Biology subjects

Evans, C. M.

Publications and source records attributed to Evans, C. M..

14 recordsLinked to original sources

bioRxiv2026

Muc5ac mediates anti-viral immunity and virus-induced parasympathetic nerve dysfunction

Respiratory viruses can induce excessive bronchoconstriction in both asthmatic and healthy airways. Airway mucins such as Muc5ac form the first line of defense against inhaled pathogens. However, when produced in excess, they can also contribute to airway narrowing and mucus plug formation in asthma. In this study, we investigated the role of airway mucins in host defense against parainfluenza virus and in virus-induced airway hyperresponsiveness using Muc5ac-deficient (Muc5ac-/-) C57BL/6 mice. Parainfluenza virus infection induced airway hyperresponsiveness to inhaled methacholine in wild-type mice, an effect that was abolished in Muc5ac-/- mice. Parainfluenza virus-induced airway hyperresponsiveness was reversed by vagotomy, demonstrating it is mediated by parasympathetic nerve dysfunction. Muc5ac-/- mice exhibited higher viral titers, increased bronchoalveolar lavage cellularity, and elevated antiviral cytokine levels, but did not develop airway hyperresponsiveness. We did not see mucus plugging in any of our animals. Together, these findings indicate that Muc5ac is important for host defense against parainfluenza virus but paradoxically is also required for virus-induced airway hyperresponsiveness.

physiology↗

bioRxiv2025

Siglec-F Protects Against Elastase-induced Lung Inflammation and Emphysema in Mice

Respiratory surfaces are exposed daily to billions of inhaled particles that could cause injury to alveolar tissues where gas exchange occurs. Accordingly, robust defense is essential but must involve minimal physiologic disruption. Airspace macrophages (AMs) protect lung surfaces while also maintaining tissue integrity through non-inflammatory homeostatic responses in health. However, in chronic obstructive pulmonary disease (COPD), AMs contribute to emphysematous alveolar destruction through mechanisms that are poorly understood. We hypothesized that damaging effects of AMs in emphysema result from loss of mechanisms that normally restrain homeostatic AMs. We found that Siglec-F, a common marker of AMs, exerts suppressive effects on tissue resident AMs (RAMs), thereby supporting alveolar integrity in mouse lungs during health and in a model of elastase-induced alveolar destruction. Siglec-F-deficient mice exhibited decreased alveolar numbers at baseline, and they had worsened alveolar damage after elastase challenge that was unexpectedly RAM-mediated. Transcriptomic profiling revealed dysregulation of key pathways involved in tissue remodeling and repair, including extracellular matrix degradation, TGF-{beta} signaling, and phagocytosis. These findings uncover previously unrecognized roles for RAMs and Siglec-F in preserving alveolar integrity. Our findings implicate RAMs and Siglecs as potential therapeutic targets for preserving alveolar integrity in health and for limiting alveolar damage in COPD.

immunology↗

bioRxiv2025

Airway Secretory Cells Contain Both a Perinuclear Golgi Ribbon and Dispersed Golgi Satellites

RationaleFinely tuned production and secretion of the polymeric mucins MUC5AC and MUCB are required for lung health, but knowledge of many details between their translation and their packaging into secretory granules is lacking. ObjectivesTo analyze the structure and function of the Golgi apparatus, a key site of mucin glycosylation, folding, polymerization and packaging, in airway epithelial secretory cells. MethodsLung tissue was obtained from mice stimulated or not with IL-13 to upregulate mucin production, and from normal human lungs. Golgi elements in mouse and human tissue were imaged by high-resolution immunofluorescence microscopy and electron microscopy. Tissue from mice with deletion of both polymeric mucins was also examined. Measurements and Main ResultsBy immunofluorescence microscopy, both mouse and human airway secretory cells contained [~]100 dispersed puncta labeled by markers of medial and trans Golgi cisternae and the trans-Golgi network (TGN), but only a few perinuclear puncta were labeled by markers of cis-Golgi cisternae. By electron microscopy, secretory cells contained both a perinuclear Golgi ribbon and numerous dispersed Golgi stacks, termed satellites. In mucous metaplastic cells, satellites were concentrated among immature mucin granules. Increasing mucin production by cytokine stimulation did not increase the number of TGN puncta, nor did preventing polymeric mucin production by gene deletion reduce TGN puncta. ConclusionsMucin-producing airway secretory cells express an unusual Golgi structure consisting of a conventional perinuclear Golgi ribbon as well as dispersed satellites. While the Golgi satellites are likely an adaptation for mucin production and packaging, their presence is specified developmentally, independent of mucin production.

