Biology subjects

Fletcher, S.

Publications and source records attributed to Fletcher, S..

11 recordsLinked to original sources

bioRxiv2026

De Novo Design and Computational Validation of a High-Affinity Peptide Inhibitor Targeting the HPV E1-E2 Interface

The oncogenic progression of high-risk Human Papillomavirus (HPV) strains relies on the cooperative interaction between the E1 replicative helicase and the E2 origin-binding protein to initiate viral DNA amplification. Disrupting this protein-protein interaction represents a promising, yet clinically unrealized, therapeutic paradigm for treating established HPV infections prior to malignant transformation. This study presents a comprehensive computational pipeline for the de novo design and evaluation of peptide inhibitors targeting the HPV E1-E2 interface, specifically a conserved arginine triad on the solvent-exposed surface of the E1 helicase. AlphaProteo was used for sequence discovery, and AlphaFold 3 for complex structural prediction, generating a candidate library that was subsequently subjected to dual-scale Molecular Dynamics (MD) simulations and MM/GBSA thermodynamic validation using GROMACS. Binder 8 emerged as the lead candidate, yielding a predicted binding free energy of -59.1 {+/-} 0.7 kcal/mol -- a statistically significant improvement over the native E1-E2 baseline (Welchs t-test, p = 8.14e-19; Cohens d = 2.21). As an implicit solvent method, MM/GBSA overestimates absolute affinities; reported values reflect effective binding enthalpy and should be interpreted as relative rankings. Per-residue energy decomposition confirms binding is anchored through multi-point interactions with the arginine triad. Physicochemical profiling via CSM-Toxin and AlgPred 2.0 confirms zero predicted toxicity and non-allergenic properties for Binder 8. Sequence alignment across 183 oncogenic Alpha-papillomavirus genotypes demonstrates near-universal conservation of the targeted triad, supporting Binder 8 as a candidate scaffold for broad-spectrum antiviral development. These findings provide a computationally validated blueprint for future in vitro validation via Bio-layer interferometry.

bioinformatics↗

bioRxiv2026

Molecular mechanics of smooth muscle contraction and relaxation modulated by caldesmon

Smooth muscle (SM) contraction is well known to be regulated by the reversible phosphorylation of the myosin regulatory light chain. However, SM force generation and relaxation are often uncoupled from myosin phosphorylation levels (e.g. the latch-state), indicating that additional regulatory mechanisms must be at play. The precise effects of the actin binding protein caldesmon (CaD) on SM force production and relaxation remain ambiguous, largely due to contradictory findings in experiments performed at the tissue level. To date, there are no studies that have measured the effects of CaD on force and relaxation at the molecular level. Here, we use a laser-trap assay to measure the force produced by SM myosin molecules in the presence and absence of CaD. Measurements were performed before and during myosin dephosphorylation, thus simulating SM contraction and relaxation in-vitro. We demonstrate that CaD inhibits force generation, most likely through competitive inhibition of actomyosin binding while simultaneously introducing a resistive load via tethering of actin and myosin. We also establish CaD as a potentiator of relaxation, increasing force decay rate during myosin dephosphorylation. Finally, we show that CaD directly modulates the dependence of myosin-actin mechanics on myosin phosphorylation levels. These findings refine our understanding of SM regulation, highlighting CaD not merely as a passive structural stabilizer, but as a critical regulatory component of force development and relaxation. Ultimately, understanding these mechanical functions offers new perspectives on pathophysiologies involving SM, such as asthma, hypertension, and gastrointestinal disorders, potentially guiding targeted therapeutic strategies. SIGNIFICANCE STATEMENTSmooth muscle (SM) is responsible for controlling the internal diameter of blood vessels and viscera. Understanding the precise regulation of SM relaxation by actin-binding proteins remains a fundamental lacuna in physiology. Using a molecular mechanics chamber to manipulate the biochemical milieu during active measurements, we demonstrate, for the first time at the molecular level, that caldesmon (CaD) acts as a mechanical modulator that inhibits force generation and accelerates relaxation of SM myosin ensembles. Our results provide a molecular basis for resolving previous contradictory findings reported in tissue-level experiments. Ultimately, understanding the role of contractile and regulatory proteins of SM will provide the basis for understanding SM disorders, such as hypertension and asthma, and guide the development of targeted therapeutic strategies.

