RESUMO
Small molecule chaperones have been exploited as therapeutics for the hundreds of diseases caused by protein misfolding. The most successful examples are the CFTR correctors, which transformed cystic fibrosis therapy. These molecules revert folding defects of the ΔF508 mutant and are widely used to treat patients. To investigate the molecular mechanism of their action, we determined cryo-electron microscopy structures of CFTR in complex with the FDA-approved correctors lumacaftor or tezacaftor. Both drugs insert into a hydrophobic pocket in the first transmembrane domain (TMD1), linking together four helices that are thermodynamically unstable. Mutating residues at the binding site rendered ΔF508-CFTR insensitive to lumacaftor and tezacaftor, underscoring the functional significance of the structural discovery. These results support a mechanism in which the correctors stabilize TMD1 at an early stage of biogenesis, prevent its premature degradation, and thereby allosterically rescuing many disease-causing mutations.
Assuntos
Aminopiridinas/metabolismo , Benzodioxóis/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Indóis/metabolismo , Dobramento de Proteína , Aminopiridinas/química , Aminopiridinas/uso terapêutico , Animais , Benzodioxóis/química , Benzodioxóis/uso terapêutico , Sítios de Ligação , Células CHO , Membrana Celular/química , Membrana Celular/metabolismo , Cricetulus , Microscopia Crioeletrônica , Fibrose Cística/tratamento farmacológico , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Indóis/química , Indóis/uso terapêutico , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/uso terapêutico , Mutação , Domínios Proteicos/genética , Células Sf9 , TransfecçãoRESUMO
Vaccines are urgently needed to control the ongoing pandemic COVID-19 and previously emerging MERS/SARS caused by coronavirus (CoV) infections. The CoV spike receptor-binding domain (RBD) is an attractive vaccine target but is undermined by limited immunogenicity. We describe a dimeric form of MERS-CoV RBD that overcomes this limitation. The RBD-dimer significantly increased neutralizing antibody (NAb) titers compared to conventional monomeric form and protected mice against MERS-CoV infection. Crystal structure showed RBD-dimer fully exposed dual receptor-binding motifs, the major target for NAbs. Structure-guided design further yielded a stable version of RBD-dimer as a tandem repeat single-chain (RBD-sc-dimer) which retained the vaccine potency. We generalized this strategy to design vaccines against COVID-19 and SARS, achieving 10- to 100-fold enhancement of NAb titers. RBD-sc-dimers in pilot scale production yielded high yields, supporting their scalability for further clinical development. The framework of immunogen design can be universally applied to other beta-CoV vaccines to counter emerging threats.
Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Desenho Universal , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/química , COVID-19 , Vacinas contra COVID-19 , Linhagem Celular Tumoral , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Coronavírus da Síndrome Respiratória do Oriente Médio/química , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/imunologia , Receptores Virais/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , SARS-CoV-2 , Células Sf9 , Organismos Livres de Patógenos Específicos , Spodoptera , Transfecção , Vacinação/métodos , Células Vero , Vacinas ViraisRESUMO
Neuronal cell types are the nodes of neural circuits that determine the flow of information within the brain. Neuronal morphology, especially the shape of the axonal arbor, provides an essential descriptor of cell type and reveals how individual neurons route their output across the brain. Despite the importance of morphology, few projection neurons in the mouse brain have been reconstructed in their entirety. Here we present a robust and efficient platform for imaging and reconstructing complete neuronal morphologies, including axonal arbors that span substantial portions of the brain. We used this platform to reconstruct more than 1,000 projection neurons in the motor cortex, thalamus, subiculum, and hypothalamus. Together, the reconstructed neurons constitute more than 85 meters of axonal length and are available in a searchable online database. Axonal shapes revealed previously unknown subtypes of projection neurons and suggest organizational principles of long-range connectivity.
