Biology subjects

Wells, L.

Publications and source records attributed to Wells, L..

18 recordsLinked to original sources

bioRxiv2026

Ethanol withdrawal induces temporally distinct alterations in oxytocinergic, opioidergic, glutamatergic and neuroimmune systems associated with recognition memory deficits

Alcohol use disorder is associated with persistent neuroadaptations and cognitive impairments, particularly during withdrawal, yet the temporal dynamics and interactions between these processes remain poorly understood. In the present study, we investigated time-dependent alterations in the oxytocin receptor (OTR), -opioid receptor (MOPr), metabotropic glutamate receptor 5 (mGlu5R), and translocator protein (TSPO) following chronic ethanol exposure and withdrawal, and their relationship to behavioral impairments. Male C57BL/6J mice were exposed to a 10-day escalating-dose ethanol liquid diet paradigm followed by 1, 4, or 7 days of withdrawal. Motor coordination and recognition memory were assessed using rotarod and novel object recognition tests, and receptor binding was quantified using autoradiography across multiple brain regions. Chronic ethanol exposure induced motor deficits that persisted during early withdrawal, whereas recognition memory impairments emerged after 7 days of withdrawal. Ethanol exposure and withdrawal produced region- and time-dependent alterations across all systems examined, with increased OTR binding in septal regions and transient reductions in basomedial amygdala OTR binding during intermediate withdrawal that normalized following prolonged withdrawal, transient increases in MOPr binding in striatal regions at 4 days of withdrawal, increased mGlu5R binding in the nucleus accumbens core, basolateral amygdala, and hippocampus, and early transient increases in TSPO binding indicative of neuroimmune activation. Notably, mGlu5R binding in the caudate-putamen and amygdala was associated with recognition memory deficits in a withdrawal stage-dependent manner. Bioinformatic analysis of human postmortem hippocampal transcriptomic datasets from individuals with alcohol use disorder revealed no consistent changes in GRM5 expression, suggesting that mGlu5R dysregulation may be driven by post-transcriptional mechanisms. These findings demonstrate that ethanol withdrawal is characterized by temporally distinct neuroadaptations across multiple systems and identify mGlu5R as a key correlate of withdrawal-associated memory impairment, highlighting its potential as a translational therapeutic target.

pharmacology and toxicology↗

bioRxiv2026

Saturation-seq integrates single-cell saturation genome editing and RNA-seq to quantify NFE2L2 (NRF2) variant effects

Interpreting the functional consequences of variants remains one of the central unsolved problems in genomics and clinical genetics. Compounding this, most existing approaches rely on reductive, one-dimensional proxies such as cell growth to score variant effects, which can be a poor substitute for the rich, multidimensional phenotyping that is ultimately needed to understand how variants alter biology. This is especially true for variants known to act through gain-of-function/neomorphic mechanisms. We developed Saturation-seq, a high-throughput platform that combines saturation genome editing with single-cell DNA and RNA profiling to systematically map variant effects. Using CRISPR-based editing in a barcoded haploid cell line, we install hundreds of variants directly into endogenous genomic loci, testing them in multiplex and preserving the native coding and regulatory context. Single-cell amplicon and transcriptome sequencing enables direct linkage of each genomic edit to its transcriptional impact. We apply Saturation-seq to comprehensively characterize 230 variants in the recurrently mutated N-terminal region of NFE2L2 (NRF2), a master regulator of oxidative stress and an oncogene mutated in lung cancer. We define variant function with disruption scores computed from misregulation of known NRF2 targets in single-cell transcriptomes; scores separate pathogenic/benign truthset variants with >90% accuracy and enabled interpretation of TCGA and TRACERx patient tumor data, as well as a rare NFE2L2 germline variant linked to a developmental syndrome. Thus, we establish a broadly applicable high-resolution single-cell variant-to-function platform with a rich phenotypic readout.

genomics↗

bioRxiv2026

A non-canonical EZH2/TRIM28 epigenetic axis drives heparan sulfate remodeling and melanoma metastasis

