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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Type 1 diabetes (T1D) is an autoimmune disease involving T cell-mediated destruction of the insulin-producing beta cells in the pancreatic islets of Langerhans. CD8+ T cells, responding to beta cell peptides presented by class I major histocompatibility complex (MHC) molecules, are important effectors leading to beta cell elimination. Human leukocyte antigen (HLA) B*39:06, B*39:01, and B*38:01 are closely related class I MHC allotypes that nonetheless show differential association with T1D. HLA-B*39:06 is the most predisposing of all HLA class I molecules and is associated with early age at disease onset. B*39:01 is also associated with susceptibility to T1D, but to a lesser extent, though differing from B*39:06 by only two amino acids. HLA-B*38:01, in contrast, is associated with protection from the disease. Upon identifying a peptide that binds to both HLA-B*39:06 and B*39:01, we determined the respective X-ray structures of the two allotypes presenting this peptide to 1.7 Å resolution. The peptide residues available for T cell receptor contact and those serving as anchors were identified. Analysis of the F pocket of HLA-B*39:06 and B*39:01 provided an explanation for the distinct peptide C-terminus preferences of the two allotypes. Structure-based modeling of the protective HLA-B*38:01 suggested a potential reason for its peptide preferences and its reduced propensity to present 8-mer peptides compared to B*39:06. Notably, the three allotypes showed differential binding to peptides derived from beta cell autoantigens. Taken together, our findings should facilitate identification of disease-relevant candidate T cell epitopes and structure-guided therapeutics to interfere with peptide binding.
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Change history: In the HTML version of this Letter, Extended Data Fig. 4 incorrectly corresponded to Fig. 4 (the PDF version of the figure was correct). This has been corrected online.
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Viral infections continue to represent major challenges to public health, and an enhanced mechanistic understanding of the processes that contribute to viral life cycles is necessary for the development of new therapeutic strategies 1 . Viperin, a member of the radical S-adenosyl-L-methionine (SAM) superfamily of enzymes, is an interferon-inducible protein implicated in the inhibition of replication of a broad range of RNA and DNA viruses, including dengue virus, West Nile virus, hepatitis C virus, influenza A virus, rabies virus 2 and HIV3,4. Viperin has been suggested to elicit these broad antiviral activities through interactions with a large number of functionally unrelated host and viral proteins3,4. Here we demonstrate that viperin catalyses the conversion of cytidine triphosphate (CTP) to 3'-deoxy-3',4'-didehydro-CTP (ddhCTP), a previously undescribed biologically relevant molecule, via a SAM-dependent radical mechanism. We show that mammalian cells expressing viperin and macrophages stimulated with IFNα produce substantial quantities of ddhCTP. We also establish that ddhCTP acts as a chain terminator for the RNA-dependent RNA polymerases from multiple members of the Flavivirus genus, and show that ddhCTP directly inhibits replication of Zika virus in vivo. These findings suggest a partially unifying mechanism for the broad antiviral effects of viperin that is based on the intrinsic enzymatic properties of the protein and involves the generation of a naturally occurring replication-chain terminator encoded by mammalian genomes.
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Antivirais/metabolismo , Citidina Trifosfato/metabolismo , Genoma Humano/genética , Proteínas/genética , Proteínas/metabolismo , Terminação da Transcrição Genética , Animais , Antivirais/química , Chlorocebus aethiops , Citidina Trifosfato/biossíntese , Citidina Trifosfato/química , Células HEK293 , Humanos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/metabolismo , Ribonucleotídeos , Especificidade por Substrato , Células Vero , Zika virus/enzimologia , Zika virus/metabolismoRESUMO
The immune system's ability to recognize peptides on major histocompatibility molecules contributes to the eradication of cancers and pathogens. Tracking these responses in vivo could help evaluate the efficacy of immune interventions and improve mechanistic understanding of immune responses. For this purpose, we employ synTacs, which are dimeric major histocompatibility molecule scaffolds of defined composition. SynTacs, when labeled with positron-emitting isotopes, can noninvasively image antigen-specific CD8+ T cells in vivo. Using radiolabeled synTacs loaded with the appropriate peptides, we imaged human papillomavirus-specific CD8+ T cells by positron emission tomography in mice bearing human papillomavirus-positive tumors, as well as influenza A virus-specific CD8+ T cells in the lungs of influenza A virus-infected mice. It is thus possible to visualize antigen-specific CD8+ T-cell populations in vivo, which may serve prognostic and diagnostic roles.