Assuntos
Encéfalo/citologia , Encéfalo/diagnóstico por imagem , Neuritos/fisiologia , Tratos Piramidais/fisiologia , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Software , TransfecçãoRESUMO
Epitranscriptomic regulation controls information flow through the central dogma and provides unique opportunities for manipulating cells at the RNA level. However, both fundamental studies and potential translational applications are impeded by a lack of methods to target specific RNAs with effector proteins. Here, we present CRISPR-Cas-inspired RNA targeting system (CIRTS), a protein engineering strategy for constructing programmable RNA control elements. We show that CIRTS is a simple and generalizable approach to deliver a range of effector proteins, including nucleases, degradation machinery, translational activators, and base editors to target transcripts. We further demonstrate that CIRTS is not only smaller than naturally occurring CRISPR-Cas programmable RNA binding systems but can also be built entirely from human protein parts. CIRTS provides a platform to probe fundamental RNA regulatory processes, and the human-derived nature of CIRTS provides a potential strategy to avoid immune issues when applied to epitranscriptome-modulating therapies.
Assuntos
Edição de Genes/métodos , Engenharia de Proteínas/métodos , RNA Guia de Cinetoplastídeos/metabolismo , RNA/metabolismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Sistemas CRISPR-Cas/genética , Escherichia coli/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Biossíntese de Proteínas , Proteólise , RNA Interferente Pequeno , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , TransfecçãoRESUMO
In situ transgenesis methods such as viruses and electroporation can rapidly create somatic transgenic mice but lack control over copy number, zygosity, and locus specificity. Here we establish mosaic analysis by dual recombinase-mediated cassette exchange (MADR), which permits stable labeling of mutant cells expressing transgenic elements from precisely defined chromosomal loci. We provide a toolkit of MADR elements for combination labeling, inducible and reversible transgene manipulation, VCre recombinase expression, and transgenesis of human cells. Further, we demonstrate the versatility of MADR by creating glioma models with mixed reporter-identified zygosity or with "personalized" driver mutations from pediatric glioma. MADR is extensible to thousands of existing mouse lines, providing a flexible platform to democratize the generation of somatic mosaic mice. VIDEO ABSTRACT.
Assuntos
Neoplasias Encefálicas/genética , Modelos Animais de Doenças , Marcação de Genes/métodos , Loci Gênicos/genética , Glioma/genética , Mutagênese Insercional/métodos , Transgenes/genética , Animais , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Neurais/metabolismo , Recombinases/metabolismo , TransfecçãoRESUMO
T cell recognition of specific antigens mediates protection from pathogens and controls neoplasias, but can also cause autoimmunity. Our knowledge of T cell antigens and their implications for human health is limited by the technical limitations of T cell profiling technologies. Here, we present T-Scan, a high-throughput platform for identification of antigens productively recognized by T cells. T-Scan uses lentiviral delivery of antigen libraries into cells for endogenous processing and presentation on major histocompatibility complex (MHC) molecules. Target cells functionally recognized by T cells are isolated using a reporter for granzyme B activity, and the antigens mediating recognition are identified by next-generation sequencing. We show T-Scan correctly identifies cognate antigens of T cell receptors (TCRs) from viral and human genome-wide libraries. We apply T-Scan to discover new viral antigens, perform high-resolution mapping of TCR specificity, and characterize the reactivity of a tumor-derived TCR. T-Scan is a powerful approach for studying T cell responses.
Assuntos
Antígenos de Neoplasias/imunologia , Epitopos de Linfócito T/imunologia , Genes MHC Classe I/imunologia , Antígenos HLA/imunologia , Proteínas de Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/genética , Doadores de Sangue , Linfócitos T CD8-Positivos/metabolismo , Feminino , Técnicas de Inativação de Genes , Genes MHC Classe I/genética , Granzimas/metabolismo , Células HEK293 , Antígenos HLA/genética , Humanos , Proteínas de Neoplasias/genética , Transdução Genética , TransfecçãoRESUMO
Interferon-gamma (IFNG) augments immune function yet promotes T cell exhaustion through PDL1. How these opposing effects are integrated to impact immune checkpoint blockade (ICB) is unclear. We show that while inhibiting tumor IFNG signaling decreases interferon-stimulated genes (ISGs) in cancer cells, it increases ISGs in immune cells by enhancing IFNG produced by exhausted T cells (TEX). In tumors with favorable antigenicity, these TEX mediate rejection. In tumors with neoantigen or MHC-I loss, TEX instead utilize IFNG to drive maturation of innate immune cells, including a PD1+TRAIL+ ILC1 population. By disabling an inhibitory circuit impacting PD1 and TRAIL, blocking tumor IFNG signaling promotes innate immune killing. Thus, interferon signaling in cancer cells and immune cells oppose each other to establish a regulatory relationship that limits both adaptive and innate immune killing. In melanoma and lung cancer patients, perturbation of this relationship is associated with ICB response independent of tumor mutational burden.
