Biology subjects

English, J. G.

Publications and source records attributed to English, J. G..

9 recordsLinked to original sources

bioRxiv2026

Protease-Activated Receptor 1 as an Endogenous Model of Peptidergic Gαq-Gα12-Biased G Protein Signaling

G protein-coupled receptors (GPCRs) are the most widely targeted class of signaling proteins, comprising ~30% of FDA-approved drugs. Their therapeutic potential arises from their ability to translate diverse extracellular cues into intracellular signals via G proteins, arrestins, and other effectors. This signaling versatility creates opportunities for functional selectivity, where ligands preferentially engage particular pathways. However, few endogenous receptor systems display well-defined ligand-dependent divergence across multiple signaling levels. Protease-activated receptor 1 (PAR1) is a candidate model. Thrombin canonically cleaves PAR1 at Arg41, whereas activated protein C (aPC) has been reported to cleave PAR1 at Arg46 in endothelial and co-receptor-supported settings, creating distinct tethered peptide ligands. Thrombin cleavage drives canonical Gq- and G12/13-dependent prothrombotic and barrier-disruptive signaling, whereas aPC cleavage has been associated with anticoagulant, cytoprotective, and anti-inflammatory signaling. However, PAR1s transducer-wide coupling profile, transcriptional consequences, and physiological outputs remain incompletely characterized. We integrated transducer-wide biosensor assays (TRUPATH, TGF shedding, PRESTO-Tango), analysis of a PAR1 {+/-} thrombin TRE-MPRA dataset followed by targeted TRE dual-luciferase validation, and platelet activation and calcium flux studies in primary human platelets to define how protease identity reshapes signaling from proximal transducer engagement to physiological output. Thrombin produced robust PAR1 coupling to Gq and G12, whereas aPC produced detectable coupling only to G12. Neither protease generated detectable {beta}-arrestin-2 recruitment in PRESTO-Tango. Both proteases supported dose-dependent TGF shedding that was insensitive to FR900359. A PAR1 {+/-} thrombin TRE-MPRA dataset identified thrombin-responsive transcriptional response elements and nominated NF{kappa}B1 and THRB for targeted follow-up. Luciferase assays showed NF{kappa}B1 was thrombin-induced and FR900359-sensitive, whereas THRB was induced by both thrombin and aPC and was FR900359-insensitive. In primary human platelets, thrombin, but not aPC, induced P-selectin expression and a calcium response. Thrombin responses were suppressed by FR900359, supporting a requirement for Gq in these platelet activation markers. Together, these findings support PAR1 as an endogenous model of protease-dependent functional selectivity that, in our heterologous assay systems, separates signaling along a Gq-versus-G12 axis, thus providing a framework for future technologies, such as high-throughput tethered-peptide evolution platforms, to understand the principles of G protein selectivity across GPCRs.

cell biology↗

bioRxiv2026

Unraveling β2-Adrenergic Receptor Signaling Bias Between Gαs and Gα15 in Shaping Macrophage Function

Myocardial infarction and heart failure are leading global causes of mortality. Chronic {beta}-adrenergic receptor ({beta}AR) activation in cardiomyocytes promotes heart failure via Gs signaling after myocardial infarction, whereas {beta}2AR activation may also provide cardiac protection and repair through alternative pathways. Macrophages play a pivotal role in cardiac repair, and {beta}2AR has been reported to signal via the hematopoietic-specific G15 in these cells. We aimed to characterize signaling bias between Gs and G15 downstream of {beta}2AR and to elucidate their roles in macrophage polarization. Using TRUPATH triple assays, we observed that several {beta}2AR agonists activate G15 with at least an order of magnitude greater potency than Gs in this system. In addition, clinically used {beta}-blockers may exhibit differential inhibition on these two pathways. Macrophages are briefly classified into M1 and M2 polarization according to their activating stimuli and functional properties. Transcriptomic profiling of THP-1-derived macrophage-like cells treated with the {beta}2AR agonist clenbuterol revealed enrichment of M1 transcriptional profile and repair-related hallmarks. Knockdown of Gs showed M1 enrichment, whereas G15 knockdown was associated with negative M2 enrichment as well as M1 enrichment. Loss of either Gs or G15 negatively affected repair-associated hallmarks. In contrast, pharmacological intervention of the Gs-cAMP signaling produced opposing M1/M2 transcriptional responses, while suppressing repair-associated hallmarks. These in vitro findings explore the distinct pharmacological profiles of {beta}2AR ligands toward Gs and G15 and reveal how {beta}2AR agonism may modulate macrophage function through dual-transducer signaling.

