RESUMO
Longitudinal analyses of the innate immune system, including the earliest time points, are essential to understand the immunopathogenesis and clinical course of coronavirus disease (COVID-19). Here, we performed a detailed characterization of natural killer (NK) cells in 205 patients (403 samples; days 2 to 41 after symptom onset) from four independent cohorts using single-cell transcriptomics and proteomics together with functional studies. We found elevated interferon (IFN)-α plasma levels in early severe COVD-19 alongside increased NK cell expression of IFN-stimulated genes (ISGs) and genes involved in IFN-α signaling, while upregulation of tumor necrosis factor (TNF)-induced genes was observed in moderate diseases. NK cells exert anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) activity but are functionally impaired in severe COVID-19. Further, NK cell dysfunction may be relevant for the development of fibrotic lung disease in severe COVID-19, as NK cells exhibited impaired anti-fibrotic activity. Our study indicates preferential IFN-α and TNF responses in severe and moderate COVID-19, respectively, and associates a prolonged IFN-α-induced NK cell response with poorer disease outcome.
Assuntos
COVID-19/imunologia , Interferon-alfa/imunologia , Células Matadoras Naturais/imunologia , SARS-CoV-2/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Sequência de Bases , Humanos , Imunidade Inata/imunologia , Inflamação/imunologia , Interferon-alfa/sangue , Fibrose Pulmonar/patologia , RNA-Seq , Índice de Gravidade de Doença , Transcriptoma/genética , Reino Unido , Estados UnidosRESUMO
Imiquimod is a small-molecule ligand of Toll-like receptor-7 (TLR7) that is licensed for the treatment of viral infections and cancers of the skin. Imiquimod has TLR7-independent activities that are mechanistically unexplained, including NLRP3 inflammasome activation in myeloid cells and apoptosis induction in cancer cells. We investigated the mechanism of inflammasome activation by imiquimod and the related molecule CL097 and determined that K+ efflux was dispensable for NLRP3 activation by these compounds. Imiquimod and CL097 inhibited the quinone oxidoreductases NQO2 and mitochondrial Complex I. This induced a burst of reactive oxygen species (ROS) and thiol oxidation, and led to NLRP3 activation via NEK7, a recently identified component of this inflammasome. Metabolic consequences of Complex I inhibition and endolysosomal effects of imiquimod might also contribute to NLRP3 activation. Our results reveal a K+ efflux-independent mechanism for NLRP3 activation and identify targets of imiquimod that might be clinically relevant.
Assuntos
Inflamassomos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Potássio/metabolismo , RNA Nuclear Pequeno/farmacologia , Animais , Complexo I de Transporte de Elétrons/metabolismo , Camundongos , Quinases Relacionadas a NIMA/metabolismo , Quinona Redutases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptor 7 Toll-Like/metabolismoRESUMO
ER-associated degradation (ERAD) is essential for protein quality control in the ER, not only when the ER is stressed, but also at steady state. We report a new layer of homeostatic control, in which ERAD activity itself is regulated posttranscriptionally and independently of the unfolded protein response by adjusting the endogenous levels of EDEM1, OS-9, and SEL1L (ERAD enhancers). Functional UBC6e requires its precise location in the ER to form a supramolecular complex with Derlin2. This complex targets ERAD enhancers for degradation, a function that depends on UBC6e's enzymatic activity. Ablation of UBC6e causes upregulation of active ERAD enhancers and so increases clearance not only of terminally misfolded substrates, but also of wild-type glycoproteins that fold comparatively slowly in vitro and in vivo. The levels of proteins that comprise the ERAD machinery are thus carefully tuned and adjusted to prevailing needs.
Assuntos
Retículo Endoplasmático/metabolismo , Lectinas/genética , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Processamento de Proteína Pós-Traducional , Proteínas/genética , Enzimas de Conjugação de Ubiquitina/genética , Animais , Degradação Associada com o Retículo Endoplasmático , Fibroblastos/citologia , Fibroblastos/metabolismo , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glicosilação , Células HEK293 , Humanos , Lectinas/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Proteínas de Neoplasias/metabolismo , Proteínas/metabolismo , Proteólise , Enzimas de Conjugação de Ubiquitina/deficiência , Resposta a Proteínas não DobradasRESUMO
ER-resident proteins destined for degradation are dislocated into the cytosol by components of the ER quality control machinery for proteasomal degradation. Dislocation substrates are ubiquitylated in the cytosol by E2 ubiquitin-conjugating/E3 ligase complexes. UBE2J1 is one of the well-characterized E2 enzymes that participate in this process. However, the physiological function of Ube2j1 is poorly defined. We find that Ube2j1(-/-) mice have reduced viability and fail to thrive early after birth. Male Ube2j1(-/-) mice are sterile due to a defect in late spermatogenesis. Ultrastructural analysis shows that removal of the cytoplasm is incomplete in Ube2j1(-/-) elongating spermatids, compromising the release of mature elongate spermatids into the lumen of the seminiferous tubule. Our findings identify an essential function for the ubiquitin-proteasome-system in spermiogenesis and define a novel, non-redundant physiological function for the dislocation step of ER quality control.
