Biology subjects

Beveridge, R.

Publications and source records attributed to Beveridge, R..

14 recordsLinked to original sources

bioRxiv2026

Mass spectrometric detection of protein nanoclusters: the invisible intermediates of phase separation

Phase separation of biomolecules, including intrinsically disordered proteins, is an underlying physical process behind cellular condensate formation. To increase mechanistic understanding of its initial stages, we demonstrate the application of charge detection mass spectrometry to characterise the mass dispersion of protein nanoclusters that represent intermediates in this important biophysical process. We detect polydisperse clusters of 1-280 protein molecules (64 kDa-18.4 MDa), that are currently invisible to most analytical techniques.

biophysics↗

bioRxiv2026

Plate-based ISD-SPE enables dual proteome-secretome concentration-response profiling of TLR signalling in iPSC-derived macrophages

Protein secretion represents a key functional output of cellular signalling, capturing dynamic responses to stimulation and pharmacological perturbation that shape immune behaviour. In macrophages, activation of Toll-like receptors (TLRs) drives tightly regulated secretion programmes that mediate inflammatory responses and provide a biologically meaningful readout of pathway activity. Whilst mass spectrometry (MS)-based secretomics enables unbiased profiling of these processes, broader application in drug discovery remains constrained by sample preparation workflows that limit scalability. Here, we describe a plate-based in-solution digestion and solid-phase extraction (ISD-SPE) workflow that enables 96-well processing of conditioned media for integrated proteome and secretome analysis from the same sample well. Benchmarking against a precipitation-based approach demonstrated comparable proteomic depth with improved quantitative reproducibility and robust performance across multiple plates. Coupled with dia-PASEF acquisition, this workflow enabled in-depth profiling of macrophage responses to TLR activation, resolving receptor-specific secretory programmes following TLR3, TLR4 and TLR7/8 activation. Extension of the approach to concentration-response studies enabled quantitative characterisation of pharmacological perturbation across intracellular and extracellular protein landscapes, revealing both shared and compartment-specific responses to TLR inhibition, as well as differences in apparent potency linked to secretion dynamics. Together, this workflow provides a scalable strategy for integrated analysis of intracellular signalling and downstream protein secretion, enabling systems-level characterisation of inflammatory responses and compound mechanisms of action.

immunology↗

bioRxiv2026

A spatial atlas of the seaweed CO2-fixation machinery reveals a unique Rubisco condensation mechanism

Seaweeds (macroalgae) are important primary producers that sustain food webs in coastal ecosystems. Most algae accelerate inorganic carbon assimilation by actively concentrating CO2 in a Rubisco-rich specialized organelle called the pyrenoid. However, the molecular composition of this pathway is unknown in seaweeds. Here, we investigated the intracellular localization of 160 proteins associated with CO2 acquisition in the green seaweed Ulva (Sea lettuce). We assign 68 proteins to different pyrenoid subdomains and identify a consensus Ulva Rubisco binding motif revealing the molecular logic of the Ulva pyrenoid. We reveal Seaweed Ulva Pyrenoid Assembly 1 (SUPA1) as the core pyrenoid assembly factor. We show that Rubisco condensation is driven by a unique mechanism: the helical folding of SUPA1 motifs upon Rubisco binding, combined with steric hindrance that halves the available Rubisco binding sites from eight to four. Our data gives an unprecedented sub-cellular spatial understanding on seaweed carbon fixation and provides insights into the evolution of this important pathway in the global carbon cycle.

cell biology↗

bioRxiv2026

dia-PASEF Enables Rapid Profiling of the Human Secretome for Deeper Insights into Cellular Dynamics and Inflammatory Mechanisms