biophysics↗

bioRxiv2025

Inactivation of a protein hydroxylase complex impairs replication fork restart in cancer and neurodevelopmental disorders

Protein hydroxylation is a post-translational modification that is commonly catalysed by enzymes of the oxygen- and 2-oxoglutarate (2OG)-dependent oxygenase family. The Jumonji-C (JmjC)-only sub-family of 2OG-oxygenases catalyse the hydroxylation of protein and tRNA substrates involved in fundamental cellular processes. Jumonji-domain 5 (JMJD5) is a highly conserved and essential 2OG-oxygenase that, thus far, is the only arginyl hydroxylase assigned in eukaryotes. We recently reported that JMJD5 hydroxylase activity is required for DNA replication fidelity, and that pathogenic variants cause replication stress (RS) and genome instability (GIN) in a novel neurodevelopmental disorder. Because of the prevalence of RS and GIN in cancer, and reported roles of JMJD5 in tumorigenesis, we here investigate the impact of JMJD5 cancer mutations on its role in replication fidelity. We describe the structural impact of cancer missense mutations on the hydroxylase activity of JMJD5 and its interaction with RCCD1, an abundant binding partner encoded by a gene associated with susceptibility to a variety of tumour types. We show that the JMJD5:RCCD1 interaction is disrupted by cancer mutations and that the complex is essential for suppressing RS and GIN in tumour cells. Finally, we describe a novel interaction of the complex with RAD51 paralogues and demonstrate the importance of the JMJD5:RCCD1 interaction for normal replication fork restart. Our findings further highlight the importance of protein hydroxylases in fundamental cellular processes and the consequences of JMJD5 and RCCD1 deregulation in human disease.

cancer biology↗

bioRxiv2021

Stem cell-derived macrophages as a new platform for studying host-pathogen interactions in livestock

Infectious diseases of farmed and wild animals pose a recurrent threat to food security and human health. The macrophage, a key component of the innate immune system, is the first line of defence against many infectious agents and plays a major role in shaping the adaptive immune response. However, this phagocyte is a target and host for many pathogens. Understanding the molecular basis of interactions between macrophages and pathogens is therefore crucial for the development of effective strategies to combat important infectious diseases. We explored how pluripotent stem cells (PSCs) can provide a limitless in vitro supply of genetically and experimentally tractable macrophages from livestock. Porcine and bovine PSC-derived macrophages (PSCdMs) exhibited molecular and functional characteristics of ex vivo primary macrophages. Pig PSCdMs were productively infected by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and African Swine Fever Virus (ASFV), two of the most economically important and devastating viruses in pig farming. Moreover, Pig PSCdMs were readily amenable to genetic modification by CRISPR/Cas9 gene editing applied in parental stem cells, or directly by lentiviral vector transduction. PSCs and differentiated derivatives therefore provide a useful and ethical experimental platform to investigate the genetic and molecular basis of host-pathogen interactions in livestock.

immunology↗

bioRxiv2021

Secreted SARS-CoV-2 ORF8 modulates the cytokine expression profile of human macrophages

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still adapting to its new human host. Attention has focussed on the viral spike protein, but substantial variation has been seen in the ORF8 gene. Here, we show that SARS-CoV-2 ORF8 protein undergoes signal peptide-mediated processing through the endoplasmic reticulum and is secreted as a glycosylated, disulphide-linked dimer. The secreted protein from the prototype SARS-CoV-2 virus had no major effect on viability of a variety of cell types, or on IFN or NF-{kappa}B signalling. However, it modulated cytokine expression from primary CSF1-derived human macrophages, most notably by decreasing IL-6 and IL-8 secretion. Furthermore, a sequence polymorphism L84S that appeared early in the pandemic associated with the Clade S lineage of virus, showed a markedly different effect, of increasing IL-6 production. We conclude that ORF8 sequence polymorphisms can potentially affect SARS-CoV-2 virulence and should therefore be monitored in sequencing-based surveillance.

microbiology↗

bioRxiv2021

Pseudohypoxic HIF pathway activation dysregulates collagen structure-function in human lung fibrosis

