Biology subjects

Hester, S.

Publications and source records attributed to Hester, S..

16 recordsLinked to original sources

bioRxiv2026

Spatial Tumour Tissue Distribution Dynamics of a Bicycle Peptide-MMAE Toxin Conjugate Profiled by Targeted Mass Spectrometry

A highly specific-NECTIN-4 binding bicyclic peptide covalently linked to the cytotoxin monomethyl auristatin E (MMAE) via a valine-citrulline cleavable linker (zelenectide pevedotin, BT8009) is a promising modality to treat cancer patients with solid tumours. Although the pharmacokinetics and biodistribution of zelenectide pevedotin have been well characterised, a more precise view of tumour delivery and intra-tumoural distribution of its released payload is lacking. To address this, we have developed an approach to profile the spatial tumour and healthy tissue distribution of this anti-cancer agent using laser capture microdissection coupled to targeted liquid chromatography parallel reaction monitoring (LCM-LC-PRM) mass spectrometry. This LCM-assisted spatial pharmacology workflow enables the precise isolation of specific tissue regions, followed by high-resolution analysis of MMAE distribution at sub-tissue-specific levels. By identifying and optimising MS/MS-specific fragmentation ions for accurate and sensitive detection and quantitation of MMAE released from zelenectide pevedotin within tumour tissues, we observed progressive accumulation and sustained retention of MMAE in mouse tumour tissues over a period of 24 h. Conversely, in plasma, liver and muscle tissues, MMAE levels generally declined following early exposure, although inter-animal variability was observed. LCM-based intra-tumour spatial profiling revealed heterogeneous MMAE distribution between tumour cell-rich and stroma-rich regions, with distinct temporal patterns across compartments. Together, our spatial targeted MS method provided valuable insights into zelenectide pevedotin's payload distribution dynamics in vivo, thereby enhancing our understanding of its therapeutic potential and guiding the development of next-generation Bicycle Drug Conjugate (BDC).

cancer biology↗

bioRxiv2026

Controlled human infection with Plasmodium falciparum-infected mosquito bites elicits antibodies against mosquito salivary protein SG1L3

Human malaria infections begin with the injection of Plasmodium sporozoites via mosquito saliva. Whole sporozoite immunizations have been used as a model to study immune responses to malaria parasites, having culminated in circumsporozoite protein (CSP)-targeting vaccines and monoclonal antibodies (mAbs). However, antibody responses targeting non-CSP antigens on the sporozoite surface remain poorly characterized. Here, we isolated single B cells from a human volunteer immunized by Plasmodium falciparum-infected mosquito bites, who had acquired non-CSP-specific antibodies that recognize sporozoites. We identified two mAbs that recognize the surface of P. falciparum sporozoites, but do not bind to CSP. Using immunoprecipitation followed by mass-spectrometry, we found that the target of these mAbs is not a P. falciparum protein but the mosquito salivary protein SG1L3. We observed that recombinant SG1L3 binds to P. falciparum sporozoites. However, the SG1L3-specific mAbs and SG1L3-specific polyclonal antibodies from this volunteer, as well as polyclonal antibodies raised against recombinant SG1L3 in rabbits, fail to block liver stage infection in vitro, making this an unlikely target for functional antibodies. We observed that inhabitants from an area with intense Anopheles exposure in Burkina Faso can have antibodies against SG1L3, and that antibody titers increase with age. In conclusion, we identified the first human mAbs against a mosquito saliva protein that binds to the surface of sporozoites. Future work should assess whether naturally acquired antibodies against this protein may be used as a serological marker of mosquito exposure.