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Linfócitos T CD8-Positivos/fisiologia , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/virologia , Papillomaviridae/imunologia , Tomografia por Emissão de Pósitrons/métodos , Animais , Antígenos , Clonagem Molecular , Epitopos/genética , Epitopos/metabolismo , Feminino , Regulação da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe I/classificação , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoglobulina G/classificação , Imunoglobulina G/imunologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/imunologiaRESUMO
The clinical outcome of SARS-CoV-2 infection varies widely between individuals. Machine learning models can support decision making in healthcare by assessing fatality risk in patients that do not yet show severe signs of COVID-19. Most predictive models rely on static demographic features and clinical values obtained upon hospitalization. However, time-dependent biomarkers associated with COVID-19 severity, such as antibody titers, can substantially contribute to the development of more accurate outcome models. Here we show that models trained on immune biomarkers, longitudinally monitored throughout hospitalization, predicted mortality and were more accurate than models based on demographic and clinical data upon hospital admission. Our best-performing predictive models were based on the temporal analysis of anti-SARS-CoV-2 Spike IgG titers, white blood cell (WBC), neutrophil and lymphocyte counts. These biomarkers, together with C-reactive protein and blood urea nitrogen levels, were found to correlate with severity of disease and mortality in a time-dependent manner. Shapley additive explanations of our model revealed the higher predictive value of day post-symptom onset (PSO) as hospitalization progresses and showed how immune biomarkers contribute to predict mortality. In sum, we demonstrate that the kinetics of immune biomarkers can inform clinical models to serve as a powerful monitoring tool for predicting fatality risk in hospitalized COVID-19 patients, underscoring the importance of contextualizing clinical parameters according to their time post-symptom onset.
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Anticorpos Antivirais/sangue , COVID-19 , SARS-CoV-2/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , COVID-19/diagnóstico , COVID-19/epidemiologia , COVID-19/imunologia , COVID-19/terapia , Biologia Computacional , Diagnóstico por Computador , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto JovemRESUMO
Machupo virus, known to cause hemorrhagic fevers, enters human cells via binding with its envelope glycoprotein to transferrin receptor 1 (TfR). Similarly, the receptor interactions have been explored in biotechnological applications as a molecular system to ferry therapeutics across the cellular membranes and through the impenetrable blood-brain barrier that effectively blocks any such delivery into the brain. Study of the experimental structure of Machupo virus glycoprotein 1 (MGP1) in complex with TfR and glycoprotein sequence homology has identified some residues at the interface that influence binding. There are, however, no studies that have attempted to optimize the binding potential between MGP1 and TfR. In pursuits for finding therapeutic solutions for the New World arenaviruses, and to gain a greater understanding of MGP1 interactions with TfR, it is crucial to understand the structure-sequence relationship driving the interface formation. By displaying MGP1 on yeast surface we have examined the contributions of individual residues to the binding of solubilized ectodomain of TfR. We identified MGP1 binding hot spot residues, assessed the importance of posttranslational N-glycan modifications, and used a selection with random mutagenesis for affinity maturation. We show that the optimized MGP1 variants can bind more strongly to TfR than the native MGP1, and there is an MGP1 sequence that retains binding in the absence of glycosylation, but with the addition of further amino acid substitutions. The engineered variants can be used to probe cellular internalization or the blood-brain barrier crossing to achieve greater understanding of TfR mediated internalization.
