RESUMO
Memory T cells are critical for the immune response to recurring infections. Their instantaneous reactivity to pathogens is empowered by the persistent expression of cytokine-encoding mRNAs. How the translation of proteins from pre-formed cytokine-encoding mRNAs is prevented in the absence of infection has remained unclear. Here we found that protein production in memory T cells was blocked via a 3' untranslated region (3' UTR)-mediated process. Germline deletion of AU-rich elements (AREs) in the Ifng-3' UTR led to chronic cytokine production in memory T cells. This aberrant protein production did not result from increased expression and/or half-life of the mRNA. Instead, AREs blocked the recruitment of cytokine-encoding mRNA to ribosomes; this block depended on the ARE-binding protein ZFP36L2. Thus, AREs mediate repression of translation in mouse and human memory T cells by preventing undesirable protein production from pre-formed cytokine-encoding mRNAs in the absence of infection.
Assuntos
Regiões 3' não Traduzidas/genética , Elementos Ricos em Adenilato e Uridilato/genética , Interferon gama/genética , RNA Mensageiro/genética , Linfócitos T/imunologia , Animais , Células Cultivadas , Repressão Epigenética , Memória Imunológica , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Elongação Traducional da Cadeia Peptídica , Ribossomos/metabolismo , Tristetraprolina/genética , Tristetraprolina/metabolismoRESUMO
Resident memory T (TRM) cells have been recently established as an important subset of memory T cells that provide early and essential protection against reinfection in the absence of circulating memory T cells. Recent findings showing that TRM expand in vivo after repeated antigenic stimulation indicate that these memory T cells are not terminally differentiated. This suggests an opportunity for in vitro TRM expansion to apply in an immunotherapy setting. However, it has also been shown that TRM may not maintain their identity and form circulating memory T cells after in vivo restimulation. Therefore, we set out to determine how TRM respond to antigenic activation in culture. Using Listeria monocytogenes and LCMV infection models, we found that TRM from the intraepithelial compartment of the small intestine expand in vitro after antigenic stimulation and subsequent resting in homeostatic cytokines. A large fraction of the expanded TRM retained their phenotype, including the expression of key TRM markers CD69 and CD103 (ITGAE). The optimal culture of TRM required low O2 pressure to maintain the expression of these and other TRM-associated molecules. Expanded TRM retained their effector capacity to produce cytokines after restimulation, but did not acquire a highly glycolytic profile indicative of effector T cells. The proteomic analysis confirmed TRM profile retention, including expression of TRM-related transcription factors, tissue retention factors, adhesion molecules, and enzymes involved in fatty acid metabolism. Collectively, our data indicate that limiting oxygen conditions supports in vitro expansion of TRM cells that maintain their TRM phenotype, at least in part, suggesting an opportunity for therapeutic strategies that require in vitro expansion of TRM.
Assuntos
Memória Imunológica , Listeria monocytogenes , Células T de Memória , Animais , Células T de Memória/imunologia , Memória Imunológica/imunologia , Camundongos , Listeria monocytogenes/imunologia , Antígenos CD/metabolismo , Antígenos CD/imunologia , Cadeias alfa de Integrinas/metabolismo , Camundongos Endogâmicos C57BL , Listeriose/imunologia , Lectinas Tipo C/metabolismo , Lectinas Tipo C/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Citocinas/metabolismo , Citocinas/imunologia , Ativação Linfocitária/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Mucosa Intestinal/imunologia , Linfócitos T CD8-Positivos/imunologia , Intestino Delgado/imunologia , Células CultivadasRESUMO
Memory CD8+ T cells are indispensable for maintaining long-term immunity against intracellular pathogens and tumors. Despite their presence at oxygen-deprived infected tissue sites or in tumors, the impact of local oxygen pressure on memory CD8+ T cells remains largely unclear. We sought to elucidate how oxygen pressure impacts memory CD8+ T cells arising after infection with Listeria monocytogenes-OVA. Our data revealed that reduced oxygen pressure during in vitro culture switched CD8+ T cell metabolism from oxidative phosphorylation to a glycolytic phenotype. Quantitative proteomic analysis showed that limiting oxygen conditions increased the expression of glucose transporters and components of the glycolytic pathway, while decreasing TCA cycle and mitochondrial respiratory chain proteins. The altered CD8+ T cell metabolism did not affect the expansion potential, but enhanced the granzyme B and IFN-γ production capacity. In vivo, memory CD8+ T cells cultured under low oxygen pressure provided protection against bacterial rechallenge. Taken together, our study indicates that strategies of cellular immune therapy may benefit from reducing oxygen during culture to develop memory CD8+ T cells with superior effector functions.