Melanoma progression and metastasis are driven not only by oncogenic alterations but also by epigenetic programs that dynamically remodel the tumor microenvironment. Heparan sulfate (HS) proteoglycans are key extracellular matrix components that integrate growth factor signaling, cell-matrix interactions, and migratory behavior by controlling ligand availability and receptor engagement, yet how chromatin-associated factors regulate HS remodeling in cancer remains poorly defined. Here, we identify the histone methyltransferase EZH2 as a key regulator of HS biosynthesis in melanoma. Integrated bioinformatic and genomic analyses revealed enrichment of EZH2 and additional Polycomb Repressive Complex (PRC) factors at regulatory regions of HS biosynthetic genes. CRISPR-mediated loss of EZH2 altered expression of multiple HS-modifying enzymes, most notably the secreted endosulfatases SULF1 and SULF2, resulting in enhanced HS 6-O sulfation and altered ligand binding at the cell surface. Unexpectedly, EZH2 promoted SULF1 expression through a methyltransferase-independent mechanism via a non-canonical interaction with TRIM28, whereas SULF2 was regulated through canonical PRC2-mediated repression. Functionally, SULF1 depletion impaired melanoma cell migration and invasion in vitro and reduced spontaneous metastasis in an orthotopic xenograft model. Together, these findings define an epigenetic axis linking chromatin regulation to extracellular glycan remodeling and identify HS-modifying enzymes as candidate targets to limit melanoma metastasis.

molecular biology↗

bioRxiv2025

Mucin-type O-glycans regulate proteoglycan stability and chondrocyte maturation

O-glycosylation is a ubiquitous post-translational modification essential for protein stability, cell signaling, and tissue organization, yet how distinct O-glycan subclasses coordinate tissue development remains unclear. Here, we identify functional crosstalk between extended mucin-type O-glycans and heparan sulfate proteoglycans (HSPGs). Genetic ablation of {beta}-1,3-galactosyltransferase 1 (C1GALT1) or its chaperone COSMC in human chondrocytes reduced cell surface HSPGs and fibroblast growth factor (FGF) binding, leading to impaired MAPK/ERK signaling, which was recapitulated in hypomorphic COSMC-CDG patient cells. Transcriptomic and secretome analyses revealed selective loss of proteoglycan expression and broader extracellular matrix remodeling in COSMC- and C1GALT1-deficient cells. Mechanistically, truncation of O-GalNAc glycans reduced Syndecan-1 levels at the cell surface via enhancing its lysosome-dependent degradation and diminished CD44v3-mediated FGF1 binding. Functionally, loss of complex O-GalNAc glycans disrupted chondrogenesis of growth plate-like chondroprogenitors. Overall, these findings reveal previously unrecognized roles for mucin-type O-glycans in maintaining proteoglycan stability and function, highlighting cross-regulatory mechanisms of glycosylation crucial for normal development that likely contribute to the pathophysiology of glycosylation-related disorders.

cell biology↗

bioRxiv2025

Rapid elicitation of a new class of neutralizing N332-glycan independent V3-glycan antibodies against HIV-1 in nonhuman primates.

Sequential immunization is a promising approach to elicit broadly neutralizing antibodies (bNAbs) against the HIV-1 Envelope (Env). However, available protocols are inefficient and involve multiple immunizations over long periods of time. Here, we present WIN332, a new engineered Env-immunogen that induces a new class of neutralizing N332-glycan-independent antibodies to the conserved V3-glycan epitope of Env after a single bolus immunization in nonhuman primates. WIN332 binds to precursors of canonical human N332-glycan-dependent (Type-I) V3-glycan bNAbs but also of a first-of-its-class N332-glycan-independent (Type-II) V3-glycan bNAb. A single immunization elicits neutralizing serum and monoclonal antibodies that are boosted and affinity matured with a heterologous immunogen. EMPEM analysis of serum antibodies, antibody cloning and cryo-EM analysis reveal that WIN332 elicits N332-glycan-independent antibodies with remarkable sequence and binding similarities with the most potent human type-I and type-II V3-glycan bNAbs. Thus, WIN332 is a promising vaccine candidate to streamline V3-glycan bNAb elicitation.

