Biology subjects

Burnap, S. A.

Publications and source records attributed to Burnap, S. A..

12 recordsLinked to original sources

bioRxiv2026

Human Choroid Plexus Organoids Progress to Postnatal-like Maturation and Reveal a Closing Window of Developmental Plasticity

The choroid plexus (ChP) produces cerebrospinal fluid (CSF) and forms the blood-CSF barrier, yet the developmental programme driving its maturation in humans remains poorly understood. Using a 15-timepoint single-cell RNA sequencing atlas of human ChP organoids benchmarked against human tissue, we resolve an ordered differentiation process comprising cell cycle exit, ciliogenesis and ion transporter expression. Maturation culminates in a transcriptional state with features of human adult ChP, establishing the first in vitro ChP model to reach postnatal-like milestones. Centrosome proteomics identified previously uncharacterised, stage-specific regulators of ciliogenesis. To probe developmental plasticity, we perturbed ChP organoids with valproic acid (VPA), a clinically used antiepileptic and established teratogen. Early VPA-mediated disruption of multiciliogenesis and secretion recovered upon drug withdrawal, whereas late treatment washout resulted in persistent impairment of mature transporters. Together, these findings resolve the developmental programme by which the ChP reaches functionally mature identity and identify a stage-dependent plasticity window during its maturation.

developmental biology↗

bioRxiv2026

Mechanism of spike opening in a mammalian gammacoronavirus

Coronavirus spike proteins mediate receptor binding and fusion and are key determinants of viral host range. To bind protein receptors, some CoV spikes undergo conformational changes termed opening, which expose the receptor-binding surface. Yet, the prerequisites and mechanism of spike opening remain poorly understood, especially outside of beta-CoVs, and many CoV spikes have been observed only in the closed conformation. The receptor specificities of gamma-CoVs, for which birds are natural hosts, await in-depth characterisation and their spikes have not been observed to open. Here, we report structures of the spike from an unusual mammalian gamma-CoV isolated from a beluga whale (BwCoV), in closed and open conformations, the latter with two S1B domains erect. BwCoV spike opening is associated with changes throughout S1 that resemble those observed in beta-CoV spikes and similarly propagate into destabilisation of the fusogenic S2 core. Furthermore, the BwCoV-S structure contains an additional density likely corresponding to a fatty acid, consistent with lipid binding stabilising the closed conformation through a pocket distinct from that identified for beta-CoVs. These structural insights reveal conserved principles regulating spike dynamics across coronavirus genera and support lipid binding as a potential modulatory mechanism of spike opening.

biochemistry↗

bioRxiv2026

An integrated mass spectrometry strategy for quantifying the proteoform diversity of the extensively modified O-glycoprotein Osteopontin

O-glycosylation is among the most abundant and structurally diverse post-translational modifications in eukaryotes, yet its heterogeneity renders O-glycoproteins exceptionally difficult to characterize. To overcome these challenges, we have developed an integrated mass spectrometry (MS) strategy to define the proteoform landscape of O-glycoproteins and applied it to human osteopontin (OPN). OPN is a disease-associated extracellular matrix protein subject to extensive modification. By combining native MS with serial exoglycosidase digestions, we directly resolved truncation, phosphorylation, sulfation, and O-glycosylation of OPN. Matched glycoproteomic analyses, using tailored (glyco)protease combinations, allowed us to quantify glycan heterogeneity inaccessible to conventional trypsin-based approaches or protein-centric methods. We integrated these datasets using forward compositional simulations to infer the intact OPN proteoform distribution and benchmarked the resulting models against an experimental intact-mass distribution obtained by proton-transfer charge-reduction MS. This comparison revealed that bottom-up O-glycoproteomics systematically underestimates the true extent of glycan sialylation, whereas assuming (near-)complete sialylation accurately reproduced the experimental intact OPN mass distribution. Together, these results provide a comprehensive, quantitative view of OPN compositional diversity and demonstrate how intact-protein and peptide-level measurements can be reconciled to resolve highly heterogeneous glycoform populations. The workflow establishes a broadly applicable framework for characterizing extensively O-glycosylated and multiply modified proteins.

biophysics↗

bioRxiv2026

A proteomic feasibility study connecting metabolic and synaptic pathway alterations in serum and extracellular vesicles to characterize treatment-resistant depression

Treatment-resistant depression (TRD) remains a major clinical challenge, yet the biological processes distinguishing TRD from non-treatment-resistant depression (nTRD) are incompletely defined. While circulating serum proteomes reflect broad systemic alterations associated with depression, extracellular vesicles (EVs) could provide a more selective representation of intercellular signaling relevant to treatment resistance. Here, we carried out a pilot study to evaluate the extent that parallel proteomic profiling of serum and serum-derived EVs could distinguish healthy controls (CON), nTRD, and TRD individuals. In this exploratory and hypothesis-generating study, serum proteomes exhibited robust global differences between depression groups and controls, largely reflecting shared systemic biology across nTRD and TRD. In contrast, EV proteomes showed limited global separation but revealed subtype-associated pathway differences. Relative to controls, nTRD EVs were enriched for immune and inflammatory pathways. By contrast, TRD EVs were characterized by enrichment of mitochondrial metabolism, oxidative phosphorylation, translational initiation, and MYC-regulated pathways, together with depletion of synaptic signalling, membrane trafficking, and cytoskeletal pathways. Comparative analysis of pathways significant in both contrasts revealed that these bioenergetic and translational signatures were selectively amplified in TRD relative to nTRD. Our exploratory analyses identified that the circulating EV cargo may reflect a treatment-resistance-specific reorganization of biological pathways not apparent in bulk serum proteomics. This study highlights parallel serum and EV proteomics as a complementary approach for molecular stratification in antidepressant resistance.

