Biology subjects

Chen, S.

Publications and source records attributed to Chen, S..

At least 379 records · Page 21Linked to original sources

bioRxiv2024

Wheat TaNADPO promotes spot blotch resistance

Bipolaris sorokiniana is a common soil-borne fungal pathogen that can infect various organs of wheat (Triticum aestivum L.), causing diseases such as spot blotch, common root rot, head blight, and black point. The genetic basis of wheat resistance to B. sorokiniana is not yet fully understood. In this study, a natural population of 1,302 global common wheat germplasms was established and inoculated with B. sorokiniana at the seedling stage in a greenhouse. Resistance to spot blotch was assessed, revealing that only about 3.8% of the germplasms exhibited moderate or higher resistance levels. A genome-wide association study (GWAS) using high-density 660K single nucleotide polymorphism (SNP) data identified a region on chromosome 1BL (621.2-674.0 Mb) with 9 SNPs significantly associated (p < 10e-4) with spot blotch resistance, designated as Qsb.hebau-1BL. RNA sequencing and qRT-PCR assays showed that the gene TraesCS1B02G410300, encoding nicotinamide-adenine dinucleotide phosphate-binding oxidoreductase (TaNADPO), was significantly induced by B. sorokiniana. Five SNP variations were found in the promoter region of TaNADPO in wheat lines with or without Qsb.hebau-1BL. Transient expression of TaNADPO in Nicotiana benthamiana leaves showed a cytoplasmic subcellular localization of the fusion protein with a green fluorescent protein (GFP) tag. Wheat transgenic lines overexpressing TaNADPO exhibited significantly enhanced resistance to spot blotch compared to wildtype plants, with higher accumulation of reactive oxygen species (ROS). The knockout EMS mutant of Triticum turgidum NADPO (tdnadpo-K2561, Gln125*) showed significantly reduced resistance to spot blotch and lower ROS accumulation compared to wildtype plants. In summary, TaNADPO has been identified as a crucial gene for resistance to B. sorokiniana, providing valuable insights for developing spot blotch-resistant wheat varieties through molecular breeding techniques.

genetics↗

bioRxiv2024

Reinforcement learning guides single-cell sequencing in decoding lineage and cell fate decisions

The rapid development of single-cell sequencing offers an unparalleled opportunity to delineate the heterogeneity of individual cells. However, current methods struggle to pinpoint the states of cell fate decisions. In this study, we introduce a novel approach called Single-cell Reinforcement Learning (scRL), which integrates reinforcement learning into single-cell data analysis using an actor-critic architecture. Among existing dimensionality reduction methods, we identified one with the best interpretability. Based on this latent space and combined with our scRL algorithm, we assessed the intensity of fate decisions at the single-cell level. Extensive evaluations demonstrate that scRL outperforms existing techniques, as well as their variants and alternative approaches, in assessing cell fate decisions. Moreover, scRL offers an alternative method for evaluating the intensity of cell lineage differentiation which shows competitive interpretability as well. The superiority of scRL in assessing fate decisions is confirmed across several types of single-cell datasets.

bioinformatics↗

bioRxiv2024

Diffusion Basis Spectrum Imaging of White Matter in Schizophrenia and Bipolar Disorder

BackgroundMultiple studies point to the role of neuroinflammation in the pathophysiology of schizophrenia (SCZ), however, there have been few in vivo tools for imaging brain inflammation. Diffusion basis spectrum imaging (DBSI) is an advanced diffusion-based MRI method developed to quantitatively assess microstructural alternations relating to neuroinflammation, axonal fiber, and other white matter (WM) pathologies. MethodsWe acquired one-hour-long high-directional diffusion MRI data from young control (CON, n =27), schizophrenia (SCZ, n =21), and bipolar disorder (BPD, n =21) participants aged 18-30. We applied Tract-based Spatial Statistics (TBSS) to allow whole-brain WM analyses and compare DBSI-derived isotropic and anisotropic diffusion measures between groups. Clinical relationships of DBSI metrics with clinical symptoms were assessed across SCZ and control participants. ResultsIn SCZ participants, we found a generalized increase in DBSI-derived cellularity (a putative marker of neuroinflammation), a decrease in restricted fiber fraction (a putative marker of apparent axonal density), and an increase in extra-axonal water (a putative marker of vasogenic edema) across several WM tracts. There were only minimal WM abnormalities noted in BPD, mainly in regions of the corpus callosum (increase in DTI-derived RD and extra-axonal water). DBSI metrics showed significant partial correlations with psychosis and mood symptoms across groups. ConclusionOur findings suggest that SCZ involves generalized white matter neuroinflammation, decreased fiber density, and demyelination, which is not seen in bipolar disorder. Larger studies are needed to identify medication-related effects. DBSI metrics could help identify high-risk groups requiring early interventions to prevent the onset of psychosis and improve outcomes.