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Antígenos CD , Arenavirus do Novo Mundo/química , Receptores da Transferrina , Proteínas do Envelope Viral , Antígenos CD/química , Antígenos CD/genética , Antígenos CD/metabolismo , Humanos , Modelos Moleculares , Mutação , Engenharia de Proteínas , Receptores da Transferrina/química , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Herpes simplex virus (HSV) infections manifest as recurrent oral or genital mucosal lesions, meningoencephalitis, corneal blindness, and perinatal disease. Subunit vaccines have advanced into the clinic without success. None were tested preclinically in male mice. We compared a single-cycle candidate vaccine deleted in HSV-2 glycoprotein D (ΔgD-2) and subunit gD-2 or gD-1 protein vaccines in a male murine skin model. The ΔgD-2 provided complete protection against 10 times the lethal dose of HSV-1 or HSV-2 clinical isolates, and no latent virus was detected, whereas gD-1- and gD-2-adjuvanted proteins provided little or no protection. Protection correlated with Fc receptor activating but not neutralizing antibody titers.
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Herpes Simples/prevenção & controle , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 2/imunologia , Vacinas contra Herpesvirus/administração & dosagem , Vacinas contra Herpesvirus/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Modelos Animais de Doenças , Deleção de Genes , Herpesvirus Humano 2/genética , Masculino , Camundongos Endogâmicos C57BL , Receptores Fc/metabolismo , Análise de Sobrevida , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Proteínas Estruturais Virais/genéticaRESUMO
Mycobacterium tuberculosis remains a threat to global health, and a more efficacious vaccine is needed to prevent disease caused by M. tuberculosis We previously reported that the mycobacterial ribosome is a major target of CD4+ T cells in mice immunized with a genetically modified Mycobacterium smegmatis strain (IKEPLUS) but not in mice immunized with Mycobacterium bovis BCG. Two specific ribosomal proteins, RplJ and RpsA, were identified as cross-reactive targets of M. tuberculosis, but the breadth of the CD4+ T cell response to M. tuberculosis ribosomes was not determined. In the present study, a library of M. tuberculosis ribosomal proteins and in silico-predicted peptide libraries were used to screen CD4+ T cell responses in IKEPLUS-immunized mice. This identified 24 out of 57 M. tuberculosis ribosomal proteins distributed over both large and small ribosome subunits as specific CD4+ T cell targets. Although BCG did not induce detectable responses against ribosomal proteins or peptide epitopes, the M. tuberculosis ribosomal protein RplJ produced a robust and multifunctional Th1-like CD4+ T cell population when administered as a booster vaccine to previously BCG-primed mice. Boosting of BCG-primed immunity with the M. tuberculosis RplJ protein led to significantly reduced lung pathology compared to that in BCG-immunized animals and reductions in the bacterial burdens in the mediastinal lymph node compared to those in naive and standard BCG-vaccinated mice. These results identify the mycobacterial ribosome as a potential source of cryptic or subdominant antigenic targets of protective CD4+ T cell responses and suggest that supplementing BCG with ribosomal antigens may enhance protective vaccination against M. tuberculosis.
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Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Mycobacterium tuberculosis/química , Proteínas Ribossômicas/imunologia , Tuberculose/imunologia , Animais , Vacina BCG/imunologia , Feminino , Imunização Secundária , Interferon gama/imunologia , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/imunologia , Biblioteca de Peptídeos , Tuberculose/prevenção & controle , Vacinas contra a Tuberculose/imunologiaRESUMO
Large-scale activity profiling of enzyme superfamilies provides information about cellular functions as well as the intrinsic binding capabilities of conserved folds. Herein, the functional space of the ubiquitous haloalkanoate dehalogenase superfamily (HADSF) was revealed by screening a customized substrate library against >200 enzymes from representative prokaryotic species, enabling inferred annotation of â¼35% of the HADSF. An extremely high level of substrate ambiguity was revealed, with the majority of HADSF enzymes using more than five substrates. Substrate profiling allowed assignment of function to previously unannotated enzymes with known structure, uncovered potential new pathways, and identified iso-functional orthologs from evolutionarily distant taxonomic groups. Intriguingly, the HADSF subfamily having the least structural elaboration of the Rossmann fold catalytic domain was the most specific, consistent with the concept that domain insertions drive the evolution of new functions and that the broad specificity observed in HADSF may be a relic of this process.