immunology↗

bioRxiv2025

Novel HIV-1 fusion peptide immunogens using glycan-engineered alphavirus-like particles

Immunofocusing on conserved, subdominant epitopes is critical for vaccines against highly diverse viruses such as HIV-1, influenza, and SARS-CoV-2. The eight-residue N-terminus of the HIV-1 fusion peptide (FP) is one such example of a promising yet small target. We developed new FP immunogens using three alphavirus-like particles (VLPs) and introduced additional glycans to mask shared carrier-specific epitopes. In two independent guinea pig studies, sequential immunization with heterologous carriers enhanced FP-directed antibody titers, which were further improved with glycan engineering. Separately, using diverse FP variants sharing the same N-terminal six amino acids increased neutralizing antibody titers. When combined, these two strategies led to higher FP-directed titers and, after Env trimer boosting, induced FP-directed neutralizing antibodies against multi-clade wild-type HIV-1 in nearly all animals. These findings established the importance of minimizing recurrent off-target epitopes across immunizations and support the engineered VLPs as a promising platform for peptide immunization. HighlightsO_LINovel HIV-1 fusion peptide immunogens using glycan-engineered alphavirus-like particles C_LIO_LIImproved FP-directed response by minimizing recurrent carrier-specific epitopes across immunizations C_LIO_LIImproved neutralizing response by sequential immunization with diverse FP variants C_LIO_LIFP-directed antibodies neutralizing multi-clade wildtype viruses in nearly all animals C_LI

immunology↗

bioRxiv2023

Taste papilla cell differentiation requires tongue mesenchyme via ALK3-BMP signaling to regulate the production of secretory proteins

Taste papillae are specialized organs each of which is comprised of an epithelial wall hosting taste buds and a core of mesenchymal tissue. In the present study, we report that during the early stages of embryonic development, bone morphogenetic protein (BMP) signaling mediated by type 1 receptor ALK3 in the tongue mesenchyme is required for the epithelial Wnt/{beta}-catenin activity and taste papilla cell differentiation. Mesenchyme-specific knockout (cKO) of Alk3 using Wnt1-Cre and Sox10-Cre resulted in an absence of taste papillae at E12.0. Biochemical and cell differentiation analyses demonstrated that mesenchymal ALK3-BMP signaling governs the production of previously unappreciated secretory proteins, i.e., suppresses those that inhibiting and facilitates those promoting taste cell differentiation. Bulk RNA-Sequencing analysis revealed many more differentially expressed genes (DEGs) in the tongue epithelium than in the mesenchyme in Alk3 cKO vs control. Moreover, we detected a down-regulated epithelial Wnt/{beta}-catenin signaling, and taste papilla development in the Alk3 cKO was rescued by GSK3{beta} inhibitor LiCl, but not Wnt3a. Our findings demonstrate for the first time the requirement of tongue mesenchyme in taste papilla cell differentiation. Summary statementThis is the first set of data to implicate the requirement of tongue mesenchyme in taste papilla cell differentiation.

developmental biology↗

bioRxiv2022

Sequential in vitro enzymatic N-glycoprotein modification reveals site-specific rates of glycoenzyme processing

