RESUMEN
In response to pathogenic threats, naive T cells rapidly transition from a quiescent to an activated state, yet the underlying mechanisms are incompletely understood. Using a pulsed SILAC approach, we investigated the dynamics of mRNA translation kinetics and protein turnover in human naive and activated T cells. Our datasets uncovered that transcription factors maintaining T cell quiescence had constitutively high turnover, which facilitated their depletion following activation. Furthermore, naive T cells maintained a surprisingly large number of idling ribosomes as well as 242 repressed mRNA species and a reservoir of glycolytic enzymes. These components were rapidly engaged following stimulation, promoting an immediate translational and glycolytic switch to ramp up the T cell activation program. Our data elucidate new insights into how T cells maintain a prepared state to mount a rapid immune response, and provide a resource of protein turnover, absolute translation kinetics and protein synthesis rates in T cells ( https://www.immunomics.ch ).
Asunto(s)
Activación de Linfocitos/fisiología , Biosíntesis de Proteínas/inmunología , Linfocitos T/inmunología , Humanos , ARN Mensajero/inmunología , ARN Mensajero/metabolismo , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismoRESUMEN
Metabolic activity is intimately linked to T cell fate and function. Using high-resolution mass spectrometry, we generated dynamic metabolome and proteome profiles of human primary naive T cells following activation. We discovered critical changes in the arginine metabolism that led to a drop in intracellular L-arginine concentration. Elevating L-arginine levels induced global metabolic changes including a shift from glycolysis to oxidative phosphorylation in activated T cells and promoted the generation of central memory-like cells endowed with higher survival capacity and, in a mouse model, anti-tumor activity. Proteome-wide probing of structural alterations, validated by the analysis of knockout T cell clones, identified three transcriptional regulators (BAZ1B, PSIP1, and TSN) that sensed L-arginine levels and promoted T cell survival. Thus, intracellular L-arginine concentrations directly impact the metabolic fitness and survival capacity of T cells that are crucial for anti-tumor responses.
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Arginina/metabolismo , Linfocitos T CD4-Positivos/inmunología , Inmunomodulación , Activación de Linfocitos , Melanoma Experimental/inmunología , Neoplasias Cutáneas/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Linfocitos T CD4-Positivos/metabolismo , Proteínas de Unión al ADN/metabolismo , Técnicas de Inactivación de Genes , Glucólisis , Humanos , Memoria Inmunológica , Metaboloma , Ratones , Ratones Endogámicos BALB C , Fosforilación Oxidativa , Proteoma , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The immune system is unique in its dynamic interplay between numerous cell types. However, a system-wide view of how immune cells communicate to protect against disease has not yet been established. We applied high-resolution mass-spectrometry-based proteomics to characterize 28 primary human hematopoietic cell populations in steady and activated states at a depth of >10,000 proteins in total. Protein copy numbers revealed a specialization of immune cells for ligand and receptor expression, thereby connecting distinct immune functions. By integrating total and secreted proteomes, we discovered fundamental intercellular communication structures and previously unknown connections between cell types. Our publicly accessible (http://www.immprot.org/) proteomic resource provides a framework for the orchestration of cellular interplay and a reference for altered communication associated with pathology.
