RESUMO
Methods for the targeted disruption of protein function have revolutionized science and greatly expedited the systematic characterization of genes. Two main approaches are currently used to disrupt protein function: DNA knockout and RNA interference, which act at the genome and mRNA level, respectively. A method that directly alters endogenous protein levels is currently not available. Here, we present Trim-Away, a technique to degrade endogenous proteins acutely in mammalian cells without prior modification of the genome or mRNA. Trim-Away harnesses the cellular protein degradation machinery to remove unmodified native proteins within minutes of application. This rapidity minimizes the risk that phenotypes are compensated and that secondary, non-specific defects accumulate over time. Because Trim-Away utilizes antibodies, it can be applied to a wide range of target proteins using off-the-shelf reagents. Trim-Away allows the study of protein function in diverse cell types, including non-dividing primary cells where genome- and RNA-targeting methods are limited.
Assuntos
Anticorpos/química , Bioquímica/métodos , Transporte Proteico , Proteólise , AnimaisRESUMO
During pathogen infection, antibodies can be carried into the infected cell, where they are detected by the ubiquitously expressed cytosolic antibody receptor TRIM21. Here we found that recognition of intracellular antibodies by TRIM21 activated immune signaling. TRIM21 catalyzed the formation of Lys63 (K63)-linked ubiquitin chains and stimulated the transcription factor pathways of NF-κB, AP-1, IRF3, IRF5 and IRF7. Activation resulted in the production of proinflammatory cytokines, modulation of natural killer stress ligands and induction of an antiviral state. Intracellular antibody signaling was abrogated by genetic deletion of TRIM21 and was restored by ectopic expression of TRIM21. The sensing of antibodies by TRIM21 was stimulated after infection by DNA or RNA nonenveloped viruses or intracellular bacteria. Thus, the antibody-TRIM21 detection system provides potent, comprehensive activation of the innate immune system independently of known pattern-recognition receptors.
Assuntos
Anticorpos Antivirais/imunologia , Espaço Intracelular/imunologia , Espaço Intracelular/metabolismo , Receptores Fc/metabolismo , Ribonucleoproteínas/imunologia , Transdução de Sinais , Vírus/imunologia , Adenoviridae/imunologia , Animais , Anticorpos Antivirais/química , Anticorpos Antivirais/metabolismo , Complexo Antígeno-Anticorpo/imunologia , Complexo Antígeno-Anticorpo/metabolismo , Bactérias/imunologia , Linhagem Celular , Reações Cruzadas , Citocinas/biossíntese , Humanos , Mediadores da Inflamação/metabolismo , Fatores Reguladores de Interferon/metabolismo , Camundongos , Simulação de Acoplamento Molecular , NF-kappa B/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores de Reconhecimento de Padrão/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Fator de Transcrição AP-1/metabolismoRESUMO
Antibodies mediate the majority of their effects in the extracellular domain, or in intracellular compartments isolated from the cytosol. Under a growing list of circumstances, however, antibodies are found to gain access to the cytoplasm. Cytosolic immune complexes are bound by the atypical antibody receptor TRIM21, which mediates the rapid degradation of the immune complexes at the proteasome. These discoveries have informed the development of TRIM-Away, a technique to selectively deplete proteins using delivery of antibodies into cells. A range of related approaches that elicit selective protein degradation using intracellular constructs linking antibody fragments to degradative effector functions have also been developed. These methods hold promise for inducing the degradation of proteins as both research tools and as a novel therapeutic approach. Protein aggregates are a pathophysiological feature of neurodegenerative diseases and are considered to have a causal role in pathology. Immunotherapy is emerging as a promising route towards their selective targeting, and a role of antibodies in the cytosol has been demonstrated in cell-based assays. This review will explore the mechanisms by which therapeutic antibodies engage and eliminate intracellularly aggregated proteins. We will discuss how future developments in intracellular antibody technology may enhance the therapeutic potential of such antibody-derived therapies.
