Biology subjects

Johnson, J. L.

Publications and source records attributed to Johnson, J. L..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Evolution of host protease interactions among SARS-CoV-2 variants of concern and related coronaviruses

Previously, we showed that coagulation factors directly cleave SARS-CoV-2 spike and promote viral entry (Kastenhuber et al., 2022). Here, we show that substitutions in the S1/S2 cleavage site observed in SARS-CoV-2 variants of concern (VOCs) exhibit divergent interactions with host proteases, including factor Xa and furin. Nafamostat remains effective to block coagulation factor-mediated cleavage of variant spike sequences. Furthermore, host protease usage has likely been a selection pressure throughout coronavirus evolution, and we observe convergence of distantly related coronaviruses to attain common host protease interactions, including coagulation factors. Interpretation of genomic surveillance of emerging SARS-CoV-2 variants and future zoonotic spillover is supported by functional characterization of recurrent emerging features.

microbiology↗

bioRxiv2022

A global atlas of substrate specificities for the human serine/threonine kinome

Protein phosphorylation is one of the most widespread post-translational modifications in biology. With the advent of mass spectrometry-based phosphoproteomics, more than 200,000 sites of serine and threonine phosphorylation have been reported, of which several thousand have been associated with human diseases and biological processes. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein Ser/Thr kinases encoded in the human genome is responsible. Here, we utilize synthetic peptide libraries to profile the substrate sequence specificity of nearly every functional human Ser/Thr kinase. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. Our kinome-wide dataset was used to computationally annotate and identify the most likely protein kinases for every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites where the protein kinases involved have been previously identified, our predictions were in excellent agreement. When this approach was applied to examine the signaling response of tissues and cell lines to hormones, growth factors, targeted inhibitors, and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the full extent of substrate specificity of the human Ser/Thr kinome, illuminate cellular signaling responses, and provide a rich resource to link unannotated phosphorylation events to biological pathways.

systems biology↗

bioRxiv2022

New system for serial crystallography data collection at SBC APS

Serial synchrotron crystallography enables studies of protein structures under physiological temperature and reduced radiation damage by collection of data from thousands of crystals. The Structural Biology Center at Sector 19 of the Advanced Photon Source has implemented a fixed-target approach with a new 3D printed mesh-holder optimized for sample handling. The holder immobilizes a crystal suspension or droplet emulsion on a nylon mesh, trapping and sealing a near-monolayer of crystals in its mother liquor between two thin mylar films. Data can be rapidly collected in scan mode and analyzed in near real-time using piezoelectric linear stages assembled in an XYZ arrangement, controlled with a graphical user interface and analyzed by using a high-performance computing pipeline. Here, the system was applied to two {beta}-lactamases: a class D serine {beta}-lactamase from Chitinophaga pinensis DSM 2588 and L1 metallo-{beta} -lactamase from Stenotrophomonas maltophilia K279a.

molecular biology↗

bioRxiv2021

Indisulam synergizes with palbociclib to induce senescence through inhibition of CDK2 kinase activity

Inducing senescence in cancer cells is emerging as a new therapeutic strategy. In order to find ways to enhance senescence induction by palbociclib, a CDK4/6 inhibitor approved for treatment of metastatic breast cancer, we performed functional genetic screens in palbociclib-resistant cells. Using this approach, we found that loss of CDK2 results in strong senescence induction in palbociclib-treated cells. Treatment with the CDK2 inhibitor indisulam, which phenocopies genetic CDK2 inactivation, led to sustained senescence induction when combined with palbociclib in various cell lines and lung cancer xenografts. Treating cells with indisulam led to downregulation of cyclin H, which prevented CDK2 activation. Combined treatment with palbociclib and indisulam induced a senescence program and sensitized cells to senolytic therapy. Our data indicate that inhibition of CDK2 through indisulam treatment can enhance senescence induction by CDK4/6 inhibition.

