RESUMO
The human embryo breaks symmetry to form the anterior-posterior axis of the body. As the embryo elongates along this axis, progenitors in the tail bud give rise to tissues that generate spinal cord, skeleton, and musculature. This raises the question of how the embryo achieves axial elongation and patterning. While ethics necessitate in vitro studies, the variability of organoid systems has hindered mechanistic insights. Here, we developed a bioengineering and machine learning framework that optimizes organoid symmetry breaking by tuning their spatial coupling. This framework enabled reproducible generation of axially elongating organoids, each possessing a tail bud and neural tube. We discovered that an excitable system composed of WNT/FGF signaling drives elongation by inducing a neuromesodermal progenitor-like signaling center. We discovered that instabilities in the excitable system are suppressed by secreted WNT inhibitors. Absence of these inhibitors led to ectopic tail buds and branches. Our results identify mechanisms governing stable human axial elongation.
Assuntos
Padronização Corporal , Mesoderma , Humanos , Via de Sinalização Wnt , Embrião de Mamíferos , OrganoidesRESUMO
Paired mapping of single-cell gene expression and electrophysiology is essential to understand gene-to-function relationships in electrogenic tissues. Here, we developed in situ electro-sequencing (electro-seq) that combines flexible bioelectronics with in situ RNA sequencing to stably map millisecond-timescale electrical activity and profile single-cell gene expression from the same cells across intact biological networks, including cardiac and neural patches. When applied to human-induced pluripotent stem-cell-derived cardiomyocyte patches, in situ electro-seq enabled multimodal in situ analysis of cardiomyocyte electrophysiology and gene expression at the cellular level, jointly defining cell states and developmental trajectories. Using machine-learning-based cross-modal analysis, in situ electro-seq identified gene-to-electrophysiology relationships throughout cardiomyocyte development and accurately reconstructed the evolution of gene expression profiles based on long-term stable electrical measurements. In situ electro-seq could be applicable to create spatiotemporal multimodal maps in electrogenic tissues, potentiating the discovery of cell types and gene programs responsible for electrophysiological function and dysfunction.
Assuntos
Eletrônica , Análise de Sequência de RNA , Humanos , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/metabolismo , Análise de Célula Única , Transcriptoma , Eletrônica/métodosRESUMO
Immune-checkpoint blockade has revolutionized cancer treatment, but some cancers, such as acute myeloid leukemia (AML), do not respond or develop resistance. A potential mode of resistance is immune evasion of T cell immunity involving aberrant major histocompatibility complex class I (MHC-I) antigen presentation (AP). To map such mechanisms of resistance, we identified key MHC-I regulators using specific peptide-MHC-I-guided CRISPR-Cas9 screens in AML. The top-ranked negative regulators were surface protein sushi domain containing 6 (SUSD6), transmembrane protein 127 (TMEM127), and the E3 ubiquitin ligase WWP2. SUSD6 is abundantly expressed in AML and multiple solid cancers, and its ablation enhanced MHC-I AP and reduced tumor growth in a CD8+ T cell-dependent manner. Mechanistically, SUSD6 forms a trimolecular complex with TMEM127 and MHC-I, which recruits WWP2 for MHC-I ubiquitination and lysosomal degradation. Together with the SUSD6/TMEM127/WWP2 gene signature, which negatively correlates with cancer survival, our findings define a membrane-associated MHC-I inhibitory axis as a potential therapeutic target for both leukemia and solid cancers.
Assuntos
Antígenos de Histocompatibilidade Classe I , Neoplasias , Evasão Tumoral , Humanos , Apresentação de Antígeno , Linfócitos T CD8-Positivos , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos HLA , Neoplasias/imunologia , Ubiquitina-Proteína Ligases/genéticaRESUMO
CD8+ T cell homeostasis is maintained by the cytokines IL-7 and IL-15. Here we show that transcription factors Tcf1 and Lef1 were intrinsically required for homeostatic proliferation of CD8+ T cells. Multiomics analyses showed that Tcf1 recruited the genome organizer CTCF and that homeostatic cytokines induced Tcf1-dependent CTCF redistribution in the CD8+ T cell genome. Hi-C coupled with network analyses indicated that Tcf1 and CTCF acted cooperatively to promote chromatin interactions and form highly connected, dynamic interaction hubs in CD8+ T cells before and after cytokine stimulation. Ablating CTCF phenocopied the proliferative defects caused by Tcf1 and Lef1 deficiency. Tcf1 and CTCF controlled a similar set of genes that regulated cell cycle progression and promoted CD8+ T cell homeostatic proliferation in vivo. These findings identified CTCF as a Tcf1 cofactor and uncovered an intricate interplay between Tcf1 and CTCF that modulates the genomic architecture of CD8+ T cells to preserve homeostasis.