Assuntos
Doenças Neurodegenerativas , Complexo Antígeno-Anticorpo/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ribonucleoproteínas/metabolismoRESUMO
Progressive supranuclear palsy is a primary tauopathy affecting both neurons and glia and is responsible for both motor and cognitive symptoms. Recently, it has been suggested that progressive supranuclear palsy tauopathy may spread in the brain from cell to cell in a 'prion-like' manner. However, direct experimental evidence of this phenomenon, and its consequences on brain functions, is still lacking in primates. In this study, we first derived sarkosyl-insoluble tau fractions from post-mortem brains of patients with progressive supranuclear palsy. We also isolated the same fraction from age-matched control brains. Compared to control extracts, the in vitro characterization of progressive supranuclear palsy-tau fractions demonstrated a high seeding activity in P301S-tau expressing cells, displaying after incubation abnormally phosphorylated (AT8- and AT100-positivity), misfolded, filamentous (pentameric formyl thiophene acetic acid positive) and sarkosyl-insoluble tau. We bilaterally injected two male rhesus macaques in the supranigral area with this fraction of progressive supranuclear palsy-tau proteopathic seeds, and two other macaques with the control fraction. The quantitative analysis of kinematic features revealed that progressive supranuclear palsy-tau injected macaques exhibited symptoms suggestive of parkinsonism as early as 6 months after injection, remaining present until euthanasia at 18 months. An object retrieval task showed the progressive appearance of a significant dysexecutive syndrome in progressive supranuclear palsy-tau injected macaques compared to controls. We found AT8-positive staining and 4R-tau inclusions only in progressive supranuclear palsy-tau injected macaques. Characteristic pathological hallmarks of progressive supranuclear palsy, including globose and neurofibrillary tangles, tufted astrocytes and coiled bodies, were found close to the injection sites but also in connected brain regions that are known to be affected in progressive supranuclear palsy (striatum, pallidum, thalamus). Interestingly, while glial AT8-positive lesions were the most frequent near the injection site, we found mainly neuronal inclusions in the remote brain area, consistent with a neuronal transsynaptic spreading of the disease. Our results demonstrate that progressive supranuclear palsy patient-derived tau aggregates can induce motor and behavioural impairments in non-human primates related to the prion-like seeding and spreading of typical pathological progressive supranuclear palsy lesions. This pilot study paves the way for supporting progressive supranuclear palsy-tau injected macaque as a relevant animal model to accelerate drug development targeting this rare and fatal neurodegenerative disease.
Assuntos
Doenças Neurodegenerativas , Paralisia Supranuclear Progressiva , Tauopatias , Animais , Masculino , Paralisia Supranuclear Progressiva/patologia , Proteínas tau/metabolismo , Doenças Neurodegenerativas/patologia , Macaca mulatta/metabolismo , Projetos Piloto , Tauopatias/patologia , Encéfalo/patologiaRESUMO
INTRODUCTION: Signatures of a type-I interferon (IFN-I) response are observed in the post mortem brain in Alzheimer's disease (AD) and other tauopathies. However, the effect of the IFN-I response on pathological tau accumulation remains unclear. METHODS: We examined the effects of IFN-I signaling in primary neural culture models of seeded tau aggregation and P301S-tau transgenic mouse models in the context of genetic deletion of the IFN-I receptor (IFNAR). RESULTS: Polyinosinic:polycytidylic acid (PolyI:C), a synthetic analog of viral nucleic acids, evoked a potent cytokine response that enhanced seeded aggregation of tau in an IFN-I-dependent manner. IFN-I-induced vulnerability could be pharmacologically prevented and was intrinsic to neurons. Aged P301S-tau mice lacking Ifnar1 had significantly reduced tau pathology compared to mice with intact IFN signaling. DISCUSSION: We identify a critical role for IFN-I in potentiating tau aggregation. IFN-I is therefore identified as a potential therapeutic target in AD and other tauopathies. HIGHLIGHTS: Type-I IFN (IFN-I) promotes seeded tau aggregation in neural cultures. IFNAR inhibition prevents IFN-I driven sensitivity to tau aggregation. IFN-I driven vulnerability is intrinsic to neurons. Tau pathology is significantly reduced in aged P301S-tau mice lacking IFNAR.