cancer biology↗

bioRxiv2021

Benchmarking 50 classification algorithms on 50 gene-expression datasets

By classifying patients into subgroups, clinicians can provide more effective care than using a uniform approach for all patients. Such subgroups might include patients with a particular disease subtype, patients with a good (or poor) prognosis, or patients most (or least) likely to respond to a particular therapy. Diverse types of biomarkers have been proposed for assigning patients to subgroups. For example, DNA variants in tumors show promise as biomarkers; however, tumors exhibit considerable genomic heterogeneity. As an alternative, transcriptomic measurements reflect the downstream effects of genomic and epigenomic variations. However, high-throughput technologies generate thousands of measurements per patient, and complex dependencies exist among genes, so it may be infeasible to classify patients using traditional statistical models. Machine-learning classification algorithms can help with this problem. However, hundreds of classification algorithms exist--and most support diverse hyperparameters--so it is difficult for researchers to know which are optimal for gene-expression biomarkers. We performed a benchmark comparison, applying 50 classification algorithms to 50 gene-expression datasets (143 class variables). We evaluated algorithms that represent diverse machine-learning methodologies and have been implemented in general-purpose, open-source, machine-learning libraries. When available, we combined clinical predictors with gene-expression data. Additionally, we evaluated the effects of performing hyperparameter optimization and feature selection in nested cross-validation folds. Kernel- and ensemble-based algorithms consistently outperformed other types of classification algorithms; however, even the top-performing algorithms performed poorly in some cases. Hyperparameter optimization and feature selection typically improved predictive performance, and univariate feature-selection algorithms outperformed more sophisticated methods. Together, our findings illustrate that algorithm performance varies considerably when other factors are held constant and thus that algorithm selection is a critical step in biomarker studies. Author Summary

bioinformatics↗

bioRxiv2021

Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry

Coagulopathy is a significant aspect of morbidity in COVID-19 patients. The clotting cascade is propagated by a series of proteases, including factor Xa and thrombin. While certain host proteases, including TMPRSS2 and furin, are known to be important for cleavage activation of SARS-CoV-2 spike to promote viral entry in the respiratory tract, other proteases may also contribute. Using biochemical and cell-based assays, we demonstrate that factor Xa and thrombin can also directly cleave SARS-CoV-2 spike, enhancing viral entry. A drug-repurposing screen identified a subset of protease inhibitors that promiscuously inhibited spike cleavage by both transmembrane serine proteases as well as coagulation factors. The mechanism of the protease inhibitors nafamostat and camostat may extend beyond inhibition of TMPRSS2 to coagulation-induced spike cleavage. Anticoagulation is critical in the management of COVID-19, and early intervention could provide collateral benefit by suppressing SARS-CoV-2 viral entry. We propose a model of positive feedback whereby infection-induced hypercoagulation exacerbates SARS-CoV-2 infectivity.

microbiology↗

bioRxiv2020

Th17-skewed inflammation due to genetic deficiency of a cadherin stress sensor

Desmoglein 1 (Dsg1) is a cadherin restricted to stratified tissues of terrestrial vertebrates, which serve as essential physical and immune barriers. Dsg1s importance in epidermal integrity is underscored by genetic, autoimmune and bacterial toxin-mediated disorders interfering with Dsg1 function. Dsg1 loss-of-function mutations in humans result not only in skin lesions, but also multiple allergies, and isolated patient keratinocytes exhibit increased pro-allergic cytokine expression. However, the mechanism by which genetic deficiency of Dsg1 causes chronic inflammation is unknown. To determine the systemic response to Dsg1 loss, we deleted the three tandem Dsg1 genes in mice using CRISPR/Cas9. Whole transcriptome analysis of E18.5 Dsg1-/- skin showed changes consistent with the observed aberrant differentiation and barrier impairment. Comparing epidermal transcriptomes from E18.5 Dsg1-deficient mice and humans with Dsg1 mutations revealed a shared psoriatic-like IL-17-skewed inflammatory signature and less so a pro-allergic IL-4/13 signature. Although the impaired intercellular adhesion observed in Dsg1-/- mice resembles that resulting from autoimmune anti-Dsg1 pemphigus foliaceus antibodies, transcriptomic analysis of pemphigus skin lesions lacks a prominent IL-17 signature. Thus, beyond impairing the physical barrier, chronic loss of Dsg1 function through gene mutation results in a psoriatic-like inflammatory signature before birth, possibly predisposing to skin inflammation.