Assuntos
Linfócitos T CD8-Positivos , Transdução de Sinais , Linfócitos T CD8-Positivos/metabolismo , Citocinas/metabolismo , Genômica , HomeostaseRESUMO
In eukaryotic cells, organelles and the cytoskeleton undergo highly dynamic yet organized interactions capable of orchestrating complex cellular functions. Visualizing these interactions requires noninvasive, long-duration imaging of the intracellular environment at high spatiotemporal resolution and low background. To achieve these normally opposing goals, we developed grazing incidence structured illumination microscopy (GI-SIM) that is capable of imaging dynamic events near the basal cell cortex at 97-nm resolution and 266 frames/s over thousands of time points. We employed multi-color GI-SIM to characterize the fast dynamic interactions of diverse organelles and the cytoskeleton, shedding new light on the complex behaviors of these structures. Precise measurements of microtubule growth or shrinkage events helped distinguish among models of microtubule dynamic instability. Analysis of endoplasmic reticulum (ER) interactions with other organelles or microtubules uncovered new ER remodeling mechanisms, such as hitchhiking of the ER on motile organelles. Finally, ER-mitochondria contact sites were found to promote both mitochondrial fission and fusion.
Assuntos
Retículo Endoplasmático/metabolismo , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Microscopia de FluorescênciaRESUMO
Despite mounting evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) engagement with immune cells, most express little, if any, of the canonical receptor of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2). Here, using a myeloid cell receptor-focused ectopic expression screen, we identified several C-type lectins (DC-SIGN, L-SIGN, LSECtin, ASGR1, and CLEC10A) and Tweety family member 2 (TTYH2) as glycan-dependent binding partners of the SARS-CoV-2 spike. Except for TTYH2, these molecules primarily interacted with spike via regions outside of the receptor-binding domain. Single-cell RNA sequencing analysis of pulmonary cells from individuals with coronavirus disease 2019 (COVID-19) indicated predominant expression of these molecules on myeloid cells. Although these receptors do not support active replication of SARS-CoV-2, their engagement with the virus induced robust proinflammatory responses in myeloid cells that correlated with COVID-19 severity. We also generated a bispecific anti-spike nanobody that not only blocked ACE2-mediated infection but also the myeloid receptor-mediated proinflammatory responses. Our findings suggest that SARS-CoV-2-myeloid receptor interactions promote immune hyperactivation, which represents potential targets for COVID-19 therapy.
Assuntos
COVID-19/metabolismo , COVID-19/virologia , Interações Hospedeiro-Patógeno , Lectinas Tipo C/metabolismo , Proteínas de Membrana/metabolismo , Células Mieloides/imunologia , Células Mieloides/metabolismo , Proteínas de Neoplasias/metabolismo , SARS-CoV-2/fisiologia , Enzima de Conversão de Angiotensina 2/metabolismo , Sítios de Ligação , COVID-19/genética , Linhagem Celular , Citocinas , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Mediadores da Inflamação/metabolismo , Lectinas Tipo C/química , Proteínas de Membrana/química , Modelos Moleculares , Proteínas de Neoplasias/química , Ligação Proteica , Conformação Proteica , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Relação Estrutura-AtividadeRESUMO
The membrane-integrated synthase FKS is involved in the biosynthesis of ß-1,3-glucan, the core component of the fungal cell wall1,2. FKS is the target of widely prescribed antifungal drugs, including echinocandin and ibrexafungerp3,4. Unfortunately, the mechanism of action of FKS remains enigmatic and this has hampered development of more effective medicines targeting the enzyme. Here we present the cryo-electron microscopy structures of Saccharomyces cerevisiae FKS1 and the echinocandin-resistant mutant FKS1(S643P). These structures reveal the active site of the enzyme at the membrane-cytoplasm interface and a glucan translocation path spanning the membrane bilayer. Multiple bound lipids and notable membrane distortions are observed in the FKS1 structures, suggesting active FKS1-membrane interactions. Echinocandin-resistant mutations are clustered at a region near TM5-6 and TM8 of FKS1. The structure of FKS1(S643P) reveals altered lipid arrangements in this region, suggesting a drug-resistant mechanism of the mutant enzyme. The structures, the catalytic mechanism and the molecular insights into drug-resistant mutations of FKS1 revealed in this study advance the mechanistic understanding of fungal ß-1,3-glucan biosynthesis and establish a foundation for developing new antifungal drugs by targeting FKS.