neuroscience↗

bioRxiv2026

Selective miniprotein inhibitors of Aurora-A kinase designed using interaction-motif scaffolding

Targeting kinase ATP-binding sites has produced many successful therapeutics, but selectivity remains a major challenge. We hypothesised that substrate-recognition surfaces could provide an alternative route to selective kinase inhibition. Here, we designed and tested structure-guided miniprotein inhibitors of Aurora-A using N-Myc as a template, a natural weak binder of the P+1 pocket. The most potent designs bind Aurora-A with single-digit nanomolar affinity, more than a thousand fold higher than the starting template, and selectively inhibit Aurora-A over Aurora-B in kinase assays and mitotic cells. Despite diverse architectures, successful binders targeting the substrate-recognition surface converged on a common strategy, coupling extensive engagement of the G helix with activation loop stabilisation. Together these results show that selective kinase inhibition can be achieved by exploiting structurally well-defined active-state conformations rather than kinase-specific inactive states. Our work establishes a framework for converting weak substrate-recognition surface interactors into potent, selective kinase inhibitors through structure-guided protein design.

molecular biology↗

bioRxiv2026

Molecular dissection of protein complexes isolated from sections of human brain

Molecular studies of brain receptors and transporters have typically relied on recombinant systems, limiting insight into their organization in native tissue. Here, we develop nanobody-based immunoprecipitation coupled with native mass spectrometry to interrogate endogenous protein assemblies from post-mortem mouse and human brain sections. We exemplify our approach by characterizing the synaptic proteins VGluT1 and mGluR2. From a single mouse brain, we discover mGluR2/3 heterodimers, alongside mGluR2 homodimers. Considering regions of human brain heterodimeric mGluR2/3 is highly abundant in the OFC and sgACC ([~]70% and 50%, respectively) and forms regional-specific interactions with additional synaptic proteins. In a modest cohort of biobanked human tissue, associated with depression and suicide, we find increased mGluR2/3 in the OFC. Consistent with this, mice exhibit similar associations between heterodimer levels and stress-susceptibility. Overall, our approach provides a direct means for establishing molecular-behavioural links at the level of receptor organization in brain.

biochemistry↗

bioRxiv2026

Continuous mass photometry by single molecule trapping

The dynamic choreography of biomolecular interactions underpins the processes of life, but its direct observation remains challenging. Here, we introduce confined diffusion mass photometry, enabling hour-long, mass-resolved observation of individual biomolecules, their complexes and interactions with up to sub-kDa mass precision and ms temporal resolution. Our approach represents a quantitative time-resolved single-molecule measurement modality for studying complex biomolecular mechanisms in action.

biophysics↗

bioRxiv2026

Iterative Bump-and-hole engineering creates a bioorthogonal reporter for N-acetylglucosaminyltransferase I

Asparagine-linked protein glycosylation is among the most frequent modifications of proteins trafficking through the secretory pathway. These glycans are manufactured in an assembly line process to a common precursor that is then subject to individual modifications with different levels of complexity. An important biosynthetic modulator is the incorporation of N-acetylglucosamine (GlcNAc) at distinct positions in N-linked glycan biosynthesis, commencing with the activity of the glycosyltransferase MGAT1. While mapping of N-glycans to their corresponding protein attachment sites is generally possible, not much is known about the glycoprotein substrate choice for MGAT1 and related transferases. Analogs of GlcNAc with small bioorthogonal tags can be incorporated into N-glycans. However, due to the promiscuity of some GlcNAc transferases, incorporation is of little specificity towards individual positions. Here, we report an iterative bump-and-hole approach in the design of a bioorthogonal precision tool for the activity of MGAT1 in mammalian cells. Structure-informed protein engineering abrogated the activity of MGAT1 towards the nucleotide-sugar UDP-GlcNAc while retaining activity towards bumped, azide-modified analogs. Kinetic and computational analyses using a neural network approach informed the synthesis of a tailored UDP-GlcNAc analog with preferential acceptance by the engineered enzyme. Following substrate biosynthesis, the strategy allowed selective incorporation of a chemical tag on MGAT1 substrate proteins in living mammalian cells with little background incorporation by other GlcNAc transferases. Our work expands the toolbox for glycan-based reporter compounds.

biochemistry↗

bioRxiv2025

Structural basis for synergistic antibody protection against the essential malaria invasion complex protein RIPR