Protein secretion is a fundamental mechanism for cellular coordination and signalling, with its dysregulation leading to widespread physiological dysfunction and disease. Immunoassay formats that utilise secondary antibody readouts are the current gold standard for measuring secreted proteins, offering high specificity and sensitivity, but relying on predefined protein panels that constrain the discovery of novel biology. We present a scalable mass spectrometry-based workflow that combines data-independent acquisition with ion mobility and parallel fragmentation to deliver rapid, global profiling of the secretome. Using a translationally relevant human iPSC-derived macrophage model, our approach identified over 1200 proteins in under 15 minutes of acquisition time, delivering exceptional reproducibility across a large sample set. We applied this approach to profile pro-inflammatory phenotypes, confirming robust identification of key cytokines and chemokines whilst revealing non-canonical immune responses absent from both targeted panels and the intracellular proteome. In particular, we identified a unique cholesterol efflux signature, marked by the secretion of APOA1 and PON1, in response to Mycobacterium Tuberculosis, consistent with the metabolic reprogramming that takes place during infection. Furthermore, temporal profiling of macrophage responses to lipopolysaccharide over 24 hours resolved dynamic secretion trajectories that distinguish between acute and chronic inflammatory states. The extended time period facilitated the observation of distinct cytokine-dependent secretion phenotypes, with early secretion of TNF and IL6 initiating downstream signalling cascades that resulted in the delayed secretion of chemokines such as CXCL10 and CCL8. Collectively, these findings establish a robust, scalable platform for global characterisation of secretory networks. Beyond macrophage biology, this workflow offers broad utility for biomarker discovery, mechanistic studies of disease progression and evaluation of new therapeutic interventions, providing a powerful tool for advancing precision medicine.

immunology↗

bioRxiv2023

Native Mass Spectrometry of Complexes Formed by Molecular Glues Revealed Stoichiometric Rearrangement of the DCAF15 E3 Ligase

In this application of native mass spectrometry (nMS) to investigate complexes formed by molecular glues (MGs), we have demonstrated its efficiency in delineating stoichiometric rearrangements of E3 ligases that occur during targeted protein degradation (TPD). MGs stabilise interactions between an E3 ligase and a protein of interest (POI) targeted for degradation, and these ternary interactions are challenging to characterise. We have shown that nMS can unambiguously identify complexes formed between the CRBN:DDB1 E3 ligase and the POI GSPT1 upon the addition of lenalidomide, pomalidomide or thalidomide. Ternary complex formation was also identified involving the DCAF15:DDA1:DDB1 E3 ligase in the presence of MG (E7820 or indisulam) and POI RBM39. Moreover, we uncovered that the DCAF15:DDA1:DDB1 E3 ligase self-associates into dimers and trimers when analysed alone at low salt concentrations (100 mM ammonium acetate) which dissociate into single copies of the complex at higher salt concentrations (500 mM ammonium acetate), or upon the addition of MG and POI, forming a 1:1:1 ternary complex. This work demonstrates the strength of nMS in TPD research, reveals novel binding mechanisms of the DCAF15 E3 ligase, and highlights the potential effect of salt concentrations on protein complexes during structural analysis.

biochemistry↗

bioRxiv2022

Ion mobility mass spectrometry unveils global protein conformations in response to conditions that promote and reverse liquid-liquid phase separation

Liquid-liquid phase separation (LLPS) is a process by which biomacromolecules, particularly proteins, condense into a dense phase that resembles liquid droplets. Dysregulation of LLPS is implicated in disease, yet the relationship between protein conformational changes and LLPS remain difficult to discern. This is due to the high flexibility and disordered nature of many proteins that phase separate under physiological conditions, and their tendency to oligomerise. Here we demonstrate that ion mobility mass spectrometry (IM-MS) overcomes these limitations. We used IM-MS to investigate the conformational states of full-length ubiquilin-2 (UBQLN2) protein, LLPS of which is driven by high salt concentration and reversed by noncovalent interactions with ubiquitin (Ub). IM-MS revealed that UBQLN2 exists as a mixture of monomers and dimers, and that increasing salt concentration causes the UBQLN2 dimers to undergo a subtle shift towards extended conformations. UBQLN2 binds to Ub in 2:1 and 2:2 UBQLN2:Ub complexes which have compact geometries compared to free UBQLN2 dimers. Together, these results suggest that extended conformations of UBQLN2 are correlated with UBQLN2s ability to phase separate. Overall, delineating protein conformations that are implicit in LLPS will greatly increase understanding of the phase separation process, both in normal cell physiology and disease states.