neuroscience↗

bioRxiv2024

Sensitive detection of synthetic response to cancer immunotherapy driven by gene paralog pairs

Emerging immunotherapies such as immune checkpoint blockade (ICB) and chimeric antigen receptor T-cell (CAR-T) therapy have revolutionized cancer treatment and have improved the survival of patients with multiple cancer types. Despite this success many patients are unresponsive to these treatments or relapse following treatment. CRISPR activation and knockout (KO) screens have been used to identify novel single gene targets that can enhance effector T cell function and promote immune cell targeting and eradication of tumors. However, cancer cells often employ multiple genes to promote an immunosuppressive pathway and thus modulating individual genes often has a limited effect. Paralogs are genes that originate from common ancestors and retain similar functions. They often have complex effects on a particular phenotype depending on factors like gene family similarity, each individual genes expression and the physiological or pathological context. Some paralogs exhibit synthetic lethal interactions in cancer cell survival; however, a thorough investigation of paralog pairs that could enhance the efficacy of cancer immunotherapy is lacking. Here we introduce a sensitive computational approach that uses sgRNA sets enrichment analysis to identify cancer-intrinsic paralog pairs which have the potential to synergistically enhance T cell-mediated tumor destruction. We have further developed an ensemble learning model that uses an XGBoost classifier and incorporates features such as gene characteristics, sequence and structural similarities, protein-protein interaction (PPI) networks, and gene coevolution data to predict paralog pairs that are likely to enhance immunotherapy efficacy. We experimentally validated the functional significance of these predicted paralog pairs using double knockout (DKO) of identified paralog gene pairs as compared to single gene knockouts (SKOs). These data and analyses collectively provide a sensitive approach to identify previously undetected paralog pairs that can enhance cancer immunotherapy even when individual genes within the pair has a limited effect.

bioinformatics↗

bioRxiv2024

Scalable Hypothalamic Arcuate Neuron Differentiation from Human Pluripotent Stem Cells Suitable for Modeling Metabolic and Reproductive Disorders

The hypothalamus, composed of several nuclei, is essential for maintaining our bodys homeostasis. The arcuate nucleus (ARC), located in the mediobasal hypothalamus, contains neuronal populations with eminent roles in energy and glucose homeostasis as well as reproduction. These neuronal populations are of great interest for translational research. To fulfill this promise, we used a robotic cell culture platform to provide a scalable and chemically defined approach for differentiating human pluripotent stem cells (hPSCs) into pro-opiomelanocortin (POMC), somatostatin (SST), tyrosine hydroxylase (TH) and gonadotropin-releasing hormone (GnRH) neuronal subpopulations with an ARC-like signature. This robust approach is reproducible across several distinct hPSC lines and exhibits a stepwise induction of key ventral diencephalon and ARC markers in transcriptomic profiling experiments. This is further corroborated by direct comparison to human fetal hypothalamus, and the enriched expression of genes implicated in obesity and type 2 diabetes (T2D). Genome-wide chromatin accessibility profiling by ATAC-seq identified accessible regulatory regions that can be utilized to predict candidate enhancers related to metabolic disorders and hypothalamic development. In depth molecular, cellular, and functional experiments unveiled the responsiveness of the hPSC-derived hypothalamic neurons to hormonal stimuli, such as insulin, neuropeptides including kisspeptin, and incretin mimetic drugs such as Exendin-4, highlighting their potential utility as physiologically relevant cellular models for disease studies. In addition, differential glucose and insulin treatments uncovered adaptability within the generated ARC neurons in the dynamic regulation of POMC and insulin receptors. In summary, the establishment of this model represents a novel, chemically defined, and scalable platform for manufacturing large numbers of hypothalamic arcuate neurons and serves as a valuable resource for modeling metabolic and reproductive disorders.