cell biology↗

bioRxiv2026

Designing Rigid Protein Fiducials to Visualize GPCR Conformational States

G-protein coupled receptors (GPCRs) mediate precise ligand-specific signaling profiles, yet structural visualization of how ligands alter receptor conformational landscapes in the absence of signaling partners or mimetics has proven incredibly challenging. Here we show that by combining generative protein design with deep-learning based conformational ensemble prediction we can reliably design fiducial markers to facilitate cryogenic electron microscopy (cryoEM) of GPCRs at arbitrary fusion points, enabling the visualization of previously intractable states. We validate the approach with high-throughput determination of inactive state structures of four pharmaceutically relevant GPCRs, allowing for key details of receptor pharmacology to be resolved in each case. We then engineered an extracellular fiducial marker for the prototypical {beta}2-adrenergic receptor that enabled direct structural characterization of the rearrangement of key intracellular motifs in the absence of G-protein. Comparison with recent co-folding models highlights gaps in current methods for predicting ligand-induced GPCR conformational changes. These results present a generalizable framework for accessing traditionally inaccessible structural states of small, dynamic proteins.

biophysics↗

bioRxiv2025

ConSeqUMI, an error-free nanopore sequencing pipeline to identify and extract individual nucleic acid molecules from heterogeneous samples

Nanopore sequencing has revolutionized genetic analysis by offering linkage information across megabase-scale genomes. However, the high intrinsic error rate of nanopore sequencing impedes the analysis of complex heterogeneous samples, such as viruses, bacteria, complex libraries, and edited cell lines. Achieving high accuracy in single-molecule sequence identification would significantly advance the study of diverse genomic populations, where clonal isolation is traditionally employed for complete genomic frequency analysis. Here, we introduce ConSeqUMI, an innovative experimental and analytical pipeline designed to address long-read sequencing error rates using unique molecular indices for precise consensus sequence determination. ConSeqUMI processes nanopore sequencing data without the need for reference sequences, enabling accurate assembly of individual molecular sequences from complex mixtures. We establish robust benchmarking criteria for this platforms performance and demonstrate its utility across diverse experimental contexts, including mixed plasmid pools, recombinant adeno-associated virus genome integrity, and CRISPR/Cas9-induced genomic alterations. Furthermore, ConSeqUMI enables detailed profiling of human pathogenic infections, as shown by our analysis of SARS-CoV-2 spike protein variants, revealing substantial intra-patient genetic heterogeneity. Lastly, we demonstrate how individual clonal isolates can be extracted directly from sequencing libraries at low cost, allowing for post-sequencing identification and validation of observed variants. Our findings highlight the robustness of ConSeqUMI in processing sequencing data from UMI-labeled molecules, offering a critical tool for advancing genomic research. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/647077v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1a81075org.highwire.dtl.DTLVardef@d07f82org.highwire.dtl.DTLVardef@634beborg.highwire.dtl.DTLVardef@10d4b25_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Molecular basis of proton-sensing by G protein-coupled receptors

Three proton-sensing G protein-coupled receptors (GPCRs), GPR4, GPR65, and GPR68, respond to changes in extracellular pH to regulate diverse physiology and are implicated in a wide range of diseases. A central challenge in determining how protons activate these receptors is identifying the set of residues that bind protons. Here, we determine structures of each receptor to understand the spatial arrangement of putative proton sensing residues in the active state. With a newly developed deep mutational scanning approach, we determined the functional importance of every residue in proton activation for GPR68 by generating [~]9,500 mutants and measuring effects on signaling and surface expression. This unbiased screen revealed that, unlike other proton-sensitive cell surface channels and receptors, no single site is critical for proton recognition in GPR68. Instead, a network of titratable residues extend from the extracellular surface to the transmembrane region and converge on canonical class A GPCR activation motifs to activate proton-sensing GPCRs. More broadly, our approach integrating structure and unbiased functional interrogation defines a new framework for understanding the rich complexity of GPCR signaling. One-sentence summaryThe protonation networks governing activation of human pH-sensing GPCRs are uncovered by integrative cryo-EM and deep mutational scanning.

biochemistry↗

bioRxiv2023

A Biotin Targeting Chimera (BioTAC) System to Map Small Molecule Interactomes in situ

Unbiased chemical biology strategies for direct readout of protein interactome remodelling by small molecules provide advantages over target-focused approaches, including the ability to detect previously unknown targets, and the inclusion of chemical off-compete controls leading to high-confidence identifications. We describe the BioTAC system, a small-molecule guided proximity labelling platform, to rapidly identify both direct and complexed small molecule binding proteins. The BioTAC system overcomes a limitation of current approaches, and supports identification of both inhibitor bound and molecular glue bound complexes.