microbiology↗

bioRxiv2026

Time resolved proteomic analysis of hepatitis B virus infection

Hepatitis B virus (HBV) infection can result in progressive inflammatory liver diseases that are associated with the development of hepatocellular carcinoma. Our understanding of the HBV infection proteome is limited, and we used label-free quantitative (LFQ) proteomics to address this gap in knowledge. Kinetic sampling of mock and HBV infected human hepatoma HepG2 cells showed a time-dependent expression of the viral core (C) and Surface (S) proteins. Concomitantly, we observed altered host cell proteome dynamics, including cytoskeletal proteins, collagens, fibrinogen subunits, extracellular matrix receptor interaction and focal adhesion pathways, as well as an elevated abundance of complement proteins. Specifically, we noted evidence for elevated C5 complement protein abundance in the HBV infected cells, highlighting selective complement adaptations during infection.

microbiology↗

bioRxiv2026

Proteomic Signatures of Mitochondrial Dysfunction Associated with Atrial Fibrillation in Goats

Atrial fibrillation (AF) increases energy demand in atrial myocytes, yet the mitochondrial mechanisms underlying this stress remain poorly defined. Using previously published proteomic data from left atrial tissue of AF and sham-operated goats, we performed organelle-specific bioinformatic analyses of the mitochondrial fraction. Over-representation and consensus pathway analyses consistently highlighted enrichment of oxidative phosphorylation (OXPHOS) subunits. Gene set enrichment and network analyses implicated Heat Shock Protein Family A Member 9 (HSPA9) as a potentially central regulatory hub coordinating the dysregulation of Complex I and III subunits, with 69% of regulatory relationships showing pathway concordance. These results indicate a coordinated, system-wide mitochondrial adaptation in AF, integrating energy production, proteostasis, and respiratory chain regulation.

molecular biology↗

bioRxiv2024

A resource for extracellular vesicles from activated CD4 T cells that relay pro-inflammatory signals

CD4 T helper cells (TH cells) play a vital role in coordinating and amplifying the immune response to specific pathogens. They constitutively produce different kinds of extracellular vesicles (EVs), which mediate cell-to cell communication and play diverse roles in immune regulation and inflammatory processes. Here we provide a resource documenting the composition of activated TH cell EVs and demonstrating their ability to instigate pro-inflammatory response in antigen-presenting cells (APCs). EVs were characterized by lipidomics, proteomics, and NanoFCM. The activated TH cells derived EVs (act-EVs) were found to be enriched in TH cell-specific proteins, transmembrane and cytosolic EV marker proteins, and HLA proteins relative to resting CD4 T cells EVs (rest-EVs). The pro-inflammatory effect of act-EVs vs rest-EVs on donor matched APCs were characterized by chemokine and cytokine profiling and flow cytometry analysis. There was no discernible contrast in endotoxin levels between act-EVs and rest-EVs. Functional distinctions were seen to arise from variations in the content and composition of these EVs. Moreover, we validated our findings with an in-vivo investigation in mice, demonstrating the recruitment of monocytes, dendritic cells (DCs), neutrophils, and NK cells in the spleen, accompanied by the release of pro-inflammatory cytokines in the serum after administering act-EVs. In summary, this study sheds light on the role of TH cell released EVs in modulating the immune response during pro-inflammatory responses and this resource provides a foundation for development of novel therapeutics on EV based scaffolds.

immunology↗

bioRxiv2024

Lysosomal signalling pathways influence heart rhythm, and regulate atrial function

In the heart, endogenous nicotinic acid adenine dinucleotide phosphate (NAADP) triggers lysosomal calcium (Ca2+) release to augment sarcoplasmic reticulum (SR) Ca2+ sequestration, producing larger Ca2+ transients. However, the role of lysosomal Ca2+ signals in pacemaker activity, a distinct Ca2+-operated function of the sinoatrial node (SAN), or in the atrial myocardium has not been investigated. Pharmacological or genetic ablation of the NAADP pathway inhibits the spontaneous beating rate response to beta-adrenergic stimulation in intact SAN. We found intracellular signaling microdomains between lysosomes and neighboring SR or mitochondria in mouse, and goat tissue. The spatial relationship between lysosomes and other Ca2+-handling organelles are altered in goat atrial fibrillation. Furthermore, we demonstrate atrial myocytes produce cAMP in response to lysosomal signaling, adding a novel trigger for cyclic nucleotide signaling. Our findings support the hypothesis that lysosomal Ca2+ signaling contributes to regulation of cardiomyocyte cAMP levels and pacemaker activity.

