Biology subjects

Kelly, B.

Publications and source records attributed to Kelly, B..

12 recordsLinked to original sources

bioRxiv2026

The function of human PIF1 in G quadruplex formation and replication stress response at ALT telomeres

Cancers maintain their telomeres through two telomere maintenance mechanisms: 85-90% of cancers rely on telomerase (TEL+), while 10-15% of cancers adopt the Alternative Lengthening of Telomeres (ALT) pathway. The Break-Induced Replication (BIR) pathway plays a critical role in maintaining telomere length in the ALT+ cells. In both yeast and human, PIF1, a 5' to 3' helicase, is required for the robust activity of BIR. However, the extent of human PIF1 (hPIF1) involvement in the ALT pathway remains unknown. Here we showed that hPIF1 can be recruited to damaged telomeres in ALT+ cells. In addition, we demonstrated that inhibition of hPIF1 induced DNA damage and G quadruplex (G4) accumulation at ALT telomeres, leading to a moderate reduction of the mean telomere length. Most interestingly, we demonstrated that inhibition of hPIF1 also attenuates checkpoint activation, BLM recruitment, single-stranded DNA (ssDNA) formation, DNA damage, and G4s at telomeres in the FANCM deficient ALT+ cells. Finally, we showed that inactivation of hPIF1 affects the viability of both ALT+ and TEL+ cancers, suggesting that hPIF1 is a potential drug target for cancer therapy.

molecular biology↗

bioRxiv2026

Distinct control of T cell proliferation and effector function by partitioning of intracellular sulfur from cysteine

Delineating how acquired nutrients are partitioned into different intracellular pathways, and how these various fates support distinct functions in T cells is limited. We show that CD8+ T cells acquire cysteine to serve both as a substrate for glutathione (GSH) production, which modulates effector functions, and to cede its sulfur for NFS1-dependent FeS-cluster synthesis, which supports proliferation. NFS1 deletion in activated CD8+ T cells promotes exhaustion and dampens anti-cancer immunity, while blocking cysteine flux into GSH, or enforcing FeS metabolism, enhance tumor control. This role for disrupted FeS metabolism in T cell exhaustion is echoed in data from human HCC. Elucidating how different intracellular pathways use cysteine enables targeted control of cysteine flux to retain beneficial effects of cysteine while abolishing those that restrain function. We illustrate this concept for one metabolite, cysteine, but it is likely to apply to other metabolites relevant for immune cell function.

immunology↗

bioRxiv2026

TET2 and TP53 Mutations Cooperatively Modulate the Response to Inflammation to Promote Leukemic Transformation

TET2 is a commonly mutated gene in hematologic malignancies, including as an initiating event in clonal hematopoiesis (CH). Its mutation alters hematopoietic self-renewal, differentiation, and systemic inflammation responses. TP53 mutations co-occur with TET2 mutations and are also observed in patients with high-risk clonal hematopoiesis and hematologic malignancies. Using a murine model, we found that HSPCs with both mutations initially promoted a myeloproliferative phenotype. Over time these double mutant HSPCs acquire additional genomic alternations, leading to disease progression to acute leukemias including B-ALL. We observed enhanced inflammatory signatures at transformation and identified NLRP1 as a target of TP53 activation. Decreased response to an inflammatory cell death pathway in the setting of TP53 mutation allows cells to tolerate inflammatory stress. This pathway also modifies response to chemotherapies that induce protein translational stalling. Our results identify a hematopoietic stem cell stress response pathway with implications on adaptation to inflammation and chemotherapy tolerance. SignificanceTET2 and TP53 mutations co-operate leading to advanced hematologic malignancy. TET2 mutations promote an inflammatory environment and TP53 mutation supports tolerance to this inflammatory stress.

cancer biology↗

bioRxiv2025

Glutamine-Dependent Slc25a39-Nrf2 Axis Couples Mitochondrial Dynamics with Metabolic Reprogramming to Establish Myogenic Commitment