N-glycosylation is an essential eukaryotic post-translational modification that affects various glycoprotein properties, including folding, solubility, protein-protein interactions, and half-life. N-glycans are processed in the secretory pathway to form varied ensembles of structures, and diversity at a single site on a glycoprotein is termed microheterogeneity. To understand the factors that influence glycan microheterogeneity, we hypothesized that local steric and electrostatic factors surrounding each site influences glycan availability to enzymatic modification. We tested this hypothesis by expression of a panel of reporter N-linked glycoproteins in MGAT1- null HEK293 cells to produce immature Man5GlcNAc2 glycoforms (38 glycan sites total). These glycoproteins were then sequentially modified in vitro from high-mannose to hybrid and on to biantennary, core fucosylated, complex structures by a panel of N-glycosylation enzymes and each reaction time-course was quantified by LC-MS/MS. Substantial differences in rates of in vitro enzymatic modification were observed between glycan sites on the same protein and differences in modification rates varied depending on the glycoenzyme being evaluated. By comparison, proteolytic digestion of the reporters prior to N-glycan processing eliminated differences in in vitro enzymatic modification. Comparison of in vitro rates of enzymatic modification with the glycan structures found on the mature reporters expressed in wild type cells correlate well with the enzymatic bottlenecks found in vitro. These data suggest that higher-order local structures surrounding each glycosylation site contribute to the efficiency of modification both in vitro and in vivo to establish the spectrum of site-specific microheterogeneity found on N-linked glycoproteins.

biochemistry↗

bioRxiv2021

Role of the G-Protein Coupled Receptor 3-Salt Inducible Kinase 2 Pathway in Human β CellProliferation

Loss of pancreatic {beta} cells is the hallmark of type 1 diabetes (T1D) 1, for which provision of insulin is the standard of care. While regenerative and stem cell therapies hold the promise of generating single-source or host-matched tissue to obviate immune-mediated complications2-4, these will still require surgical intervention and immunosuppression. Thus, methods that harness the innate capacity of {beta}-cells to proliferate to increase {beta} cell mass in vivo are considered vital for future T1D treatment5, 6. However, early in life {beta} cells enter what appears to be a permanent state of quiescence 7-10, directed by an evolutionarily selected genetic program that establishes a {beta} cell mass setpoint to guard against development of fatal endocrine tumours. Here we report the development of a high-throughput RNAi screening approach to identify upstream pathways that regulate adult human {beta} cell quiescence and demonstrate in a screen of the GPCRome that silencing G-protein coupled receptor 3 (GPR3) leads to human pancreatic {beta} cell proliferation. Loss of GPR3 leads to activation of Salt Inducible Kinase 2 (SIK2), which is necessary and sufficient to drive cell cycle entry, increase {beta} cell mass, and enhance insulin secretion in mice. Taken together, targeting the GPR3-SIK2 pathway represents a novel avenue to stimulate the regeneration of {beta} cells.

cell biology↗

bioRxiv2021

Cell Surface Glycan Engineering Reveals that Matriglycan Alone can Recapitulate Dystroglycan Binding and Function

-Dystroglycan (-DG) is uniquely modified on O-mannose sites by a repeating disaccharide (-Xyl1,3-GlcA{beta}1,3-)n termed matriglycan, which is a receptor for laminin-G domain-containing proteins and employed by old-world arenaviruses for infection. Using chemoenzymatically synthesized matriglycans printed as a microarray, we demonstrated length-dependent binding to Laminin, Lassa virus GP1, and the clinically-important antibody IIH6. Utilizing an enzymatic engineering approach, an N-linked glycoprotein was converted into a IIH6-positive Laminin-binding glycoprotein. Engineering of the surface of cells deficient for either -DG or O-mannosylation with matriglycans of sufficient length recovered infection with a Lassa-pseudovirus. Finally free matriglycan in a dose and length dependent manner inhibited viral infection of wildtype cells. These results indicate that matriglycan alone is necessary and sufficient for IIH6 staining, Laminin and LASV GP1 binding, and Lassa-pseudovirus infection and support a model in which it is a tunable receptor for which increasing chain length enhances ligand-binding capacity. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY Graphical Abstract C_FIG_DISPLAY