biophysics↗

bioRxiv2022

Shuffled ATG8 interacting motifs form an ancestral bridge between UFMylation and C53-mediated autophagy

UFMylation mediates the covalent modification of substrate proteins with UFM1 (Ubiquitin-fold modifier 1) and regulates the selective degradation of endoplasmic reticulum (ER) via autophagy (ER-phagy) to maintain ER homeostasis. Specifically, collisions of the ER-bound ribosomes trigger ribosome UFMylation, which in turn activates C53-mediated autophagy that clears the toxic incomplete polypeptides. C53 has evolved non-canonical shuffled ATG8 interacting motifs (sAIMs) that are essential for ATG8 interaction and autophagy initiation. Why these non-canonical motifs were selected during evolution, instead of canonical ATG8 interacting motifs remains unknown. Here, using a phylogenomics approach, we show that UFMylation is conserved across the eukaryotes and secondarily lost in fungi and some other species. Further biochemical assays have confirmed those results and showed that the unicellular algae, Chlamydomonas reinhardtii has a functional UFMylation machinery, overturning the assumption that this process is linked to multicellularity. Our conservation analysis also revealed that UFM1 co-evolves with the sAIMs in C53, reflecting a functional link between UFM1 and the sAIMs. Using biochemical and structural approaches, we confirmed the interaction of UFM1 with the C53 sAIMs and found that UFM1 and ATG8 bound to the sAIMs in a different mode. Conversion of sAIMs into canonical AIMs prevented binding of UFM1 to C53, while strengthening ATG8 interaction. This led to the autoactivation of the C53 pathway and sensitized Arabidopsis thaliana to ER stress. Altogether, our findings reveal an ancestral toggle switch embodied in the sAIMs that regulates C53-mediated autophagy to maintain ER homeostasis.

plant biology↗

bioRxiv2022

SARS-CoV-2 mutations affect proteasome processing to alter CD8+ T cell responses

Viral CD8+ epitopes are generated by the cellular turnover of viral proteins, predominantly by the proteasome. Mutations located within viral epitopes can result in escape from memory T cells but the contribution of mutations in flanking regions of epitopes in SARS-CoV-2 has not been investigated. Focusing on two of the most dominant SARS-CoV-2 nucleoprotein CD8+ epitopes, we identified mutations in epitope flanking regions and investigated the contribution of these mutations to antigen processing and T cell activation using SARS-CoV-2 nucleoprotein transduced B cell lines and in vitro proteasomal processing of peptides. We found that decreased NP9-17-B*27:05 CD8+ T cell responses to the NP-Q7K mutation correlated with lower epitope surface expression, likely due to a lack of efficient epitope production by the proteasome, suggesting immune escape caused by this mutation. In contrast, NP-P6L and NP-D103N/Y mutations flanking the NP9-17-B*27:05 and NP105-113-B*07:02 epitopes, respectively, increased CD8+ T cell responses associated with enhanced epitope production by the proteasome. Our results provide evidence that SARS-CoV-2 mutations outside the epitope could have a significant impact on antigen processing and presentation, thereby contributing to escape from immunodominant T cell responses. Alternatively, mutations could enhance antigen processing and efficacy of T cell recognition, opening new avenues for improving future vaccine designs. One Sentence SummaryNatural mutations in the flanking regions of known immunodominant SARS-CoV-2 nucleoprotein epitopes can decrease CD8+ T cell responses leading to partial escape.

immunology↗

bioRxiv2022

Identification and Age-dependent Increase of Platelet Biased Human Hematopoietic Stem Cells