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Família Multigênica , Monoéster Fosfórico Hidrolases/metabolismo , Ensaios de Triagem em Larga Escala , Cinética , Reprodutibilidade dos Testes , Especificidade por SubstratoRESUMO
TYRO3, AXL, and MER receptors (TAMs) are three homologous type I receptor-tyrosine kinases that are activated by endogenous ligands, protein S (PROS1) and growth arrest-specific gene 6 (GAS6). These ligands can either activate TAMs as soluble factors, or, in turn, opsonize phosphatidylserine (PS) on apoptotic cells (ACs) and serve as bridging molecules between ACs and TAMs. Abnormal expression and activation of TAMs have been implicated in promoting proliferation and survival of cancer cells, as well as in suppressing anti-tumor immunity. Despite the fact that TAM receptors share significant similarity, little is known about the specificity of interaction between TAM receptors and their ligands, particularly in the context of ACs, and about the functional diversity of TAM receptors. To study ligand-mediated activation of TAMs, we generated a series of reporter cell lines expressing chimeric TAM receptors. Using this system, we found that each TAM receptor has a unique pattern of interaction with and activation by GAS6 and PROS1, which is also differentially affected by the presence of ACs, PS-containing lipid vesicles and enveloped virus. We also demonstrated that γ-carboxylation of ligands is essential for the full activation of TAMs and that soluble immunoglobulin-like TAM domains act as specific ligand antagonists. These studies demonstrate that, despite their similarity, TYRO3, AXL, and MER are likely to perform distinct functions in both immunoregulation and the recognition and removal of ACs.
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Apoptose/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Proteínas Sanguíneas/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células Jurkat , Proteína S , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Estomatite Vesicular/genética , c-Mer Tirosina Quinase , Receptor Tirosina Quinase AxlRESUMO
MERTK, a member of the TAM (TYRO3, AXL, and MERTK) receptor tyrosine kinases, has complex and diverse roles in cell biology. On the one hand, knock-out of MERTK results in age-dependent autoimmunity characterized by failure of apoptotic cell clearance, while on the other, MERTK overexpression in cancer drives classical oncogene pathways leading to cell transformation. To better understand the interplay between cell transformation and efferocytosis, we stably expressed MERTK in human MCF10A cells, a non-tumorigenic breast epithelial cell line devoid of endogenous MERTK. While stable expression of MERTK in MCF10A resulted in enhanced motility and AKT-mediated chemoprotection, MERTK-10A cells did not form stable colonies in soft agar, or enhance proliferation compared with parental MCF10A cells. Concomitant to chemoresistance, MERTK also stimulated efferocytosis in a gain-of-function capacity. However, unlike AXL, MERTK activation was highly dependent on apoptotic cells, suggesting MERTK may preferentially interface with phosphatidylserine. Consistent with this idea, knockdown of MERTK in breast cancer cells MDA-MB 231 reduced efferocytosis, while transient or stable expression of MERTK stimulated apoptotic cell clearance in all cell lines tested. Moreover, human breast cancer cells with higher endogenous MERTK showed higher levels of efferocytosis that could be blocked by soluble TAM receptors. Finally, through MERTK, apoptotic cells induced PD-L1 expression, an immune checkpoint blockade, suggesting that cancer cells may adopt MERTK-driven efferocytosis as an immune suppression mechanism for their advantage. These data collectively identify MERTK as a significant link between cancer progression and efferocytosis, and a potentially unrealized tumor-promoting event when MERTK is overexpressed in epithelial cells.