Extracellular matrix (ECM) stiffening with downstream activation of mechanosensitive pathways is strongly implicated in fibrosis. We previously reported that altered collagen nanoarchitecture is a key determinant of pathogenetic ECM structure-function in human fibrosis (Jones et al., 2018). Here, through human tissue, bioinformatic and ex vivo studies we show that hypoxia-inducible factor (HIF) pathway activation is a critical pathway for this process regardless of oxygen status (pseudohypoxia). Whilst TGF{beta} increased rate of fibrillar collagen synthesis, HIF pathway activation was required to dysregulate post-translational modification of fibrillar collagen, promoting bone-type cross-linking, altering collagen nanostructure, and increasing tissue stiffness. In vitro, knock down of Factor Inhibiting HIF (FIH) or oxidative stress caused pseudohypoxic HIF activation in normal fibroblasts. In contrast, endogenous FIH activity was reduced in fibroblasts from patients with lung fibrosis in association with significantly increased normoxic HIF pathway activation. In human lung fibrosis tissue, HIF mediated signalling was increased at sites of active fibrogenesis whilst subpopulations of IPF lung mesenchymal cells had increases in both HIF and oxidative stress scores. Our data demonstrate that oxidative stress can drive pseudohypoxic HIF pathway activation which is a critical regulator of pathogenetic collagen structure-function in fibrosis.

cell biology↗

bioRxiv2021

MicroMator: Open and Flexible Software for Reactive Microscopy

Microscopy image analysis has recently made enormous progress both in terms of accuracy and speed thanks to machine learning methods. This greatly facilitates the online adaptation of microscopy experimental plans using real-time information of the observed systems and their environments. Here we report MicroMator, an open and flexible software for defining and driving reactive microscopy experiments, and present applications to single-cell control and single-cell recombination.

systems biology↗

bioRxiv2020

Lack of evidence of ACE2 expression and replicative infection by SARS-CoV-2 in human endothelial cells

A striking feature of severe COVID-19 is thrombosis in large as well as small vessels of multiple organs. This has led to the assumption that SARS-CoV-2 virus directly infects and damages the vascular endothelium. However, endothelial expression of ACE2, the cellular receptor for SARS-CoV-2, has not been convincingly demonstrated. Interrogating human bulk and single-cell transcriptomic data, we found ACE2 expression in endothelial cells to be extremely low or absent in vivo and not upregulated by exposure to inflammatory agents in vitro. Also, the endothelial chromatin landscape at the ACE2 locus showed presence of repressive and absence of activation marks, suggesting that the gene is inactive in endothelial cells. Finally, we failed to achieve infection and replication of SARS-CoV-2 in cultured human endothelial cells, which were permissive to productive infection by coronavirus 229E that uses CD13 as the receptor. Our data suggest that SARS-Cov-2 is unlikely to infect endothelial cells directly; these findings are consistent with a scenario where endothelial injury is indirectly caused by the infection of neighbouring epithelial cells and/or due to systemic effects mediated by immune cells, platelets, complement activation, and/or proinflammatory cytokines.

pathology↗

bioRxiv2020

PolyA-modulating antisense oligonucleotides reveal opposing functions for long non-coding RNA NEAT1 isoforms in neuroblastoma