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Células Sanguíneas/fisiología , Inmunidad Celular , Mapas de Interacción de Proteínas , Proteoma , Proteómica , Animales , Secreciones Corporales , Comunicación Celular , Simulación por Computador , Humanos , Espectrometría de Masas , Apoyo SocialRESUMEN
Some Plasmodium falciparum repetitive interspersed families of polypeptides (RIFINs)-variant surface antigens that are expressed on infected erythrocytes1-bind to the inhibitory receptor LAIR1, and insertion of DNA that encodes LAIR1 into immunoglobulin genes generates RIFIN-specific antibodies2,3. Here we address the general relevance of this finding by searching for antibodies that incorporate LILRB1, another inhibitory receptor that binds to ß2 microglobulin and RIFINs through their apical domains4,5. By screening plasma from a cohort of donors from Mali, we identified individuals with LILRB1-containing antibodies. B cell clones isolated from three donors showed large DNA insertions in the switch region that encodes non-apical LILRB1 extracellular domain 3 and 4 (D3D4) or D3 alone in the variable-constant (VH-CH1) elbow. Through mass spectrometry and binding assays, we identified a large set of RIFINs that bind to LILRB1 D3. Crystal and cryo-electron microscopy structures of a RIFIN in complex with either LILRB1 D3D4 or a D3D4-containing antibody Fab revealed a mode of RIFIN-LILRB1 D3 interaction that is similar to that of RIFIN-LAIR1. The Fab showed an unconventional triangular architecture with the inserted LILRB1 domains opening up the VH-CH1 elbow without affecting VH-VL or CH1-CL pairing. Collectively, these findings show that RIFINs bind to LILRB1 through D3 and illustrate, with a naturally selected example, the general principle of creating novel antibodies by inserting receptor domains into the VH-CH1 elbow.
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Anticuerpos/química , Anticuerpos/inmunología , Antígenos de Protozoos/química , Antígenos de Protozoos/inmunología , Microscopía por Crioelectrón , Receptor Leucocitario Tipo Inmunoglobulina B1/química , Plasmodium falciparum/química , Plasmodium falciparum/inmunología , Adolescente , Adulto , Secuencia de Aminoácidos , Anticuerpos/ultraestructura , Especificidad de Anticuerpos , Antígenos de Protozoos/ultraestructura , Sitios de Unión de Anticuerpos , Niño , Preescolar , Estudios de Cohortes , Humanos , Lactante , Receptor Leucocitario Tipo Inmunoglobulina B1/inmunología , Malí , Modelos Moleculares , Plasmodium falciparum/genética , Plasmodium falciparum/ultraestructura , Dominios Proteicos , Adulto JovenRESUMEN
The availability of L-arginine in tumours is a key determinant of an efficient anti-tumour T cell response1-4. Consequently, increases of typically low L-arginine concentrations within the tumour may greatly potentiate the anti-tumour responses of immune checkpoint inhibitors, such as programmed death-ligand 1 (PD-L1)-blocking antibodies5. However, currently no means are available to locally increase intratumoural L-arginine levels. Here we used a synthetic biology approach to develop an engineered probiotic Escherichia coli Nissle 1917 strain that colonizes tumours and continuously converts ammonia, a metabolic waste product that accumulates in tumours6, to L-arginine. Colonization of tumours with these bacteria increased intratumoural L-arginine concentrations, increased the number of tumour-infiltrating T cells and had marked synergistic effects with PD-L1 blocking antibodies in the clearance of tumours. The anti-tumour effect of these bacteria was mediated by L-arginine and was dependent on T cells. These results show that engineered microbial therapies enable metabolic modulation of the tumour microenvironment leading to enhanced efficacy of immunotherapies.
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Inmunoterapia/métodos , Ingeniería Metabólica , Microorganismos Modificados Genéticamente , Neoplasias Experimentales/terapia , Traslado Adoptivo , Animales , Arginina/metabolismo , Antígeno B7-H1/antagonistas & inhibidores , Línea Celular Tumoral , Escherichia coli , Femenino , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/microbiología , Probióticos , Proteoma , Biología Sintética , Linfocitos T/inmunología , Microambiente Tumoral/inmunologíaRESUMEN
Cell-cell interactions are essential for the proper functioning of multicellular organisms. For example, T cells interact with antigen-presenting cells (APCs) through specific T-cell receptor (TCR)-antigen interactions during an immune response. Fluorescence-activated droplet sorting (FADS) is a high-throughput technique for efficiently screening cellular interaction events. Unfortunately, current droplet sorting instruments have significant limitations, most notably related to analytical throughput and complex operation. In contrast, commercial fluorescence-activated cell sorters offer superior speed, sensitivity, and multiplexing capabilities, although their use as droplet sorters is poorly defined and underutilized. Herein, we present a universally applicable and simple-to-implement workflow for generating double emulsions and performing multicolor cell sorting using a commercial FACS instrument. This workflow achieves a double emulsion detection rate exceeding 90%, enabling multicellular encapsulation and high-throughput immune cell activation sorting for the first time. We anticipate that the presented droplet sorting strategy will benefit cell biology laboratories by providing access to an advanced microfluidic toolbox with minimal effort and cost investment.