developmental biology↗

bioRxiv2024

GeneLLM: A Large cfRNA Language Model for Cancer Screening from Raw Reads

We present GeneLLM, a novel large language model that offers a transformative approach to non-invasive cancer detection and biomarker discovery by directly interpreting plasma cell-free RNA (cfRNA) sequences. Unlike traditional annotation-dependent methods, GeneLLM operates without prior knowledge, achieving significantly improved multi-cancer detection accuracy. Critically, GeneLLM identifies novel cfRNAs ( pseudo-biomarkers) originating from previously unannotated genomic regions-overlooked by existing methods-offering new therapeutic targets and insights into intercellular communication. This innovative, cost-effective approach bypasses traditional bioinformatics tools, generating novel pseudo-biomarkers that outperform existing methods even with low-depth sequencing data. Consequently, GeneLLM opens new avenues for biomarker discovery and expands our understanding of the extracellular transcriptomes role in cancer development.

bioinformatics↗

bioRxiv2024

ClOneHORT: Approaches for Improved Fidelity in Generative Models of Synthetic Genomes

MotivationDeep generative models have the potential to overcome difficulties in sharing individual-level genomic data by producing synthetic genomes that preserve the genomic associations specific to a cohort while not violating the privacy of any individual cohort member. However, there is significant room for improvement in the fidelity and usability of existing synthetic genome approaches. ResultsWe demonstrate that when combined with plentiful data and with population-specific selection criteria, deep generative models can produce synthetic genomes and cohorts that closely model the original populations. Our methods improve fidelity in the site-frequency spectra and linkage disequilibrium decay and yield synthetic genomes that can be substituted in downstream local ancestry inference analysis, recreating results with .91 to .94 accuracy. AvailabilityThe model described in this paper is freely available at github.com/rlaboulaye/clonehort.

genomics↗

bioRxiv2024

De novo whole-genome assembly and annotation of a high-quality coffee variety from the primary origin of coffee, Coffea arabica var. Geisha

Geisha coffee is recognized for its unique aromas and flavors and accordingly, has achieved the highest prices in the specialty coffee markets. We report the development of a chromosome-level, well-annotated, genome assembly of Coffea arabica var. Geisha, considered an Ethiopian landrace thatrepresents germplasm from the Ethiopian center of origin of coffee. We used a hybrid de novo assembly approach combining two long-reads single molecule sequencing technologies, Oxford Nanopore and Pacific Biosciences, together with scaffolding with Hi-C libraries. The final assembly is 1.03GB in size with BUSCO assessment of the assembly completeness of 97.7% of single-copy orthologs clusters. RNAseq and IsoSeq data were used as transcriptional experimental evidence for annotation and gene prediction revealing the presence of 47,062 gene loci encompassing 53,273 protein-coding transcripts. Comparison of the assembly to the progenitor subgenomes, separated the set of chromosome sequences inherited from C. canephora from those of C. eugenioides., Corresponding orthologs between Geisha and Red Bourbon had a 99.67% median identity, higher than what we observe with the progenitor assemblies (median 97.28%). Both, Geisha and Red Bourbon contain an inversion on Chromosome 10 relative to the pseudomolecules of the genetic material inherited from the two progenitors that must have happened before the separation in the geographical migration of the two varieties. Lending support of a single allopolyploidization event that gave origin to C. arabica after the hybridization event with the two progenitor lines. Broadening the availability of high-quality genome assemblies of Coffea arabica varieties, paves the way for understanding the evolution and domestication of coffee, as well as the genetic basis and environmental interactions of why a variety like Geisha is capable of producing beans with such exceptional and unique high-quality.

genomics↗

bioRxiv2024

Insect metamorphosis is regulated differently between sexes by members of a microRNA cluster

Insects comprise the majority of all described animal species and dominate the terrestrial habitats. The evolution of insect metamorphosis played a profound role in their successful adaptation and radiation. Insect metamorphosis is dependent on hormones ecdysteroids and sesquiterpenoids such as juvenile hormone. Despite the fact there are genuine differences between sexes during insect metamorphosis which facilitate their successful mating, how such sexual dimorphism in metamorphosis is being controlled is poorly known. We first generated transcriptomic profiles of male and female flies in late larvae and early pupae stages. Using a combination of genome-wide prediction and in vitro dual-luciferase validations, members of a microRNA cluster miR-277/34 were found to potentially regulate the neuropeptide receptor (AstC-R1) that when activated inhibits the sesquiterpenoid pathway and a juvenile hormone-dependent transcription factor (Kr-h1) in fly Drosophila melanogaster. Loss-of-function mutants were created deleting either miR-277 or miR-34, and expression levels of both AstC-R1 and Kr-h1 as well as ecdysteroid and sesquiterpenoid hormone titres were altered. Further comparison of transcriptomes of the late larvae and early pupae of both sexes revealed differential gene pathways being regulated by members of miR-277/34 between sexes during metamorphosis. This study highlights how members of a microRNA cluster control hormonal and developmental gene pathways in different sexes of insects during metamorphosis.