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Neoplasias da Mama/genética , Transformação Celular Neoplásica/genética , Células Epiteliais/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Apoptose/genética , Neoplasias da Mama/patologia , Movimento Celular/genética , Células Epiteliais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Fagocitose/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , c-Mer Tirosina Quinase , Receptor Tirosina Quinase AxlRESUMO
There is an unmet need for monoclonal antibodies (mAbs) for prevention or as adjunctive treatment of herpes simplex virus (HSV) disease. Most vaccine and mAb efforts focus on neutralizing antibodies, but for HSV this strategy has proven ineffective. Preclinical studies with a candidate HSV vaccine strain, ΔgD-2, demonstrated that non-neutralizing antibodies that activate Fcγ receptors (FcγRs) to mediate antibody-dependent cellular cytotoxicity (ADCC) provide active and passive protection against HSV-1 and HSV-2. We hypothesized that this vaccine provides a tool to identify and characterize protective mAbs. We isolated HSV-specific mAbs from germinal center and memory B cells and bone marrow plasmacytes of ΔgD-2-vaccinated mice and evaluated these mAbs for binding, neutralizing, and FcγR-activating activity and for protective efficacy in mice. The most potent protective mAb, BMPC-23, was not neutralizing but activated murine FcγRIV, a biomarker of ADCC. The cryo-electron microscopic structure of the Fab-glycoprotein B (gB) assembly identified domain IV of gB as the epitope. A single dose of BMPC-23 administered 24 hours before or after viral challenge provided significant protection when configured as mouse IgG2c and protected mice expressing human FcγRIII when engineered as a human IgG1. These results highlight the importance of FcR-activating antibodies in protecting against HSV.
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Herpes Simples , Herpesvirus Humano 1 , Animais , Humanos , Camundongos , Anticorpos Neutralizantes , Herpes Simples/prevenção & controle , Anticorpos Antivirais , Glicoproteínas , Anticorpos Monoclonais , Proteínas do Envelope Viral/genéticaRESUMO
Antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target multiple epitopes on different domains of the spike protein, and other SARS-CoV-2 proteins. We developed a SARS-CoV-2 multi-antigen protein microarray with the nucleocapsid, spike and its domains (S1, S2), and variants with single (D614G, E484K, N501Y) or double substitutions (N501Y/Deletion69/70), allowing a more detailed high-throughput analysis of the antibody repertoire following infection. The assay was demonstrated to be reliable and comparable to ELISA. We analyzed antibodies from 18 COVID-19 patients and 12 recovered convalescent donors. The S IgG level was higher than N IgG in most of the COVID-19 patients, and the receptor-binding domain of S1 showed high reactivity, but no antibodies were detected against the heptad repeat domain 2 of S2. Furthermore, antibodies were detected against S variants with single and double substitutions in COVID-19 patients who were infected with SARS-CoV-2 early in the pandemic. Here we demonstrated that the SARS-CoV-2 multi-antigen protein microarray is a powerful tool for detailed characterization of antibody responses, with potential utility in understanding the disease progress and assessing current vaccines and therapies against evolving SARS-CoV-2.
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COVID-19 , SARS-CoV-2 , Humanos , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Formação de Anticorpos/genética , Formação de Anticorpos/imunologia , COVID-19/genética , COVID-19/imunologia , COVID-19/virologia , Imunoglobulina G , Análise Serial de Proteínas , SARS-CoV-2/genética , Glicoproteína da Espícula de CoronavírusRESUMO
Pre-steady state kinetic analysis was utilized for biochemical evaluation of a series of cyclobutyl adenosine nucleotide analogs with HIV-1 RT(WT). The phosphonyl-diphosphate form of the cyclobutyl nucleotide, 5, was the most efficiently incorporated of the series. Nucleotide 5 was fourfold more efficiently incorporated than the FDA approved TFV-DP by RT(WT). The kinetics of incorporation for 5 using the drug resistant mutant enzyme K65R was also determined. Compound 5 was threefold more efficiently incorporated compared to TFV-DP with RT(K65R). These results demonstrate cyclobutyl adenosine analogs can act as substrates for incorporation by HIV-1 RT and be a potential scaffold for HIV inhibitors.