pharmacology and toxicology↗

bioRxiv2023

Discovery and Validation of Context-Dependent Synthetic Mammalian Promoters

Cellular transcription enables cells to adapt to various stimuli and maintain homeostasis. Transcription factors bind to transcription response elements (TREs) in gene promoters, initiating transcription. Synthetic promoters, derived from natural TREs, can be engineered to control exogenous gene expression using endogenous transcription machinery. This technology has found extensive use in biological research for applications including reporter gene assays, biomarker development, and programming synthetic circuits in living cells. However, a reliable and precise method for selecting minimally-sized synthetic promoters with desired background, amplitude, and stimulation response profiles has been elusive. In this study, we introduce a massively parallel reporter assay library containing 6184 synthetic promoters, each less than 250 bp in length. This comprehensive library allows for rapid identification of promoters with optimal transcriptional output parameters across multiple cell lines and stimuli. We showcase this librarys utility to identify promoters activated in unique cell types, and in response to metabolites, mitogens, cellular toxins, and agonism of both aminergic and non-aminergic GPCRs. We further show these promoters can be used in luciferase reporter assays, eliciting 50-100 fold dynamic ranges in response to stimuli. Our platform is effective, easily implemented, and provides a solution for selecting short-length promoters with precise performance for a multitude of applications.

synthetic biology↗

bioRxiv2019

Deschloroclozapine: a potent and selective chemogenetic actuator enables rapid neuronal and behavioral modulations in mice and monkeys

The chemogenetic technology Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) affords remotely reversible control of cellular signaling, neuronal activity and behavior. Although the combination of muscarinic-based DREADDs with clozapine-N-oxide (CNO) has been widely used, sluggish kinetics, metabolic liabilities, and potential off-target effects of CNO represent areas for improvement. Here we provide a new high affinity and selective agonist deschloroclozapine (DCZ) for muscarinic-based DREADDs. Positron emission tomography revealed that DCZ selectively bound to and occupied DREADDs in both mice and monkeys. Systemic delivery of low doses of DCZ (1 or 3 g/kg) enhanced neuronal activity via hM3Dq within minutes in mice and monkeys. Intramuscular injections of DCZ (100 g/kg) reversibly induced spatial working memory deficits in monkeys expressing hM4Di in the prefrontal cortex. DCZ represents the most potent, selective, metabolically stable and fast-acting DREADD agonist reported with utility in both mice and non-human primates for a variety of applications.

neuroscience↗

bioRxiv2017

Cell-type selective deletion of RSK2 reveals insights into altered signaling in Coffin-Lowry Syndrome

Coffin-Lowry syndrome (CLS) is an X-linked syndromic form of mental retardation characterized by various skeletal dysmorphisms, moderate to severe mental retardation, and in some cases, psychosis. CLS is caused by loss-of-function mutations of the p90 ribosomal S6 kinase 2 (RPS6KA3) gene encoding a growth factor-regulated serine/threonine kinase, ribosomal S6 kinase 2 (RSK2). We previously identified RSK2 as a novel interacting protein that tonically inhibits 5-HT2A receptor signaling by phosphorylating Ser-314 within the third intracellular loop. To determine if RSK2 inhibits 5-HT2A receptor signaling in vivo and whether disruption of RSK2 could lead to schizophrenia-like behaviors - as is seen in some CLS patients - we genetically disrupted the function of RSK2 either globally or selectively in forebrain pyramidal neurons in mice. Both global and forebrain-selective RSK2 deletion augmented the locomotor responses to the psychotomimetic drugs phencyclidine (PCP) and amphetamine (AMPH). Significantly, forebrain-selective deletion of RSK2 augmented 5-HT2A receptor signaling as exemplified by enhanced 5-HT2A-mediated c-fos activation and head-twitch response without altering the levels or distribution of 5-HT2A receptor protein. Thus, RSK2 modulates 5HT2A receptor function in vivo, and disruption of RSK2 leads to augmented psychostimulant-induced responses reminiscent of those seen in many animal models of schizophrenia. These findings strengthen the association between 5-HT2A receptor dysfunction and psychosis, and provide a potential mechanism underlying the schizophrenia-like symptoms present in some CLS patients.\n\nHighlightsO_LIGlobal and cell-type-specific RSK2 knock-out mice were assessed behaviorally and pharmacologically\nC_LIO_LIAugmentation of amphetamine and PCP locomotor responses were seen in both global and forebrain-specific RSK2 KO mice\nC_LIO_LIAugmentation of 5-HT2A serotonin receptor function but not number was also observed\nC_LIO_LIThese alterations reveal insights into mechanisms potentially responsible for behavioral sequlae of Coffin-Lowry Syndrome\nC_LI

neuroscience↗