physiology↗

bioRxiv2024

Activation of IP3R in atrial cardiomyocytes leads to generation of cytosolic cAMP

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Excessive stimulation of the IP3 signaling pathway has been linked to AF through abnormal calcium handling. However, little is known about the mechanisms involved in this process. We expressed Fluorescence resonance energy transfer (FRET) based cytosolic cAMP sensor EPAC-SH187 in neonatal rat atrial myocytes (NRAMs) and neonatal rat ventricular myocytes (NRVMs). In NRAMs, addition of the alpha ()-1 agonist phenylephrine (PE, 3{micro}M) resulted in a bi-phasic FRET change (R1) 21.20 {+/-} 7.43% and (R2) 9.67 {+/-} 4.23% and addition of membrane permeant IP3 derivative, 2,3,6-tri-O-Butyryl-myo-IP3(1,4,5)-hexakis(acetoxymethyl)ester (IP3-AM, 20M) resulted in a peak of 20.31 {+/-} 6.74%. These FRET changes imply an increase in cAMP. Prior application of IP3 receptor (IP3R) inhibitors 2-Aminoethyl diphenylborinate (2-APB, 2.5M) or Xestospongin-C (0.3M) significantly inhibited the change in FRET in NRAMs in response to PE. Xestospongin-C (0.3M) significantly inhibited the change in FRET in NRAMs in response to IP3-AM. The FRET change in response to PE in NRVMs were not inhibited by 2-APB or Xestospongin-C. Finally, the localisation of cAMP signals was tested by expressing the FRET-based cAMP sensor, AKAP79-CUTie, which targets the intracellular surface of the plasmalemma. We found in NRAMs that PE led to FRET change corresponding to an increase in cAMP that was inhibited by 2-APB and Xestospongin C. This data support further investigation of the pro-arrhythmic nature and components of IP3 induced cAMP signalling to identify potential pharmacological targets. NEW & NOTEWORTHYThis study shows that indirect activation of the IP3 pathway in atrial myocytes using phenylephrine and direct activation using IP3-AM leads to an increase in cAMP and is in-part localized to the cell membrane. These changes can be pharmacologically inhibited using IP3R inhibitors. However, the cAMP rise in ventricular myocytes is independent of IP3R calcium release. Our data support further investigation into the pro-arrhythmic nature of IP3-induced cAMP signaling.

physiology↗

bioRxiv2023

Extracellular vesicles from patients with coronary artery disease (CAD) demonstrate enhanced pro-coagulatory activity.

AimsExtracellular vesicles (EVs) carry unique repertoires of biologically active cargo that hold promise as novel biomarkers and future interventional targets for cardiovascular diseases (CVDs). However, it is unclear as to how the number, location, cellular origin, and size of these EVs within the circulation relate to the development of CVDs such as coronary artery disease (CAD). The current study compared these novel markers in arterial and venous blood samples in individuals undergoing coronary assessment with angiography.. EVs were then characterized from those presenting with and without CAD. Methods and ResultsArterial and venous blood samples were collected from individuals with confirmed CAD following coronary angiography and a matched cohort in whom there was no evidence of disease. EV fractions were isolated from 500{micro}L of platelet free plasma (PFP) by size exclusion chromatography. EVs were analyzed by Nanoparticle Tracking Analysis and flow cytometry to characterize number, size and cellular origin. A thrombin generation assay was used to assess the pro-coagulatory activity of the isolated circulating EVs. Proteomics was used to determine the protein cargo carried within the EVs. Coagulatory activity of EVs isolated from CAD patients was significantly higher in when compared to controls, although the numbers of EVs did not differ. There were higher numbers of endothelial-derived EVs in arterial blood compared with venous blood. Linear regression models revealed that plasma triacylglycerol concentration and age independently predicted circulating EV numbers in CAD patients. Proteomics revealed several proteins associated with coagulation upregulated in patients with CAD. ConclusionsAlthough the absolute numbers of EVs in CAD patients were not elevated, EVs in CAD patients had greater pro-coagulant activity, highlighting a potentially important role for EVs in the pathogenesis of CAD. Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABSO_LICAD patients displayed a greater EV-induced thrombin generation capability when compated to those with no disease. C_LIO_LIThe proteomic profile of arterial-derived EVs in patients with CAD demonstrated several proteins associated with coagulation that may be a risk factor for developing a future event. C_LI What are the clinical implications?O_LIThe identification of EVs in patients with CAD with upregulated coagulation proteins may be a novel biomarker for the development of future events and may identify a high risk patient group that may benefit for more aggressive cardiovascular risk intervention. C_LI

