RESUMO
Antibodies can block immune receptor engagement or trigger the receptor machinery to initiate signaling. We hypothesized that antibody agonists trigger signaling by sterically excluding large receptor-type protein tyrosine phosphatases (RPTPs) such as CD45 from sites of receptor engagement. An agonist targeting the costimulatory receptor CD28 produced signals that depended on antibody immobilization and were sensitive to the sizes of the receptor, the RPTPs, and the antibody itself. Although both the agonist and a non-agonistic anti-CD28 antibody locally excluded CD45, the agonistic antibody was more effective. An anti-PD-1 antibody that bound membrane proximally excluded CD45, triggered Src homology 2 domain-containing phosphatase 2 recruitment, and suppressed systemic lupus erythematosus and delayed-type hypersensitivity in experimental models. Paradoxically, nivolumab and pembrolizumab, anti-PD-1-blocking antibodies used clinically, also excluded CD45 and were agonistic in certain settings. Reducing these agonistic effects using antibody engineering improved PD-1 blockade. These findings establish a framework for developing new and improved therapies for autoimmunity and cancer.
Assuntos
Proteínas Tirosina Fosfatases , Transdução de Sinais , Proteínas Tirosina Fosfatases/metabolismo , Antígenos CD28 , Receptores ImunológicosRESUMO
Neural migration is a critical step during brain development that requires the interactions of cell-surface guidance receptors. Cancer cells often hijack these mechanisms to disseminate. Here, we reveal crystal structures of Uncoordinated-5 receptor D (Unc5D) in complex with morphogen receptor glypican-3 (GPC3), forming an octameric glycoprotein complex. In the complex, four Unc5D molecules pack into an antiparallel bundle, flanked by four GPC3 molecules. Central glycan-glycan interactions are formed by N-linked glycans emanating from GPC3 (N241 in human) and C-mannosylated tryptophans of the Unc5D thrombospondin-like domains. MD simulations, mass spectrometry and structure-based mutants validate the crystallographic data. Anti-GPC3 nanobodies enhance or weaken Unc5-GPC3 binding and, together with mutant proteins, show that Unc5/GPC3 guide migrating pyramidal neurons in the mouse cortex, and cancer cells in an embryonic xenograft neuroblastoma model. The results demonstrate a conserved structural mechanism of cell guidance, where finely balanced Unc5-GPC3 interactions regulate cell migration.
Assuntos
Movimento Celular , Glipicanas/química , Receptores de Netrina/química , Animais , Glipicanas/metabolismo , Humanos , Camundongos , Proteínas Mutantes , Receptores de Netrina/metabolismo , Receptores de Superfície Celular/metabolismo , Anticorpos de Domínio Único , TrombospondinasRESUMO
CD28 and CTLA-4 (CD152) play essential roles in regulating T cell immunity, balancing the activation and inhibition of T cell responses, respectively. Although both receptors share the same ligands, CD80 and CD86, the specific requirement for two distinct ligands remains obscure. In the present study, we demonstrate that, although CTLA-4 targets both CD80 and CD86 for destruction via transendocytosis, this process results in separate fates for CTLA-4 itself. In the presence of CD80, CTLA-4 remained ligand bound, and was ubiquitylated and trafficked via late endosomes and lysosomes. In contrast, in the presence of CD86, CTLA-4 detached in a pH-dependent manner and recycled back to the cell surface to permit further transendocytosis. Furthermore, we identified clinically relevant mutations that cause autoimmune disease, which selectively disrupted CD86 transendocytosis, by affecting either CTLA-4 recycling or CD86 binding. These observations provide a rationale for two distinct ligands and show that defects in CTLA-4-mediated transendocytosis of CD86 are associated with autoimmunity.