Assuntos
Imunidade Adaptativa/imunologia , Imunidade Inata/imunologia , Interferon gama/genética , Interferon gama/metabolismo , Neoplasias Pulmonares/imunologia , Melanoma/imunologia , Transferência Adotiva , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4/antagonistas & inibidores , Linhagem Celular Tumoral , Estudos de Coortes , Feminino , Técnicas de Inativação de Genes , Humanos , Interferon gama/antagonistas & inibidores , Células Matadoras Naturais/imunologia , Neoplasias Pulmonares/tratamento farmacológico , Melanoma/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Intervalo Livre de Progressão , RNA-Seq , TransfecçãoRESUMO
Loss of BRCA1 p220 function often results in basal-like breast cancer (BLBC), but the underlying disease mechanism is largely opaque. In mammary epithelial cells (MECs), BRCA1 interacts with multiple proteins, including NUMB and HES1, to form complexes that participate in interstrand crosslink (ICL) DNA repair and MEC differentiation control. Unrepaired ICL damage results in aberrant transdifferentiation to a mesenchymal state of cultured, human basal-like MECs and to a basal/mesenchymal state in primary mouse luminal MECs. Loss of BRCA1, NUMB, or HES1 or chemically induced ICL damage in primary murine luminal MECs results in persistent DNA damage that triggers luminal to basal/mesenchymal transdifferentiation. In vivo single-cell analysis revealed a time-dependent evolution from normal luminal MECs to luminal progenitor-like tumor cells with basal/mesenchymal transdifferentiation during murine BRCA1 BLBC development. Growing DNA damage accompanied this malignant transformation.
Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/genética , Transdiferenciação Celular/genética , Dano ao DNA/genética , Reparo do DNA/genética , Glândulas Mamárias Animais/patologia , Animais , Proteína BRCA1/metabolismo , Neoplasias da Mama/induzido quimicamente , Neoplasias da Mama/patologia , Diferenciação Celular/genética , Transformação Celular Neoplásica , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Feminino , Células HEK293 , Humanos , Células MCF-7 , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição HES-1/metabolismo , TransfecçãoRESUMO
RLR-mediated type I IFN production plays a pivotal role in elevating host immunity for viral clearance and cancer immune surveillance. Here, we report that glycolysis, which is inactivated during RLR activation, serves as a barrier to impede type I IFN production upon RLR activation. RLR-triggered MAVS-RIG-I recognition hijacks hexokinase binding to MAVS, leading to the impairment of hexokinase mitochondria localization and activation. Lactate serves as a key metabolite responsible for glycolysis-mediated RLR signaling inhibition by directly binding to MAVS transmembrane (TM) domain and preventing MAVS aggregation. Notably, lactate restoration reverses increased IFN production caused by lactate deficiency. Using pharmacological and genetic approaches, we show that lactate reduction by lactate dehydrogenase A (LDHA) inactivation heightens type I IFN production to protect mice from viral infection. Our study establishes a critical role of glycolysis-derived lactate in limiting RLR signaling and identifies MAVS as a direct sensor of lactate, which functions to connect energy metabolism and innate immunity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína DEAD-box 58/antagonistas & inibidores , Proteína DEAD-box 58/metabolismo , Ácido Láctico/farmacologia , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/metabolismo , Animais , Feminino , Glicólise , Células HEK293 , Humanos , Interferon beta/metabolismo , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células RAW 264.7 , Receptores Imunológicos , Transdução de Sinais/efeitos dos fármacos , TransfecçãoRESUMO
Single-cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single-cell phenotypes have not been well defined. Combining single-cell RNA and protein analytics in studying the role of stromal cancer-associated fibroblasts (CAFs) in modulating heterogeneity in pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) model systems, we have identified significant single-cell population shifts toward invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes linked with mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling. Using high-content digital imaging of RNA in situ hybridization in 195 PDAC tumors, we quantified these EMT and PRO subpopulations in 319,626 individual cancer cells that can be classified within the context of distinct tumor gland "units." Tumor gland typing provided an additional layer of intratumoral heterogeneity that was associated with differences in stromal abundance and clinical outcomes. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.