RESUMEN
Plasmodium falciparum antigens expressed on the surface of infected erythrocytes are important targets of naturally acquired immunity against malaria, but their high number and variability provide the pathogen with a powerful means of escape from host antibodies. Although broadly reactive antibodies against these antigens could be useful as therapeutics and in vaccine design, their identification has proven elusive. Here we report the isolation of human monoclonal antibodies that recognize erythrocytes infected by different P. falciparum isolates and opsonize these cells by binding to members of the RIFIN family. These antibodies acquired broad reactivity through a novel mechanism of insertion of a large DNA fragment between the V and DJ segments. The insert, which is both necessary and sufficient for binding to RIFINs, encodes the entire 98 amino acid collagen-binding domain of LAIR1, an immunoglobulin superfamily inhibitory receptor encoded on chromosome 19. In each of the two donors studied, the antibodies are produced by a single expanded B-cell clone and carry distinct somatic mutations in the LAIR1 domain that abolish binding to collagen and increase binding to infected erythrocytes. These findings illustrate, with a biologically relevant example, a novel mechanism of antibody diversification by interchromosomal DNA transposition and demonstrate the existence of conserved epitopes that may be suitable candidates for the development of a malaria vaccine.
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Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Variación Antigénica/inmunología , Antígenos de Protozoos/inmunología , Malaria/inmunología , Mutagénesis Insercional/genética , Plasmodium falciparum/inmunología , Receptores Inmunológicos/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/uso terapéutico , Linfocitos B/citología , Linfocitos B/inmunología , Células Clonales/citología , Células Clonales/inmunología , Colágeno/inmunología , Colágeno/metabolismo , Secuencia Conservada/inmunología , Elementos Transponibles de ADN/genética , Elementos Transponibles de ADN/inmunología , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Eritrocitos/inmunología , Eritrocitos/metabolismo , Eritrocitos/parasitología , Humanos , Kenia , Malaria/parasitología , Vacunas contra la Malaria/química , Vacunas contra la Malaria/inmunología , Proteínas de la Membrana/química , Proteínas de la Membrana/inmunología , Datos de Secuencia Molecular , Estructura Terciaria de Proteína/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/inmunología , Receptores Inmunológicos/química , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismoRESUMEN
DNA double-strand breaks are repaired by end-joining or homologous recombination. A key-committing step of recombination is DNA end resection. In resection, phosphorylated CtIP first promotes the endonuclease of MRE11-RAD50-NBS1 (MRN). Subsequently, CtIP also stimulates the WRN/BLM-DNA2 pathway, coordinating thus both short and long-range resection. The structure of CtIP differs from its orthologues in yeast, as it contains a large internal unstructured region. Here, we conducted a domain analysis of CtIP to define the function of the internal region in DNA end resection. We found that residues 350-600 were entirely dispensable for resection in vitro. A mutant lacking these residues was unexpectedly more efficient than full-length CtIP in DNA end resection and homologous recombination in vivo, and consequently conferred resistance to lesions induced by the topoisomerase poison camptothecin, which require high MRN-CtIP-dependent resection activity for repair. This suggested that the internal CtIP region, further mapped to residues 550-600, may mediate a negative regulatory function to prevent over resection in vivo. The CtIP internal deletion mutant exhibited sensitivity to other DNA-damaging drugs, showing that upregulated resection may be instead toxic under different conditions. These experiments together identify a region within the central CtIP domain that negatively regulates DNA end resection.