cell biology↗

bioRxiv2024

Siglec-F Deficiency Prevents Fibrosis After Bleomycin-Induced Acute Lung Injury

Injury to the lungs causes acute inflammation that can lead to pathological lung fibrosis. Airspace macrophages (AMs) are critical for repair of injured tissue, but they can contribute fibrosis through mechanisms that are incompletely understood. Siglecs are expressed by immune cells. In mice, Siglec-F is chiefly expressed AMs where it is considered inflammosuppressive. We hypothesized that its deletion would worsen lung injury and fibrosis in response to intratracheal bleomycin challenge. We evaluated Siglec-F expression and function in mice challenged with bleomycin on days 7, 14, and 21 post-challenge (2.5 U/kg). AMs were the predominant inflammatory cells at all timepoints, and they included resident (RAM) and recruited (RecAM) subsets. Siglec-F deficiency prevented fibrosis than in Siglecf-/- mouse lungs, as evident from biochemical and histologic readouts. We performed RNAseq on pooled RAMs and RecAMs from wild type and Siglec-F deficient mice. Lung fibrosis 21 d after bleomycin challenge was associated with differentially expressed genes (DEGs) related to cholesterol synthesis and metabolism. In AMs from healthy lung lavage fluid and idiopathic pulmonary fibrosis patient tissues, the human paralogs Siglec-7 and Siglec-9 were expressed. Findings here identify novel mechanisms that control protective and detrimental functions of AMs after lung injury.

physiology↗

bioRxiv2024

Requirement for Fucosyltransferase 2 in Allergic Airway Hyperreactivity and Mucus Obstruction

Mucus hypersecretion is an important pathological problem in respiratory diseases. Mucus accumulates in the airways of people with asthma, and it contributes to airflow limitation by forming plugs that occlude airways. Current treatments have minimal effects on mucus or its chief components, the polymeric mucin glycoproteins MUC5AC and MUC5B. This treatment gap reflects a poor molecular understanding of mucins that could be used to determine how they contribute to airway obstruction. Due to the prominence of glycosylation as a defining characteristic of mucins, we investigated characteristics of mucin glycans in asthma and in a mouse model of allergic asthma. Mucin fucosylation was observed in asthma, and in healthy mice it was induced as part of a mucous metaplastic response to allergic inflammation. In allergically inflamed mouse airways, mucin fucosylation was dependent on the enzyme fucosyltransferase 2 (Fut2). Fut2 gene deficient mice were protected from asthma-like airway hyperreactivity and mucus plugging. These findings provide mechanistic and translational links between observations in human asthma and a mouse model that may help improve therapeutic targeting of airway mucus.

physiology↗

bioRxiv2022

Head and Neck Cancer-derived small extracellular vesicles sensitize TRPV1+ neurons to mediate cancer pain

Severe pain is often experienced by patients with head and neck cancer and is associated with a poor prognosis. Despite its frequency and severity, current treatments fail to adequately control cancer-associated pain, because of our lack of mechanistic understanding. Cancer-derived small extracellular vesicles (Cancer-sEVs) are well- positioned to function as mediators of communication between cancer cells and neurons. Inhibition of Cancer-sEV release attenuated pain in tumor-bearing mice. Injection of purified Cancer-sEVs is sufficient to induce pain hypersensitivity in naive mice. Cancer-sEVs triggered calcium influx in nociceptors and inhibition or ablation of nociceptors protect against cancer pain. Interrogation of published sequencing data of human sensory neurons exposed to human Cancer-sEVs suggested a stimulation of protein translation in neurons. Induction of translation by Cancer-sEVs was validated in our mouse model and its inhibition alleviated cancer pain in mice. These findings define a role of Cancer-sEVs in cancer pain and identify several druggable targets. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/506411v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@2a13a2org.highwire.dtl.DTLVardef@6f305dorg.highwire.dtl.DTLVardef@1d0417corg.highwire.dtl.DTLVardef@17b36df_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2022