physiology↗

bioRxiv2023

Loss of FAM111B protease mutated in hereditary fibrosing poikiloderma syndrome negatively regulates telomere length

Hereditary fibrosing poikiloderma (HFP) is a rare human dominant negative disorder caused by mutations in the FAM111B gene that encodes a nuclear trypsin-like serine protease. HFP patients present with symptoms including skin abnormalities, tendon contractures, myopathy and lung fibrosis. We characterised the cellular roles of human FAM111B using U2OS and MCF7 cell lines and report here that the protease interacts with components of the nuclear pore complex. Loss of FAM111B expression resulted in abnormal nuclear shape and reduced telomeric DNA content suggesting that FAM111B protease is required for normal telomere length; we show that this function is independent of telomerase or recombination driven telomere extension. Even though FAM111B-deficient cells were proficient in DNA repair, they showed hallmarks of genomic instability such as increased levels of micronuclei and ultra-fine DNA bridges. Interestingly, FAM111B variants, including mutations that cause HFP, showed more frequent localisation to the nuclear lamina suggesting that accumulation of mutant FAM111B at the nuclear periphery may drive the disease pathology.

cell biology↗

bioRxiv2022

MR1 dependent MAIT cell activation is regulated by autophagy associated proteins

The antigen presenting molecule, MR1, presents microbial metabolites to MAIT cells, a population of innate-like, anti-microbial T cells. It also presents an unidentified ligand to MR-1 restricted T cells in the setting of cancer. The cellular co-factors that regulate MR1 antigen presentation have yet to be fully defined. We performed a mass spectrometry-based proteomics screen to identify MR1 interacting proteins and discovered the selective autophagy receptor SQSTM1/p62. CRISPR-Cas9-mediated knock out of SQSTM1/p62 increased MAIT cell activation in the presence of E.coli but not the synthetic ligand 5-OP-RU whereas depletion of Atg5 and Atg7, key autophagy proteins, increased MAIT activation irrespective of the ligand used. This regulation appears to occur at an early step in the MR1 trafficking pathway. This data implicates distinct roles for autophagy associated proteins in the regulation of MR1 activity and highlights the contribution of the autophagy pathway to regulation of cellular antigen presentation.

cell biology↗

bioRxiv2021

Interplay Between KRAS and LZTR1 Protein Turnover, Controlled by CUL3/LZTR1 E3 Ubiquitin Ligase, is Disrupted by KRAS Mutations

KRAS is a proto-oncogene encoding a small GTPase. Mutations contribute up to 30% of human solid tumours including lung adenocarcinoma, pancreatic and colorectal carcinomas. Most KRAS activating mutations interfere with GTP hydrolysis, essential for its role as a molecular switch, leading to alterations in their molecular environment and oncogenic signalling. Here, APEX-2 proximity labelling was used to profile the molecular environment of wild type and G12D, G13D and Q61H activating mutants of KRAS under both, starvation and stimulation conditions. We demonstrate by quantitative proteomics the presence of known interactors of KRAS including a-RAF and LZTR1, which varied in abundance with wildtype and KRAS mutants. Notably, the KRAS mutations G12D, G13D and Q61H abrogate association with LZTR1. Wildtype KRAS and LZTR1, as part of the CUL3 ubiquitin E3 ligase complex, affect each others protein stability, revealing a direct feedback loop mechanism. KRAS mutations disconnect this regulatory circuit, thereby contributing to oncogenesis.

molecular biology↗

bioRxiv2021

CDK9 and PP2A regulate the link between RNA polymerase II transcription termination and RNA maturation.