Assuntos
Microscopia Crioeletrônica , Glucosiltransferases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Antifúngicos/farmacologia , beta-Glucanas/metabolismo , Domínio Catalítico , Membrana Celular/química , Membrana Celular/metabolismo , Farmacorresistência Fúngica/efeitos dos fármacos , Farmacorresistência Fúngica/genética , Equinocandinas/farmacologia , Glucosiltransferases/antagonistas & inibidores , Glucosiltransferases/química , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Glucosiltransferases/ultraestrutura , Testes de Sensibilidade Microbiana , Mutação , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestruturaRESUMO
Spatially charting molecular cell types at single-cell resolution across the 3D volume is critical for illustrating the molecular basis of brain anatomy and functions. Single-cell RNA sequencing has profiled molecular cell types in the mouse brain1,2, but cannot capture their spatial organization. Here we used an in situ sequencing method, STARmap PLUS3,4, to profile 1,022 genes in 3D at a voxel size of 194 × 194 × 345 nm3, mapping 1.09 million high-quality cells across the adult mouse brain and spinal cord. We developed computational pipelines to segment, cluster and annotate 230 molecular cell types by single-cell gene expression and 106 molecular tissue regions by spatial niche gene expression. Joint analysis of molecular cell types and molecular tissue regions enabled a systematic molecular spatial cell-type nomenclature and identification of tissue architectures that were undefined in established brain anatomy. To create a transcriptome-wide spatial atlas, we integrated STARmap PLUS measurements with a published single-cell RNA-sequencing atlas1, imputing single-cell expression profiles of 11,844 genes. Finally, we delineated viral tropisms of a brain-wide transgene delivery tool, AAV-PHP.eB5,6. Together, this annotated dataset provides a single-cell resource that integrates the molecular spatial atlas, brain anatomy and the accessibility to genetic manipulation of the mammalian central nervous system.
Assuntos
Sistema Nervoso Central , Imageamento Tridimensional , Análise de Célula Única , Transcriptoma , Animais , Camundongos , Encéfalo/anatomia & histologia , Encéfalo/citologia , Encéfalo/metabolismo , Sistema Nervoso Central/anatomia & histologia , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Análise de Célula Única/métodos , Medula Espinal/anatomia & histologia , Medula Espinal/citologia , Medula Espinal/metabolismo , Transcriptoma/genética , Análise da Expressão Gênica de Célula Única , Tropismo Viral , Conjuntos de Dados como Assunto , Transgenes/genética , Imageamento Tridimensional/métodosRESUMO
Incompletely synthesized nascent chains obstructing large ribosomal subunits are targeted for degradation by ribosome-associated quality control (RQC). In bacterial RQC, RqcH marks the nascent chains with C-terminal alanine (Ala) tails that are directly recognized by proteasome-like proteases, whereas in eukaryotes, RqcH orthologs (Rqc2/NEMF [nuclear export mediator factor]) assist the Ltn1/Listerin E3 ligase in nascent chain ubiquitylation. Here, we study RQC-mediated proteolytic targeting of ribosome stalling products in mammalian cells. We show that mammalian NEMF has an additional, Listerin-independent proteolytic role, which, as in bacteria, is mediated by tRNA-Ala binding and Ala tailing. However, in mammalian cells Ala tails signal proteolysis indirectly, through a pathway that recognizes C-terminal degrons; we identify the CRL2KLHDC10 E3 ligase complex and the novel C-end rule E3, Pirh2/Rchy1, as bona fide RQC pathway components that directly bind to Ala-tailed ribosome stalling products and target them for degradation. As Listerin mutation causes neurodegeneration in mice, functionally redundant E3s may likewise be implicated in molecular mechanisms of neurodegeneration.