biochemistry↗

bioRxiv2021

ROMO1 is essential for glucose coupling in the pancreatic beta cell of male mice

Aims/HypothesisReactive oxygen species modulator 1 (ROMO1) is a highly conserved inner mitochondrial membrane protein that senses reactive oxygen species and regulates mitochondrial dynamics. ROMO1 is required for mitochondrial fusion in vitro, and silencing ROMO1 increases sensitivity to cell death stimuli. The physiological role of ROMO1 remains unclear. MethodsTo determine the role of ROMO1 in vivo, we used gene targeting in mice to ablate ROMO1 in the whole mouse and to conditionally knock out ROMO1 in the pancreatic beta cell. Mitochondrial functional analyses were performed on isolated mouse and human islets lacking ROMO1. ResultsWe show that ROMO1 is essential for embryonic development, as ROMO1-null mice die before embryonic day 8.5, earlier than GTPases OPA1 or MFN1/2 that catalyze mitochondrial inner and outer membrane fusion. Knockout of ROMO1 in adult pancreatic beta cells results in impaired glucose homeostasis in young male mice due to an insulin secretion defect. Isolated islets from male, but not female, mice showed impaired glucose-stimulated insulin secretion. While mitochondria from female mice were morphologically normal, mitochondria in Romo1 adult beta cell knockout (RABKO) cells from male mice were swollen and fragmented, with a reduction in mtDNA content. Knockout of ROMO1 did not affect basal respiration in males or females, but deletion of ROMO1 in both sexes in mice and isolated human islets reduced spare respiratory capacity (SRC), which involved the specific loss of respiratory activity at Complex II/SDH. Aging of female ROMO1 KO mice resulted in loss of spare respiratory capacity and glucose intolerance. Conclusions/InterpretationOur data demonstrate that ROMO1 is a key regulator of mitochondrial bioenergetics and SRC and is required for effective nutrient coupling to insulin secretion in the beta cell. These observations point to a critical role for spare respiratory capacity in the maintenance of euglycemia and to the potential for targeting ROMO1-complex II to promote glucose coupling in settings of insulin insufficiency. Research in ContextWhat is already known about this subject? O_LIROMO1 is required for mitochondrial fusion C_LIO_LIGlucose coupling to insulin secretion is accomplished in part via generation of NADH during the oxidation of glycolytic metabolites in the TCA cycle C_LIO_LISpare respiratory capacity is lost in aging C_LI What is the key question? O_LIWhat is the physiological role of ROMO1 in the whole animal and the pancreatic beta cell? C_LI What are the new findings? O_LIROMO1 is essential for mouse development C_LIO_LIROMO1 is required to maintain spare respiratory capacity (SRC) and to promote insulin secretion in the beta cells of mice and humans C_LIO_LIAblation of the Romo1 gene in the pancreatic beta cell leads to glucose coupling defects and glucose intolerance in young males and aged females C_LIO_LIAging highlights the importance of SRC in the beta cell for maintaining euglycemia C_LI How might this impact clinical practice in the foreseeable future? O_LIAging is a significant risk factor for T2D. Human males, but not females, experience a loss of insulin secretion with age; designing strategies that enhance ROMO1 and complex II activity to promote SRC may help to reverse these effects. C_LI

cell biology↗

bioRxiv2020

Separation and identification of permethylated glycan isomers by reversed phase nanoLC-NSI-MSn