evolutionary biology↗

bioRxiv2024

Cryo-electron tomography reveals the microtubule-bound form of inactive LRRK2

Parkinsons Disease (PD) is the second most common neurodegenerative disorder. Mutations in leucine-rich repeat kinase 2 (LRRK2), a multi-domain protein containing both a kinase and a GTPase, are a leading cause of the familial form of PD. Pathogenic LRRK2 mutations increase LRRK2 kinase activity. While the bulk of LRRK2 is found in the cytosol, the protein associates with membranes where its Rab GTPase substrates are found, and under certain conditions, with microtubules. Integrative structural studies using single-particle cryo-electron microscopy (cryo-EM) and in situ cryo-electron tomography (cryo-ET) have revealed the architecture of microtubule-associated LRRK2 filaments, and that formation of these filaments requires LRRK2s kinase to be in the active-like conformation. However, whether LRRK2 can interact with and form filaments on microtubules in its autoinhibited state, where the kinase domain is in the inactive conformation and the N-terminal LRR domain covers the kinase active site, was not known. Using cryo-ET, we show that full-length LRRK2 can oligomerize on microtubules in its autoinhibited state. Both WT-LRRK2 and PD-linked LRRK2 mutants formed filaments on microtubules. While these filaments are stabilized by the same interfaces seen in the active-LRRK2 filaments, we observed a new interface involving the N-terminal repeats that were disordered in the active-LRRK2 filaments. The helical parameters of the autoinhibited-LRRK2 filaments are different from those reported for the active-LRRK2 filaments. Finally, the autoinhibited-LRRK2 filaments are shorter and less regular, suggesting they are less stable.

biophysics↗

bioRxiv2024

PTBP1 mediates Sertoli cell actin cytoskeleton organization by regulating alternative splicing of actin regulators

Spermatogenesis is a biological process within the testis that produces haploid spermatozoa for the continuity of species. Sertoli cells are somatic cells in the seminiferous epithelium that orchestrate spermatogenesis. Cyclic reorganization of Sertoli cell actin cytoskeleton is vital for spermatogenesis, but the underlying mechanism remains largely unclear. Here, we report that RNA-binding protein PTBP1 controls Sertoli cell actin cytoskeleton reorganization by programming alternative splicing of actin cytoskeleton regulators. This splicing control enables ectoplasmic specializations, the actin-based adhesion junctions, to maintain the blood-testis barrier and support spermatid transport and transformation. Particularly, we show that PTBP1 promotes actin bundle formation by repressing the inclusion of exon 14 of Tnik, a kinase present at the ectoplasmic specialization. Our results thus reveal a novel mechanism wherein Sertoli cell actin cytoskeleton dynamics is controlled post-transcriptionally by utilizing functionally distinct isoforms of actin regulatory proteins, and PTBP1 is a critical regulatory factor in generating such isoforms.

molecular biology↗

bioRxiv2024

Cross-modality representation and multi-sample integration of spatially resolved omics data

Spatially resolved sequencing technologies have revolutionized our understanding of biological regulatory processes within the microenvironment by accessing the states of genomic regions, genes and proteins as well as spatial coordinates of cells. However, discrepancies between different modalities and samples hinder the analysis of spatial omics data, necessitating the development of advanced computational methods. In this article, we propose PRESENT, an effective and scalable contrastive learning framework, for the cross-modality representation and multi-sample integration of spatial multi-omics, epigenomics and transcriptomics data. Through comprehensive experiments on spatial datasets, PRESENT demonstrates superior performance across various species, tissues, and technologies. Specifically, PRESENT effectively integrates spatial dependency and omics information simultaneously, facilitating the detection of spatially functional domains and the exploration of biological regulatory mechanisms. Furthermore, PRESENT can be extended for the integrative analysis of tissue samples across different dissected regions or developmental stages, promoting the identification of hierarchical structures from systematic and spatiotemporal perspectives.