Assuntos
Listeria monocytogenes , Listeriose , Neoplasias , Animais , Camundongos , Linfócitos T CD8-Positivos , Proteômica , Neoplasias/patologia , Oxigênio/metabolismo , Glicólise , Memória Imunológica , Camundongos Endogâmicos C57BLRESUMO
Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is an autoimmune disorder caused by the development of autoantibodies targeting different domains of ADAMTS13. Profiling studies have shown that residues R568, F592, R660, Y661, and Y665 within exosite-3 of the spacer domain provide an immunodominant region of ADAMTS13 for pathogenic autoantibodies that develop in patients with iTTP. Modification of these 5 core residues with the goal of reducing autoantibody binding revealed a significant tradeoff between autoantibody resistance and proteolytic activity. Here, we employed structural bioinformatics to identify a larger epitope landscape on the ADAMTS13 spacer domain. Models of spacer-antibody complexes predicted that residues R568, L591, F592, K608, M609, R636, L637, R639, R660, Y661, Y665, and L668 contribute to an expanded epitope within the spacer domain. Based on bioinformatics-guided predictions, we designed a panel of N-glycan insertions in this expanded epitope to reduce the binding of spacer domain autoantibodies. One N-glycan variant (NGLY3-ADAMTS13, containing a K608N substitution) showed strongly reduced reactivity with TTP patient sera (28%) as compared with WT-ADAMTS13 (100%). Insertion of an N-glycan at amino acid position 608 did not interfere with processing of von Willebrand factor, positioning the resulting NGLY3-ADAMTS13 variant as a potential novel therapeutic option for treatment of iTTP.
Assuntos
Proteína ADAMTS13/imunologia , Complexo Antígeno-Anticorpo/química , Reações Antígeno-Anticorpo , Autoanticorpos/imunologia , Autoantígenos/imunologia , Polissacarídeos/imunologia , Púrpura Trombocitopênica Trombótica/imunologia , Proteína ADAMTS13/química , Proteína ADAMTS13/metabolismo , Substituição de Aminoácidos , Aminoácidos , Anticorpos Monoclonais/imunologia , Complexo Antígeno-Anticorpo/imunologia , Autoanticorpos/metabolismo , Autoantígenos/química , Autoantígenos/metabolismo , Epitopos/imunologia , Epitopos/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Fator de von Willebrand/metabolismoRESUMO
BACKGROUND: Biomonitoring may provide important insights into the impact of a whole blood donation for individual blood donors. STUDY DESIGN AND METHODS: Here, we used unbiased mass spectrometry (MS)-based proteomics to assess longitudinal changes in the global plasma proteome, after a single blood donation for new and regular donors. Subsequently, we compared plasma proteomes of 76 male and female whole blood donors, that were grouped based on their ferritin and hemoglobin (Hb) levels. RESULTS: The longitudinal analysis showed limited changes in the plasma proteomes of new and regular donors after a whole blood donation during a 180-day follow-up period, apart from a significant short-term decrease in fibronectin. No differences were observed in the plasma proteomes of donors with high versus low Hb and/or ferritin levels. Plasma proteins with the highest variation between and within donors included pregnancy zone protein, which was associated with sex, Alfa 1-antitrypsin which was associated with the allelic variation, and Immunoglobulin D. Coexpression analysis revealed clustering of proteins that are associated with platelet, red cell, and neutrophil signatures as well as with the complement system and immune responses, including a prominent correlating cluster of immunoglobulin M (IgM), immunoglobulin J chain (JCHAIN), and CD5 antigen-like (CD5L). DISCUSSION: Overall, our proteomic approach shows that whole blood donation has a limited impact on the plasma proteins measured. Our findings suggest that plasma profiling can be successfully employed to consistently detect proteins and protein complexes that reflect the functionality and integrity of platelets, red blood cells, and immune cells in blood donors and thus highlights its potential use for donor health monitoring.