immunology↗

bioRxiv2020

GR chaperone cycle mechanism revealed by cryo-EM: inactivation of GR by GR:Hsp90:Hsp70:Hop client-loading complex

Maintaining a healthy proteome is fundamental for organism survival1,2. Integral to this are Hsp90 and Hsp70 molecular chaperones that together facilitate the folding, remodeling and maturation of Hsp90s many "client" proteins3-7. The glucocorticoid receptor (GR) is a model client strictly dependent upon Hsp90/Hsp70 for activity8-13. Chaperoning GR involves a cycle of inactivation by Hsp70, formation of an inactive GR:Hsp90:Hsp70:Hop "loading" complex, conversion to an active GR:Hsp90:p23 "maturation" complex, and subsequent GR release14. Unfortunately, a molecular understanding of this intricate chaperone cycle is lacking for any client. Here, we report the cryo-EM structure of the GR loading complex, in which Hsp70 loads GR onto Hsp90, revealing the molecular basis of direct Hsp90/Hsp70 coordination. The structure reveals two Hsp70s--one delivering GR and the other scaffolding Hop. Unexpectedly, the Hop cochaperone interacts with all components of the complex including GR, poising Hsp90 for subsequent ATP hydrolysis. GR is partially unfolded and recognized via an extended binding pocket composed of Hsp90, Hsp70 and Hop, revealing the mechanism of GR loading and inactivation. Together with the GR maturation complex (Noddings et al., 2020), we present the first complete molecular mechanism of chaperone-dependent client remodeling, establishing general principles of client recognition, inhibition, transfer and activation.

biochemistry↗

bioRxiv2020

EfgA is a conserved formaldehyde sensor that halts bacterial translation in response to elevated formaldehyde

Normal cellular processes give rise to toxic metabolites that cells must mitigate. Formaldehyde is a universal stressor and potent metabolic toxin that is generated in organisms from bacteria to humans. Methylotrophic bacteria such as Methylorubrum extorquens face an acute challenge due to their production of formaldehyde as an obligate central intermediate of single-carbon metabolism. Mechanisms to sense and respond to formaldehyde were speculated to exist in methylotrophs for decades but had never been discovered. Here we identify a member of the DUF336 domain family, named efgA for enhanced formaldehyde growth, that plays an important role in endogenous formaldehyde stress response in M. extorquens PA1 and is found almost exclusively in methylotrophic taxa. Our experimental analyses reveal that EfgA is a formaldehyde sensor that inhibits translation in response to elevated levels of formaldehyde. Heterologous expression of EfgA in Escherichia coli increases formaldehyde resistance, indicating that its interaction partners are widespread and conserved and may include translational machinery. EfgA represents the first example of a formaldehyde stress response system that does not involve enzymatic detoxification. Thus, EfgA comprises a unique stress response mechanism in bacteria, whereby a single protein directly senses elevated levels of a toxic intracellular metabolite and modulates translational activity.

microbiology↗

bioRxiv2020

DYNC1LI2 regulates localization of the chaperone-mediated autophagy-receptor LAMP2A and improves cellular homeostasis in cystinosis

The dynein motor protein complex is required for retrograde transport but the functions of the intermediate-light chains that form the cargo-binding complex are not elucidated and the importance of individual subunits in the maintenance of cellular homeostasis is unknown. Here, using mRNA arrays and protein analysis, we show that the dynein subunit, intermediate chain 2 (DYNC1LI2) is downregulated in cystinosis, a lysosomal storage disorder caused by genetic defects in the lysosomal cystine transporter, cystinosin. Reconstitution of the expression of DYNC1LI2 in Ctns-/- cells re-established endolysosomal dynamics. Defective vesicular trafficking in cystinotic cells was rescued by DYNC1LI2 expression which correlated with decreased endoplasmic reticulum stress manifested as decreased expression levels of the chaperone Grp78. Mitochondrial fragmentation in cystinotic fibroblasts was also rescued by DYNC1LI2. Survival of cystinotic cells to oxidative stress insult was increased by DYNC1LI2 reconstitution but not by its paralog DYNC1LI1, which also failed to decrease ER stress levels and mitochondrial fragmentation. Restoring DYNC1LI2 expression rescued the localization of the chaperone-mediated autophagy receptor, LAMP2A, and restored cellular homeostasis of cystinotic proximal tubule cells, the primary cell type affected in cystinosis. DYNC1LI2 failed to rescue phenotypes in cystinotic cells when LAMP2A was downregulated or when co-expressed with dominant negative (DN) RAB7 or DN-RAB11, which impair LAMP2A trafficking. DYNC1LI2 emerges as a new target to repair underlying trafficking and CMA defects in cystinosis, a mechanism that is not restored by currently used lysosomal depletion therapies.