Assuntos
Espermatogênese , Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linhagem Celular , Retículo Endoplasmático/metabolismo , Imunoglobulinas/metabolismo , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , Camundongos , Espermátides/citologia , Espermátides/patologia , Enzimas de Conjugação de Ubiquitina/deficiência , Resposta a Proteínas não Dobradas , Regulação para CimaRESUMO
Antigen presenting cells (APCs) that express a catalytically inactive version of the deubiquitylase YOD1 (YOD1-C160S) present exogenous antigens more efficiently to CD8(+) T cells, both in vitro and in vivo. Compared with controls, immunization of YOD1-C160S mice led to greater expansion of specific CD8(+) T cells and showed improved control of infection with a recombinant -herpes virus, MHV-68, engineered to express SIINFEKL peptide, the ligand for the ovalbumin-specific TCR transgenic OT-I cells. Enhanced expansion of specific CD8(+) T cells was likewise observed on infection of YOD1-C160S mice with a recombinant influenza A virus expressing SIINFEKL. YOD1-C160S APCs retained antigen longer than did control APCs. Enhanced crosspresentation by YOD1-C160S APCs was transporter associated with antigen processing (TAP1)-independent but sensitive to inclusion of inhibitors of acidification and of the proteasome. The activity of deubiquitylating enzymes may thus help control antigenspecific CD8(+) T-cell responses during immunization.
Assuntos
Apresentação Cruzada , Mutação de Sentido Incorreto , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/imunologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/deficiência , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/imunologia , Transferência Adotiva , Animais , Células Apresentadoras de Antígenos/enzimologia , Células Apresentadoras de Antígenos/imunologia , Brefeldina A/farmacologia , Linfócitos T CD8-Positivos/imunologia , Apresentação Cruzada/efeitos dos fármacos , Apresentação Cruzada/genética , Feminino , Concentração de Íons de Hidrogênio , Imunização , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina/imunologia , Fragmentos de Peptídeos/imunologia , Rhadinovirus/imunologia , Rhadinovirus/patogenicidade , Ubiquitina Tiolesterase/metabolismoRESUMO
Multiple sclerosis (MS) is an autoimmune disease that affects the CNS. One approved treatment for relapsing forms of MS is YEAK, a random copolymer of the amino acids tyrosine, glutamic acid, alanine, and lysine. YFAK, a second-generation copolymer composed of tyrosine, phenylalanine, alanine, and lysine, is more successful in treating experimental autoimmune encephalomyelitis, a mouse model of MS. Although originally designed and optimized based on the autoantigen myelin basic protein (MBP) and the MBP-derived peptide MBP85-99 presented to the MS-associated class II MHC molecule HLA-DR2, YEAK and YFAK also stimulate cytokine and chemokine production in APCs that lack class II MHC products. How YEAK and YFAK copolymers interact with APCs remains enigmatic. We used biotinylated YFAK to affinity-purify YFAK-interacting proteins from RAW264.7 cells and tested APCs from mice deficient in several of the newly identified interactors for their capacity to secrete CCL22 in response to YEAK and YFAK. We propose that initial contact of YFAK with cells is mediated mainly by electrostatic interactions, and find that interaction of YFAK with host proteins is strongly dependent on ionic strength. Cells deficient in enzymes involved in sulfation of proteins and proteoglycans showed strongly reduced binding of biotinylated YFAK. Lastly, cells stimulated with YFAK in the presence of heparin, structurally similar to heparan sulfates, failed to produce CCL22. We conclude that charge-dependent interactions of copolymers that alleviate MS/experimental autoimmune encephalomyelitis are critical for their effects exerted on APCs and may well be the main initial mediators of these therapeutically active copolymers.