Asunto(s)
Reparación del ADN , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/fisiología , Proteína BRCA1/metabolismo , Camptotecina/toxicidad , Línea Celular , Roturas del ADN de Doble Cadena , ADN Helicasas/metabolismo , Endodesoxirribonucleasas/genética , Humanos , Dominios Proteicos , Eliminación de Secuencia , Proteína 1 de Unión al Supresor Tumoral P53/metabolismoRESUMEN
BACKGROUND: Primary antibody deficiencies (PADs) are the most frequent primary immunodeficiencies in human subjects. The genetic causes of PADs are largely unknown. Sec61 translocon alpha 1 subunit (SEC61A1) is the major subunit of the Sec61 complex, which is the main polypeptide-conducting channel in the endoplasmic reticulum membrane. SEC61A1 is a target gene of spliced X-box binding protein 1 and strongly induced during plasma cell (PC) differentiation. OBJECTIVE: We identified a novel genetic defect and studied its pathologic mechanism in 11 patients from 2 unrelated families with PADs. METHODS: Whole-exome and targeted sequencing were conducted to identify novel genetic mutations. Functional studies were carried out ex vivo in primary cells of patients and in vitro in different cell lines to assess the effect of SEC61A1 mutations on B-cell differentiation and survival. RESULTS: We investigated 2 families with patients with hypogammaglobulinemia, severe recurrent respiratory tract infections, and normal peripheral B- and T-cell subpopulations. On in vitro stimulation, B cells showed an intrinsic deficiency to develop into PCs. Genetic analysis and targeted sequencing identified novel heterozygous missense (c.254T>A, p.V85D) and nonsense (c.1325G>T, p.E381*) mutations in SEC61A1, segregating with the disease phenotype. SEC61A1-V85D was deficient in cotranslational protein translocation, and it disturbed the cellular calcium homeostasis in HeLa cells. Moreover, SEC61A1-V85D triggered the terminal unfolded protein response in multiple myeloma cell lines. CONCLUSION: We describe a monogenic defect leading to a specific PC deficiency in human subjects, expanding our knowledge about the pathogenesis of antibody deficiencies.
Asunto(s)
Síndromes de Inmunodeficiencia/genética , Mutación/genética , Células Plasmáticas/patología , Canales de Translocación SEC/genética , Agammaglobulinemia/genética , Agammaglobulinemia/metabolismo , Agammaglobulinemia/patología , Linfocitos B/metabolismo , Linfocitos B/patología , Calcio/metabolismo , Diferenciación Celular/genética , Línea Celular , Línea Celular Tumoral , Exoma/genética , Células HEK293 , Células HeLa , Heterocigoto , Humanos , Síndromes de Inmunodeficiencia/metabolismo , Células Plasmáticas/metabolismo , Transporte de Proteínas/genética , Infecciones del Sistema Respiratorio/genética , Infecciones del Sistema Respiratorio/metabolismo , Infecciones del Sistema Respiratorio/patología , Linfocitos T/metabolismo , Linfocitos T/patología , Respuesta de Proteína Desplegada/genéticaRESUMEN
Cholera toxin enters cells via an unusual pathway that involves trafficking through endosomes to the endoplasmic reticulum (ER). Whether the toxin induces its own pathway or travels along a physiological retrograde route is not known. To study its trafficking, we labeled cholera toxin B (CTB) or endogenous plasma membrane proteins with a small chemical compound, benzylguanine, which covalently reacts with the protein SNAP-tag. Using ER-targeted SNAP-tag as reporter, we found that transport of CTB to the ER depends on dynamin-2 and syntaxin 5. Plasma membrane proteins and a fluid-phase marker added to the medium were also transported to the ER. This flux was not affected by exposing cells to CTB but was inhibited by depleting syntaxin 5 and increased by depleting dynamin-2. As a control for confined intracellular localization of ER-targeted SNAP-tag we used adenovirus-5, which traffics to endosomes and then escapes into the cytosol. The virus did not react with ER-targeted SNAP but with cytosolic SNAP. Together, our results establish a new method (SNAP-trap) to study trafficking of different cargo to the ER and the cytosol and provide evidence for the existence of a constitutive pathway from the cell surface to the ER.