AbstractCDK9 is a kinase critical for the productive transcription of protein-coding genes by RNA polymerase II (pol II). As part of P-TEFb, CDK9 phosphorylates the carboxyl-terminal domain (CTD) of pol II and elongation factors, which allows pol II to elongate past the early elongation checkpoint (EEC) encountered soon after initiation. We show that, in addition to halting pol II at the EEC, loss of CDK9 activity causes premature termination of transcription across the last exon, loss of polyadenylation factors from chromatin, and loss of polyadenylation of nascent transcripts. Inhibition of the phosphatase PP2A abrogates the premature termination and loss of polyadenylation caused by CDK9 inhibition, indicating that this kinase/phosphatase pair regulates transcription elongation and RNA processing at the end of protein-coding genes. We also confirm the splicing factor SF3B1 as a target of CDK9 and show that SF3B1 in complex with polyadenylation factors is lost from chromatin after CDK9 inhibition. These results emphasize the important roles that CDK9 plays in coupling transcription elongation and termination to RNA maturation downstream of the EEC.

molecular biology↗

bioRxiv2021

Synthetic Antigen Presenting cells reveal the diversity and functional specialization of extracellular vesicles composing the fourth signal of T cell immunological synapses.

The T cell Immunological Synapse (IS) is a pivotal hub for the regulation of adaptive immunity by endowing the exchange of information between cells engaged in physical contacts. Beyond the integration of antigen (signal one), co-stimulation (signal two), and cytokines (signal three), the IS facilitates the delivery of T-cell effector assemblies including supramolecular attack particles (SMAPs) and extracellular vesicles (EVs). How these particulate outputs differ among T -cell subsets and how subcellular compartments and signals exchanged at the synapse contribute to their composition is not fully understood. Here we harnessed bead-supported lipid bilayers (BSLBs) as a tailorable and versatile technology for the study of synaptic particle biogenesis and composition in different T-cell subsets, including CART. These synthetic antigen-presenting cells (APCs) facilitated the characterisation of trans-synaptic vesicles (tSV) as a heterogeneous population of EVs comprising among others PM-derived synaptic ectosomes and CD63+ exosomes. We harnessed BSLB to unveil the factors influencing the vesicular release of CD40L, as a model effector, identifying CD40 trans presentation, T-cell activation, ESCRT upregulation/recruitment, antigen density/potency, co-repression by PD-1 ligands, and its processing by ADAM10 as major determinants. Further, BSLB made possible the comparison of microRNA (miR) species associated with tSV and steadily released EVs. Altogether, our data provide evidence for a higher specialisation of tSV which are enriched not only in effector immune receptors but also in miR and RNA-binding proteins. Considering the molecular uniqueness and functional complexity of the tSV output, which is also accompanied by SMAPs, we propose their classification as signal four. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/445691v3_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@100f3aorg.highwire.dtl.DTLVardef@57adfforg.highwire.dtl.DTLVardef@6052fcorg.highwire.dtl.DTLVardef@1e85c21_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIBead Supported Lipid Bilayers (BSLB) reconstituting antigen-presenting cells support synapse assembly by T cells and the release of effector particles. C_LIO_LIBSLB facilitate the dissection of the cellular machineries and synapse composition shaping the released tSV. C_LIO_LItSV and their steadily released counterparts have a different composition. TSV show a higher enrichment of effectors including immune receptors, miR, RNA- and other nucleic acid-binding proteins, than EVs. C_LI

immunology↗

bioRxiv2019

System-wide analyses of the fission yeast poly(A)+ RNA interactome reveal insights into organisation and function of RNA-protein complexes