Assuntos
Doença de Alzheimer , Interferon Tipo I , Tauopatias , Camundongos , Animais , Proteínas tau/genética , Interferon Tipo I/uso terapêutico , Tauopatias/patologia , Camundongos Transgênicos , Doença de Alzheimer/patologia , Modelos Animais de DoençasRESUMO
INTRODUCTION: The "prion-like" features of Alzheimer's disease (AD) tauopathy and its relationship with amyloid-ß (Aß) have never been experimentally studied in primates phylogenetically close to humans. METHODS: We injected 17 macaques in the entorhinal cortex with nanograms of seeding-competent tau aggregates purified from AD brains or control extracts from aged-matched healthy brains, with or without intracerebroventricular co-injections of oligomeric-Aß. RESULTS: Pathological tau injection increased cerebrospinal fluid (CSF) p-tau181 concentration after 18 months. Tau pathology spreads from the entorhinal cortex to the hippocampal trisynaptic loop and the cingulate cortex, resuming the experimental progression of Braak stage I to IV. Many AD-related molecular networks were impacted by tau seeds injections regardless of Aß injections in proteomic analyses. However, we found mature neurofibrillary tangles, increased CSF total-tau concentration, and pre- and postsynaptic degeneration only in Aß co-injected macaques. DISCUSSION: Oligomeric-Aß mediates the maturation of tau pathology and its neuronal toxicity in macaques but not its initial spreading. HIGHLIGHTS: This study supports the "prion-like" properties of misfolded tau extracted from AD brains. This study empirically validates the Braak staging in an anthropomorphic brain. This study highlights the role of oligomeric Aß in driving the maturation and toxicity of tau pathology. This work establishes a novel animal model of early sporadic AD that is closer to the human pathology.
Assuntos
Doença de Alzheimer , Príons , Animais , Humanos , Idoso , Doença de Alzheimer/patologia , Macaca/metabolismo , Proteômica , Proteínas tau/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/patologiaRESUMO
Hyperphosphorylation and aggregation of the protein tau play key roles in the development of Alzheimer's disease (AD). While the molecular structure of the filamentous tau aggregates has been determined to atomic resolution, there is far less information available about the smaller, soluble aggregates, which are believed to be more toxic. Traditional techniques are limited to bulk measures and struggle to identify individual aggregates in complex biological samples. To address this, we developed a novel single-molecule pull-down-based assay (MAPTau) to detect and characterize individual tau aggregates in AD and control post-mortem brain and biofluids. Using MAPTau, we report the quantity, as well as the size and circularity of tau aggregates measured using super-resolution microscopy, revealing AD-specific differences in tau aggregate morphology. By adapting MAPTau to detect multiple phosphorylation markers in individual aggregates using two-color coincidence detection, we derived compositional profiles of the individual aggregates. We find an AD-specific phosphorylation profile of tau aggregates with more than 80 % containing multiple phosphorylations, compared to 5 % in age-matched non-AD controls. Our results show that MAPTau is able to identify disease-specific subpopulations of tau aggregates phosphorylated at different sites, that are invisible to other methods and enable the study of disease mechanisms and diagnosis.
Assuntos
Doença de Alzheimer , Agregados Proteicos , Proteínas tau , Humanos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/diagnóstico , Proteínas tau/metabolismo , Proteínas tau/química , Proteínas tau/análise , Fosforilação , Imagem Individual de Molécula/métodos , Encéfalo/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/patologiaRESUMO
During the early stages of infection, the HIV-1 capsid protects viral components from cytosolic sensors and nucleases such as cGAS and TREX, respectively, while allowing access to nucleotides for efficient reverse transcription. Here we show that each capsid hexamer has a size-selective pore bound by a ring of six arginine residues and a 'molecular iris' formed by the amino-terminal ß-hairpin. The arginine ring creates a strongly positively charged channel that recruits the four nucleotides with on-rates that approach diffusion limits. Progressive removal of pore arginines results in a dose-dependent and concomitant decrease in nucleotide affinity, reverse transcription and infectivity. This positively charged channel is universally conserved in lentiviral capsids despite the fact that it is strongly destabilizing without nucleotides to counteract charge repulsion. We also describe a channel inhibitor, hexacarboxybenzene, which competes for nucleotide binding and efficiently blocks encapsidated reverse transcription, demonstrating the tractability of the pore as a novel drug target.