Asunto(s)
Citosol/metabolismo , Endocitosis , Retículo Endoplásmico/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Toxina del Cólera/genética , Toxina del Cólera/metabolismo , Dinamina II/metabolismo , Endosomas/metabolismo , Colorantes Fluorescentes , Humanos , Transporte de Proteínas , Proteínas Qa-SNARE/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Bacterial therapeutics have emerged as promising delivery systems to target tumors. These engineered live therapeutics can be harnessed to modulate the tumor microenvironment or to deliver and selectively release therapeutic payloads to tumors. A major challenge is to deliver bacteria systemically without causing widespread inflammation, which is critical for the many tumors that are not accessible to direct intratumoral injection. We describe potential strategies to address this challenge, along with approaches for specific payload delivery and biocontainment to ensure safety. These strategies will pave the way for the development of cost-effective, widely applicable next-generation cancer therapeutics.
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Inmunoterapia , Neoplasias , Humanos , Neoplasias/terapia , Bacterias , Microambiente TumoralRESUMEN
BACKGROUND: Angioimmunoblastic T-cell lymphoma (AITL) is a malignancy with very poor survival outcome, in urgent need of more specific therapeutic strategies. The drivers of malignancy in this disease are CD4+ follicular helper T cells (Tfh). The metabolism of these malignant Tfh cells was not yet elucidated. Therefore, we decided to identify their metabolic requirements with the objective to propose a novel therapeutic option. METHODS: To reveal the prominent metabolic pathways used by the AITL lymphoma cells, we relied on metabolomic and proteomic analysis of murine AITL (mAITL) T cells isolated from our established mAITL model. We confirmed these results using AITL patient and healthy T cell expression data. RESULTS: Strikingly, the mAITL Tfh cells were highly dependent on the second branch of the Kennedy pathway, the choline lipid pathway, responsible for the production of the major membrane constituent phosphatidylcholine. Moreover, gene expression data from Tfh cells isolated from AITL patient tumors, confirmed the upregulation of the choline lipid pathway. Several enzymes involved in this pathway such as choline kinase, catalyzing the first step in the phosphatidylcholine pathway, are upregulated in multiple tumors other than AITL. Here we showed that treatment of our mAITL preclinical mouse model with a fatty acid oxydation inhibitor, significantly increased their survival and even reverted the exhausted CD8 T cells in the tumor into potent cytotoxic anti-tumor cells. Specific inhibition of Chokα confirmed the importance of the phosphatidylcholine production pathway in neoplastic CD4 + T cells, nearly eradicating mAITL Tfh cells from the tumors. Finally, the same inhibitor induced in human AITL lymphoma biopsies cell death of the majority of the hAITL PD-1high neoplastic cells. CONCLUSION: Our results suggest that interfering with choline metabolism in AITL reveals a specific metabolic vulnerability and might represent a new therapeutic strategy for these patients.
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Linfadenopatía Inmunoblástica , Linfoma de Células T , Linfoma , Humanos , Animales , Ratones , Proteómica , Linfocitos T Colaboradores-Inductores/metabolismo , Linfocitos T Colaboradores-Inductores/patología , Linfadenopatía Inmunoblástica/genética , Linfadenopatía Inmunoblástica/metabolismo , Linfadenopatía Inmunoblástica/patología , Linfoma de Células T/genética , Linfoma de Células T/metabolismo , Linfoma de Células T/patología , Fosfatidilcolinas/metabolismo , Linfoma/metabolismo , Linfoma/patologíaRESUMEN
Castration-resistant prostate cancer (CRPC) is a frequently occurring disease with adverse clinical outcomes and limited therapeutic options. Here, we identify methionine adenosyltransferase 2a (MAT2A) as a critical driver of the androgen-indifferent state in ERG fusion-positive CRPC. MAT2A is upregulated in CRPC and cooperates with ERG in promoting cell plasticity, stemness and tumorigenesis. RNA, ATAC and ChIP-sequencing coupled with histone post-translational modification analysis by mass spectrometry show that MAT2A broadly impacts the transcriptional and epigenetic landscape. MAT2A enhances H3K4me2 at multiple genomic sites, promoting the expression of pro-tumorigenic non-canonical AR target genes. Genetic and pharmacological inhibition of MAT2A reverses the transcriptional and epigenetic remodeling in CRPC models and improves the response to AR and EZH2 inhibitors. These data reveal a role of MAT2A in epigenetic reprogramming and provide a proof of concept for testing MAT2A inhibitors in CRPC patients to improve clinical responses and prevent treatment resistance.