The Colon Mucosal Sialylglycome Is Redox-Regulated by the Golgi Enzyme QSOX1

Mucus shields the intestinal epithelium from pathogens and provides a supportive environment for commensal bacteria. Mucus is composed of enormous, heavily glycosylated proteins called mucins, which become disulfide crosslinked in a multi-step biosynthetic pathway culminating in the Golgi apparatus and secretory granules of goblet cells. We observed that knockout mice lacking the Golgi-localized disulfide catalyst QSOX1 produced poorly protective colon mucus, were hypersensitive to induced colitis, and had an altered microbiome. The initial hypothesis arising from these observations was that QSOX1 catalyzes disulfide crosslinking of mucins. Contrary to this hypothesis, the disulfide-mediated polymerization of mucins and related glycoproteins proceeded normally without QSOX1. Instead, we found that QSOX1 forms regulatory disulfides in Golgi glycosyltransferases and thereby promotes effective sialylation of the colon glycome. Our findings reveal that enzymatic control of Golgi redox state impacts glycan elaboration in goblet cells, and that this pathway is crucial for maintaining mucosal function.

cell biology↗

bioRxiv2022

Breasi-CRISPR: an efficient genome editing method to interrogate protein localization and protein-protein interactions in the embryonic mouse cortex

In developing tissues, knowing the localization and interactors of proteins of interest is key to understanding their function. This can be challenging when the researched protein lacks reliable antibodies. Here, we combine Easi-CRISPR with in utero electroporation to tag endogenous proteins within embryonic mouse brains. This method is called Breasi-CRISPR (Brain Easi-CRISPR), and enables knock-in of both short and long epitope tag sequences in genes of interest with high efficiency. Using Breasi-CRISPR, we were able to visualize epitope tagged proteins known to have either high or low expression levels, such as ACTB, LMNB1, EMD, FMRP, NOTCH1, and RPL22. Detection was possible by immunohistochemistry as soon as one day after electroporation at embryonic day 13 (E13). Two and five days after electroporation, we observed efficient gene editing in up to 50% of electroporated cells. Moreover, tagged proteins could be detected by immunoblotting in lysates from individual cortices two days after electroporation. Next, we demonstrated that Breasi-CRISPR enables the tagging of proteins with fluorophores in an efficient manner, allowing the visualization of endogenous proteins via live-imaging in organotypic brain slices two days after electroporation. Finally, we used Breasi-CRISPR to perform co-IP mass-spectrometry analyses of tagged autism-related protein FMRP to discover its interactome in the embryonic cortex. Together, these data show Breasi-CRISPR is a powerful tool with diverse applications that will propel the understanding of protein function in neurodevelopment.