cell biology↗

bioRxiv2020

Broadly reactive influenza antibodies are not limited by germinal center competition with high affinity antibodies

Enhancing the generation of broadly reactive influenza antibodies is a pertinent goal towards developing a universal influenza vaccine. While antibodies that bind conserved influenza epitopes have been identified in humans, the frequency of these antibodies is typically very low. The predominant theory is that antibodies specific for conserved influenza epitopes are limited in germinal centers by competition with high affinity antibodies specific for the variable region of the virus. Here, we show that reducing germinal center formation and removing competition with high affinity antibodies was not sufficient to increase broadly reactive influenza antibodies or enhance protection against distinct influenza subtypes. These data disprove the prevailing hypothesis that broadly reactive influenza antibodies are rare due to competition in germinal centers. Additionally, levels of IgM antibodies specific for the variable region of HA persisted in mice in the absence of germinal centers, further demonstrating that immunodominance can be established independent of germinal centers. Our data also highlight the protective capacity of germinal center-independent IgM antibodies, which are not typically considered when testing correlates of protection, and offer an alternate target for delivering a universal influenza vaccine. IMPORTANCEIt is estimated that 250,000 - 650,000 individuals worldwide die each year from seasonal influenza infections. Current vaccines provide little protection against newly emerging strains. Thus, considerable effort is focused on enhancing the generation of broadly reactive influenza antibodies in order to develop a universal influenza vaccine. However, broadly reactive antibodies are rare and the factors that limit their generation are not completely understood. Our data disprove the prevailing hypothesis that broadly reactive influenza antibodies are rare due to completion in the germinal centers with antibodies to the variable HA head. Understanding the factors limiting antibodies specific for conserved regions of the influenza virus is imperative for developing a universal vaccine, which could potentially circumvent a global pandemic

immunology↗

bioRxiv2020

Identification of Candidate COVID-19 Therapeutics using hPSC-derived Lung Organoids

Summary ParagraphThe SARS-CoV-2 virus has caused already over 3.5 million COVID-19 cases and 250,000 deaths globally. There is an urgent need to create novel models to study SARS-CoV-2 using human disease-relevant cells to understand key features of virus biology and facilitate drug screening. As primary SARS-CoV-2 infection is respiratory-based, we developed a lung organoid model using human pluripotent stem cells (hPSCs) that could be adapted for drug screens. The lung organoids, particularly aveolar type II cells, express ACE2 and are permissive to SARS-CoV-2 infection. Transcriptomic analysis following SARS-CoV-2 infection revealed a robust induction of chemokines and cytokines with little type I/III interferon signaling, similar to that observed amongst human COVID-19 pulmonary infections. We performed a high throughput screen using hPSC-derived lung organoids and identified FDA-approved drug candidates, including imatinib and mycophenolic acid, as inhibitors of SARS-CoV-2 entry. Pre- or post-treatment with these drugs at physiologically relevant levels decreased SARS-CoV-2 infection of hPSC-derived lung organoids. Together, these data demonstrate that hPSC-derived lung cells infected by SARS-CoV-2 can model human COVID-19 disease and provide a valuable resource to screen for FDA-approved drugs that might be repurposed and should be considered for COVID-19 clinical trials.