Asunto(s)
Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Metionina Adenosiltransferasa , Neoplasias de la Próstata Resistentes a la Castración , Regulador Transcripcional ERG , Masculino , Humanos , Regulador Transcripcional ERG/genética , Regulador Transcripcional ERG/metabolismo , Metionina Adenosiltransferasa/genética , Metionina Adenosiltransferasa/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Animales , Andrógenos/metabolismo , Epigenoma , Ratones , Histonas/metabolismo , Receptores Androgénicos/metabolismo , Receptores Androgénicos/genética , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidoresRESUMEN
Tumor progression is accompanied by fibrosis, a condition of excessive extracellular matrix accumulation, which is associated with diminished antitumor immune infiltration. Here we demonstrate that tumor-associated macrophages (TAMs) respond to the stiffened fibrotic tumor microenvironment (TME) by initiating a collagen biosynthesis program directed by transforming growth factor-ß. A collateral effect of this programming is an untenable metabolic milieu for productive CD8+ T cell antitumor responses, as collagen-synthesizing macrophages consume environmental arginine, synthesize proline and secrete ornithine that compromises CD8+ T cell function in female breast cancer. Thus, a stiff and fibrotic TME may impede antitumor immunity not only by direct physical exclusion of CD8+ T cells but also through secondary effects of a mechano-metabolic programming of TAMs, which creates an inhospitable metabolic milieu for CD8+ T cells to respond to anticancer immunotherapies.
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Neoplasias de la Mama , Linfocitos T CD8-positivos , Colágeno , Microambiente Tumoral , Macrófagos Asociados a Tumores , Microambiente Tumoral/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Femenino , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Colágeno/metabolismo , Animales , Macrófagos Asociados a Tumores/inmunología , Macrófagos Asociados a Tumores/metabolismo , Humanos , Ratones , Factor de Crecimiento Transformador beta/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Reprogramación MetabólicaRESUMEN
MYC is a pleiotropic transcription factor involved in cancer, cell proliferation, and metabolism. Its regulation and function in NK cells, which are innate cytotoxic lymphocytes important to control viral infections and cancer, remain poorly defined. Here, we show that mice deficient for Myc in NK cells presented a severe reduction in these lymphocytes. Myc was required for NK cell development and expansion in response to the key cytokine IL-15, which induced Myc through transcriptional and posttranslational mechanisms. Mechanistically, Myc ablation in vivo largely impacted NK cells' ribosomagenesis, reducing their translation and expansion capacities. Similar results were obtained by inhibiting MYC in human NK cells. Impairing translation by pharmacological intervention phenocopied the consequences of deleting or blocking MYC in vitro. Notably, mice lacking Myc in NK cells exhibited defective anticancer immunity, which reflected their decreased numbers of mature NK cells exerting suboptimal cytotoxic functions. These results indicate that MYC is a central node in NK cells, connecting IL-15 to translational fitness, expansion, and anticancer immunity.