Myogenic commitment is a decisive and irreversible step in skeletal muscle regeneration, necessitating proliferating myoblasts to integrate metabolic cues with nuclear transcriptional programs. Among amino acids, glutamine is uniquely positioned to influence this transition by coupling energy production to macromolecule biosynthesis and epigenetic regulation. We reasoned that myoblasts must sense glutamine availability to ensure orderly progression toward commitment, and we tested this by examining the molecular consequences of acute glutamine withdrawal. We find that continued glutamine oxidation is required to sustain glycolysis, maintain mitochondrial fission, and preserve a redox balance that supports progression towards myogenic commitment. In its absence, myoblasts undergo a reductive shift, characterized by mitochondrial elongation, membrane depolarization, and suppression of glycolysis, ultimately leading to growth arrest. Transcriptomic profiling reveals reduced MyoD and MKi67, accompanied by increased Sprouty1 levels, defining a reversible non-proliferative state that resembles but is distinct from quiescent and reserve cells. We term this state Poised Metabolic Arrest (PMA), a cellular response to glutamine limitation during myogenic progression. Mechanistically, PMA is driven by Nrf2-dependent increased glutathione (GSH) biosynthesis and upregulation of mitochondrial GSH carrier Slc25a39 when glutamine is limited. Depleting mitochondrial glutathione or silencing Slc25a39 forces exit from PMA. However, this premature exit compromises subsequent differentiation potential, indicating PMA serves to preserve differentiation competence when glutamine is limited. Consistent with this, both loss and overexpression of Slc25a39 impair myoblast differentiation in vitro and disrupt regeneration in vivo. Together, these data suggest that a reciprocal Slc25a39-Nrf2 redox axis functions as a nutrient-dependent checkpoint, coupling glutamine availability to mitochondrial remodeling and metabolic reprogramming, necessary to establish irreversible myogenic commitment.

biochemistry↗

bioRxiv2025

Missing data in single-cell transcriptomes reveals transcriptional shifts

Profiling thousands of single cell transcriptomes is routine, yet cell prioritization based on response to biological perturbations is challenging and confounded by clustering, normalization and dimensionality reduction strategies. We developed a scoring approach independent of these obstacles that unbiasedly identifies distinct transcriptomes within a set based on missing data patterns, allowing cell prioritization and feature selection for downstream analysis. Our method applied to D. discoideum reveals a metabolic shift that marks the transition between the amoeboid and aggregated states of this model organism.

bioinformatics↗

bioRxiv2025

REMEDY: REpair of heterozygous Mutations independent of Exogenous Donor template with high efficiency using allele specific CRISPR targeting and HDR enhancers

We report the development of REMEDY (REpair of heterozygous Mutations independent of Exogenous Donor template with high efficiencY), a genome editing strategy that allows efficient repair of heterozygous mutations in human and mouse cells without necessitating an exogenous donor DNA template. Here, we used in-PAM or near-PAM CRISPR strategies to induce a double-strand break (DSB) in mutant alleles. Following the DSB, the wild-type homologous chromosome itself serves as an endogenous DNA donor template and initiates the correction of the mutant allele. Concurrently treating the cells with HDR enhancers, such as AZD7648, further improved the efficiency of the correction. We demonstrated the utility of REMEDY in the context of six different diseases with heterozygous mutations such as IBMPFD, cystic fibrosis, progeria, ITPR3-associated combined immunodeficiency, ACTA1, and TBCD in human patient derived primary cells and complementary mouse model cell lines. O_FIG O_LINKSMALLFIG WIDTH=91 HEIGHT=200 SRC="FIGDIR/small/641201v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@5f6c41org.highwire.dtl.DTLVardef@bc50d5org.highwire.dtl.DTLVardef@15edca2org.highwire.dtl.DTLVardef@e74ada_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

genetics↗

bioRxiv2024

Untangling structural molecular details of the endocytic adaptor protein CALM upon binding with phosphatidylinositol 4,5-bisphosphate-containing model membranes