Production, function, and turnover of mRNA are orchestrated by multi-subunit machineries that play a central role in gene expression. Within these molecular machines, interactions with the target mRNA are mediated by RNA-binding proteins (RBPs), and the accuracy and dynamics of these RNA-protein interactions are essential for their function. Here, we show that fission yeast whole cell poly(A)+ RNA-protein crosslinking data provides system-wide information on the organisation and function of the RNA-protein complexes. We evaluate relative enrichment of cellular RBPs on poly(A)+ RNA to identify interactors with high RNA-binding activity and provide key information about the RNA-binding properties of large multi-protein complexes, such as the mRNA 3 end processing machinery (cleavage and polyadenylation factor, CPF) and the RNA exosome. We demonstrate that different functional modules within CPF differ in their ability to interact with RNA. Importantly, we reveal that CPF forms additional contacts with RNA via the Fip1 subunit of the polyadenylation module and two subunits of the nuclease module. In addition, our data highlights the central role of the RNA helicase Mtl1 in RNA degradation by the exosome as mutations in Mtl1 lead to disengagement of the exosome from RNA. We examine how routes of substrate access to the complex are affected upon mutation of exosome subunits. Our results provide important insights into how different components of the exosome contribute to engagement of the complex with substrate RNA. Overall, our data uncover how multi-subunit cellular machineries interact with RNA, on a proteome-wide scale.

molecular biology↗

bioRxiv2019

Lysine acetylation promotes PALB2 chromatin association to maintain genome stability

The tumour suppressor PALB2 stimulates error-free repair of DNA breaks, whilst its steady-state chromatin association protects active genes from genotoxic stress. Here, we report that the lysine acetyltransferases 2A and 2B (KAT2A/B), commonly known to promote transcriptional activation, acetylate the PALB2 chromatin association motif (ChAM), providing a dynamic regulatory mechanism for PALB2. ChAM acetylation within a cluster of seven lysine residues (7K), detected in the chromatin-enriched fraction in undamaged cells, enhanced its association with nucleosomes while decreasing its non-specific binding to naked DNA. DNA damage triggered a rapid deacetylation of ChAM and a concomitant increase in PALB2 mobility. Significantly, a 7K-null mutation, which hindered ChAM binding to both nucleosomes and DNA, conferred deficiency in DNA repair and hypersensitivity to the anti-cancer drug olaparib. Thus, our study reveals a unique mechanism mediated by KAT2A/B-dependent acetylation of a non-histone protein, which fine-tunes the DNA damage response and hence promotes genome stability.

cell biology↗

bioRxiv2018

Understanding RNP remodelling uncovers RBPs functionally required for viral replication

The compendium of RNA-binding proteins (RBPome) has been greatly expanded by the development of RNA-interactome capture (RNA-IC). However, it remains unknown how responsive is the RBPome and whether these responses are biologically relevant. To answer these questions, we created comparative RNA-IC to analyse cells challenged with an RNA virus, called sindbis (SINV). Strikingly, the virus altered the activity of 245 RBPs, many of which were newly discovered by RNA-IC. Mechanistically, alterations in RNA binding upon SINV infection are caused by changes in the subcellular localisation of RBPs and RNA availability. Moreover, RBPome responses are crucial, as perturbation of dynamic RBPs modulates the capacity of the virus to infect the cell. For example, ablation of XRN1 causes cells to be refractory to infection, while GEMIN5 moonlights as a novel antiviral factor. Therefore, RBPome remodelling provides a mechanism by which cells can extensively rewire gene expression in response to physiological cues.\n\nHIGHLIGHTSO_LIA quarter of the RBPome remodels upon SINV infection.\nC_LIO_LIThe remodelling is caused by changes in protein localisation and RNA availability.\nC_LIO_LIRewiring of the RBPome is crucial for viral infection efficacy.\nC_LIO_LIWe discover RBPs with previously unknown anti- or pro-viral activity.\nC_LI

molecular biology↗