microbiology↗

bioRxiv2020

ISG15 drives immune pathology and respiratory failure during viral infection

Cytokine storm during respiratory viral infection is an indicator of disease severity and poor prognosis. Type 1 interferon (IFN-I) production and signaling has been reported to be causal in cytokine storm-associated pathology in several respiratory viral infections, however, the mechanisms by which IFN-I promotes disease pathogenesis remain poorly understood. Here, using Usp18-deficient, USP18 enzymatic-inactive and Isg15-deficient mouse models, we report that lack of deISGylation during persistent viral infection leads to severe immune pathology characterized by hematological disruptions, cytokine amplification, lung vascular leakage and death. This pathology requires T cells but not T cell-intrinsic deletion of Usp18. However, lack of Usp18 in myeloid cells mimicked the pathological manifestations observed in Usp18-/- or Usp18C61A mice which were dependent on Isg15. We further mechanistically demonstrate that interrupting the ISGylation/deISGylation circuit increases extracellular levels of ISG15 which is accompanied by inflammatory neutrophil accumulation to the lung. Importantly, neutrophil depletion reversed morbidity and mortality in Usp18C61A mice. In summary, we reveal that the enzymatic function of Usp18 is crucial for regulating extracellular release of ISG15. This is accompanied by altered neutrophil differentiation, cytokine amplification and mortality following persistent viral infection. Moreover, our results suggest that extracellular ISG15 may drive the inflammatory pathology observed and could be both a prospective predictor of disease outcome and a therapeutic target during severe respiratory viral infections.

immunology↗

bioRxiv2019

A pivotal role for Nrf2 in endothelial detachment- implications for endothelial erosion of stenotic plaques

Endothelial erosion of atherosclerotic plaques and resulting thrombosis causes approximately 30% of acute coronary syndromes (ACS). As changes in the haemodynamic environment strongly influence endothelial function and contribute to plaque development, we reconstructed the coronary artery geometries of plaques with thrombi overlying intact fibrous caps from 17 ACS patients and performed computational fluid dynamic analysis. The results demonstrated that erosions frequently occur within areas of stenosis exposed to elevated flow. We recapitulated this flow environment in vitro, exposing human coronary artery endothelial cells to elevated flow and modelled smoking (a risk factor for erosion) by exposure to a combination of aqueous cigarette smoke extract and TNF. This treatment induced endothelial detachment, which increased with pharmacological activation of the antioxidant system controlled by transcription factor Nrf2 (encoded by NFE2L2). The expression of Oxidative Stress Growth INhibitor genes OSGIN1 and OSGIN2 increased under these conditions and also in the aortas of mice exposed to cigarette smoke. Sustained high level expression of OSGIN1+2 resulted in cell cycle arrest, induction of senescence, loss of focal adhesions and actin stress fibres, and dysregulation of autophagy. Overexpression of either Nrf2 or OSGIN1+2 induced cell detachment, which did not depend on apoptosis, and could be partially rescued by inhibition of HSP70 using VER-155008, or AMP kinase activation using metformin. These findings demonstrate that under elevated flow, smoking-induced hyperactivation of Nrf2 can trigger endothelial cell detachment, highlighting a novel mechanism that could contribute to ACS involving endothelial erosion overlying stenotic plaques.

cell biology↗

bioRxiv2018

RMI-chip: a microfluidics setup for functional imaging of microbial interactions with tree roots

Coupling microfludics with microscopy has emerged as a powerful approach to study at cellular resolution the dynamics in plant physiology and root-microbe interactions. Most devices have been designed to study the model plant Arabidopsis thaliana at higher throughput than conventional methods. However, there is a need for microfluidic devices which enable in vivo studies of root development and root-microbe interactions in woody plants. Here, we developed the RMI-chip, a simple microfluidic setup in which Populus tremuloides (aspen tree) seedlings can grow for over a month, allowing continuous microscopic observation of interactions between live roots and rhizobacteria. We find that the colonization of growing aspen roots by Pseudomonas fluorescens in the RMI-chip involves dynamic biofilm formation and dispersal, in keeping with previous observations in a different experimental set-up. Also, we find that whole-cell biosensors based on the rhizobacterium Bacillus subtilis can be used to monitor compositional changes in the rhizosphere but that the application of these biosensors is limited by their efficiency at colonizing aspen roots and persisting. These results indicate that functional imaging of dynamic root-bacteria interactions in the RMI-chip requires careful matching between the host plant and the bacterial root colonizer.