Assuntos
Doação de Sangue , Proteoma , Humanos , Masculino , Feminino , Proteômica , Eritrócitos/química , Doadores de Sangue , Ferritinas , Hemoglobinas/análiseRESUMO
Cytotoxic CD8+ T cells can effectively kill target cells by producing cytokines, chemokines, and granzymes. Expression of these effector molecules is however highly divergent, and tools that identify and preselect CD8+ T cells with a cytotoxic expression profile are lacking. Human CD8+ T cells can be divided into IFN-γ- and IL-2-producing cells. Unbiased transcriptomics and proteomics analysis on cytokine-producing fixed CD8+ T cells revealed that IL-2+ cells produce helper cytokines, and that IFN-γ+ cells produce cytotoxic molecules. IFN-γ+ T cells expressed the surface marker CD29 already prior to stimulation. CD29 also marked T cells with cytotoxic gene expression from different tissues in single-cell RNA-sequencing data. Notably, CD29+ T cells maintained the cytotoxic phenotype during cell culture, suggesting a stable phenotype. Preselecting CD29-expressing MART1 TCR-engineered T cells potentiated the killing of target cells. We therefore propose that CD29 expression can help evaluate and select for potent therapeutic T cell products.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica/imunologia , Integrina beta1/metabolismo , Interferon gama/metabolismo , Interleucina-2/metabolismo , Melanoma/patologia , Linfócitos T Citotóxicos/imunologia , Perfilação da Expressão Gênica , Humanos , Melanoma/imunologia , Melanoma/metabolismo , Prognóstico , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Taxa de SobrevidaRESUMO
The assembly of the enzyme-activated factor IX (FIXa) with its cofactor, activated factor VIII (FVIIIa) is a crucial event in the coagulation cascade. The absence or dysfunction of either enzyme or cofactor severely compromises hemostasis and causes hemophilia. FIXa is a notoriously inefficient enzyme that needs FVIIIa to drive its hemostatic potential, by a mechanism that has remained largely elusive to date. In this study, we employed hydrogen-deuterium exchange-mass spectrometry (HDX-MS) to investigate how FIXa responds to assembly with FVIIIa in the presence of phospholipids. This revealed a complex pattern of changes that partially overlaps with those changes that occur upon occupation of the substrate-binding site by an active site-directed inhibitor. Among the changes driven by both cofactor and substrate, HDX-MS highlighted several surface loops that have been implicated in allosteric networks in related coagulation enzymes. Inspection of FVIIIa-specific changes indicated that 3 helices are involved in FIXa-FVIIIa assembly. These are part of a basic interface that is also known as exosite II. Mutagenesis of basic residues herein, followed by functional studies, identified this interface as an extended FVIIIa-interactive patch. HDX-MS was also applied to recombinant FIXa variants that are associated with severe hemophilia B. This revealed that single amino acid substitutions can silence the extended network of FVIIIa-driven allosteric changes. We conclude that HDX-MS has the potential to visualize the functional impact of disease-associated mutations on enzyme-cofactor complexes in the hemostatic system.