bioinformatics↗

bioRxiv2024

A Mechanistic Understanding of the Activity-Stability Trade-off in PETase

Enzymatic degradation of poly(ethylene terephthalate) (PET) has garnered considerable interest in plastic recycling efforts. Despite numerous descriptions of both natural and engineered enzymes, the fundamental mechanism underlying PETase-catalyzed PET depolymerization at the solid-liquid interface remains elusive. This lack of understanding hampers the rational design of highly efficient depolymerases. Here, we employ multiscale simulations and experiments to elucidate the complete catalytic pathway of IsPETase, from enzyme adsorption at the interface to PET fragment capture, conformational refinement, and ester bond cleavage. Both endo- and exo-cleavage modes of the enzyme are identified, indicating its capacity for endo- and exo-lytic activities. We discover that the trade-off between the activity and stability of IsPETases PET-capturing pliers brings compromises to its PET depolymerization performance. Reshaping the loop dynamics of the enzyme can break this trade-off and enhance its stability and activity simultaneously, as demonstrated by the evolved variant HotPETase. Overall, our study offers comprehensive details into how PETase functions at the interface and provides valuable insights for engineering efficient plastic-degrading enzymes.

biochemistry↗

bioRxiv2024

At the onset of active whisking, the input layer of barrel cortex exhibits a 24 h window of increased excitability that depends on prior experience.

The development of motor control over sensory organs is a critical milestone, enabling active explo-ration and shaping of the sensory environment. Whether the onset of sensory organ motor control directly influences the development of corresponding sensory cortices remains unknown. Here, we confirm and exploit the late onset of whisking behavior in mice to address this question in the somatosensory system. Using ex vivo electrophysiology, we describe a transient increase in the intrinsic excitability of excitatory neurons in layer IV of the barrel cortex, which processes whisker input, immediately following the onset of active whisking around on postnatal days 13 and 14. This increase in neuronal gain is specific to layer IV, independent of changes in synaptic strength, and requires prior sensory experience. Further, these effects are not expressed in inhibitory in-terneurons in barrel cortex. The transient increase in excitability is not evident in layer II/III of barrel cortex or in the visual cortex upon eye opening, suggesting a unique interaction between the development of active sensing and the thalamocortical input layer in the somatosensory iso-cortex. Predictive modeling indicates that, immediately following the onset of active whisking, changes in active membrane conductances alone can reliably distinguish neurons in control but not whisker-deprived hemispheres. Our findings demonstrate an experience-dependent, lamina-specific refinement of neuronal excitability tightly linked to the emergence of active whisking. This tran-sient increase in the gain of the thalamic input layer coincides with a critical period for synaptic plasticity in downstream layers, suggesting a role in cortical maturation and sensory processing.

neuroscience↗

bioRxiv2024

The intestinal microbiota contributes to the development of immune-mediated cardiovascular inflammation and vasculitis in mice.

Alterations in the intestinal microbiota contribute to the pathogenesis of various cardiovascular disorders, but how they affect the development of Kawasaki disease (KD), an acute pediatric vasculitis, remains unclear. We report that depleting the gut microbiota reduces the development of cardiovascular inflammation in a murine model mimicking KD vasculitis. The development of cardiovascular lesions was associated with alterations in the intestinal microbiota composition and, notably, a decreased abundance of Akkermansia muciniphila and Faecalibacterium prausnitzii. Oral supplementation with either of these live or pasteurized individual bacteria, or with short-chain fatty acids (SCFAs) produced by them, attenuated cardiovascular inflammation. Treatment with Amuc_1100, the TLR-2 signaling outer membrane protein from A. muciniphila, also decreased the severity of vascular inflammation. This study reveals an underappreciated gut microbiota-cardiovascular inflammation axis in KD vasculitis pathogenesis and identifies specific intestinal commensals that regulate vasculitis in mice by producing metabolites or via extracellular proteins acting on gut barrier function. IN BRIEFIt remains unclear whether changes in the intestinal microbiota composition are involved in the development of cardiovascular lesions associated with Kawasaki disease (KD), an immune-mediated vasculitis. Jena et al. observe alterations in the intestinal microbiota composition of mice developing vasculitis, characterized by reduced A. muciniphila and F. prausnitzii. Oral supplementation with either of these bacteria, live or pasteurized, or with bacteria-produced short-chain fatty acids (SCFAs) or Amuc_1100, the TLR-2 signaling outer membrane protein of A. muciniphila, was sufficient to alleviate the development of cardiovascular lesions in mice by promoting intestinal barrier function. HIGHLIGHTSO_LIAbsence or depletion of the microbiota decreases the severity of vasculitis in a murine model mimicking KD vasculitis. C_LIO_LISupplementation of B. wadsworthia and B. fragilis promotes murine KD vasculitis. C_LIO_LIDecreased abundances of F. prausnitzii and A. muciniphila are associated with the development of cardiovascular lesions in mice. C_LIO_LISupplementation with either live or pasteurized A. muciniphila and F. prausnitzii, or the TLR-2 signaling Amuc_1100, reduces the severity of vasculitis by promoting gut barrier function. C_LI