Assuntos
Capsídeo/metabolismo , Replicação do DNA , DNA Viral/biossíntese , HIV-1/metabolismo , Nucleotídeos/metabolismo , Arginina/metabolismo , Benzoatos/farmacologia , Ligação Competitiva/efeitos dos fármacos , Transporte Biológico Ativo/efeitos dos fármacos , Capsídeo/química , Capsídeo/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Difusão , Células HEK293 , HIV-1/efeitos dos fármacos , HIV-1/genética , HIV-1/crescimento & desenvolvimento , Células HeLa , Humanos , Cinética , Modelos Moleculares , Porosidade/efeitos dos fármacos , Transcrição Reversa/efeitos dos fármacosRESUMO
The accumulation of amyloid Tau aggregates is implicated in Alzheimer's disease (AD) and other tauopathies. Molecular chaperones are known to maintain protein homeostasis. Here, we show that an ATP-dependent human chaperone system disassembles Tau fibrils in vitro We found that this function is mediated by the core chaperone HSC70, assisted by specific cochaperones, in particular class B J-domain proteins and a heat shock protein 110 (Hsp110)-type nucleotide exchange factor (NEF). The Hsp70 disaggregation machinery processed recombinant fibrils assembled from all six Tau isoforms as well as Sarkosyl-resistant Tau aggregates extracted from cell cultures and human AD brain tissues, demonstrating the ability of the Hsp70 machinery to recognize a broad range of Tau aggregates. However, the chaperone activity released monomeric and small oligomeric Tau species, which induced the aggregation of self-propagating Tau conformers in a Tau cell culture model. We conclude that the activity of the Hsp70 disaggregation machinery is a double-edged sword, as it eliminates Tau amyloids at the cost of generating new seeds.
Assuntos
Doença de Alzheimer , Amiloide , Encéfalo , Proteínas de Choque Térmico HSP70 , Proteínas tau , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Amiloide/química , Amiloide/genética , Amiloide/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Células HEK293 , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Proteínas tau/química , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Alzheimer's disease (AD) and other neurodegenerative disorders are associated with the cytoplasmic aggregation of microtubule-associated protein tau. Recent evidence supports transcellular transfer of tau misfolding (seeding) as the mechanism of spread within an affected brain, a process reminiscent of viral infection. However, whereas microbial pathogens can be recognized as nonself by immune receptors, misfolded protein assemblies evade detection, as they are host-derived. Here, we show that when misfolded tau assemblies enter the cell, they can be detected and neutralized via a danger response mediated by tau-associated antibodies and the cytosolic Fc receptor tripartite motif protein 21 (TRIM21). We developed fluorescent, morphology-based seeding assays that allow the formation of pathological tau aggregates to be measured in situ within 24 h in the presence of picomolar concentrations of tau seeds. We found that anti-tau antibodies accompany tau seeds into the cell, where they recruit TRIM21 shortly after entry. After binding, TRIM21 neutralizes tau seeds through the activity of the proteasome and the AAA ATPase p97/VCP in a similar manner to infectious viruses. These results establish that intracellular antiviral immunity can be redirected against host-origin endopathogens involved in neurodegeneration.