Assuntos
Medição da Troca de Deutério , Fator IXa/química , Fator VIII/química , Espectrometria de Massas , Mutação , Regulação Alostérica/genética , Fator IXa/genética , Fator IXa/metabolismo , Fator VIII/genética , Fator VIII/metabolismo , Hemofilia B/genética , Hemofilia B/metabolismo , Humanos , Conformação Proteica em alfa-Hélice , Domínios ProteicosRESUMO
Megakaryoblastic leukemia 1 (MKL1) promotes the regulation of essential cell processes, including actin cytoskeletal dynamics, by coactivating serum response factor. Recently, the first human with MKL1 deficiency, leading to a novel primary immunodeficiency, was identified. We report a second family with 2 siblings with a homozygous frameshift mutation in MKL1. The index case died as an infant from progressive and severe pneumonia caused by Pseudomonas aeruginosa and poor wound healing. The younger sibling was preemptively transplanted shortly after birth. The immunodeficiency was marked by a pronounced actin polymerization defect and a strongly reduced motility and chemotactic response by MKL1-deficient neutrophils. In addition to the lack of MKL1, subsequent proteomic and transcriptomic analyses of patient neutrophils revealed actin and several actin-related proteins to be downregulated, confirming a role for MKL1 as a transcriptional coregulator. Degranulation was enhanced upon suboptimal neutrophil activation, whereas production of reactive oxygen species was normal. Neutrophil adhesion was intact but without proper spreading. The latter could explain the observed failure in firm adherence and transendothelial migration under flow conditions. No apparent defect in phagocytosis or bacterial killing was found. Also, monocyte-derived macrophages showed intact phagocytosis, and lymphocyte counts and proliferative capacity were normal. Nonhematopoietic primary fibroblasts demonstrated defective differentiation into myofibroblasts but normal migration and F-actin content, most likely as a result of compensatory mechanisms of MKL2, which is not expressed in neutrophils. Our findings extend current insight into the severe immune dysfunction in MKL1 deficiency, with cytoskeletal dysfunction and defective extravasation of neutrophils as the most prominent features.
Assuntos
Citoesqueleto de Actina/metabolismo , Mutação da Fase de Leitura , Neutrófilos/fisiologia , Doenças da Imunodeficiência Primária/genética , Doenças da Imunodeficiência Primária/metabolismo , Transativadores/deficiência , Transativadores/genética , Citoesqueleto de Actina/química , Movimento Celular/genética , Movimento Celular/fisiologia , Consanguinidade , Feminino , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Humanos , Lactente , Masculino , Linhagem , Polimerização , Doenças da Imunodeficiência Primária/terapia , Proteômica , Fatores de Transcrição/metabolismoRESUMO
Von Willebrand factor (VWF) is a multimeric hemostatic protein primarily synthesized in endothelial cells. VWF is stored in endothelial storage organelles, the Weibel-Palade bodies (WPB), whose biogenesis strongly depends on VWF anterograde trafficking and Golgi architecture. Elongated WPB morphology is correlated to longer VWF strings with better adhesive properties. We previously identified the SNARE SEC22B, which is involved in anterograde endoplasmic reticulum-to-Golgi transport, as a novel regulator of WPB elongation. To elucidate novel determinants of WPB morphology we explored endothelial SEC22B interaction partners in a mass spectrometry-based approach, identifying the Golgi SNARE Syntaxin 5 (STX5). We established STX5 knockdown in endothelial cells using shRNA-dependent silencing and analyzed WPB and Golgi morphology, using confocal and electron microscopy. STX5-depleted endothelial cells exhibited extensive Golgi fragmentation and decreased WPB length, which was associated with reduced intracellular VWF levels, and impaired stimulated VWF secretion. However, the secretion-incompetent organelles in shSTX5 cells maintained WPB markers such as Angiopoietin 2, P-selectin, Rab27A, and CD63. In brief, we identified SNARE protein STX5 as a novel regulator of WPB biogenesis.
Assuntos
Corpos de Weibel-Palade , Fator de von Willebrand , Tamanho Corporal , Células Cultivadas , Células Endoteliais/metabolismo , Exocitose , Humanos , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Corpos de Weibel-Palade/metabolismo , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismoRESUMO
Osteolineage cell-derived extracellular vesicles (EVs) play a regulatory role in hematopoiesis and have been shown to promote the ex vivo expansion of human hematopoietic stem and progenitor cells (HSPCs). Here, we demonstrate that EVs from different human osteolineage sources do not have the same HSPC expansion promoting potential. Comparison of stimulatory and non-stimulatory osteolineage EVs by next-generation sequencing and mass spectrometry analyses revealed distinct microRNA and protein signatures identifying EV-derived candidate regulators of ex vivo HSPC expansion. Accordingly, the treatment of umbilical cord blood-derived CD34+ HSPCs with stimulatory EVs-altered HSPC transcriptome, including genes with known roles in cell proliferation. An integrative bioinformatics approach, which connects the HSPC gene expression data with the candidate cargo in stimulatory EVs, delineated the potentially targeted biological functions and pathways during hematopoietic cell expansion and development. In conclusion, our study gives novel insights into the complex biological role of EVs in osteolineage cell-HSPC crosstalk and promotes the utility of EVs and their cargo as therapeutic agents in regenerative medicine.