immunology↗

bioRxiv2024

PEX14 acts as the interface linking optineurin to cell type-selective pexophagy

Pexophagy, the selective degradation of peroxisomes, is essential for removing excess or dysfunctional peroxisomes, and its dysregulation is linked to various diseases. Previous research has shown that optineurin (OPTN), an autophagy receptor involved in mitophagy, aggrephagy, and xenophagy, can induce pexophagy in HEK-293 cells. However, the underlying mechanism remains unclear. In this study, we used proximity labeling to identify PEX14, a peroxisomal membrane protein, as a neighboring partner of OPTN. Biochemical analyses revealed that PEX14 and OPTN interact through their respective coiled-coil and ubiquitin-binding domains. Further analyses demonstrated that the C-terminal half of overexpressed OPTN triggers pexophagy, likely by forming oligomers with endogenous OPTN. The co-localization of PEX14-OPTN complexes with LC3, combined with the suppression of OPTN-mediated peroxisome degradation by bafilomycin A1, supports a model in which PEX14 acts as a docking site for OPTN on the peroxisomal membrane, enabling the recruitment of the autophagic machinery for OPTN-mediated pexophagy. SummaryThis study uncovers and defines the peroxisomal membrane protein PEX14 as a key player in optineurin-driven pexophagy, advancing our mechanistic understanding of this cellular process. These findings open new avenues for developing therapeutic strategies targeting diseases associated with defective pexophagy.

cell biology↗

bioRxiv2024

The genomic and epigenomic dynamics of hemizygous genes across crops with contrasting mating systems

Hemizygous genes, which present on only one of the two homologous chromosomes of diploid organisms, have been mainly studied in the context of sex chromosomes and sex-linked genes. However, structural variants (SVs), such as a deletion/insertion of one allele, can lead to hemizygous genes, a phenomenon largely unexplored in plants. Here, we investigated the genomic and epigenomic landscapes of hemizygous genes across 22 genomes with varying propagation histories: eleven clonal lineages, seven outcrossed samples and four inbred and putatively homozygous genomes. We identified SVs leading to genic hemizygosity. As expected, very few genes (0.01%-1.2%) were hemizygous in the homozygous genomes, representing negative controls. Hemizygosity was appreciable among outcrossed lineages, averaging 8.7% of genes, but consistently elevated for the clonal samples at 13.8% genes, likely reflecting heterozygous SVs accumulation during clonal propagation. Compared to diploid genes, hemizygous genes were more often situated in centromeric than telomeric regions and underlying weaker purifying selection. They also had reduced levels of expression, averaging [~]20% of the expression levels of diploid genes, violating the evolutionary model of dosage compensation. We also detected higher DNA methylation levels, in hemizygous genes and transposable elements, which may contribute to the reduced expression of hemizygous genes. Finally, expression profiles showed that hemizygous genes were more specifically expressed in contexts related to fruit development, organ differentiation and stress responses. Overall, hemizygous genes accumulate in clonally propagated lineages and display distinct genetic, and epigenetic features compared to diploid genes, shedding new insights on genetic studies and breeding programs of clonal crops.

plant biology↗

bioRxiv2024

The hyperphosphorylation of SARS-CoV-2s nucleocapsid protein by GSK-3 involves a complex and redundant priming mechanism

Upon entry into host cells, the nucleocapsid (N) protein of coronavirus becomes heavily phosphorylated within its central Ser-Arg domain by glycogen synthase kinase-3 (GSK-3). Most substrates for GSK-3 need to be phosphorylated, or primed, by another Ser/Thr kinase four residues downstream of the GSK-3 phosphorylation site. It was widely assumed that the hyperphosphorylation of SARS-CoV-2s N protein requires independent priming at Ser-188 and Ser-206, which initiates sequential phosphorylation by GSK-3. Here we present biochemical and mass spectrometry data that contradicts this simple model, revealing instead a much more complex and highly cooperative mechanism where redundant priming, as well as exosite docking, enables N proteins efficient phosphorylation by GSK-3. The R203M and R203K/G204R mutations, found in the recent delta and omicron variants of concern, both hinder N hyperphosphorylation by GSK-3. The mechanistic insights generated in this study suggest a novel approach to treat COVID-19 by combining multiple classes of pharmacological agents to inhibit N hyperphosphorylation.

biochemistry↗