Biology subjects

Gordon, D. E.

Publications and source records attributed to Gordon, D. E..

9 recordsLinked to original sources

bioRxiv2026

PQBP1 couples HIV-1 capsid recognition to cGAS recruitment through conformational remodeling

Pattern recognition receptors (PRRs) must selectively engage pathogen-derived signals to potentiate inflammation and antimicrobial responses. Polyglutamine-binding protein 1 (PQBP1) acts upstream of cyclic GMP-AMP synthase (cGAS) during HIV-1 infection through the recognition of viral capsid. However, the mechanism by which capsid binding enables cGAS recruitment remains unclear. As an intrinsically disordered protein, PQBP1 samples an ensemble of conformational states whose distribution depends on ligand engagement. Here we show that capsid engagement shifts this population toward a conformation competent for cGAS binding. Capsid binding at the N-terminus of PQBP1 redistributes conformational sampling within the WW domain and distal polar-rich domain (PRD). Alanine substitutions within these capsid-responsive regions retain capsid binding yet disrupt infection-dependent cGAS recruitment, indicating that capsid binding and cGAS recruitment depend on distinct regions of PQBP1. Together, these findings define a mechanism by which HIV-1 capsid engagement remodels PQBP1 into a cGAS-competent state, coupling capsid recognition to innate immune activation. IMPORTANCEPolyglutamine-binding protein 1 (PQBP1) initiates innate immune detection of HIV-1 by recognizing the incoming viral capsid. We show that capsid engagement reshapes PQBP1 conformational dynamics, and we identify distal regions required for infection-dependent cGAS association. These findings provide a mechanistic framework for how pathogen recognition is coupled to downstream innate immune activation.

molecular biology↗

bioRxiv2026

Interleukin-6 restricts pre-thymic T cell lineage commitment of progenitors driving loss of SIV control

Effective T cell reconstitution in people living with HIV is central to durable immune control and cure strategies. Sustained thymic output underpins T cell recovery and requires continuous seeding by T cell-committed progenitors originating in the bone marrow (BM). Using the SIV/rhesus macaque model, we identified a thymus-seeding progenitor (TSP; CD4-CD8-CD34CD38-CD7) in BM declining rapidly following SIV infection. This loss closely associated with reduction in T cell lineage committed differentiation of BM-derived hematopoietic stem and progenitor cells (HSPCs). Importantly, both the decline in TSPs and the impairment of pre-thymic T cell potential were strongly associated with early loss of viral control, independent of peripheral T cell dynamics. Plasma interleukin-6 (IL-6) levels robustly predicted the magnitude of TSP loss and the restriction of T cell-biased HSPC differentiation. Integrated transcriptomic and proteomic analyses revealed inflammatory imprinting of HSPCs characterized by activation of the IL-6-JAK-STAT axis, inflammasome engagement, and coordinated suppression of key T cell specification factors, including RUNX1, FYN, and ZAP70. In a nonanimal model of thymopoiesis, IL-6 exposure of rhesus macaque and human HSPCs inhibited their transition from DN1 (CD38-) to DN2 (CD38) TSP states, indicating an early block in T cell lineage commitment. Conversely, ex vivo IL-6 receptor blockade restored thymocyte differentiation to levels comparable to untreated controls. Collectively, these findings demonstrate that pathogenic inflammation restricts pre-thymic T cell development early after infection, directly contributing to loss of viral control. These findings have important implications for understanding the mediators of anti-viral T cell immunity and HIV cure.

immunology↗

bioRxiv2026

Microbiome-Derived Metabolites Shape CD4⁺ T-Cell Differentiations and Immune Aging in Chronic HIV-1 Infection