Assuntos
Alanina/metabolismo , Mamíferos/metabolismo , Proteólise , Ribossomos/metabolismo , Animais , Antígenos de Neoplasias/metabolismo , Células HeLa , Humanos , Modelos Biológicos , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores de Citocinas/metabolismo , Proteínas Salivares Ricas em Prolina/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Phosphoglycerate mutase 1 (PGAM1) is a key node enzyme that diverts the metabolic reactions from glycolysis into its shunts to support macromolecule biosynthesis for rapid and sustainable cell proliferation. It is prevalent that PGAM1 activity is upregulated in various tumors; however, the underlying mechanism remains unclear. Here, we unveil that pyruvate kinase M2 (PKM2) moonlights as a histidine kinase in a phosphoenolpyruvate (PEP)-dependent manner to catalyze PGAM1 H11 phosphorylation, that is essential for PGAM1 activity. Moreover, monomeric and dimeric but not tetrameric PKM2 are efficient to phosphorylate and activate PGAM1. In response to epidermal growth factor signaling, Src-catalyzed PGAM1 Y119 phosphorylation is a prerequisite for PKM2 binding and the subsequent PGAM1 H11 phosphorylation, which constitutes a discrepancy between tumor and normal cells. A PGAM1-derived pY119-containing cell-permeable peptide or Y119 mutation disrupts the interaction of PGAM1 with PKM2 and PGAM1 H11 phosphorylation, dampening the glycolysis shunts and tumor growth. Together, these results identify a function of PKM2 as a histidine kinase, and illustrate the importance of enzyme crosstalk as a regulatory mode during metabolic reprogramming and tumorigenesis.
Assuntos
Glicólise , Fosfoglicerato Mutase , Hormônios Tireóideos , Humanos , Fosfoglicerato Mutase/metabolismo , Fosfoglicerato Mutase/genética , Fosforilação , Animais , Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/genética , Camundongos , Proteínas de Ligação a Hormônio da Tireoide , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Linhagem Celular Tumoral , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genéticaRESUMO
Cervical cancer is caused by human papillomavirus (HPV) infection, has few approved targeted therapeutics, and is the most common cause of cancer death in low-resource countries. We characterized 19 cervical and four head and neck cancer cell lines using long-read DNA and RNA sequencing and identified the HPV types, HPV integration sites, chromosomal alterations, and cancer driver mutations. Structural variation analysis revealed telomeric deletions associated with DNA inversions resulting from breakage-fusion-bridge (BFB) cycles. BFB is a common mechanism of chromosomal alterations in cancer, and our study applies long-read sequencing to this important chromosomal rearrangement type. Analysis of the inversion sites revealed staggered ends consistent with exonuclease digestion of the DNA after breakage. Some BFB events are complex, involving inter- or intra-chromosomal insertions or rearrangements. None of the BFB breakpoints had telomere sequences added to resolve the dicentric chromosomes, and only one BFB breakpoint showed chromothripsis. Five cell lines have a chromosomal region 11q BFB event, with YAP1-BIRC3-BIRC2 amplification. Indeed, YAP1 amplification is associated with a 10-year-earlier age of diagnosis of cervical cancer and is three times more common in African American women. This suggests that individuals with cervical cancer and YAP1-BIRC3-BIRC2 amplification, especially those of African ancestry, might benefit from targeted therapy. In summary, we uncovered valuable insights into the mechanisms and consequences of BFB cycles in cervical cancer using long-read sequencing.