High performance liquid chromatography has been employed for decades to enhance detection sensitivity and quantification of complex analytes within biological mixtures. Among these analytes, glycans released from glycoproteins and glycolipids have been characterized as underivatized or fluorescently tagged derivatives by HPLC coupled to various detection methods. These approaches have proven extremely useful for profiling the structural diversity of glycoprotein and glycolipid glycosylation but require the availability of glycan standards and secondary orthogonal degradation strategies to validate structural assignments. A robust method for HPLC separation of glycans as their permethylated derivatives, coupled with in-line MSn fragmentation to assign structural features independent of standards, would significantly enhance the depth of knowledge obtainable from biological samples. Here, we report an optimized workflow for LC-MS analysis of permethylated glycans that includes sample preparation, mobile phase optimization, and MSn method development to resolve structural isomers on-the-fly. We report baseline separation and MSn fragmentation of isomeric N- and O-glycan structures, aided by supplementing mobile phases with Li+, which simplifies adduct heterogeneity and facilitates cross-ring fragmentation to obtain valuable monosaccharide linkage information. Our workflow has been adapted from standard proteomics-based workflows and, therefore, provides opportunities for laboratories with expertise in proteomics to acquire glycomic data with minimal deviation from existing buffer systems, chromatography media, and instrument configurations. Furthermore, our workflow does not require a mass spectrometer with high-resolution/accurate mass capabilities. The rapidly evolving appreciation of the biological significance of glycans for human health and disease requires the implementation of high-throughput methods to identify and quantify glycans harvested from sample sets of sufficient size to achieve appropriately powered statistical significance. The LC-MSn approach we report generates glycan isomeric separations, robust structural characterization, and is amenable to auto-sampling with associated throughput enhancements.

biochemistry↗

bioRxiv2020

Generation of an Unbiased Interactome for the Tetratricopeptide Repeat Domain of O-GlcNAc Transferase Indicates a Role for the Enzyme in Intellectual Disability

The O-GIcNAc transferase (OGT) is localized to the nucleus and cytoplasm where it regulates nucleocytoplasmic proteins by modifying serine and threonine residues with a non-extended monosaccharide, {beta}-N-Acetyl-Glucosamine (O-GlcNAc). With thousands of known O-GlcNAc modified proteins but only one OGT encoded in the mammalian genome, a prevailing question is how OGT selects its substrates. Prior work has indicated that the N-terminal tetratricopeptide repeat (TPR) domain of OGT, rather than its C-terminal catalytic domain, is responsible for subcellular targeting and substrate selection. An additional impetus for exploring the OGT TPR domain interactome is the fact that missense mutations in OGT associated with X-linked intellectual disability (XLID) are primarily localized to the TPR domain without substantial impact on activity or stability of the enzyme. Therefore, we adapted the BioID labeling method to identify interactors of a TPR-BirA* fusion protein in HeLa cells. We identified 115 high confidence interactors representing both known and novel O-GlcNAc modified proteins and OGT interactors. The TPR interactors are highly enriched in processes in which OGT has a known role (e.g. chromatin remodeling, cellular survival of heat stress, circadian rhythm), as well as processes i n which OGT has yet to be implicated (e.g. pre-mRNA processing). Importantly, the identified TPR interactors are involved in several disease states but most notably are highly enriched in pathologies featuring intellectual disability. These proteins represent candidate interactors that may underlie the mechanism by which mutations in OGT lead to XLID. Furthermore, the identified interactors provide additional evidence of the importance of the TPR domain for OGT targeting and/or substrate selection. Thus, this defined interactome for the TPR domain of OGT serves as a jumping off point for future research exploring the role of OGT, the TPR domain, and its protein interactors in multiple cellular processes and disease mechanisms, including intellectual disability.

biochemistry↗

bioRxiv2020

Virus-Receptor Interactions of Glycosylated SARS-CoV-2 Spike and Human ACE2 Receptor

The current COVID-19 pandemic is caused by the SARS-CoV-2 betacoronavirus, which utilizes its highly glycosylated trimeric Spike protein to bind to the cell surface receptor ACE2 glycoprotein and facilitate host cell entry. We utilized glycomics-informed glycoproteomics to characterize site-specific microheterogeneity of glycosylation for a recombinant trimer Spike mimetic immunogen and for a soluble version of human ACE2. We combined this information with bioinformatic analyses of natural variants and with existing 3D-structures of both glycoproteins to generate molecular dynamics simulations of each glycoprotein alone and interacting with one another. Our results highlight roles for glycans in sterically masking polypeptide epitopes and directly modulating Spike-ACE2 interactions. Furthermore, our results illustrate the impact of viral evolution and divergence on Spike glycosylation, as well as the influence of natural variants on ACE2 receptor glycosylation that, taken together, can facilitate immunogen design to achieve antibody neutralization and inform therapeutic strategies to inhibit viral infection.