Assuntos
Diferenciação Celular , Linhagem da Célula , Vesículas Extracelulares/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/citologia , Osteoblastos/citologia , Antígenos CD34/metabolismo , Proliferação de Células , Células Cultivadas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Osteoblastos/metabolismo , TranscriptomaRESUMO
Megakaryopoiesis is the process during which megakaryoblasts differentiate to polyploid megakaryocytes that can subsequently shed thousands of platelets in the circulation. Megakaryocytes accumulate mRNA during their maturation, which is required for the correct spatio-temporal production of cytoskeletal proteins, membranes and platelet-specific granules, and for the subsequent shedding of thousands of platelets per cell. Gene expression profiling identified the RNA binding protein ATAXIN2 (ATXN2) as a putative novel regulator of megakaryopoiesis. ATXN2 expression is high in CD34+/CD41+ megakaryoblasts and sharply decreases upon maturation to megakaryocytes. ATXN2 associates with DDX6 suggesting that it may mediate repression of mRNA translation during early megakaryopoiesis. Comparative transcriptome and proteome analysis on megakaryoid cells (MEG-01) with differential ATXN2 expression identified ATXN2 dependent gene expression of mRNA and protein involved in processes linked to hemostasis. Mice deficient for Atxn2 did not display differences in bleeding times, but the expression of key surface receptors on platelets, such as ITGB3 (carries the CD61 antigen) and CD31 (PECAM1), was deregulated and platelet aggregation upon specific triggers was reduced.
Assuntos
Ataxina-2/genética , Perfilação da Expressão Gênica/métodos , Células Progenitoras de Megacariócitos/citologia , Animais , Antígenos CD34/genética , Ataxina-2/metabolismo , Diferenciação Celular , Linhagem Celular , RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica , Humanos , Camundongos , Glicoproteína IIb da Membrana de Plaquetas/genética , Proteínas Proto-Oncogênicas/genéticaRESUMO
Dominant-negative mutations in the transcription factor Growth Factor Independence-1B (GFI1B), such as GFI1BQ287*, cause a bleeding disorder characterized by a plethora of megakaryocyte and platelet abnormalities. The deregulated molecular mechanisms and pathways are unknown. Here we show that both normal and Q287* mutant GFI1B interacted most strongly with the lysine specific demethylase-1 - REST corepressor - histone deacetylase (LSD1-RCOR-HDAC) complex in megakaryoblasts. Sequestration of this complex by GFI1BQ287* and chemical separation of GFI1B from LSD1 induced abnormalities in normal megakaryocytes comparable to those seen in patients. Megakaryocytes derived from GFI1BQ287*-induced pluripotent stem cells also phenocopied abnormalities seen in patients. Proteome studies on normal and mutant-induced pluripotent stem cell-derived megakaryocytes identified a multitude of deregulated pathways downstream of GFI1BQ287* including cell division and interferon signaling. Proteome studies on platelets from GFI1BQ287* patients showed reduced expression of proteins implicated in platelet function, and elevated expression of proteins normally downregulated during megakaryocyte differentiation. Thus, GFI1B and LSD1 regulate a broad developmental program during megakaryopoiesis, and GFI1BQ287* deregulates this program through LSD1-RCOR-HDAC sequestering.