Assuntos
Receptores Fc/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas tau/metabolismo , Animais , Anticorpos Neutralizantes/metabolismo , Células Cultivadas , Citosol/metabolismo , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Degeneração Neural/imunologia , Degeneração Neural/metabolismo , Degeneração Neural/prevenção & controle , Neurônios/imunologia , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Agregados Proteicos , Agregação Patológica de Proteínas/imunologia , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/prevenção & controle , Deficiências na Proteostase/metabolismo , Deficiências na Proteostase/prevenção & controle , Receptores Fc/deficiência , Receptores Fc/genética , Ribonucleoproteínas/deficiência , Ribonucleoproteínas/genética , Proteínas tau/química , Proteínas tau/imunologiaRESUMO
Encapsidation is a strategy almost universally employed by viruses to protect their genomes from degradation and from innate immune sensors. We show that TRIM21, which targets antibody-opsonized virions for proteasomal destruction, circumvents this protection, enabling the rapid detection and degradation of viral genomes before their replication. TRIM21 triggers an initial wave of cytokine transcription that is antibody, rather than pathogen, driven. This early response is augmented by a second transcriptional program, determined by the nature of the infecting virus. In this second response, TRIM21-induced exposure of the viral genome promotes sensing of DNA and RNA viruses by cGAS and RIG-I. This mechanism allows early detection of an infection event and drives an inflammatory response in mice within hours of viral challenge.
Assuntos
RNA Helicases DEAD-box/fisiologia , Genoma Viral , Nucleotidiltransferases/fisiologia , Fagocitose , Ribonucleoproteínas/fisiologia , Viroses/imunologia , Infecções por Adenovirus Humanos/imunologia , Animais , Proteína DEAD-box 58 , Células HeLa , Humanos , Imunidade Inata , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Picornaviridae/imunologia , Receptores Imunológicos , RhinovirusRESUMO
IgA is the most prevalent antibody type on mucosal surfaces and the second most prevalent antibody in circulation, yet its role in immune defense is not fully understood. Here we show that IgA is carried inside cells during virus infection, where it activates intracellular virus neutralization and innate immune signaling. Cytosolic IgA-virion complexes colocalize with the high-affinity antibody receptor tripartite motif-containing protein 21 (TRIM21) and are positive for lysine-48 ubiquitin chains. IgA neutralizes adenovirus infection in a TRIM21- and proteasome-dependent manner in both human and mouse cells. Translocated IgA also potently activates NF-κB signaling pathways in cells expressing TRIM21, whereas viral infection in the absence of antibody or TRIM21 is undetected. TRIM21 recognizes an epitope in IgG Fc that is not conserved in IgA; however, fluorescence anisotropy experiments demonstrate that direct binding to IgA is maintained. We use molecular modeling to show that TRIM21 forms a nonspecific hydrophobic seal around a ß-loop structure that is present in IgG, IgM, and IgA, explaining how TRIM21 achieves such remarkable broad antibody specificity. The findings demonstrate that the antiviral protection afforded by IgA extends to the intracellular cytosolic environment.
Assuntos
Infecções por Adenoviridae/imunologia , Anticorpos Neutralizantes/metabolismo , Imunidade Inata , Imunoglobulina A/metabolismo , Testes de Neutralização , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Citocinas/metabolismo , Células HeLa , Humanos , Imunoglobulina A/sangue , Imunoglobulina M/metabolismo , Espaço Intracelular/metabolismo , Camundongos , Modelos Moleculares , NF-kappa B/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Proteína com Valosina , Replicação ViralRESUMO
The HIV-1 capsid is involved in all infectious steps from reverse transcription to integration site selection, and is the target of multiple host cell and pharmacologic ligands. However, structural studies have been limited to capsid monomers (CA), and the mechanistic basis for how these ligands influence infection is not well understood. Here we show that a multi-subunit interface formed exclusively within CA hexamers mediates binding to linear epitopes within cellular cofactors NUP153 and CPSF6, and is competed for by the antiretroviral compounds PF74 and BI-2. Each ligand is anchored via a shared phenylalanine-glycine (FG) motif to a pocket within the N-terminal domain of one monomer, and all but BI-2 also make essential interactions across the N-terminal domain: C-terminal domain (NTD:CTD) interface to a second monomer. Dissociation of hexamer into CA monomers prevents high affinity interaction with CPSF6 and PF74, and abolishes binding to NUP153. The second interface is conformationally dynamic, but binding of NUP153 or CPSF6 peptides is accommodated by only one conformation. NUP153 and CPSF6 have overlapping binding sites, but each makes unique CA interactions that, when mutated selectively, perturb cofactor dependency. These results reveal that multiple ligands share an overlapping interface in HIV-1 capsid that is lost upon viral disassembly.