Assuntos
Fibroblastos Associados a Câncer/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Microambiente Tumoral , Animais , Proliferação de Células , Técnicas de Cocultura , Transição Epitelial-Mesenquimal , Feminino , Células HEK293 , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Quinases Ativadas por Mitógeno/metabolismo , RNA-Seq , Fator de Transcrição STAT3/metabolismo , Células Estromais/metabolismo , TransfecçãoRESUMO
Long-distance RNA transport enables local protein synthesis at metabolically-active sites distant from the nucleus. This process ensures an appropriate spatial organization of proteins, vital to polarized cells such as neurons. Here, we present a mechanism for RNA transport in which RNA granules "hitchhike" on moving lysosomes. In vitro biophysical modeling, live-cell microscopy, and unbiased proximity labeling proteomics reveal that annexin A11 (ANXA11), an RNA granule-associated phosphoinositide-binding protein, acts as a molecular tether between RNA granules and lysosomes. ANXA11 possesses an N-terminal low complexity domain, facilitating its phase separation into membraneless RNA granules, and a C-terminal membrane binding domain, enabling interactions with lysosomes. RNA granule transport requires ANXA11, and amyotrophic lateral sclerosis (ALS)-associated mutations in ANXA11 impair RNA granule transport by disrupting their interactions with lysosomes. Thus, ANXA11 mediates neuronal RNA transport by tethering RNA granules to actively-transported lysosomes, performing a critical cellular function that is disrupted in ALS.
Assuntos
Anexinas/metabolismo , Transporte Axonal/fisiologia , Grânulos Citoplasmáticos/metabolismo , Lisossomos/metabolismo , RNA/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Animais , Animais Geneticamente Modificados , Anexinas/genética , Axônios/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Mutação , Ligação Proteica , Ratos/embriologia , Ratos Sprague-Dawley , Transfecção , Peixe-ZebraRESUMO
Intrinsic and acquired drug resistance and induction of secondary malignancies limit successful chemotherapy. Because mutagenic translesion synthesis (TLS) contributes to chemoresistance as well as treatment-induced mutations, targeting TLS is an attractive avenue for improving chemotherapeutics. However, development of small molecules with high specificity and in vivo efficacy for mutagenic TLS has been challenging. Here, we report the discovery of a small-molecule inhibitor, JH-RE-06, that disrupts mutagenic TLS by preventing recruitment of mutagenic POL ζ. Remarkably, JH-RE-06 targets a nearly featureless surface of REV1 that interacts with the REV7 subunit of POL ζ. Binding of JH-RE-06 induces REV1 dimerization, which blocks the REV1-REV7 interaction and POL ζ recruitment. JH-RE-06 inhibits mutagenic TLS and enhances cisplatin-induced toxicity in cultured human and mouse cell lines. Co-administration of JH-RE-06 with cisplatin suppresses the growth of xenograft human melanomas in mice, establishing a framework for developing TLS inhibitors as a novel class of chemotherapy adjuvants.