The role of aromatic gut-derived bacterial metabolites (GDBMs) in shaping immune cell metabolism and function remains poorly explored. Using ex vivo metabolomic profiling of paired plasma and CD4 T-cells from people living with HIV-1 (PLWH), we identified a network of aromatic GDBMs whose cell-associated abundance, rather than systemic levels, was linked to broad alterations in CD4 T-cell metabolic and functional states. Among these metabolites, p-cresol sulfate (PCS) emerged as a mechanistic prototype investigated in depth. Ex vivo flow cytometry and single-cell RNA sequencing of CD4 T-cells stratified by cell-associated PCS levels revealed dose-dependent enrichment of transcriptional programs associated with impaired differentiation capacity, regulatory-like identity, and cellular senescence. Consistently, in vitro transcriptomic and proteomic analyses of PCS-exposed CD4 T cells demonstrated induction of cell-cycle arrest, mitochondrial dysfunction, and senescence-associated programs, including upregulation of p16 and p21. Integration of these immunometabolic features with measurements of HIV-1 reservoir size in PLWH revealed that CD4 T-cell states defined by cell-associated GDBMs track with intact proviral DNA levels in vivo. Together, these findings define a microbiome-derived axis that reshapes CD4 T-cell metabolism and fate and promotes immune aging-associated states in PLWH. Our data suggest that cell-associated GDBMs may foster immunometabolic CD4 T-cell states previously linked to long-term HIV-1 reservoir persistence in vivo. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/699280v1_figa1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@faf44aorg.highwire.dtl.DTLVardef@1bc590aorg.highwire.dtl.DTLVardef@79c557org.highwire.dtl.DTLVardef@8b0a64_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract.C_FLOATNO PCS-driven metabolic reprogramming and senescence promoting CD4+ T-cell immune cell aging.Dietary proteins are metabolized by proteolytic gut microbiota into p-cresol, which is absorbed and converted in the liver to PCS. Circulating PCS accumulates in CD4 T-cells, where it activates the aryl hydrocarbon receptor (AhR). AhR signaling reduces glycolysis and mTOR activity, while enhancing TGF-, Wnt/-catenin, and TCF7 pathways, driving a regulatory-like and stem-like transcriptional program. These changes are associated with increased expression of p16 and p21, leading to cell cycle arrest and cellular senescence promoting CD4+ T-cell immune cell aging. C_FIG

systems biology↗

bioRxiv2025

A scalable proteogenomic framework for dissecting phospho-signaling pathways in primary immune cells

Signaling networks modulated by post-translational modifications orchestrate cellular responses to external cues. Traditional approaches to study these pathways lack the throughput to systematically capture the causal architecture of these signaling pathways at scale. Here, we present an integrated proteogenomic framework that combines saturating genetic perturbations with high-throughput proteomics to systematically map cytokine-induced signaling in primary human T cells. Supporting this framework is simplePhos, a streamlined, low-input phosphoproteomics workflow that enables scalable, time-resolved analysis without the requirement for specialized equipment or robotics. We extensively validate the simplePhos pipeline by applying inflammatory stimuli, including type I and II interferons, lipopolysaccharide, and Sendai virus to primary T cells and myeloid cells, establishing foundational datasets in these treatment contexts. Ultimately, using type I interferon signaling in genetically modified T cells as a model, we demonstrate that combined application of genetic alterations and proteomic analyses can map key signaling nodes in primary immune cells. This represents a powerful strategy to mechanistically interrogate phospho-signaling networks in human immune cells, with broad applications in translational immunology and therapeutic development.

systems biology↗

bioRxiv2024

Longitudinal proteomic analysis of T cell differentiation in vivo reveals dynamic changes of the interactome

CD8 T cell exhaustion impedes immune responses to cancer and chronic infections, and a biochemical understanding of exhaustion is essential to improving immunotherapy. Here, we present the first longitudinal protein abundance and phosphoproteomic analysis of antigen-specific CD8 T cells undergoing differentiation in vivo during acute (LCMV-Armstrong) and chronic (LCMV-Clone 13) infection. Comparing protein abundance across the two infection conditions identified over 180 known and novel exhaustion-associated proteins, including proteins missed by transcriptional analyses. Phosphoproteomic analysis identified >900 differentially regulated phosphosites on >400 proteins, including known inhibitory phosphosites on PD1, PAG1, SHP-1/PTPN6, SLAMF1/CD150. We also calculated phosphosite conservation across mammals, to direct follow-up studies towards sites with likely essential function. Lastly, our analysis uncovers exhaustion-associated kinases with clinical-stage inhibitors, underscoring the translational utility of our dataset to guide immunotherapy development. Together, our datasets define a biochemical atlas of T cell exhaustion in vivo, shedding light on the molecular mechanisms of T cell dysfunction.

immunology↗

bioRxiv2020

SARS-CoV-2 ORF9c Is a Membrane-Associated Protein that Suppresses Antiviral Responses in Cells