Assuntos
Proteínas do Capsídeo/metabolismo , Capsídeo/química , Infecções por HIV/virologia , HIV-1/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Fármacos Anti-HIV/farmacologia , Sítios de Ligação , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Infecções por HIV/tratamento farmacológico , HIV-1/genética , HIV-1/metabolismo , Humanos , Indóis/farmacologia , Ligantes , Modelos Moleculares , Modelos Estruturais , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Compostos Policíclicos/farmacologia , Polimerização , Ligação Proteica , Estrutura Terciária de Proteína , Transcrição Reversa/efeitos dos fármacos , Vírion , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
Tripartite motif-containing 21 (TRIM21) is a cytosolic IgG receptor that mediates intracellular virus neutralization by antibody. TRIM21 targets virions for destruction in the proteasome, but it is unclear how a substrate as large as a viral capsid is degraded. Here, we identify the ATPase p97/valosin-containing protein (VCP), an enzyme with segregase and unfoldase activity, as a key player in this process. Depletion or catalytic inhibition of VCP prevents capsid degradation and reduces neutralization. VCP is required concurrently with the proteasome, as addition of inhibitor after proteasomal degradation has no effect. Moreover, our results suggest that it is the challenging nature of virus as a substrate that necessitates involvement of VCP, since intracellularly expressed IgG Fc is degraded in a VCP-independent manner. These results implicate VCP as an important host factor in antiviral immunity.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Testes de Neutralização , Ribonucleoproteínas/fisiologia , Catálise , Humanos , Proteína com ValosinaRESUMO
Antibodies allow the immune system to target pathogens despite their tremendous diversity and rapid evolution. Once bound to a pathogen, antibodies induce a broad range of effector mechanisms, including phagocytosis and complement. However, these mechanisms are all initiated in the extracellular space, meaning that pathogens like viruses evade them upon infection of their target cells. Recently, it has been shown that, in addition to mediating extracellular immune responses, antibodies also activate immunity inside infected cells. Antibodies that are bound to the surface of non-enveloped viruses or bacteria are carried into the cell during pathogen entry. Once inside the cell, these pathogen-attached antibodies are recognised by a highly conserved, high affinity cytosolic antibody receptor called TRIM21. TRIM21 initiates both sensor and effector responses that reduce viral replication and induce an antiviral state. These responses are an important part of antiviral immunity and the removal of TRIM21 results in uncontrolled viraemia and death in a mouse model of infection.
Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Imunidade Humoral , Infecções/imunologia , Espaço Intracelular , Ribonucleoproteínas/metabolismo , Animais , Antígenos de Bactérias/metabolismo , Antígenos Virais/metabolismo , Modelos Animais de Doenças , Humanos , Espaço Intracelular/imunologia , Camundongos , Camundongos Knockout , Ribonucleoproteínas/genéticaRESUMO
Protection against bacterial and viral pathogens by antibodies has always been thought to end at the cell surface. Once inside the cell, a pathogen was understood to be safe from humoral immunity. However, it has now been found that antibodies can routinely enter cells attached to viral particles and mediate an intracellular immune response. Antibody-coated virions are detected inside the cell by means of an intracellular antibody receptor, TRIM21, which directs their degradation by recruitment of the ubiquitin-proteasome system. In this article we assess how this discovery alters our view of the way in which antibodies neutralise viral infection. We also consider the antiviral function of TRIM21 in the context of its other reported roles in immune signalling and autoimmunity. Finally, we discuss the conceptual implications of intracellular antibody immunity and how it alters our view of the discrete separation of extracellular and intracellular environments.