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Fármacos Anti-HIV/síntese química , Ciclobutanos/síntese química , Nucleotídeos de Desoxiadenina/síntese química , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Inibidores da Transcriptase Reversa/síntese química , Adenina/análogos & derivados , Adenina/farmacologia , Fármacos Anti-HIV/farmacologia , Ciclobutanos/farmacologia , Primers do DNA , Nucleotídeos de Desoxiadenina/farmacologia , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/genética , HIV-1/genética , Humanos , Cinética , Mutação , Técnicas de Amplificação de Ácido Nucleico , Organofosfonatos/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , TenofovirRESUMO
Human transferrin receptor 1 (TfR) is necessary for the delivery of the iron carrier protein transferrin into cells and can be utilized for targeted delivery across cellular membranes. Binding of transferrin to the receptor is regulated by hereditary hemochromatosis protein (HFE), an iron regulatory protein that partly shares a binding site with transferrin on TfR. Here, we derived essential binding interactions from HFE and computationally grafted these into a library of small protein scaffolds. One of the designed proteins, TB08, was further optimized computationally and experimentally to identify variants with improved binding to TfR. The optimized variant, TB08 S3.1, expressed well in the E. coli expression system and had an affinity to TfR in the low micromolar range, Kd ≈ 1 µm, as determined by surface plasmon resonance. A binding competition assay with transferrin further confirmed the interaction of the evolved variant to TfR at the shared binding surface. Additionally, the GFP-tagged evolved variant of TB08 demonstrated cellular internalization as determined by fluorescent and confocal microscopy in HeLa cells. The designed protein is small, allows for robust cargo tagging, and interacts specifically with TfR, thus making it a valuable tool for the characterization of TfR-mediated cellular transport mechanisms and for the assessment of engineering strategies for cargo delivery across cell membranes.
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Receptores da Transferrina , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Domínios Proteicos , Engenharia de Proteínas , Receptores da Transferrina/química , Receptores da Transferrina/genética , Transferrina/químicaRESUMO
Cell surface receptors, ligands, and adhesion molecules underlie development, circuit formation, and synaptic function of the central nervous system and represent important therapeutic targets for many neuropathologies. The functional contributions of interactions between cell surface proteins of neurons and nonneuronal cells have not been fully addressed. Using an unbiased protein-protein interaction screen, we showed that the human immunomodulatory ligand B7-1 (hB7-1) interacts with the p75 neurotrophin receptor (p75NTR) and that the B7-1:p75NTR interaction is a recent evolutionary adaptation present in humans and other primates, but absent in mice, rats, and other lower mammals. The surface of hB7-1 that engages p75NTR overlaps with the hB7-1 surface involved in CTLA-4/CD28 recognition, and these molecules directly compete for binding to p75NTR. Soluble or membrane-bound hB7-1 altered dendritic morphology of cultured hippocampal neurons, with loss of the postsynaptic protein PSD95 in a p75NTR-dependent manner. Abatacept, an FDA-approved therapeutic (CTLA-4-hFc fusion) inhibited these processes. In vivo injection of hB7-1 into the murine subiculum, a hippocampal region affected in Alzheimer's disease, resulted in p75NTR-dependent pruning of dendritic spines. Here, we report the biochemical interaction between B7-1 and p75NTR, describe biological effects on neuronal morphology, and identify a therapeutic opportunity for treatment of neuroinflammatory diseases.
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Antígeno B7-1 , Neurônios , Receptor de Fator de Crescimento Neural , Receptores de Fator de Crescimento Neural , Sinapses , Animais , Humanos , Camundongos , Ratos , Antígeno CTLA-4/metabolismo , Neurônios/metabolismo , Receptor de Fator de Crescimento Neural/genética , Receptor de Fator de Crescimento Neural/metabolismo , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Antígeno B7-1/metabolismo , Sinapses/metabolismoRESUMO
Aerobic organisms have a tricarboxylic acid (TCA) cycle that is functionally distinct from those found in anaerobic organisms. Previous reports indicate that the aerobic pathogen Mycobacterium tuberculosis lacks detectable alpha-ketoglutarate (KG) dehydrogenase activity and drives a variant TCA cycle in which succinyl-CoA is replaced by succinic semialdehyde. Here, we show that M. tuberculosis expresses a CoA-dependent KG dehydrogenase activity, albeit one that is typically found in anaerobic bacteria. Unlike most enzymes of this family, the M. tuberculosis KG: ferredoxin oxidoreductase (KOR) is extremely stable under aerobic conditions. This activity is absent in a mutant strain deleted for genes encoding a previously uncharacterized oxidoreductase, and this strain is impaired for aerobic growth in the absence of sufficient amounts of CO(2). Interestingly, inhibition of the glyoxylate shunt or exclusion of exogenous fatty acids alleviates this growth defect, indicating the presence of an alternate pathway that operates in the absence of beta-oxidation. Simultaneous disruption of KOR and the first enzyme of the succinic semialdehyde pathway (KG decarboxylase; KGD) results in strict dependence upon the glyoxylate shunt for growth, demonstrating that KG decarboxylase is also functional in M. tuberculosis intermediary metabolism. These observations demonstrate that unlike most organisms M. tuberculosis utilizes two distinct TCA pathways from KG, one that functions concurrently with beta-oxidation (KOR-dependent), and one that functions in the absence of beta-oxidation (KGD-dependent). As these pathways are regulated by metabolic cues, we predict that their differential utilization provides an advantage for growth in different environments within the host.