Hematopoietic stem cells (HSC) reconstitute multi-lineage human hematopoiesis after clinical bone marrow transplantation and are the cells-of-origin of hematological malignancies. Though HSC provide multi-lineage engraftment, individual murine HSCs are lineage-biased and contribute unequally to blood cell lineages. Now, by combining xenografting of molecularly barcoded adult human bone marrow (BM) HSCs and high-throughput single cell RNA sequencing we demonstrate that human individual BM HSCs are also functionally and transcriptionally lineage biased. Specifically, we identify platelet-biased and multi-lineage human HSCs. Quantitative comparison of transcriptomes from single HSCs from young, and aged, BM show that both the proportion of platelet-biased HSCs, and their level of transcriptional platelet priming, increases with age. Therefore, platelet-biased HSCs, as well as their increased prevalence and elevated transcriptional platelet priming during ageing, are conserved between human and murine hematopoiesis. One-Sentence SummaryIn vivo barcoding and single cell RNA sequencing identifies platelet-biased human bone marrow HSCs.

cell biology↗

bioRxiv2020

Phenotypic analysis of an MLL-AF4 gene regulatory network reveals indirect CASP9 repression as a mode of inducing apoptosis resistance.

Regulatory interactions mediated by transcription factors (TFs) make up complex networks that control cellular behavior. Fully understanding these gene regulatory networks (GRNs) offers greater insight into the consequences of disease-causing perturbations than studying single TF binding events in isolation. Chromosomal translocations of the Mixed Lineage Leukemia gene (MLL) produce MLL fusion proteins such as MLL-AF4, causing poor prognosis acute lymphoblastic leukemias (ALLs). MLL-AF4 is thought to drive leukemogenesis by directly binding to genes and inducing aberrant overexpression of key gene targets, including anti-apoptotic factors such as BCL-2. However, this model minimizes the potential for circuit generated regulatory outputs, including gene repression. To better understand the MLL-AF4 driven regulatory landscape, we integrated ChIP-seq, patient RNA-seq and CRISPR essentiality screens to generate a model GRN. This GRN identified several key transcription factors, including RUNX1, that regulate target genes using feed-forward loop and cascade motifs. We used CRISPR screening in the presence of the BCL-2 inhibitor venetoclax to identify functional impacts on apoptosis. This identified an MLL-AF4:RUNX1 cascade that represses CASP9, perturbation of which disrupts venetoclax induced apoptosis. This illustrates how our GRN can be used to better understand potential mechanisms of drug resistance acquisition. Graphical abstract caption O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/179796v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@cca8f2org.highwire.dtl.DTLVardef@67b289org.highwire.dtl.DTLVardef@1fa44aeorg.highwire.dtl.DTLVardef@1b32487_HPS_FORMAT_FIGEXP M_FIG A network model of the MLL-AF4 regulatory landscape identifies feed-forward loop and cascade motifs. Functional screening using CRISPR and venetoclax identified an MLL-AF4:RUNX1:CASP9 repressive cascade that impairs drug-induced cell death. C_FIG

genomics↗

bioRxiv2020

A cross-kingdom conserved ER-phagy receptor maintains endoplasmic reticulum homeostasis during stress

Eukaryotes have evolved various quality control mechanisms to promote proteostasis in the ER. Selective removal of certain ER domains via autophagy (termed as ER-phagy) has emerged as a major quality control mechanism. However, the degree to which ER-phagy is employed by other branches of ER-quality control remains largely elusive. Here, we identify a cytosolic protein, C53, that is specifically recruited to autophagosomes during ER-stress, in both plant and mammalian cells. C53 interacts with ATG8 via a distinct binding epitope, featuring a shuffled ATG8 interacting motif (sAIM). C53 senses proteotoxic stress in the ER lumen by forming a tripartite receptor complex with the ER-associated ufmylation ligase UFL1 and its membrane adaptor DDRGK1. The C53/UFL1/DDRGK1 receptor complex is activated by stalled ribosomes and induces the degradation of internal or passenger proteins in the ER. Consistently, the C53 receptor complex and ufmylation mutants are highly susceptible to ER stress. Thus, C53 forms an ancient quality control pathway that bridges selective autophagy with ribosome-associated quality control at the ER.