Assuntos
Aminoácidos/metabolismo , Células Apresentadoras de Antígenos/química , Células Apresentadoras de Antígenos/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/prevenção & controle , Heparitina Sulfato/metabolismo , Glicoproteínas de Membrana/metabolismo , Mapeamento de Interação de Proteínas/métodos , Aminoácidos/química , Aminoácidos/farmacologia , Animais , Células Apresentadoras de Antígenos/imunologia , Biopolímeros/química , Biopolímeros/metabolismo , Biopolímeros/farmacologia , Biotinilação , Linhagem Celular , Encefalomielite Autoimune Experimental/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/metabolismo , Glicoproteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Cultura Primária de Células , Distribuição AleatóriaRESUMO
Tripartite ATP-independent periplasmic (TRAP) transporters are found widely in bacteria and archaea and consist of three structural domains, a soluble substrate-binding protein (P-domain), and two transmembrane domains (Q- and M-domains). HiSiaPQM and its homologs are TRAP transporters for sialic acid and are essential for host colonization by pathogenic bacteria. Here, we reconstitute HiSiaQM into lipid nanodiscs and use cryo-EM to reveal the structure of a TRAP transporter. It is composed of 16 transmembrane helices that are unexpectedly structurally related to multimeric elevator-type transporters. The idiosyncratic Q-domain of TRAP transporters enables the formation of a monomeric elevator architecture. A model of the tripartite PQM complex is experimentally validated and reveals the coupling of the substrate-binding protein to the transporter domains. We use single-molecule total internal reflection fluorescence (TIRF) microscopy in solid-supported lipid bilayers and surface plasmon resonance to study the formation of the tripartite complex and to investigate the impact of interface mutants. Furthermore, we characterize high-affinity single variable domains on heavy chain (VHH) antibodies that bind to the periplasmic side of HiSiaQM and inhibit sialic acid uptake, providing insight into how TRAP transporter function might be inhibited in vivo.
Assuntos
Proteínas de Bactérias , Ácido N-Acetilneuramínico , Trifosfato de Adenosina/metabolismo , Archaea/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Ácido N-Acetilneuramínico/metabolismoRESUMO
The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. In this study, we generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. We used x-ray crystallography and cryo-electron microscopy to define two distinct binding epitopes. On the basis of these structures, we engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion and rendered the virions noninfectious.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Substituição de Aminoácidos , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/metabolismo , Afinidade de Anticorpos , Antígenos Virais/imunologia , Sítios de Ligação de Anticorpos , COVID-19/virologia , Linhagem Celular , Microscopia Crioeletrônica , Epitopos , Humanos , Fusão de Membrana , Mutação , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Receptores de Coronavírus/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Replicação ViralRESUMO
Achieving durable clinical responses to immune checkpoint inhibitors remains a challenge. Here, we demonstrate that immunotherapy with anti-CTLA-4 and its combination with anti-PD-1 rely on tumor cell-intrinsic activation of the cytosolic RNA receptor RIG-I. Mechanistically, tumor cell-intrinsic RIG-I signaling induced caspase-3-mediated tumor cell death, cross-presentation of tumor-associated antigen by CD103+ dendritic cells, subsequent expansion of tumor antigen-specific CD8+ T cells, and their accumulation within the tumor tissue. Consistently, therapeutic targeting of RIG-I with 5'- triphosphorylated RNA in both tumor and nonmalignant host cells potently augmented the efficacy of CTLA-4 checkpoint blockade in several preclinical cancer models. In humans, transcriptome analysis of primary melanoma samples revealed a strong association between high expression of DDX58 (the gene encoding RIG-I), T cell receptor and antigen presentation pathway activity, and prolonged overall survival. Moreover, in patients with melanoma treated with anti-CTLA-4 checkpoint blockade, high DDX58 RIG-I transcriptional activity significantly associated with durable clinical responses. Our data thus identify activation of RIG-I signaling in tumors and their microenvironment as a crucial component for checkpoint inhibitor-mediated immunotherapy of cancer.
Assuntos
Proteína DEAD-box 58/imunologia , Melanoma/imunologia , Animais , Linhagem Celular Tumoral , Estudos de Coortes , Proteína DEAD-box 58/genética , Modelos Animais de Doenças , Humanos , Imunoterapia , Melanoma/patologia , Melanoma/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microambiente TumoralRESUMO
Clearance of invading microbes requires phagocytes of the innate immune system. However, successful pathogens have evolved sophisticated strategies to evade immune killing. The opportunistic human fungal pathogen Candida albicans is efficiently phagocytosed by macrophages, but causes inflammasome activation, host cytolysis, and escapes after hypha formation. Previous studies suggest that macrophage lysis by C. albicans results from early inflammasome-dependent cell death (pyroptosis), late damage due to glucose depletion and membrane piercing by growing hyphae. Here we show that Candidalysin, a cytolytic peptide toxin encoded by the hypha-associated gene ECE1, is both a central trigger for NLRP3 inflammasome-dependent caspase-1 activation via potassium efflux and a key driver of inflammasome-independent cytolysis of macrophages and dendritic cells upon infection with C. albicans. This suggests that Candidalysin-induced cell damage is a third mechanism of C. albicans-mediated mononuclear phagocyte cell death in addition to damage caused by pyroptosis and the growth of glucose-consuming hyphae.