Assuntos
Transtornos da Coagulação Sanguínea/patologia , Plaquetas/patologia , Regulação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/patologia , Megacariócitos/patologia , Mutação , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Transtornos da Coagulação Sanguínea/genética , Transtornos da Coagulação Sanguínea/metabolismo , Plaquetas/metabolismo , Diferenciação Celular , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Megacariócitos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Mapas de Interação de Proteínas , Proteoma/análise , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismoRESUMO
The classical central macrophage found in erythroblastic islands plays an important role in erythroblast differentiation, proliferation and enucleation in the bone marrow. Convenient human in vitro models to facilitate the study of erythroid-macrophage interactions are desired. Recently, we demonstrated that cultured monocytes/macrophages enhance in vitro erythropoiesis by supporting hematopoietic stem and progenitor cell survival. Herein, we describe that these specific macrophages also support erythropoiesis. Human monocytes cultured in serum-free media supplemented with stem cell factor, erythropoietin, lipids and dexamethasone differentiate towards macrophages expressing CD16, CD163, CD169, CD206, CXCR4 and the phagocytic TAM-receptor family. Phenotypically, they resemble both human bone marrow and fetal liver resident macrophages. This differentiation is dependent on glucocorticoid receptor activation. Proteomic studies confirm that glucocorticoid receptor activation differentiates monocytes to anti-inflammatory tissue macrophages with a M2 phenotype, termed GC-macrophages. Proteins involved in migration, tissue residence and signal transduction/receptor activity are upregulated whilst lysosome and hydrolase activity GO-categories are downregulated. Functionally, we demonstrate that GC-macrophages are highly mobile and can interact to form clusters with erythroid cells of all differentiation stages and phagocytose the expelled nuclei, recapitulating aspects of erythroblastic islands. In conclusion, glucocorticoid-directed monocyte differentiation to macrophages represents a convenient model system to study erythroid-macrophage interactions.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Glucocorticoides/farmacologia , Macrófagos/citologia , Monócitos/citologia , Comunicação Celular , Células Cultivadas , Células Eritroides/citologia , Eritropoese , Humanos , Monócitos/efeitos dos fármacos , ProteômicaRESUMO
OBJECTIVE: Thrombin is the key serine protease of the coagulation cascade and mediates cellular responses by activation of PARs (protease-activated receptors). The predominant thrombin receptor is PAR1, and in endothelial cells (ECs), thrombin dynamically regulates a plethora of phosphorylation events. However, it has remained unclear whether thrombin signaling is exclusively mediated through PAR1. Furthermore, mechanistic insight into activation and inhibition of PAR1-mediated EC signaling is lacking. In addition, signaling networks of biased PAR1 activation after differential cleavage of the PAR1 N terminus have remained an unresolved issue. APPROACH AND RESULTS: Here, we used a quantitative phosphoproteomics approach to show that classical and peptide activation of PAR1 induce highly similar signaling, that low thrombin concentrations initiate only limited phosphoregulation, and that the PAR1 inhibitors vorapaxar and parmodulin-2 demonstrate distinct antagonistic properties. Subsequent analysis of the thrombin-regulated phosphosites in the presence of PAR1 inhibitors revealed that biased activation of PAR1 is not solely linked to a specific G-protein downstream of PAR1. In addition, we showed that only the canonical thrombin PAR1 tethered ligand induces extensive early phosphoregulation in ECs. CONCLUSIONS: Our study provides detailed insight in the signaling mechanisms downstream of PAR1. Our data demonstrate that thrombin-induced EC phosphoregulation is mediated exclusively through PAR1, that thrombin and thrombin-tethered ligand peptide induce similar phosphoregulation, and that only canonical PAR1 cleavage by thrombin generates a tethered ligand that potently induces early signaling. Furthermore, platelet PAR1 inhibitors directly affect EC signaling, indicating that it will be a challenge to design a PAR1 antagonist that will target only those pathways responsible for tissue pathology.
