAP labeling of DiOlistically-labeled cells with astrocyte morphologies was used to investigate cell identity. 3D models of astrocytes were created from confocal image stacks for quantification of morphological features. DiOlistic labeling revealed fine details of LC astrocyte morphologies including somas, primary branches, higher-order branches, and end-feet. Labeled cells with astrocyte morphologies were GFAP+. Astrocytes were visible across seven distinct color channels, allowing high labeling density while still distinguishing individual cells from their neighbors. MuDi was capable of revealing tens to hundreds of collagenous LC astrocytes, in situ, with a single application. 3D astrocyte models allowed automated quantification of morphological features including branch number, length, thickness, hierarchy, and straightness as well as Sholl analysis. MuDi labeling provides an opportunity to investigate morphologies of collagenous LC astrocytes, providing both qualitative and quantitative detail, in healthy tissues. This approach may open doors for research of glaucoma, where astrocyte morphological alterations are thought to coincide with key functional changes related to disease progression.

bioengineering↗

bioRxiv2022

Structure of the Inmazeb cocktail and resistance to escape against Ebola virus

Monoclonal antibodies can provide important pre- or post-exposure protection against disease for those not yet vaccinated or in individuals that fail to mount a protective immune response after vaccination. A key concern in use of monotherapy monoclonal antibody products lies in the high risk of mutagenic escape. Inmazeb (REGN-EB3), a three-antibody cocktail against Ebola virus, demonstrated efficacy in lessening disease course and improving survival in a randomized, controlled trial. Here we present the cryoEM structure at 3.1 [A] of the Ebola virus glycoprotein, determined without symmetry averaging, in a simultaneous complex with eight Fab fragments of antibodies in the Inmazeb cocktail. This structure allows modeling of previously disordered portions of the glycan cap, maps the non-overlapping epitopes of Inmazeb, and illuminates the basis for complementary activities, as well as residues that are critical for resistance to escape by each component of this cocktail and other clinically relevant antibodies. We also provide direct evidence that, unlike monotherapy treatments, including those targeting conserved epitopes, the Inmazeb protects against the rapid emergence of EBOV escape mutants and supports the benefit of the combination approach.

biophysics↗