Assuntos
Antineoplásicos/uso terapêutico , Cisplatino/uso terapêutico , Mutagênese/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Quinolinas/uso terapêutico , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/efeitos adversos , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA , Feminino , Técnicas de Silenciamento de Genes , Humanos , Proteínas Mad2/metabolismo , Camundongos , Camundongos Nus , Camundongos Transgênicos , Neoplasias/metabolismo , Neoplasias/patologia , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Quinolinas/química , Quinolinas/farmacologia , Transfecção , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Microglia and central nervous system (CNS)-associated macrophages (CAMs), such as perivascular and meningeal macrophages, are implicated in virtually all diseases of the CNS. However, little is known about their cell-type-specific roles in the absence of suitable tools that would allow for functional discrimination between the ontogenetically closely related microglia and CAMs. To develop a new microglia gene targeting model, we first applied massively parallel single-cell analyses to compare microglia and CAM signatures during homeostasis and disease and identified hexosaminidase subunit beta (Hexb) as a stably expressed microglia core gene, whereas other microglia core genes were substantially downregulated during pathologies. Next, we generated HexbtdTomato mice to stably monitor microglia behavior in vivo. Finally, the Hexb locus was employed for tamoxifen-inducible Cre-mediated gene manipulation in microglia and for fate mapping of microglia but not CAMs. In sum, we provide valuable new genetic tools to specifically study microglia functions in the CNS.
Assuntos
Encéfalo/patologia , Encefalomielite Autoimune Experimental/patologia , Traumatismos do Nervo Facial/patologia , Microglia/metabolismo , Cadeia beta da beta-Hexosaminidase/metabolismo , Animais , Encéfalo/citologia , Encéfalo/imunologia , Sistemas CRISPR-Cas/genética , Encefalomielite Autoimune Experimental/imunologia , Traumatismos do Nervo Facial/imunologia , Técnicas de Introdução de Genes , Genes Reporter/genética , Loci Gênicos/genética , Humanos , Microscopia Intravital , Substâncias Luminescentes/química , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Microglia/imunologia , Células NIH 3T3 , RNA-Seq , Análise de Célula Única , Transfecção , Cadeia beta da beta-Hexosaminidase/genética , Proteína Vermelha FluorescenteRESUMO
Aerobic glycolysis regulates T cell function. However, whether and how primary cancer alters T cell glycolytic metabolism and affects tumor immunity in cancer patients remains a question. Here we found that ovarian cancers imposed glucose restriction on T cells and dampened their function via maintaining high expression of microRNAs miR-101 and miR-26a, which constrained expression of the methyltransferase EZH2. EZH2 activated the Notch pathway by suppressing Notch repressors Numb and Fbxw7 via trimethylation of histone H3 at Lys27 and, consequently, stimulated T cell polyfunctional cytokine expression and promoted their survival via Bcl-2 signaling. Moreover, small hairpin RNA-mediated knockdown of human EZH2 in T cells elicited poor antitumor immunity. EZH2(+)CD8(+) T cells were associated with improved survival in patients. Together, these data unveil a metabolic target and mechanism of cancer immune evasion.