Assuntos
Antígenos CD , Antígenos CD28 , Antígenos CD/metabolismo , Antígenos de Diferenciação/metabolismo , Antígeno B7-1 , Antígeno B7-2/genética , Antígenos CD28/metabolismo , Antígeno CTLA-4/genética , Moléculas de Adesão Celular , Ligantes , Ativação LinfocitáriaRESUMO
The SLC15 family of proton-coupled solute carriers PepT1 and PepT2 play a central role in human physiology as the principal route for acquiring and retaining dietary nitrogen. A remarkable feature of the SLC15 family is their extreme substrate promiscuity, which has enabled the targeting of these transporters for the improvement of oral bioavailability for several prodrug molecules. Although recent structural and biochemical studies on bacterial homologs have identified conserved sites of proton and peptide binding, the mechanism of peptide capture and ligand promiscuity remains unclear for mammalian family members. Here, we present the cryo-electron microscopy structure of the outward open conformation of the rat peptide transporter PepT2 in complex with an inhibitory nanobody. Our structure, combined with molecular dynamics simulations and biochemical and cell-based assays, establishes a framework for understanding peptide and prodrug recognition within this pharmaceutically important transporter family.
Assuntos
Pró-Fármacos , Simportadores , Animais , Microscopia Crioeletrônica , Mamíferos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Transportador 1 de Peptídeos/química , Peptídeos/metabolismo , Prótons , RatosRESUMO
Folates (also known as vitamin B9) have a critical role in cellular metabolism as the starting point in the synthesis of nucleic acids, amino acids and the universal methylating agent S-adenylsmethionine1,2. Folate deficiency is associated with a number of developmental, immune and neurological disorders3-5. Mammals cannot synthesize folates de novo; several systems have therefore evolved to take up folates from the diet and distribute them within the body3,6. The proton-coupled folate transporter (PCFT) (also known as SLC46A1) mediates folate uptake across the intestinal brush border membrane and the choroid plexus4,7, and is an important route for the delivery of antifolate drugs in cancer chemotherapy8-10. How PCFT recognizes folates or antifolate agents is currently unclear. Here we present cryo-electron microscopy structures of PCFT in a substrate-free state and in complex with a new-generation antifolate drug (pemetrexed). Our results provide a structural basis for understanding antifolate recognition and provide insights into the pH-regulated mechanism of folate transport mediated by PCFT.
Assuntos
Microscopia Crioeletrônica , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/metabolismo , Pemetrexede/química , Pemetrexede/metabolismo , Transportador de Folato Acoplado a Próton/química , Transportador de Folato Acoplado a Próton/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestrutura , Transporte Biológico , Humanos , Modelos Moleculares , Transportador de Folato Acoplado a Próton/ultraestrutura , PrótonsRESUMO
There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic.
Assuntos
Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , Peptídeos/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/imunologia , Animais , Anticorpos Bloqueadores/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , COVID-19/imunologia , Linhagem Celular , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , SuínosRESUMO
Clinical development of the COVID-19 vaccine candidate ChAdOx1 nCoV-19, a replication-deficient simian adenoviral vector expressing the full-length SARS-CoV-2 spike (S) protein was initiated in April 2020 following non-human primate studies using a single immunisation. Here, we compared the immunogenicity of one or two doses of ChAdOx1 nCoV-19 in both mice and pigs. Whilst a single dose induced antigen-specific antibody and T cells responses, a booster immunisation enhanced antibody responses, particularly in pigs, with a significant increase in SARS-CoV-2 neutralising titres.
RESUMO
Platelet activation is regulated by both positive and negative signals. G6B-b is an inhibitory platelet receptor with an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). The molecular basis of inhibition by G6B-b is currently unknown but thought to involve the SH2 domain-containing tyrosine phosphatase SHP-1. Here we show that G6B-b also associates with SHP-2, as well as SHP-1, in human platelets. Using a number of biochemical approaches, we found these interactions to be direct and that the tandem SH2 domains of SHP-2 demonstrated a binding affinity for G6B-b 100-fold higher than that of SHP-1. It was also observed that while SHP-1 has an absolute requirement for phosphorylation at both motifs to bind, SHP-2 can associate with G6B-b when only one motif is phosphorylated, with the N-terminal SH2 domain and the ITIM being most important for the interaction. A number of other previously unreported SH2 domain-containing proteins, including Syk and PLCγ2, also demonstrated specificity for G6B-b phosphomotifs and may serve to explain the observation that G6B-b remains inhibitory in the absence of both SHP-1 and SHP-2. In addition, the presence of dual phosphorylated G6B-b in washed human platelets can reduce the EC(50) for both CRP and collagen.