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Ciclo do Ácido Cítrico/fisiologia , Complexo Cetoglutarato Desidrogenase/fisiologia , Mycobacterium tuberculosis/fisiologia , Piruvato Sintase/fisiologia , Anaerobiose/fisiologia , OxirreduçãoRESUMO
We present a direct injection mass spectrometry (DI-MS) platform that accurately, precisely, and quickly quantitates global levels of DNA cytidine methylation (5 mC) and hydroxymethylation (5hmC). Our platform combines an Advion TriVersa NanoMate coupled online to a Thermo Scientific Orbitrap Fusion Lumos. Following digestion to nucleosides, the DNA samples are analyzed at the rate of <1 min per injection with comparable detection limits of 0.63 ng/µL and 0.31 ng/µL, respectively. In contrast, the detection limits for 5 mC and 5hmC in state-of-art nano liquid chromatography (LC) coupled to online mass spectrometry (nLC-MS) are notably different (0.04 ng/µL and 2.5 ng/µL, respectively). The high sensitivity of DI-MS is achieved by optimizing sample buffer composition, the source fragmentation energy, and the radio frequency of the instrument ion funnel. DI-MS accurately reports the relative abundance of 5 mC and 5hmC over a range of 1%-7% (R2 > 0.98) and 0.13%-1.75% (R2 > 0.99), respectively. Accurate measurement of C, 5 mC and 5hmC is achieved by optimizing in-source fragmentation to obtain a population of up to 93% of just the nucleoside base. This protocol minimizes base dimer formation and partial base-deoxyribose dissociation in gas phase and greatly improves modified base quantitation. We also demonstrate that DI-MS overcomes biases in differential chromatographic retention and issues of sample degradation in the autosampler due to its high throughput. Finally, we present an application of our workflow to quantify DNA modifications on a batch of 81 samples in about 1.5 h.
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Metilação de DNA , DNA , Viés , Cromatografia Líquida , Espectrometria de MassasRESUMO
To delineate the in vivo role of different costimulatory signals in activating and expanding highly functional virus-specific cytotoxic CD8+ T cells, we designed synTacs, infusible biologics that recapitulate antigen-specific T cell activation signals delivered by antigen-presenting cells. We constructed synTacs consisting of dimeric Fc-domain scaffolds linking CD28- or 4-1BB-specific ligands to HLA-A2 MHC molecules covalently tethered to HIV- or CMV-derived peptides. Treatment of HIV-infected donor PBMCs with synTacs bearing HIV- or CMV-derived peptides induced vigorous and selective ex vivo expansion of highly functional HIV- and/or CMV-specific CD8+ T cells, respectively, with potent antiviral activities. Intravenous injection of HIV- or CMV-specific synTacs into immunodeficient mice intrasplenically engrafted with donor PBMCs markedly and selectively expanded HIV-specific (32-fold) or CMV-specific (46-fold) human CD8+ T cells populating their spleens. Notably, these expanded HIV- or CMV-specific CD8+ T cells directed potent in vivo suppression of HIV or CMV infections in the humanized mice, providing strong rationale for consideration of synTac-based approaches as a therapeutic strategy to cure HIV and treat CMV and other viral infections. The synTac platform flexibility supports facile delineation of in vivo effects of different costimulatory signals on patient-derived virus-specific CD8+ T cells, enabling optimization of individualized therapies, including HIV cure strategies.