Assuntos
Proteínas Fúngicas/toxicidade , Inflamassomos/metabolismo , Leucócitos Mononucleares/citologia , Micotoxinas/toxicidade , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fagócitos/citologia , Actinas/metabolismo , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Caspase 1/metabolismo , Morte Celular/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Feminino , Humanos , Inflamação/patologia , Interleucina-1beta/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Necrose , Fagócitos/efeitos dos fármacos , Fagócitos/metabolismo , Fagossomos/efeitos dos fármacos , Fagossomos/metabolismo , Potássio/farmacologiaRESUMO
Kinase inhibitors are important cancer therapeutics. Polypharmacology is commonly observed, requiring thorough target deconvolution to understand drug mechanism of action. Using chemical proteomics, we analyzed the target spectrum of 243 clinically evaluated kinase drugs. The data revealed previously unknown targets for established drugs, offered a perspective on the "druggable" kinome, highlighted (non)kinase off-targets, and suggested potential therapeutic applications. Integration of phosphoproteomic data refined drug-affected pathways, identified response markers, and strengthened rationale for combination treatments. We exemplify translational value by discovering SIK2 (salt-inducible kinase 2) inhibitors that modulate cytokine production in primary cells, by identifying drugs against the lung cancer survival marker MELK (maternal embryonic leucine zipper kinase), and by repurposing cabozantinib to treat FLT3-ITD-positive acute myeloid leukemia. This resource, available via the ProteomicsDB database, should facilitate basic, clinical, and drug discovery research and aid clinical decision-making.
Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas/métodos , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/farmacologia , Proteômica/métodos , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Citocinas/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/enzimologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Camundongos , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidoresRESUMO
Fungal infections are major causes of morbidity and mortality, especially in immunocompromised individuals. The innate immune system senses fungal pathogens through Syk-coupled C-type lectin receptors (CLRs), which signal through the conserved immune adaptor Card9. Although Card9 is essential for antifungal defense, the mechanisms that couple CLR-proximal events to Card9 control are not well defined. Here, we identify Vav proteins as key activators of the Card9 pathway. Vav1, Vav2, and Vav3 cooperate downstream of Dectin-1, Dectin-2, and Mincle to engage Card9 for NF-κB control and proinflammatory gene transcription. Although Vav family members show functional redundancy, Vav1/2/3-/- mice phenocopy Card9-/- animals with extreme susceptibility to fungi. In this context, Vav3 is the single most important Vav in mice, and a polymorphism in human VAV3 is associated with susceptibility to candidemia in patients. Our results reveal a molecular mechanism for CLR-mediated Card9 regulation that controls innate immunity to fungal infections.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/genética , Candida/metabolismo , Candidemia/genética , Imunidade Inata/genética , Lectinas Tipo C/genética , Animais , Antifúngicos/administração & dosagem , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Candida/genética , Candida/patogenicidade , Candidemia/microbiologia , Candidemia/patologia , Humanos , Lectinas Tipo C/metabolismo , Proteínas de Membrana/genética , Camundongos , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-vav/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
THE TOPOLOGICAL BARRIERS DEFINED BY BIOLOGICAL MEMBRANES ARE NOT IMPERMEABLE: from small solutes to intact proteins, specialized transport and translocation mechanisms adjust to the cell's needs. Here, we review the removal of unwanted proteins from the endoplasmic reticulum (ER) and emphasize the need to extend observations from tissue culture models and simple eukaryotes to studies in whole animals. The variation in protein production and composition that characterizes different cell types and tissues requires tailor-made solutions to exert proper control over both protein synthesis and breakdown. The ER is an organelle essential to achieve and maintain such homeostasis.
RESUMO
To study the CD8(+) T cell response against a mouse γ-herpes virus, we generated K(b)-MHV-68-ORF8(604-612)RAG(-/-) CD8(+) T cell receptor transnuclear (TN) mice as a source of virus-specific CD8(+) T cells. K(b)-ORF8-Tet(+) CD8(+) T cells, expanded in the course of a resolving MHV-68 infection, served as a source of nucleus donors. Various in vivo and ex vivo assay criteria demonstrated the fine specificity and functionality of TN cells. TN cells proliferated extensively in response to viral infection, helped control viral burden, and exhibited a phenotype similar to that of endogenous K(b)-ORF8-Tet(+) cells. When compared to OT-1 cells, TN cells displayed distinct properties in response to lymphopenia and cognate antigen stimulation, which may be attributable to the affinity of the TCR expressed by the TN cells. The availability of MHV-68-specific CD8(+) TCR TN mice provides a new tool for investigating aspects of host-pathogen interactions unique to γ-herpes viruses.