Assuntos
Células Endoteliais/fisiologia , Receptor PAR-1/antagonistas & inibidores , Receptor PAR-1/fisiologia , Humanos , Lactonas/farmacologia , Fosforilação , Proteômica , Piridinas/farmacologia , Transdução de SinaisRESUMO
ABSTRACT: Sitosterolemia is a rare autosomal recessive genetic disorder in which patients develop hypercholesterolemia and may exhibit abnormal hematologic and/or liver test results. In this disease, dysfunction of either ABCG5 or ABCG8 results in the intestinal hyperabsorption of all sterols, including cholesterol and, more specifically, plant sterols or xenosterols, as well as in the impaired ability to excrete xenosterols into the bile. It remains unknown how and why some patients develop hematologic abnormalities. Only a few unrelated patients with hematologic abnormalities at the time of diagnosis have been reported. Here, we report on 2 unrelated pedigrees who were believed to have chronic immune thrombocytopenia as their most prominent feature. Both consanguineous families showed recessive gene variants in ABCG5, which were associated with the disease by in silico protein structure analysis and clinical segregation. Hepatosplenomegaly was absent. Thrombopoietin levels and megakaryocyte numbers in the bone marrow were normal. Metabolic analysis confirmed the presence of strongly elevated plasma levels of xenosterols. Potential platelet proteomic aberrations were longitudinally assessed following dietary restrictions combined with administration of the sterol absorption inhibitor ezetimibe. No significant effects on platelet protein content before and after the onset of treatment were demonstrated. Although we cannot exclude that lipotoxicity has a direct and platelet-specific impact in patients with sitosterolemia, our data suggest that thrombocytopenia is neither caused by a lack of megakaryocytes nor driven by proteomic aberrations in the platelets themselves.
Assuntos
Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Plaquetas , Hipercolesterolemia , Enteropatias , Erros Inatos do Metabolismo Lipídico , Fitosteróis , Proteômica , Trombocitopenia , Humanos , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Plaquetas/metabolismo , Plaquetas/patologia , Hipercolesterolemia/sangue , Hipercolesterolemia/genética , Hipercolesterolemia/complicações , Enteropatias/sangue , Enteropatias/diagnóstico , Enteropatias/genética , Enteropatias/etiologia , Enteropatias/metabolismo , Erros Inatos do Metabolismo Lipídico/diagnóstico , Erros Inatos do Metabolismo Lipídico/genética , Erros Inatos do Metabolismo Lipídico/sangue , Erros Inatos do Metabolismo Lipídico/complicações , Lipoproteínas , Linhagem , Fitosteróis/efeitos adversos , Fitosteróis/sangue , Proteoma , Proteômica/métodos , Trombocitopenia/diagnóstico , Trombocitopenia/sangue , Trombocitopenia/etiologia , Trombocitopenia/metabolismoRESUMO
Background: The importance of CD11b/CD18 expression in neutrophil effector functions is well known. Beyond KINDLIN3 and TALIN1, which are involved in the induction of the high-affinity binding CD11b/CD18 conformation, the signaling pathways that orchestrate this response remain incompletely understood. Method: We performed an unbiased screening method for protein selection by biotin identification (BioID) and investigated the KINDLIN3 interactome. We used liquid chromatography with tandem mass spectrometry as a powerful analytical tool. Generation of NB4 CD18, KINDLIN3, or SKAP2 knockout neutrophils was achieved using CRISPR-Cas9 technology, and the cells were examined for their effector function using flow cytometry, live cell imaging, microscopy, adhesion, or antibody-dependent cellular cytotoxicity (ADCC). Results: Among the 325 proteins significantly enriched, we identified Src kinase-associated phosphoprotein 2 (SKAP2), a protein involved in actin polymerization and integrin-mediated outside-in signaling. CD18 immunoprecipitation in primary or NB4 neutrophils demonstrated the presence of SKAP2 in the CD11b/CD18 complex at a steady state. Under this condition, adhesion to plastic, ICAM-1, or fibronectin was observed in the absence of SKAP2, which could be abrogated by blocking the actin rearrangements with latrunculin B. Upon stimulation of NB4 SKAP2-deficient neutrophils, adhesion to fibronectin was enhanced whereas CD18 clustering was strongly reduced. This response corresponded with significantly impaired CD11b/CD18-dependent NADPH oxidase activity, phagocytosis, and cytotoxicity against tumor cells. Conclusion: Our results suggest that SKAP2 has a dual role. It may restrict CD11b/CD18-mediated adhesion only under resting conditions, but its major contribution lies in the regulation of dynamic CD11b/CD18-mediated actin rearrangements and clustering as required for cellular effector functions of human neutrophils.