Plasmodium falciparum RH5-interacting protein (RIPR) is central to the essential PTRAMP-CSS-RIPR-CyRPA-RH5 (PCRCR)-complex; a leading target of blood-stage malaria vaccines. However, mechanisms whereby anti-RIPR antibodies inhibit parasite invasion are poorly understood. Here, we characterise 83 human IgG mAbs from RIPR-vaccinated Kymouse platform mice. Single mAbs have minimal neutralising activity, however, high-level synergistic inhibition is observed with pools of mAbs targeting the RIPR-Tail region. Structural characterisation and molecular dynamics simulations of RIPR-Tail show that mAbs targeting EGF-like domains 6-8 (RIPREGF (6-8)), but not EGF-like domains 9-10 or the C-terminal domain (RIPREGF (9-10)-CTD), synergise to constrain the RIPR-Tail conformation. The same antibodies dissociate PTRAMP-CSS from RIPR, thereby enabling anti-RIPREGF (9-10)-CTD mAbs or anti-CSS sdAbs to bind and potentiate anti-RIPREGF (6-8) IgG. Addition of these mAbs to IgG from humans immunised with the R78C (RIPREGF (7-8)-CyRPA) candidate vaccine enhances malaria growth inhibition. These data provide a framework to guide next-generation blood-stage malaria vaccine design.

immunology↗

bioRxiv2024

Structural and Functional Glycosylation of the Abdala COVID-19 Vaccine

Abdala is a COVID-19 vaccine produced in Pichia pastoris and is based on the receptor-binding domain (RBD) of the SARS-CoV-2 spike. Abdala is currently approved for use in multiple countries with clinical trials confirming its safety and efficacy in preventing severe illness and death. Although P. pastoris is used as an expression system for protein-based vaccines, yeast glycosylation remains largely uncharacterised across immunogens. Here, we characterise N-glycan structures and their site of attachment on Abdala and show how yeast-specific glycosylation decreases binding to the ACE2 receptor and a receptor-binding motif (RBM) targeting antibody compared to the equivalent mammalian-derived RBD. Reduced receptor and antibody binding is attributed to changes in conformational dynamics resulting from N-glycosylation. These data highlight the critical importance of glycosylation in vaccine design and demonstrate how individual glycans can influence host interactions and immune recognition via protein structural dynamics.

biochemistry↗

bioRxiv2023

Haptenic adducts of beta-lactam antibiotics elicit antibody responses with narrow clonality and specificity

Many classes of small-molecule drugs form protein adducts in vivo, which may elicit antibodies via a classical hapten-carrier-type response, with implications for both allergy and drug sequestration. Although {beta}-lactam antibiotics are a drug class long associated with these phenomena, the molecular determinants of drug-protein conjugation and consequent drug-specific immune responses remain incomplete. Here, we interrogated factors influencing penicilloyl adduct formation and immunogenicity, and used penicillin G (PenG) to probe the B and T cell determinants of drug-specific IgG responses in mice. We identify through deep clonotyping a dominant murine penicilloyl-specific clonal antibody class encompassing phylogenetically related IGHV1, IGHV5 and IGHV10 subgroup gene segments. Through protein NMR and x-ray structural analysis, we determined that adduct specific antibody clones--the MIL series--predominantly recognise the variable side-chain moiety (which for PenG is phenylacetamide) via a hydrophobic pocket, while secondary H-bond contacts with both thiazolidine and the adducted lysine residue is made. As a result, the cross-reactivity against other {beta}-lactam antibiotics is limited. These data demonstrate the relationship between the chemistry of protein-reactive drugs such as penicilloyls, and how their predisposition to generating B cell responses can inform the functional implications at the clonal level. Highlights- PenG readily forms immunogenic adducts on lysine sidechains of diverse self- and non-self proteins including complete serum under physiological conditions. - PenG-protein adduction in vitro or in vivo is sufficient to elicit penicillin-specific IgG responses. - Murine B cell clonotypic responses are characterised by near-uniform antibody binding modes of similar immunogenetic origin. - The dominant murine PenG-specific clonotype is dominated by benzene ring recognition and correlates with serological cross-reactivity profiles.

immunology↗

bioRxiv2023

Cooperativity and induced oligomerisation control the interaction of SARS- CoV-2 with its cellular receptor and patient-derived antibodies

Viral entry is mediated by oligomeric proteins on the virus and cell surfaces. The association is therefore open to multivalent interactions between these proteins, yet such recognition is typically rationalised as affinity between monomeric equivalents. As a result, assessment of the thermodynamic mechanisms that control viral entry has been limited. Here, we use mass photometry to overcome the analytical challenges consequent to multivalency. Examining the interaction between the spike protein of SARS-CoV-2 and the ACE2 receptor, we find that ACE2 induces oligomerisation of spike in a variant-dependent fashion. We also demonstrate that patient-derived antibodies use induced-oligomerisation as a primary inhibition mechanism or to enhance the effects of receptor-site blocking. Our results reveal that naive affinity measurements are poor predictors of potency, and introduce a novel antibody-based inhibition mechanism for oligomeric targets. One-Sentence SummaryMultivalent interactions between viral proteins, cell-surface receptors, and anti-viral antibodies regulate infection and inhibition.

biophysics↗