Assuntos
Infecções por Papillomavirus , Neoplasias do Colo do Útero , Feminino , Humanos , Neoplasias do Colo do Útero/genética , Aberrações Cromossômicas , Telômero/genética , DNARESUMO
Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor expressed on activated T cells and an emerging immunotherapy target. Domain 1 (D1) of LAG-3, which has been purported to directly interact with major histocompatibility complex class II (MHCII) and fibrinogen-like protein 1 (FGL1), has been the major focus for the development of therapeutic antibodies that inhibit LAG-3 receptor-ligand interactions and restore T cell function. Here, we present a high-resolution structure of glycosylated mouse LAG-3 ectodomain, identifying that cis-homodimerization, mediated through a network of hydrophobic residues within domain 2 (D2), is critically required for LAG-3 function. Additionally, we found a previously unidentified key protein-glycan interaction in the dimer interface that affects the spatial orientation of the neighboring D1 domain. Mutation of LAG-3 D2 residues reduced dimer formation, dramatically abolished LAG-3 binding to both MHCII and FGL1 ligands, and consequentially inhibited the role of LAG-3 in suppressing T cell responses. Intriguingly, we showed that antibodies directed against D1, D2, and D3 domains are all capable of blocking LAG-3 dimer formation and MHCII and FGL-1 ligand binding, suggesting a potential allosteric model of LAG-3 function tightly regulated by dimerization. Furthermore, our work reveals unique epitopes, in addition to D1, that can be targeted for immunotherapy of cancer and other human diseases.
Assuntos
Antígenos de Histocompatibilidade Classe II , Linfócitos T , Animais , Humanos , Camundongos , Dimerização , Fibrinogênio/metabolismo , Ligantes , MutaçãoRESUMO
African swine fever, one of the major viral diseases of swine, poses an imminent threat to the global pig industry. The high-efficient replication of the causative agent African swine fever virus (ASFV) in various organs in pigs greatly contributes to the disease. However, how ASFV manipulates the cell population to drive high-efficient replication of the virus in vivo remains unclear. Here, we found that the spleen reveals the most severe pathological manifestation with the highest viral loads among various organs in pigs during ASFV infection. By using single-cell-RNA-sequencing technology and multiple methods, we determined that macrophages and monocytes are the major cell types infected by ASFV in the spleen, showing high viral-load heterogeneity. A rare subpopulation of immature monocytes represents the major population infected at late infection stage. ASFV causes massive death of macrophages, but shifts its infection into these monocytes which significantly arise after the infection. The apoptosis, interferon response, and antigen-presentation capacity are inhibited in these monocytes which benefits prolonged infection of ASFV in vivo. Until now, the role of immature monocytes as an important target by ASFV has been overlooked due to that they do not express classical monocyte marker CD14. The present study indicates that the shift of viral infection from macrophages to the immature monocytes is critical for maintaining prolonged ASFV infection in vivo. This study sheds light on ASFV tropism, replication, and infection dynamics, and elicited immune response, which may instruct future research on antiviral strategies.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Suínos , Animais , Vírus da Febre Suína Africana/fisiologia , Baço/patologia , Replicação Viral , Macrófagos/patologiaRESUMO
SNX32 is a member of the evolutionarily conserved Phox (PX) homology domain- and Bin/Amphiphysin/Rvs (BAR) domain- containing sorting nexin (SNX-BAR) family of proteins, which play important roles in sorting and membrane trafficking of endosomal cargoes. Although SNX32 shares the highest amino acid sequence homology with SNX6, and has been believed to function redundantly with SNX5 and SNX6 in retrieval of the cation-independent mannose-6-phosphate receptor (CI-MPR) from endosomes to the trans-Golgi network (TGN), its role(s) in intracellular protein trafficking remains largely unexplored. Here, we report that it functions in parallel with SNX1 in mediating epidermal growth factor (EGF)-stimulated postendocytic trafficking of the epidermal growth factor receptor (EGFR). Moreover, SNX32 interacts directly with EGFR, and recruits SNX5 to promote sorting of EGF-EGFR into multivesicular bodies (MVBs) for lysosomal degradation. Thus, SNX32 functions distinctively from other SNX-BAR proteins to mediate signaling-coupled endolysosomal trafficking of EGFR.