physiology↗

bioRxiv2018

Keratinocyte desmoglein 1 regulates the epidermal microenvironment and tanning response

Coordinated responses to environmental stimuli within the keratinocyte:melanocyte niche are poorly understood. Desmoglein 1 (Dsg1), a keratinocyte-specific desmosomal cell-cell adhesion protein with emerging signaling roles, is reduced by ultraviolet light radiation. Loss-of-function Dsg1 mutations elevate keratinocyte cytokines in Severe dermatitis, multiple Allergies, and Metabolic wasting (SAM) syndrome. We asked whether Dsg1 regulates keratinocyte:melanocyte paracrine communication to induce the tanning response. Dsg1-silenced keratinocytes increased Pro-opiomelanocortin mRNA and cytokine secretion. Melanocytes treated with conditioned media from Dsg1-silenced keratinocytes exhibited increased Mitf and Trp1 mRNA, melanin secretion, and dendrite length. Inhibiting the melanocyte pigment-associated melanocortin 1 receptor reduced pigment secretion in response to Dsg1-deficient conditioned media. Melanocytes incorporated into Dsg1-deficient human skin equivalents relocalized suprabasally, reminiscent of early melanoma pagetoid behavior. Dsg1 decreased in keratinocytes surrounding dysplastic nevi and early melanoma, but not benign nevi. We posit Dsg1 controls keratinocyte:melanocyte communication through paracrine signaling, which goes awry upon Dsg1 loss in melanoma development.

cell biology↗

bioRxiv2018

A cosine similarity-based method to infer variability of chromatin accessibility at the single-cell level

Cellular identity between generations of developing cells is propagated through the epigenome particularly via the accessible parts of the chromatin. It is now possible to measure chromatin accessibility at single-cell resolution using single-cell assay for transposase accessible chromatin (scATAC-seq), which can reveal the regulatory variation behind the phenotypic variation. However, single-cell chromatin accessibility data are sparse, binary, and high dimensional, leading to unique computational challenges. To overcome these difficulties, we developed PRISM a computational workflow and R package (https://github.com/stanleycai123/PRISM) that quantifies cell-to-cell chromatin accessibility variation while controlling for technical biases. Using data generated in our lab or publically available, we show that PRISM outperforms an existing algorithm, which relies on the aggregate of signal across a set of genomic regions. PRISM shows robustness to noise in low accessibility cells and reveals previously masked accessibility variation where accessible sites differ between cells but total number of accessible sites is constant. We also show that PRISM, but not an existing algorithm, finds suppressed heterogeneity of accessibility at CTCF binding sites. PRISM is a novel multidimensional scaling-based method using angular cosine distance metrics coupled with distance from the spatial centroid. PRISM takes differences in accessibility at each genomic region between single cells into account. This updated approach uncovers new biological results with profound implications on the cellular heterogeneity of chromatin architecture.

genomics↗

bioRxiv2017

Genomic responses to selection for tame/aggressive behaviors in the silver fox (Vulpes vulpes)

Animal domestications have led to a shared spectrum of striking behavioral and morphological changes. To recapitulate this process, silver foxes have been selectively bred for tame and aggressive behaviors for over 50 generations at the Institute for Cytology and Genetics in Novosibirsk, Russia. To understand the genetic basis and molecular mechanisms underlying the phenotypic changes, we profiled gene expression level and coding SNP allele frequencies in two brain tissues from 12 aggressive and 12 tame foxes. Expression analysis revealed 146 genes in prefrontal cortex and 33 genes in basal forebrain that were differentially expressed (5% FDR). These candidates include genes in key pathways known to be critical to neurological processing, including the serotonin and glutamate receptor pathways. In addition, 295 of the 31,000 exonic SNPs show significant allele frequency differences between tame and aggressive population (1% FDR), including genes with a role in neural crest cell fate determination.

evolutionary biology↗