Assuntos
Regulação Neoplásica da Expressão Gênica/imunologia , MicroRNAs , Neoplasias/imunologia , Complexo Repressor Polycomb 2/imunologia , Linfócitos T/imunologia , Evasão Tumoral/imunologia , Animais , Separação Celular , Imunoprecipitação da Cromatina , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Citometria de Fluxo , Imunofluorescência , Glicólise , Humanos , Immunoblotting , Melanoma Experimental/imunologia , Camundongos Endogâmicos C57BL , Neoplasias Ovarianas/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Análise Serial de Tecidos , TransfecçãoRESUMO
Rapid advances in nucleic acid therapies highlight the immense therapeutic potential of genetic therapeutics. Lipid nanoparticles (LNPs) are highly potent nonviral transfection agents that can encapsulate and deliver various nucleic acid therapeutics, including but not limited to messenger RNA (mRNA), silencing RNA (siRNA), and plasmid DNA (pDNA). However, a major challenge of targeted LNP-mediated systemic delivery is the nanoparticles' nonspecific uptake by the liver and the mononuclear phagocytic system, due partly to the adsorption of endogenous serum proteins onto LNP surfaces. Tunable LNP surface chemistries may enable efficacious delivery across a range of organs and cell types. Here, we describe a method to electrostatically adsorb bioactive polyelectrolytes onto LNPs to create layered LNPs (LLNPs). LNP cores varying in nucleic acid cargo and component lipids were stably layered with four biologically relevant polyanions: hyaluronate (HA), poly-L-aspartate (PLD), poly-L-glutamate (PLE), and polyacrylate (PAA). We further investigated the impact of the four surface polyanions on the transfection and uptake of mRNA- and pDNA-loaded LNPs in cell cultures. PLD- and PLE-LLNPs increased mRNA transfection twofold over unlayered LNPs in immune cells. HA-LLNPs increased pDNA transfection rates by more than twofold in epithelial and immune cells. In a healthy C57BL/6 murine model, PLE- and HA-LLNPs increased transfection by 1.8-fold to 2.5-fold over unlayered LNPs in the liver and spleen. These results suggest that LbL assembly is a generalizable, highly tunable platform to modify the targeting specificity, stability, and transfection efficacy of LNPs, as well as incorporate other charged targeting and therapeutic molecules into these systems.
Assuntos
Lipossomos , Nanopartículas , Animais , Camundongos , Polieletrólitos , Adsorção , Eletricidade Estática , Transfecção , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Ácido GlutâmicoRESUMO
Despite the widespread use of ionizable lipid nanoparticles (LNPs) in clinical applications for messenger RNA (mRNA) delivery, the mRNA drug delivery system faces an efficient challenge in the screening of LNPs. Traditional screening methods often require a substantial amount of experimental time and incur high research and development costs. To accelerate the early development stage of LNPs, we propose TransLNP, a transformer-based transfection prediction model designed to aid in the selection of LNPs for mRNA drug delivery systems. TransLNP uses two types of molecular information to perceive the relationship between structure and transfection efficiency: coarse-grained atomic sequence information and fine-grained atomic spatial relationship information. Due to the scarcity of existing LNPs experimental data, we find that pretraining the molecular model is crucial for better understanding the task of predicting LNPs properties, which is achieved through reconstructing atomic 3D coordinates and masking atom predictions. In addition, the issue of data imbalance is particularly prominent in the real-world exploration of LNPs. We introduce the BalMol block to solve this problem by smoothing the distribution of labels and molecular features. Our approach outperforms state-of-the-art works in transfection property prediction under both random and scaffold data splitting. Additionally, we establish a relationship between molecular structural similarity and transfection differences, selecting 4267 pairs of molecular transfection cliffs, which are pairs of molecules that exhibit high structural similarity but significant differences in transfection efficiency, thereby revealing the primary source of prediction errors. The code, model and data are made publicly available at https://github.com/wklix/TransLNP.
Assuntos
Lipídeos , Lipossomos , Nanopartículas , RNA Mensageiro , Nanopartículas/química , RNA Mensageiro/genética , RNA Mensageiro/química , Lipídeos/química , Transfecção , Humanos , Modelos Moleculares , Sistemas de Liberação de MedicamentosRESUMO
Enzymatically inactive chitinase-like proteins (CLPs) such as BRP-39, Ym1 and Ym2 are established markers of immune activation and pathology, yet their functions are essentially unknown. We found that Ym1 and Ym2 induced the accumulation of neutrophils through the expansion of γδ T cell populations that produced interleukin 17 (IL-17). While BRP-39 did not influence neutrophilia, it was required for IL-17 production in γδ T cells, which suggested that regulation of IL-17 is an inherent feature of mouse CLPs. Analysis of a nematode infection model, in which the parasite migrates through the lungs, revealed that the IL-17 and neutrophilic inflammation induced by Ym1 limited parasite survival but at the cost of enhanced lung injury. Our studies describe effector functions of CLPs consistent with innate host defense traits of the chitinase family.