developmental biology↗

bioRxiv2021

Coordination of di-acetylated histone ligands by the ATAD2 bromodomain

The ATPase Family, AAA domain-containing protein 2 (ATAD2) bromodomain (BRD) has a canonical bromodomain structure consisting of four -helices. ATAD2 functions as a co-activator of the androgen and estrogen receptors as well as the MYC and E2F transcription factors. ATAD2 also functions during DNA replication, recognizing newly synthesized histones. In addition, ATAD2 is shown to be up regulated in multiple forms of cancer including breast, lung, gastric, endometrial, renal, and prostate. Furthermore, up-regulation of ATAD2 is strongly correlated with poor prognosis in many types of cancer, making the ATAD2 bromodomain an innovative target for cancer therapeutics. In this study, we describe the recognition of histone acetyllysine modifications by the ATAD2 bromodomain. Residue-specific information on the complex formed between the histone tail and the ATAD2 bromodomain, obtained through nuclear magnetic resonance spectroscopy (NMR) and X-ray crystallography, illustrates key residues lining the binding pocket, which are involved in coordination of di-acetylated histone tails. Analytical ultracentrifugation, NMR relaxation data, and isothermal titration calorimetry further confirm the monomeric state of the functionally active ATAD2 bromodomain in complex with di-acetylated histone ligands. Overall, we describe histone tail recognition by ATAD2 BRD and illustrate that one acetyllysine group is primarily engaged by the conserved asparagine (N1064), the "RVF" shelf residues, and the flexible ZA loop. Coordination of a second acetyllysine group also occurs within the same binding pocket, but is essentially governed by unique hydrophobic and electrostatic interactions making the di-acetyllysine histone coordination more specific than previously presumed.

biochemistry↗

bioRxiv2020

Spike mutation pipeline reveals the emergence of a more transmissible form of SARS-CoV-2

We have developed an analysis pipeline to facilitate real-time mutation tracking in SARS-CoV-2, focusing initially on the Spike (S) protein because it mediates infection of human cells and is the target of most vaccine strategies and antibody-based therapeutics. To date we have identified thirteen mutations in Spike that are accumulating. Mutations are considered in a broader phylogenetic context, geographically, and over time, to provide an early warning system to reveal mutations that may confer selective advantages in transmission or resistance to interventions. Each one is evaluated for evidence of positive selection, and the implications of the mutation are explored through structural modeling. The mutation Spike D614G is of urgent concern; it began spreading in Europe in early February, and when introduced to new regions it rapidly becomes the dominant form. Also, we present evidence of recombination between locally circulating strains, indicative of multiple strain infections. These finding have important implications for SARS-CoV-2 transmission, pathogenesis and immune interventions.

evolutionary biology↗

bioRxiv2020

The BRPF1 bromodomain is a molecular reader of di-acetyllysine

Bromodomain-containing proteins are often part of chromatin-modifying complexes, and their activity can lead to altered expression of genes that drive cancer, inflammation and neurological disorders in humans. Bromodomain-PHD finger protein 1 (BRPF1) is part of the MOZ (monocytic leukemic zinc-finger protein) HAT (histone acetyltransferase) complex, which is associated with chromosomal translocations known to contribute to the development of acute myeloid leukemia (AML). BRPF1 contains a unique combination of chromatin reader domains including two plant homeodomain (PHD) fingers separated by a zinc knuckle (PZP domain), a bromodomain, and a proline-tryptophan-tryptophan-proline (PWWP) domain. BRPF1 is known to recruit the MOZ HAT complex to chromatin by recognizing acetylated lysine residues on the N-terminal histone tail region through its bromodomain. However, histone proteins can contain several acetylation modifications on their N-terminus, and it is unknown how additional marks influence bromodomain recruitment to chromatin. Here, we identify the BRPF1 bromodomain as a selective reader of di-acetyllysine modifications on histone H4. We used ITC assays to characterize the binding of di-acetylated histone ligands to the BRPF1 bromodomain and found that the domain binds preferentially to histone peptides H4K5acK8ac and H4K5acK12ac. Analytical ultracentrifugation (AUC) experiments revealed that the monomeric state of the BRPF1 bromodomain coordinates di-acetylated histone ligands. NMR chemical shift perturbation studies, along with binding and mutational analyses, revealed non-canonical regions of the bromodomain-binding pocket that are important for histone tail recognition. Together, our findings provide critical information on how the combinatorial action of post-translational modifications can modulate BRPF1 bromodomain binding and specificity.