Assuntos
Neutrófilos , Quinases da Família src , Humanos , Neutrófilos/metabolismo , Quinases da Família src/metabolismo , Fibronectinas/metabolismo , Antígenos CD18/metabolismo , Adesão Celular , Actinas/metabolismo , Fosfoproteínas/metabolismo , Antígeno de Macrófago 1/metabolismoRESUMO
Vascular endothelial cells (ECs) form a dynamic interface between blood and tissue and play a crucial role in the progression of vascular inflammation. Here, we aim to dissect the system-wide molecular mechanisms of inflammatory endothelial-cytokine responses. Applying an unbiased cytokine library, we determined that TNFα and IFNγ induced the largest EC response resulting in distinct proteomic inflammatory signatures. Notably, combined TNFα + IFNγ stimulation induced an additional synergetic inflammatory signature. We employed a multi-omics approach to dissect these inflammatory states, combining (phospho-) proteome, transcriptome and secretome and found, depending on the stimulus, a wide-array of altered immune-modulating processes, including complement proteins, MHC complexes and distinct secretory cytokines. Synergy resulted in cooperative activation of transcript induction. This resource describes the intricate molecular mechanisms that are at the basis of endothelial inflammation and supports the adaptive immunomodulatory role of the endothelium in host defense and vascular inflammation.
Assuntos
Citocinas , Fator de Necrose Tumoral alfa , Humanos , Citocinas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Células Endoteliais/metabolismo , Proteômica , Multiômica , Inflamação/metabolismo , Endotélio VascularRESUMO
Potent T cell responses against infections and malignancies require a rapid yet tightly regulated production of toxic effector molecules. Their production level is defined by post-transcriptional events at 3' untranslated regions (3' UTRs). RNA binding proteins (RBPs) are key regulators in this process. With an RNA aptamer-based capture assay, we identify >130 RBPs interacting with IFNG, TNF, and IL2 3' UTRs in human T cells. RBP-RNA interactions show plasticity upon T cell activation. Furthermore, we uncover the intricate and time-dependent regulation of cytokine production by RBPs: whereas HuR supports early cytokine production, ZFP36L1, ATXN2L, and ZC3HAV1 dampen and shorten the production duration, each at different time points. Strikingly, even though ZFP36L1 deletion does not rescue the dysfunctional phenotype, tumor-infiltrating T cells produce more cytokines and cytotoxic molecules, resulting in superior anti-tumoral T cell responses. Our findings thus show that identifying RBP-RNA interactions reveals key modulators of T cell responses in health and disease.
Assuntos
Citocinas , Linfócitos T , Humanos , Linfócitos T/metabolismo , Regiões 3' não Traduzidas , Citocinas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fator 1 de Resposta a Butirato/genética , Fator 1 de Resposta a Butirato/metabolismoRESUMO
BACKGROUND: Inherited platelet disorders (IPDs) are a heterogeneous group of rare diseases that are caused by the defects in early megakaryopoiesis, proplatelet formation, and/or mature platelet function. Although genomic sequencing is increasingly used to identify genetic variants underlying IPD, this technique does not disclose resulting molecular changes that impact platelet function. Proteins are the functional units that shape platelet function; however, insights into how variants that cause IPDs impact platelet proteomes are limited. OBJECTIVES: The objective of this study was to profile the platelet proteomics signatures of IPDs. METHODS: We performed unbiased label-free quantitative mass spectrometry (MS)-based proteome profiling on platelets of 34 patients with IPDs with variants in 13 ISTH TIER1 genes that affect different stages of platelet development. RESULTS: In line with the phenotypical heterogeneity between IPDs, proteomes were diverse between IPDs. We observed extensive proteomic alterations in patients with a GFI1B variant and for genetic variants in genes encoding proteins that impact cytoskeletal processes (MYH9, TUBB1, and WAS). Using the diversity between IPDs, we clustered protein dynamics, revealing disrupted protein-protein complexes. This analysis furthermore grouped proteins with similar cellular function and location, classifying mitochondrial protein constituents and identifying both known and putative novel alpha granule associated proteins. CONCLUSIONS: With this study, we demonstrate a MS-based proteomics perspective to IPDs. By integrating the effects of IPDs that impact different aspects of platelet function, we dissected the biological contexts of protein alterations to gain further insights into the biology of platelet (dys)function.