Assuntos
Anticorpos Neutralizantes/imunologia , Imunidade Humoral , Ribonucleoproteínas/imunologia , Adenoviridae/imunologia , Animais , Complexo Antígeno-Anticorpo/imunologia , Autoantígenos/imunologia , Citosol/imunologia , Citosol/virologia , Humanos , Camundongos , Complexo de Endopeptidases do Proteassoma/imunologia , Transdução de Sinais , Ubiquitinação , Ligação Viral , Internalização do VírusRESUMO
Antibodies provide effective antiviral immunity despite the fact that viruses escape into cells when they infect. Here we show that antibodies remain attached to viruses after cell infection and mediate an intracellular immune response that disables virions in the cytosol. We have discovered that cells possess a cytosolic IgG receptor, tripartite motif-containing 21 (TRIM21), which binds to antibodies with a higher affinity than any other IgG receptor in the human body. TRIM21 rapidly recruits to incoming antibody-bound virus and targets it to the proteasome via its E3 ubiquitin ligase activity. Proteasomal targeting leads to rapid degradation of virions in the cytosol before translation of virally encoded genes. Infection experiments demonstrate that at physiological antibody concentrations TRIM21 neutralizes viral infection. These results reveal an intracellular arm of adaptive immunity in which the protection mediated by antibodies does not end at the cell membrane but continues inside the cell to provide a last line of defense against infection.
Assuntos
Anticorpos Neutralizantes/imunologia , Imunidade/imunologia , Espaço Intracelular/imunologia , Ribonucleoproteínas/imunologia , Linhagem Celular , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Microesferas , Testes de Neutralização , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Inativação de VírusRESUMO
During initiation of antiviral and antitumor T cell-mediated immune responses, dendritic cells (DCs) cross-present exogenous antigens on major histocompatibility complex (MHC) class I molecules. Cross-presentation relies on the unusual "leakiness" of endocytic compartments in DCs, whereby internalized proteins escape into the cytosol for proteasome-mediated generation of MHC I-binding peptides. Given that type 1 conventional DCs excel at cross-presentation, we searched for cell type-specific effectors of endocytic escape. We devised an assay suitable for genetic screening and identified a pore-forming protein, perforin-2 (Mpeg1), as a dedicated effector exclusive to cross-presenting cells. Perforin-2 was recruited to antigen-containing compartments, where it underwent maturation, releasing its pore-forming domain. Mpeg1-/- mice failed to efficiently prime CD8+ T cells to cell-associated antigens, revealing an important role for perforin-2 in cytosolic entry of antigens during cross-presentation.
Assuntos
Apresentação de Antígeno , Linfócitos T CD8-Positivos , Endocitose , Proteínas Citotóxicas Formadoras de Poros , Animais , Camundongos , Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Apresentação Cruzada/genética , Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Endocitose/genética , Endocitose/imunologia , Testes Genéticos , Antígenos de Histocompatibilidade Classe I , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , ProteóliseRESUMO
Tau is a soluble protein interacting with tubulin to stabilize microtubules. However, under pathological conditions, it becomes hyperphosphorylated and aggregates, a process that can be induced by treating cells with exogenously added tau fibrils. Here, we employ single-molecule localization microscopy to resolve the aggregate species formed in early stages of seeded tau aggregation. We report that entry of sufficient tau assemblies into the cytosol induces the self-replication of small tau aggregates, with a doubling time of 5 h inside HEK cells and 1 day in murine primary neurons, which then grow into fibrils. Seeding occurs in the vicinity of the microtubule cytoskeleton, is accelerated by the proteasome, and results in release of small assemblies into the media. In the absence of seeding, cells still spontaneously form small aggregates at lower levels. Overall, our work provides a quantitative picture of the early stages of templated seeded tau aggregation in cells.