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Interleucina-15 , Células Asesinas Naturales , Animales , Humanos , Ratones , Citocinas/metabolismo , Regulación de la Expresión Génica , Interleucina-15/genética , Interleucina-15/metabolismo , Transducción de SeñalRESUMEN
Cardiovascular diseases (CVD) can cause various conditions, including an increase in reactive oxygen species (ROS) levels that can decrease nitric oxide (NO) availability and promote vasoconstriction, leading to arterial hypertension. Physical exercise (PE) has been found to be protective against CVD by helping to maintain redox homeostasis through a decrease in ROS levels, achieved by increased expression of antioxidant enzymes (AOEs) and modulation of heat shock proteins (HSPs). Extracellular vesicles (EVs) circulating in the body are a major source of regulatory signals, including proteins and nucleic acids. Interestingly, the cardioprotective role of EVs released after PE has not been fully described. The aim of this study was to investigate the role of circulating EVs, obtained through Size Exclusion Chromatography (SEC) of plasma samples from healthy young males (age: 26.95 ± 3.07; estimated maximum oxygen consumption rate (VO2max): 51.22 ± 4.85 (mL/kg/min)) at basal level (Pre_EVs) and immediately after a single bout of endurance exercise (30' treadmill, 70% heart rate (HR) -Post_EVs). Gene ontology (GO) analysis of proteomic data from isolated EVs, revealed enrichment in proteins endowed with catalytic activity in Post_EVs, compare to Pre_EVs, with MAP2K1 being the most significantly upregulated protein. Enzymatic assays on EVs derived from Pre and Post samples showed increment in Glutathione Reductase (GR) and Catalase (CAT) activity in Post_EVs. At functional level, Post_EVs, but not Pre_EVs, enhanced the activity of antioxidant enzymes (AOEs) and reduced oxidative damage accumulation in treated human iPS-derived cardiomyocytes (hCM) at basal level and under stress conditions (Hydrogen Peroxide (H2O2) treatment), resulting in a global cardioprotective effect. In conclusion, our data demonstrated, for the first time, that a single 30-min endurance exercise is able to alter the cargo of circulating EVs, resulting in cardioprotective effect through antioxidant activity.
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Enfermedades Cardiovasculares , Vesículas Extracelulares , Masculino , Humanos , Adulto Joven , Adulto , Antioxidantes/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Peróxido de Hidrógeno/metabolismo , Proteómica , Enfermedades Cardiovasculares/metabolismoRESUMEN
Elucidating the mechanisms by which immune cells become dysfunctional in tumors is critical to developing next-generation immunotherapies. We profiled proteomes of cancer tissue as well as monocyte/macrophages, CD4+ and CD8+ T cells, and NK cells isolated from tumors, liver, and blood of 48 patients with hepatocellular carcinoma. We found that tumor macrophages induce the sphingosine-1-phospate-degrading enzyme SGPL1, which dampened their inflammatory phenotype and anti-tumor function in vivo. We further discovered that the signaling scaffold protein AFAP1L2, typically only found in activated NK cells, is also upregulated in chronically stimulated CD8+ T cells in tumors. Ablation of AFAP1L2 in CD8+ T cells increased their viability upon repeated stimulation and enhanced their anti-tumor activity synergistically with PD-L1 blockade in mouse models. Our data reveal new targets for immunotherapy and provide a resource on immune cell proteomes in liver cancer.
RESUMEN
Although bovine pancreatic RNase is one of the best characterized proteins in respect to structure and in vitro refolding, little is known about its synthesis and maturation in the endoplasmic reticulum (ER) of live cells. We expressed the RNase in live cells and analyzed its folding, quality control, and secretion using pulse-chase analysis and other cell biological techniques. In contrast to the slow in vitro refolding, the protein folded almost instantly after translation and translocation into the ER lumen (t(½) < 3 min). Despite high stability of the native protein, only about half of the RNase reached a secretion competent, monomeric form and was rapidly transported from the rough ER via the Golgi complex (t(½) = 16 min) to the extracellular space (t(½) = 35 min). The rest remained in the ER mainly in the form of dimers and was slowly degraded. The dimers were most likely formed by C-terminal domain swapping since mutation of Asn(113), a residue that stabilizes such dimers, to Ser increased the efficiency of secretion from 59 to 75%. Consistent with stringent ER quality control in vivo, the secreted RNase in the bovine pancreas was mainly monomeric, whereas the enzyme present in the cells also contained 20% dimers. These results suggest that the efficiency of secretion is not only determined by the stability of the native protein but by multiple factors including the stability of secretion-incompetent side products of folding. The presence of N-glycans had little effect on the folding and secretion process.