Assuntos
Quitinases/imunologia , Glicoproteínas/imunologia , Lectinas/imunologia , Infecções por Nematoides/imunologia , Infiltração de Neutrófilos/imunologia , beta-N-Acetil-Hexosaminidases/imunologia , Animais , Proteína 1 Semelhante à Quitinase-3 , Citotoxicidade Imunológica/imunologia , Citometria de Fluxo , Imunofluorescência , Imunidade Inata/imunologia , Inflamação/imunologia , Interleucina-17/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nematoides , Neutrófilos/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Linfócitos T/imunologia , TransfecçãoRESUMO
The NLRP3 inflammasome functions as a crucial component of the innate immune system in recognizing viral infection, but the mechanism by which viruses activate this inflammasome remains unclear. Here we found that inhibition of the serine-threonine kinases RIP1 (RIPK1) or RIP3 (RIPK3) suppressed RNA virus-induced activation of the NLRP3 inflammasome. Infection with an RNA virus initiated assembly of the RIP1-RIP3 complex, which promoted activation of the GTPase DRP1 and its translocation to mitochondria to drive mitochondrial damage and activation of the NLRP3 inflammasome. Notably, the RIP1-RIP3 complex drove the NLRP3 inflammasome independently of MLKL, an essential downstream effector of RIP1-RIP3-dependent necrosis. Together our results reveal a specific role for the RIP1-RIP3-DRP1 pathway in RNA virus-induced activation of the NLRP3 inflammasome and establish a direct link between inflammation and cell-death signaling pathways.
Assuntos
Proteínas de Transporte/imunologia , Inflamassomos/imunologia , Infecções por Vírus de RNA/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologia , Transdução de Sinais/imunologia , Animais , Linhagem Celular , Dinaminas/imunologia , Ensaio de Imunoadsorção Enzimática , GTP Fosfo-Hidrolases/imunologia , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Proteínas Associadas aos Microtúbulos/imunologia , Proteínas Mitocondriais/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR , Vírus de RNA , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , TransfecçãoRESUMO
Interactions of T cell antigen receptors (TCRs) with complexes of self peptide and major histocompatibility complex (MHC) are crucial to T cell development, but their role in peripheral T cell responses remains unclear. Specific and nonspecific stimulation of LLO56 and LLO118 T cells, which transgenically express a TCR specific for the same Listeria monocytogenes epitope, elicited distinct interleukin 2 (IL-2) and phosphorylated kinase Erk responses, the strength of which was set in the thymus and maintained in the periphery in proportion to the avidity of the binding of the TCR to the self peptide-MHC complex. Deprivation of self peptide-MHC substantially compromised the population expansion of LLO56 T cells in response to L. monocytogenes in vivo. Despite their very different self-reactivity, LLO56 T cells and LLO118 T cells bound cognate peptide-MHC with an identical affinity, which challenges associations made between these parameters. Our findings highlight a crucial role for selecting ligands encountered during thymic 'education' in determining the intrinsic functionality of CD4+ T cells.
Assuntos
Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Ativação Linfocitária/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transferência Adotiva , Animais , Separação Celular , Citometria de Fluxo , Humanos , Immunoblotting , Listeriose/imunologia , Camundongos , Camundongos Knockout , Ressonância de Plasmônio de Superfície , Timo/citologia , Timo/imunologia , TransfecçãoRESUMO
The maintenance of immunological tolerance requires the deletion of self-reactive T cells in the thymus. The expression of genes encoding tissue-specific antigens (TSAs) by thymic epithelial cells is critical for this process and depends on activity of the transcriptional regulator Aire; however, the molecular mechanisms Aire uses to target loci encoding TSAs are unknown. Here we identified two Aire-interacting proteins known to be involved in gene repression, ATF7ip and MBD1, that were required for Aire's targeting of loci encoding TSAs. Moreover, Mbd1(-/-) mice developed pathological autoimmunity and had a defect in Aire-dependent thymic expression of genes encoding TSAs, which underscores the importance of Aire's interaction with the ATF7ip-MBD1 protein complex in maintaining central tolerance.