Clathrin assembly lymphoid myeloid leukemia protein (CALM) is involved in the formation of clathrin-mediated endocytic coats by virtue of binding many proteins involved in the process, including clathrin itself and AP2 cargo adaptor complex. CALM is able to specifically recognize the inner leaflet of the plasma membrane by binding the membranes phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Here, a biophysical approach, primarily using neutron and X-ray scattering and solid-state NMR experiments, was exploited to investigate CALM interaction with PtdIns(4,5)P2-presenting model membranes. The presented experimental data reveal how the CALM folded domain is partly accommodated within the lipid membrane, directly interacting with PtdIns(4,5)P2 phosphates. Moreover, these data suggest that CALMs amphiphilic N-terminal helix buries into the membrane, not only stabilising the protein docking to the membrane but also providing a mechanism to induce membrane curvature.

biophysics↗

bioRxiv2023

Differences in white matter detected by ex vivo 9.4T MRI are associated with axonal changes in the R6/1 model of Huntington's Disease

White matter (WM) volume loss has been reported in people with Huntingtons disease (HD), but the cellular basis of this deficit remains to be elucidated. To address this, we assessed ex vivo WM microstructure in the transgenic R6/1 mouse model of HD with magnetic resonance imaging (MRI) and studied the neurobiological basis of the MRI brain signals with histological and electron microscopy analyses in a separate cohort of age- and sex-matched mice. Differences in the macromolecular proton fraction (MPF) from quantitative magnetization transfer (qMT) as a proxy myelin measure, and the intra-axonal signal fraction (FR) from the composite hindered and restricted model of diffusion (CHARMED) as a proxy marker of axon density, were assessed alongside diffusion tensor imaging (DTI) parameters. A tractometry approach was employed to inspect region-specific differences across the corpus callosum (CC). Furthermore, voxel-based morphometry (VBM) and tract-based spatial statistics (TBSS) were used to explore brain-wise WM macro- and microstructure abnormalities. To gain insight into disease-associated impairments in attentional and visuospatial processing, a third cohort of age-matched mice was assessed with the 5-choice serial reaction time task (5-CSRTT). We report cognitive impairments in R6/1 mice and, by evaluating MRI and light and electron microscopy results, we show that this HD mouse model presents disruptions in axonal morphology (i.e. less complex, thinner axons) and organization (i.e. more densely packed axons). Furthermore, we show that, at least early in disease progression, R6/1 mice present a reduction in the expression or content of myelin-associated proteins without significant alterations in the structure of myelin sheaths. Finally, our findings indicate that neuroinflammation-driven glial and axonal swelling might also affect this mouse model early in disease progression. Crucially, we demonstrate the potential of FR, an in vivo estimate of axon density, as a novel MRI biomarker of HD-associated changes in WM microstructure.

neuroscience↗

bioRxiv2023

Prostaglandin E2 controls the metabolic adaptation of T cells to the intestinal microenvironment

Immune cells must adapt to different environments during the course of an immune response. We studied the adaptation of CD8+ T cells to the intestinal microenvironment and how this process shapes their residency in the gut. CD8+ T cells progressively remodel their transcriptome and surface phenotype as they acquire gut residency, and downregulate expression of mitochondrial genes. Human and mouse gut-resident CD8+ T cells have reduced mitochondrial mass, but maintain a viable energy balance to sustain their function. We found that the intestinal microenvironment is rich in prostaglandin E2 (PGE2), which drives mitochondrial depolarization in CD8+ T cells. Consequently, these cells engage autophagy to clear depolarized mitochondria, and enhance glutathione synthesis to scavenge reactive oxygen species (ROS) that result from mitochondrial depolarization. Impairing PGE2 sensing promotes CD8+ T cell accumulation in the gut, while tampering with autophagy and glutathione negatively impacts the T cell population. Thus, a PGE2-autophagy-glutathione axis defines the metabolic adaptation of CD8+ T cells to the intestinal microenvironment, to ultimately influence the T cell pool.

immunology↗

bioRxiv2022

A cell cycle-linked mechanism for the glutamine driven establishment of stem cell fate