biochemistry↗

bioRxiv2019

Disulfide disruption reverses mucus dysfunction in allergic airway disease

Airway mucus is essential for healthy lung defense1, but excessive mucus in asthma obstructs airflow, leading to severe and potentially fatal outcomes2-5. Current asthma therapies reduce allergic inflammation and relax airway smooth muscle, but treatments are often inadequate due to their minimal effects on mucus obstruction6,7. The lack of efficacious mucus-targeted treatments stems from a poor understanding of healthy mucus function and pathological mucus dysfunction at a molecular level. The chief macromolecules in mucus, polymeric mucins, are massive glycoproteins whose sizes and biophysical properties are dictated in part by covalent disulfide bonds that link mucin molecules into assemblies of 10 or more subunits8. Once secreted, mucin glycopolymers can aggregate to form plugs that block airflow. Here we show that reducing mucin disulfide bonds depolymerizes mucus in human asthma and reverses pathological effects of mucus hypersecretion in a mouse allergic asthma model. In mice challenged with a fungal allergen, inhaled mucolytic treatment acutely loosened mucus mesh, enhanced mucociliary clearance (MCC), and abolished airway hyperreactivity (AHR) to the bronchoprovocative agent methacholine. AHR reversal was directly related to reduced mucus plugging. Furthermore, protection in mucolytic treated mice was identical to prevention observed in mice lacking Muc5ac, the polymeric mucin required for allergic AHR in murine models9. These findings establish grounds for developing novel fast-acting agents to treat mucus hypersecretion in asthma10,11. Efficacious mucolytic therapies could be used to directly improve airflow, help resolve inflammation, and enhance the effects of inhaled treatments for asthma and other respiratory conditions11,12.

physiology↗

bioRxiv2019

MUC5AC drives COPD exacerbation severity through amplification of virus-induced airway inflammation

The respiratory tract surface is protected from inhaled pathogens by a secreted layer of mucus that is rich in mucin glycoproteins. Disrupted mucus production is a cardinal feature of chronic respiratory diseases but how this alteration affect interactions between mucins and pathogens is complex and poorly understood. Here, we identify a central and unexpected role for the major airway mucin MUC5AC in pathogenesis of virus-induced exacerbations of chronic obstructive pulmonary disease (COPD). Virus induction of MUC5AC is augmented in COPD compared to healthy subjects, is enhanced in frequent exacerbators and correlates with inflammation, symptom severity and secondary bacterial infection during exacerbation. MUC5AC is functionally related to inflammation as MUC5AC-deficient (Muc5ac-/-) mice had attenuated rhinovirus-induced airway inflammation whilst exogenous MUC5AC glycoprotein administration augmented virus-induced inflammatory responses and bacterial load. Mechanistically, MUC5AC-augmentation of rhinovirus-induced inflammation occurred through release of extracellular adenosine triphosphate (ATP). Therapeutic suppression of virus-induced MUC5AC release using an epidermal growth factor receptor (EGFR) inhibitor ameliorated exaggerated pro-inflammatory responses in a mouse COPD exacerbation model. Collectively, these studies demonstrate previously unrecognised pro-inflammatory effects of MUC5AC during infection and thus highlight a key unforeseen role in driving COPD exacerbation severity.

immunology↗

bioRxiv2018

Different Munc18 Proteins Mediate Baseline and Stimulated Airway Mucin Secretion

Airway mucin secretion is necessary for ciliary clearance of inhaled particles and pathogens, but can be detrimental in pathologies such as asthma and cystic fibrosis. Exocytosis in mammals requires a Munc18 scaffolding protein, and airway secretory cells express all three Munc18 isoforms. Using conditional airway epithelial deletant mice, we found that Munc18a has the major role in baseline mucin secretion, Munc18b has the major role in stimulated mucin secretion, and Munc18c does not function in mucin secretion. In an allergic asthma model, Munc18b deletion reduced airway mucus occlusion and airflow resistance. In a cystic fibrosis model, Munc18b deletion reduced airway mucus occlusion and emphysema. Munc18b deficiency in the airway epithelium did not result in any abnormalities of lung structure, particle clearance, inflammation, or bacterial infection. Our results show that regulated secretion in a polarized epithelial cell may involve more than one exocytic machine at the apical plasma membrane, and that the protective roles of mucin secretion can be preserved while therapeutically targeting its pathologic roles.

cell biology↗