cell biology↗

bioRxiv2020

cGAMP loading enhances the immunogenicity of VLP vaccines

Cyclic GMP-AMP (cGAMP) is an immunostimulatory second messenger produced by cGAS that activates STING. Soluble cGAMP acts as an adjuvant when administered with antigens. cGAMP is also incorporated into enveloped virus particles during budding. We hypothesised that inclusion of the adjuvant cGAMP within viral vaccine vectors would promote adaptive immunity against vector antigens. We immunised mice with virus-like particles (VLPs) containing the HIV-1 Gag protein and VSV-G. Inclusion of cGAMP within these VLPs augmented splenic VLP-specific CD4 and CD8 T cell responses. It also increased VLP- and VSV-G-specific serum antibody titres and enhanced in vitro virus neutralisation. The superior antibody response was accompanied by increased numbers of T follicular helper cells in draining lymph nodes. Vaccination with cGAMP-loaded VLPs containing haemagglutinin induced high titres of influenza A virus neutralising antibodies and conferred protection following subsequent influenza A virus challenge. Together, these results show that incorporating cGAMP into VLPs enhances their immunogenicity, making cGAMP-VLPs an attractive platform for novel vaccination strategies. Short summarycGAMP is an innate immune signalling molecule that can be transmitted between cells by inclusion in enveloped virions. This study demonstrates enhanced immunogenicity of HIV-derived virus-like particles containing cGAMP. Viral vectors loaded with cGAMP may thus be potent vaccines.

immunology↗

bioRxiv2019

Native mass spectrometry can effectively predict PROTAC efficacy

Protein degraders, also known as proteolysis targeting chimeras (PROTACs), are bifunctional small molecules that bring an E3 ubiquitin ligase and a protein of interest (POI) into proximity, thus promoting ubiquitination and degradation of the targeted POI [1-3]. Despite their great promise as next-generation pharmaceutical drugs, the development of new PROTACs is challenged by the complexity of the system, which involves binary and ternary interactions between components. Here, we demonstrate the strength of native mass spectrometry (nMS), a label-free technique, to provide novel insight into PROTAC-mediated protein interactions. We show that nMS can monitor the formation of ternary E3-PROTAC-POI complexes and detect various intermediate species in a single experiment. A unique benefit of the method is its ability to reveal preferentially formed E3-PROTAC-POI combinations in competition experiments with multiple substrate proteins, thereby positioning it as an ideal high-throughput screening strategy during the development of new PROTACs.

biochemistry↗

bioRxiv2019

A synthetic peptide library for benchmarking crosslinking mass spectrometry search engines

We have created synthetic peptide libraries to benchmark crosslinking mass spectrometry search engines for different types of crosslinker. The unique benefit of using a library is knowing which identified crosslinks are true and which are false. Here we have used mass spectrometry data generated from measurement of the peptide libraries to evaluate the most frequently applied search algorithms in crosslinking mass-spectrometry. When filtered to an estimated false discovery rate of 5%, false crosslink identification ranged from 5.2% to 11.3% for search engines with inbuilt validation strategies for error estimation. When different external validation strategies were applied to one single search output, false crosslink identification ranged from 2.4% to a surprising 32%, despite being filtered to an estimated 5% false discovery rate. Remarkably, the use of MS-cleavable crosslinkers did not reduce the false discovery rate compared to non-cleavable crosslinkers, results from which have far-reaching implications in structural biology. We anticipate that the datasets acquired during this research will further drive optimisation and development of search engines and novel data-interpretation technologies, thereby advancing our understanding of vital biological interactions.

bioinformatics↗