The cell cycle offers a unique opportunity for stem cells to sample metabolic and signaling cues to establish cell fate. Molecular pathways that integrate and convey these signals to cell cycle machinery to license cell fate transitions and drive terminal differentiation remain unknown. Here, we describe a signaling role of mitochondrial glutamine metabolism in driving exit from cell cycle-linked self-renewal to generate differentiation competent progenitors. In proliferating stem cells, mitochondrial glutamine metabolism opposes the WDR5-linked self-renewal network via acetylation and nuclear translocation of its upstream regulator, PASK. Nuclear PASK disrupts the mitotic WDR5-anaphase-promoting complex (APC/C) interaction to drive exit from self-renewal. Consistent with these roles, loss of PASK or inhibition of glutamine metabolism preserves stemness in vitro and in vivo during muscle regeneration. Our results suggest a mechanism whereby the proliferative functions of glutamine metabolism are co- opted by stem cells to establish cell fate.

cell biology↗

bioRxiv2020

Sex-Dependent Shared and Non-Shared Genetic Architecture Across Mood and Psychotic Disorders

BACKGROUNDSex differences in incidence and/or presentation of schizophrenia (SCZ), major depressive disorder (MDD), and bipolar disorder (BIP) are pervasive. Previous evidence for shared genetic risk and sex differences in brain abnormalities across disorders suggest possible shared sex-dependent genetic risk. METHODSWe conducted the largest to date genome-wide genotype-by-sex (GxS) interaction of risk for these disorders, using 85,735 cases (33,403 SCZ, 19,924 BIP, 32,408 MDD) and 109,946 controls from the Psychiatric Genomics Consortium (PGC) and iPSYCH. RESULTSAcross disorders, genome-wide significant SNP-by-sex interaction was detected for a locus encompassing NKAIN2 (rs117780815; p=3.2x10-8), that interacts with sodium/potassium-transporting ATPase enzymes implicating neuronal excitability. Three additional loci showed evidence (p<1x10-6) for cross-disorder GxS interaction (rs7302529, p=1.6x10-7; rs73033497, p=8.8x10-7; rs7914279, p=6.4x10-7) implicating various functions. Gene-based analyses identified GxS interaction across disorders (p=8.97x10-7) with transcriptional inhibitor SLTM. Most significant in SCZ was a MOCOS gene locus (rs11665282; p=1.5x10-7), implicating vascular endothelial cells. Secondary analysis of the PGC-SCZ dataset detected an interaction (rs13265509; p=1.1x10-7) in a locus containing IDO2, a kynurenine pathway enzyme with immunoregulatory functions implicated in SCZ, BIP, and MDD. Pathway enrichment analysis detected significant GxS of genes regulating vascular endothelial growth factor (VEGF) receptor signaling in MDD (pFDR<0.05). CONCLUSIONSIn the largest genome-wide GxS analysis of mood and psychotic disorders to date, there was substantial genetic overlap between the sexes. However, significant sex-dependent effects were enriched for genes related to neuronal development, immune and vascular functions across and within SCZ, BIP, and MDD at the variant, gene, and pathway enrichment levels.

genetics↗

bioRxiv2018

Stable networks of water-mediated interactions are conserved in activation of diverse GPCRs

G protein-coupled receptors (GPCRs) have evolved to recognize incredibly diverse extracellular ligands while sharing a common architecture and structurally conserved intracellular signaling partners. It remains unclear how binding of diverse ligands brings about GPCR activation, the common structural change that enables intracellular signaling. Here, we identify highly conserved networks of water-mediated interactions that play a central role in activation. Using atomic-level simulations of diverse GPCRs, we show that most of the water molecules in GPCR crystal structures are highly mobile. Several water molecules near the G protein-coupling interface, however, are stable. These water molecules form two kinds of polar networks that are conserved across diverse GPCRs: (i) a network that is maintained across the inactive and the active states and (ii) a network that rearranges upon activation. Comparative analysis of GPCR crystal structures independently confirms the striking conservation of water-mediated interaction networks. These conserved water-mediated interactions near the G protein-coupling region, along with diverse water-mediated interactions with extracellular ligands, have direct implications for structure-based drug design and GPCR engineering.

biophysics↗