Assuntos
Fator de Crescimento Epidérmico , Receptores ErbB , Lisossomos , Transporte Proteico , Nexinas de Classificação , Nexinas de Classificação/metabolismo , Nexinas de Classificação/genética , Receptores ErbB/metabolismo , Lisossomos/metabolismo , Humanos , Transporte Proteico/fisiologia , Fator de Crescimento Epidérmico/metabolismo , Células HeLa , Endossomos/metabolismo , Rede trans-Golgi/metabolismo , Corpos Multivesiculares/metabolismoRESUMO
Spatiotemporal regulation of the cellular transcriptome is crucial for proper protein expression and cellular function. However, the intricate subcellular dynamics of RNA remain obscured due to the limitations of existing transcriptomics methods. Here, we report TEMPOmap-a method that uncovers subcellular RNA profiles across time and space at the single-cell level. TEMPOmap integrates pulse-chase metabolic labeling with highly multiplexed three-dimensional in situ sequencing to simultaneously profile the age and location of individual RNA molecules. Using TEMPOmap, we constructed the subcellular RNA kinetic landscape in various human cells from transcription and translocation to degradation. Clustering analysis of RNA kinetic parameters across single cells revealed 'kinetic gene clusters' whose expression patterns were shaped by multistep kinetic sculpting. Importantly, these kinetic gene clusters are functionally segregated, suggesting that subcellular RNA kinetics are differentially regulated in a cell-state- and cell-type-dependent manner. Spatiotemporally resolved transcriptomics provides a gateway to uncovering new spatiotemporal gene regulation principles.
Assuntos
RNA , Transcriptoma , Humanos , RNA/genética , Cinética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Análise de Célula Única/métodosRESUMO
Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly pathogenic tick-borne virus, prevalent in more than 30 countries worldwide. Human infection by this virus leads to severe illness, with an average case fatality of 40%. There is currently no approved vaccine or drug to treat the disease. Neutralizing antibodies are a promising approach to treat virus infectious diseases. This study generated 37 mouse-derived specific monoclonal antibodies against CCHFV Gc subunit. Neutralization assays using pseudotyped virus and authentic CCHFV identified Gc8, Gc13, and Gc35 as neutralizing antibodies. Among them, Gc13 had the highest neutralizing activity and binding affinity with CCHFV Gc. Consistently, Gc13, but not Gc8 or Gc35, showed in vivo protective efficacy (62.5% survival rate) against CCHFV infection in a lethal mouse infection model. Further characterization studies suggested that Gc8 and Gc13 may recognize a similar, linear epitope in domain II of CCHFV Gc, while Gc35 may recognize a different epitope in Gc. Cryo-electron microscopy of Gc-Fab complexes indicated that both Gc8 and Gc13 bind to the conserved fusion loop region and Gc13 had stronger interactions with sGc-trimers. This was supported by the ability of Gc13 to block CCHFV GP-mediated membrane fusion. Overall, this study provides new therapeutic strategies to treat CCHF and new insights into the interaction between antibodies with CCHFV Gc proteins.
Assuntos
Vírus da Febre Hemorrágica da Crimeia-Congo , Febre Hemorrágica da Crimeia , Animais , Camundongos , Humanos , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Anticorpos Monoclonais , Microscopia Crioeletrônica , Anticorpos Neutralizantes , EpitoposRESUMO
Sustained energy starvation leads to activation of AMP-activated protein kinase (AMPK), which coordinates energy status with numerous cellular processes including metabolism, protein synthesis, and autophagy. Here, we report that AMPK phosphorylates the histone methyltransferase EZH2 at T311 to disrupt the interaction between EZH2 and SUZ12, another core component of the polycomb repressive complex 2 (PRC2), leading to attenuated PRC2-dependent methylation of histone H3 at Lys27. As such, PRC2 target genes, many of which are known tumor suppressors, were upregulated upon T311-EZH2 phosphorylation, which suppressed tumor cell growth both in cell culture and mouse xenografts. Pathologically, immunohistochemical analyses uncovered a positive correlation between AMPK activity and pT311-EZH2, and higher pT311-EZH2 correlates with better survival in both ovarian and breast cancer patients. Our finding suggests that AMPK agonists might be promising sensitizers for EZH2-targeting cancer therapies.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Animais , Carcinogênese/genética , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/fisiologia , Epigênese Genética , Feminino , Histonas/metabolismo , Humanos , Camundongos , Proteínas de Neoplasias , Proteínas Nucleares/metabolismo , Oncogenes , Neoplasias Ovarianas/metabolismo , Fosforilação , Complexo Repressor Polycomb 2/metabolismo , Complexo Repressor Polycomb 2/fisiologia , Fatores de Transcrição , Regulação para CimaRESUMO
CD62L+ central memory CD8+ T (TCM) cells provide enhanced protection than naive cells; however, the underlying mechanism, especially the contribution of higher-order genomic organization, remains unclear. Systematic Hi-C analyses reveal that antigen-experienced CD8+ T cells undergo extensive rewiring of chromatin interactions (ChrInt), with TCM cells harboring specific interaction hubs compared with naive CD8+ T cells, as observed at cytotoxic effector genes such as Ifng and Tbx21. TCM cells also acquire de novo CTCF (CCCTC-binding factor) binding sites, which are not only strongly associated with TCM-specific hubs but also linked to increased activities of local gene promoters and enhancers. Specific ablation of CTCF in TCM cells impairs rapid induction of genes in cytotoxic program, energy supplies, transcription, and translation by recall stimulation. Therefore, acquisition of CTCF binding and ChrInt hubs by TCM cells serves as a chromatin architectural basis for their transcriptomic dynamics in primary response and for imprinting the code of "recall readiness" against secondary challenge.