biochemistry↗

bioRxiv2019

A glycogenin homolog controls Toxoplasma gondii growth via glycosylation of an E3 ubiquitin ligase

Skp1, a subunit of E3 Skp1/Cullin-1/F-box protein ubiquitin ligases, is uniquely modified in protists by an O2-dependent prolyl hydroxylase that generates the attachment site for a defined pentasaccharide. Previous studies demonstrated the importance of the core glycan for growth of the parasite Toxoplasma gondii in fibroblasts, but the significance of the non-reducing terminal sugar was unknown. Here, we find that a homolog of glycogenin, an enzyme that can initiate and prime glycogen synthesis in yeast and animals, is required to catalyze the addition of an -galactose in 3-linkage to the subterminal glucose to complete pentasaccharide assembly in cells. A strong selectivity of the enzyme (Gat1) for Skp1 in extracts is consistent with other evidence that Skp1 is the sole target of the glycosyltransferase pathway. gat1-disruption results in slow growth attesting to the importance of the terminal sugar. Molecular dynamics simulations provide an explanation for this finding and confirm the potential of the full glycan to control Skp1 organization as in the amoebozoan Dictyostelium despite the different terminal disaccharide assembled by different glycosyltransferases. Though Gat1 also exhibits low -glucosyltransferase activity like glycogenin, autoglycosylation is not detected and gat1-disruption reveals no effect on starch accumulation. A crystal structure of the ortholog from the crop pathogen Pythium ultimum explains the distinct substrate preference and regiospecificity relative to glycogenin. A phylogenetic analysis suggests that Gat1 is related to the evolutionary progenitor of glycogenin, and acquired a role in glycogen formation following the ancestral disappearance of the underlying Skp1 glycosyltransferase prior to amoebozoan emergence.

microbiology↗

bioRxiv2019

Identification of a Novel Base J Binding Protein Complex Involved in RNA Polymerase II Transcription Termination in Trypanosomes

Base J, {beta}-D-glucosyl-hydroxymethyluracil, is a modification of thymine DNA base involved in RNA Polymerase (Pol) II transcription termination in kinetoplastid protozoa. Little is understood regarding how specific thymine residues are targeted for J-modification or the mechanism of J regulated transcription termination. To identify proteins involved in J-synthesis, we expressed a tagged version of the J-glucosyltransferase (JGT) in Leishmania tarentolae, and identified four co-purified proteins by mass spectrometry: protein phosphatase (PP1), a homolog of Wdr82, a potential PP1 regulatory protein (PNUTS) and a protein containing a J-DNA binding domain (named JBP3). Gel shift studies indicate JBP3 is a J-DNA binding protein. Reciprocal tagging, co-IP and sucrose gradient analyses indicate PP1, JGT, JBP3, Wdr82 and PNUTS form a multimeric complex in kinetoplastids, similar to the mammalian PTW/PP1 complex involved in transcription termination via PP1 mediated dephosphorylation of Pol II. Using RNAi and analysis of Pol II termination by RNA-seq and RT-PCR, we demonstrate that ablation of PNUTS, JBP3 and Wdr82 lead to defects in Pol II termination at the 3-end of polycistronic gene arrays in Trypanosoma brucei. Mutants also contain increased antisense RNA levels upstream of promoters, suggesting an additional role of the complex in regulating termination of bi-directional transcription. In addition, PNUTS loss causes derepression of silent Variant Surface Glycoprotein genes important for host immune evasion. Our results provide the first direct mechanistic link between base J and regulation of Pol II termination and suggest a novel molecular model for the role of the CTD of Pol II in terminating polycistronic transcription in trypanosomatids.\n\nAuthor SummaryTrypanosoma brucei is an early-diverged parasitic protozoan that causes African sleeping sickness in humans. The genome of T. brucei is organized into polycistronic gene clusters that contain multiple genes that are co-transcribed from a single promoter. We have recently described the presence of a modified DNA base J and variant of histone H3 (H3.V) at transcription termination sites within gene clusters where the loss of base J and H3.V leads to read-through transcription and the expression of downstream genes. We now identify a novel stable multimeric complex containing a J binding protein (JBP3), base J glucosyltransferase (JGT), PP1 phosphatase, PP1 interactive-regulatory protein (PNUTS) and Wdr82, which we refer to as PJW/PP1. A similar complex (PTW/PP1) has been shown to be involved in Pol II termination in humans and yeast. We demonstrate that PNUTS, JBP3 and Wdr82 mutants lead to read-through transcription in T. brucei. Our data suggest the PJW/PP1 complex regulates termination by recruitment to termination sites via JBP3-base J interactions and dephosphorylation of specific proteins (including Pol II and termination factors) by PP1. These findings significantly expand our understanding of mechanisms underlying transcription termination in eukaryotes, including divergent organisms that utilize polycistronic transcription and novel epigenetic marks such as base J and H3.V. The studies also provide the first direct mechanistic link between J modification of DNA at termination sites and regulated Pol II termination and gene expression in kinetoplastids.