Disrupted antiviral immune responses are associated with severe COVID-19, the disease caused by SAR-CoV-2. Here, we show that the 73-amino-acid protein encoded by ORF9c of the viral genome contains a putative transmembrane domain, interacts with membrane proteins in multiple cellular compartments, and impairs antiviral processes in a lung epithelial cell line. Proteomic, interactome, and transcriptomic analyses, combined with bioinformatic analysis, revealed that expression of only this highly unstable small viral protein impaired interferon signaling, antigen presentation, and complement signaling, while inducing IL-6 signaling. Furthermore, we showed that interfering with ORF9c degradation by either proteasome inhibition or inhibition of the ATPase VCP blunted the effects of ORF9c. Our study indicated that ORF9c enables immune evasion and coordinates cellular changes essential for the SARS-CoV-2 life cycle. One-sentence summarySARS-CoV-2 ORF9c is the first human coronavirus protein localized to membrane, suppressing antiviral response, resembling full viral infection.

bioinformatics↗

bioRxiv2020

A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing

An outbreak of the novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 290,000 people since the end of 2019, killed over 12,000, and caused worldwide social and economic disruption1,2. There are currently no antiviral drugs with proven efficacy nor are there vaccines for its prevention. Unfortunately, the scientific community has little knowledge of the molecular details of SARS-CoV-2 infection. To illuminate this, we cloned, tagged and expressed 26 of the 29 viral proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), which identified 332 high confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 existing FDA-approved drugs, drugs in clinical trials and/or preclinical compounds, that we are currently evaluating for efficacy in live SARS-CoV-2 infection assays. The identification of host dependency factors mediating virus infection may provide key insights into effective molecular targets for developing broadly acting antiviral therapeutics against SARS-CoV-2 and other deadly coronavirus strains.

systems biology↗

bioRxiv2020

Efficient Generation of Isogenic Primary Human Myeloid Cells using CRISPR-Cas9 Ribonucleoproteins

Genome engineering of primary human cells with CRISPR-Cas9 has revolutionized experimental and therapeutic approaches to cell biology, but human myeloid-lineage cells have remained largely genetically intractable. We present a method for delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes by nucleofection directly into CD14+ human monocytes purified from peripheral blood, leading to high rates of precise gene knockout. These cells can be efficiently differentiated into monocyte-derived macrophages or dendritic cells. This process yields genetically-edited cells that retain critical markers of both myeloid differentiation and phagocytic function. Genetic ablation of the restriction factor SAMHD1 increased HIV-1 infection more than fifty-fold, demonstrating the power of this system for genotype-phenotype interrogation. This fast, flexible and scalable platform can be used for genetic studies of human myeloid cells in immune signaling, inflammation, cancer immunology, host-pathogen interactions, and beyond, and could facilitate development of novel myeloid cellular therapies.

genetics↗

bioRxiv2020

Calcium Sensors ALG-2 And Peflin Bind ER Exit Sites In Alternate States To Modulate Secretion In Response To Calcium Signaling

ER-to-Golgi transport is the first step in the constitutive secretory pathway which, unlike regulated secretion, is believed to proceed non-stop regardless of Ca2+ flux. Rowever, here we demonstrate that penta-EF hand (PEF) proteins ALG-2 and peflin constitute a hetero-bifunctional COPII regulator that responds to Ca2+ signaling by adjusting the ER export rate of COPII-sorted cargos up or down by ~50%. At steady-state Ca2+, ALG-2/peflin hetero-complexes bind to ER exit sites (ERES) through the ALG-2 subunit to confer a low, buffered secretion rate, while peflin-lacking ALG-2 complexes markedly stimulate secretion. During Ca2+ signaling, ALG-2 complexes lacking peflin can either increase or decrease the secretion rate depending on signaling intensity and duration-phenomena that could contribute to cellular growth and intercellular communication, following secretory increases, or protection from excitotoxicity and infection following decreases. In epithelial normal rat kidney (NRK) cells, the Ca2+-mobilizing agonist ATP causes ALG-2 to depress ER export, while in neuroendocrine PC12 cells, Ca2+ mobilization by ATP results in ALG-2-dependent enhancement of secretion. Within the NRK cell model, distinct Ca2+ signaling patterns can produce opposing ALG-2-dependent effects on secretion. Mechanistically, ALG-2-dependent depression of secretion involves decreased COPTT outer shell and increased peflin targeting to ERES, while ALG-2-dependent enhancement of secretion involves increased COPTT outer shell and decreased peflin at ERES. PEF protein complexes represent a true regulator of transport as they are dispensable for secretion yet adjust the secretion rate to physiological conditions. Their dynamics affects secretion of important physiological cargoes such as collagen T and significantly impacts ER stress.

cell biology↗