Asunto(s)
Retículo Endoplásmico/enzimología , Pliegue de Proteína , Multimerización de Proteína/fisiología , Ribonucleasa Pancreática/metabolismo , Animales , Células CHO , Bovinos , Cricetinae , Cricetulus , Retículo Endoplásmico/genética , Estabilidad de Enzimas/genética , Pancrelipasa/metabolismo , Biosíntesis de Proteínas/fisiología , Transporte de Proteínas/fisiología , Ribonucleasa Pancreática/genéticaRESUMEN
Common variable immunodeficiency (CVID) is the most prevalent form of symptomatic primary immunodeficiency in humans. The genetic cause of CVID is still unknown in about 70% of cases. Ten percent of CVID patients carry heterozygous mutations in the tumor necrosis factor receptor superfamily member 13B gene (TNFRSF13B), encoding TACI. Mutations in TNFRSF13B alone may not be sufficient for the development of CVID, as 1% of the healthy population carry these mutations. The common hypothesis is that TACI mutations are not fully penetrant and additional factors contribute to the development of CVID. To determine these additional factors, we investigated the perturbations of transcription factor (TF) binding and the transcriptome profiles in unstimulated and CD40L/IL21-stimulated naïve B cells from CVID patients harboring the C104R mutation in TNFRSF13B and compared them to their healthy relatives with the same mutation. In addition, the proteome of stimulated naïve B cells was investigated. For functional validation, intracellular protein concentrations were measured by flow cytometry. Our analysis revealed 8% less accessible chromatin in unstimulated naïve B cells and 25% less accessible chromatin in class-switched memory B cells from affected and unaffected TACI mutation carriers compared to healthy donors. The most enriched TF binding motifs in TACI mutation carriers involved members from the ETS, IRF, and NF-κB TF families. Validation experiments supported dysregulation of the NF-κB and MAPK pathways. In steady state, naïve B cells had increased cell death pathways and reduced cell metabolism pathways, while after stimulation, enhanced immune responses and decreased cell survival were detected. Using a multi-omics approach, our findings provide valuable insights into the impaired biology of naïve B cells from TACI mutation carriers.
Asunto(s)
Inmunodeficiencia Variable Común , FN-kappa B , Linfocitos B , Cromatina/metabolismo , Humanos , Mutación , FN-kappa B/metabolismoRESUMEN
Stromal infiltration is associated with poor prognosis in human colon cancers. However, the high heterogeneity of human tumor-associated stromal cells (TASCs) hampers a clear identification of specific markers of prognostic relevance. To address these issues, we established short-term cultures of TASCs and matched healthy mucosa-associated stromal cells (MASCs) from human primary colon cancers and, upon characterization of their phenotypic and functional profiles in vitro and in vivo, we identified differentially expressed markers by proteomic analysis and evaluated their prognostic significance. TASCs were characterized by higher proliferation and differentiation potential, and enhanced expression of mesenchymal stem cell markers, as compared to MASCs. TASC triggered epithelial-mesenchymal transition (EMT) in tumor cells in vitro and promoted their metastatic spread in vivo, as assessed in an orthotopic mouse model. Proteomic analysis of matched TASCs and MASCs identified a panel of markers preferentially expressed in TASCs. The expression of genes encoding two of them, calponin 1 (CNN1) and tropomyosin beta chain isoform 2 (TPM2), was significantly associated with poor outcome in independent databases and outperformed the prognostic significance of currently proposed TASC markers. The newly identified markers may improve prognostication of primary colon cancers and identification of patients at risk.