biochemistry↗

bioRxiv2019

The human UDP-galactose 4’-epimerase (GALE) is required for cell surface glycome structure and function

Glycan biosynthesis relies on nucleotide-sugars (NS), abundant metabolites that serve as monosaccharide donors for glycosyltransferases. In vivo, signal-dependent fluctuations in NS levels are required to maintain normal cell physiology and are dysregulated in disease, but how mammalian cells regulate NS levels and pathway flux remains largely uncharacterized. To address this knowledge gap, we examined uridine diphosphate (UDP)-galactose 4-epimerase (GALE), which interconverts two pairs of essential NSs. GALE deletion in human cells triggered major imbalances in its substrate NSs and consequent dramatic changes in glycolipids and glycoproteins, including a subset of integrins and the Fas death receptor. NS dysregulation also directly impacted cell signaling, as GALE-/- cells exhibit Fas hypoglycosylation and hypersensitivity to Fas ligand-induced apoptosis. Our results reveal a new role for GALE-mediated NS regulation in supporting death receptor signaling and may have implications for the molecular etiology of illnesses characterized by NS imbalances, including galactosemia and metabolic syndrome.

cell biology↗

bioRxiv2018

Enabling tools for Toxoplasma glycobiology

Infection by the protozoan parasite Toxoplasma gondii is a major health risk owing to its chronic nature, ability to reactivate to cause blindness and encephalitis, and high prevalence in human populations. Like nearly all eukaryotes, Toxoplasma glycosylates many of its proteins and lipids and assembles polysaccharides. Unlike most eukaryotes, Toxoplasma divides and differentiates in vacuoles within host cells. While their glycans resemble canonical models, they exhibit species-specific variations that have inhibited deeper investigations into their roles in parasite biology and virulence. The genome of Toxoplasma encodes a suite of likely glycogenes expected to assemble a range of N-glycans, O-glycans, a C-glycan, GPI-anchors, and polysaccharides, along with their requisite precursors and membrane transporters. To facilitate genetic approaches to investigate the roles of specific glycans, we mapped probable connections between 59 glycogenes, their enzyme products, and the glycans to which they contribute. We adapted a double-CRISPR/Cas9 strategy and a mass spectrometry-based glycomics workflow to test these relationships, and conducted infection studies in fibroblast monolayers to probe cellular functions. Through the validated disruption of 17 glycogenes, we also discovered novel Glc0-2-Man6-GlcNAc2-type N-glycans, GalNAc2- and Glc-Fuc-type O-glycans, and a nuclear O-Fuc type glycan. We describe the guide sequences, disruption constructs, and mutant strains, which are freely available to practitioners for application in the context of the relational map to investigate the roles of glycans in their favorite biological processes.

microbiology↗