Assuntos
Linfócitos T CD8-Positivos , Cromatina , Cromatina/genética , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Sítios de Ligação , GenômicaRESUMO
African swine fever, caused by the African swine fever virus (ASFV), is a viral hemorrhagic disease that affects domestic pigs and wild boars. ASFV infection causes extensive tissue damage, and the associated mechanism is poorly understood. Pyroptosis is characterized by the activation of inflammatory caspases and pore formation in the cellular plasma membrane, resulting in the release of inflammatory cytokines and cell damage. How ASFV infection regulates pyroptosis remains unclear. Here, using siRNA assay and overexpression methods, we report that ASFV infection regulated pyroptosis by cleaving the pyroptosis execution protein gasdermin A (GSDMA). ASFV infection activated caspase-3 and caspase-4, which specifically cleaved GSDMA at D75-P76 and D241-V242 to produce GSDMA into five fragments, including GSDMA-N1-75, GSDMA-N1-241, and GSDMA-N76-241 fragments at the N-terminal end of GSDMA. Only GSDMA-N1-241, which was produced in the late stage of ASFV infection, triggered pyroptosis and inhibited ASFV replication. The fragments, GSDMA-N1-75 and GSDMA-N76-241, lose the ability to induce pyroptosis. Overall ASFV infection differentially regulates pyroptosis by GSDMA in the indicated phase, which may be conducive to its own replication. Our findings reveal a novel molecular mechanism for the regulation of pyroptosis.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Caspase 3 , Caspases Iniciadoras , Piroptose , Vírus da Febre Suína Africana/metabolismo , Animais , Febre Suína Africana/metabolismo , Febre Suína Africana/virologia , Febre Suína Africana/patologia , Suínos , Caspase 3/metabolismo , Caspase 3/genética , Caspases Iniciadoras/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Ligação a Fosfato/metabolismo , Células HEK293 , Replicação ViralRESUMO
Membrane-based cells are the fundamental structural and functional units of organisms, while evidences demonstrate that liquid-liquid phase separation (LLPS) is associated with the formation of membraneless organelles, such as P-bodies, nucleoli and stress granules. Many studies have been undertaken to explore the functions of protein phase separation (PS), but these studies lacked an effective tool to identify the sequence segments that critical for LLPS. In this study, we presented a novel software called dSCOPE (http://dscope.omicsbio.info) to predict the PS-driving regions. To develop the predictor, we curated experimentally identified sequence segments that can drive LLPS from published literature. Then sliding sequence window based physiological, biochemical, structural and coding features were integrated by random forest algorithm to perform prediction. Through rigorous evaluation, dSCOPE was demonstrated to achieve satisfactory performance. Furthermore, large-scale analysis of human proteome based on dSCOPE showed that the predicted PS-driving regions enriched various protein post-translational modifications and cancer mutations, and the proteins which contain predicted PS-driving regions enriched critical cellular signaling pathways. Taken together, dSCOPE precisely predicted the protein sequence segments critical for LLPS, with various helpful information visualized in the webserver to facilitate LLPS-related research.