Many long non-coding RNAs (lncRNA) are highly dysregulated in cancer and are emerging as therapeutic targets. One example is NEAT1, which consists of two overlapping lncRNA isoforms, NEAT1_1 (3.7kb) and NEAT1_2 (23kb), that are functionally distinct. The longer NEAT1_2 is responsible for scaffolding gene-regulatory nuclear bodies termed paraspeckles, whereas NEAT1_1 is involved in paraspeckle-independent function. The NEAT1 isoform ratio is dependent on the efficient cleavage and polyadenylation of NEAT1_1 at the expense of NEAT1_2. Here we developed a targeted antisense oligonucleotide (ASO) approach to sterically block NEAT1_1 polyadenylation processing, achieving upregulation of NEAT1_2 and abundant paraspeckles. We have applied these ASOs to cells of the heterogeneous infant cancer, neuroblastoma, as we found higher NEAT1_1:NEAT1_2 ratio and lack of paraspeckles in high-risk neuroblastoma cells. These ASOs decrease NEAT1_1 levels, increase NEAT1_2/paraspeckles and concomitantly reduce cell viability in high-risk neuroblastoma specifically. In contrast, overexpression of NEAT1_1 has the opposite effect, increasing cell-proliferation. Transcriptomic analyses of high-risk neuroblastoma cells with altered NEAT1 ratios and increased paraspeckle abundance after ASO treatment showed an upregulation of differentiation pathways, as opposed to the usual aggressive neuroblastic phenotype. Thus, we have developed potential anti-cancer ASO drugs that can transiently increase growth-inhibiting NEAT1_2 RNA at the expense of growth-promoting NEAT1_1 RNA. These ASOs, unlike others that degrade lncRNAs, provide insights into the importance of altering lncRNA polyadenylation events to suppress tumorigenesis as a strategy to combat cancer.

cancer biology↗

bioRxiv2019

The Avocado Genome Informs Deep Angiosperm Phylogeny, Highlights Introgressive Hybridization, and Reveals Pathogen Influenced Gene Space Adaptation

The avocado, Persea americana, is a fruit crop of immense importance to Mexican agriculture with an increasing demand worldwide. Avocado lies in the anciently-diverged magnoliid clade of angiosperms, which has a controversial phylogenetic position relative to eudicots and monocots. We sequenced the nuclear genomes of the Mexican avocado race, P. americana var. drymifolia, and the most commercially popular hybrid cultivar, Hass, and anchored the latter to chromosomes using a genetic map. Resequencing of Guatemalan and West Indian varieties revealed that [~]39% of the Hass genome represents Guatemalan source regions introgressed into a Mexican race background. Some introgressed blocks are extremely large, consistent with the recent origin of the cultivar. The avocado lineage experienced two lineage-specific polyploidy events during its evolutionary history. Although gene-tree/species-tree phylogenomic results are inconclusive, syntenic ortholog distances to other species place avocado as sister to the enormous monocot and eudicot lineages combined. Duplicate genes descending from polyploidy augmented the transcription factor diversity of avocado, while tandem duplicates enhanced the secondary metabolism of the species. Phenylpropanoid biosynthesis, known to be elicited by Colletotrichum (anthracnose) pathogen infection in avocado, is one enriched function among tandems. Furthermore, transcriptome data show that tandem duplicates are significantly up- and down-regulated in response to anthracnose infection, whereas polyploid duplicates are not, supporting the general view that collections of tandem duplicates contribute evolutionarily recent \"tuning knobs\" in the genome adaptive landscapes of given species.\n\nSIGNIFICANCE STATEMENTAvocado is a nutritious, economically important fruit species that occupies an unresolved position near the earliest evolutionary branchings of flowering plants. Our nuclear genome sequences of Mexican and Hass variety avocados inform ancient evolutionary relationships and genome doublings, the admixed nature of Hass, and provide a look at how pathogen interactions have shaped avocados more recent genomic evolutionary history.

genomics↗

bioRxiv2018

Interaction of modified oligonucleotides with nuclear proteins, formation of novel nuclear structures and sequence-independent effects on RNA processing

Oligonucleotides and nucleic acid analogues that alter gene expression are showing therapeutic promise for selected human diseases. The modification of synthetic nucleic acids to protect against nuclease degradation and to influence drug function is common practice, however, such modifications may also confer unexpected physicochemical and biological properties. Here we report backbone-specific effects of modified oligonucleotides on subnuclear organelles, altered distribution of nuclear proteins, the appearance of novel structured nuclear inclusions, and modification of RNA processing in cultured cells transfected with antisense oligonucleotides on a phosphorothioate backbone. Phosphodiester and phosphorodiamidate morpholino oligomers elicited no such consequences. Disruption of subnuclear structures and proteins elicit severe phenotypic disturbances, revealed by transcriptomic analysis of fibroblasts exhibiting such disruption. These data suggest that the toxic effects and adverse events reported after clinical evaluation of phosphorothioate nucleic acid drugs may be mediated, at least in part, by non-specific interaction of nuclear components with the phosphorothioate backbone.

molecular biology↗