RESUMEN
Downregulation of the urea cycle enzyme argininosuccinate synthase (ASS1) in multiple tumors is associated with a poor prognosis partly because of the metabolic diversion of cytosolic aspartate for pyrimidine synthesis, supporting proliferation and mutagenesis owing to nucleotide imbalance. Here, we find that prolonged loss of ASS1 promotes DNA damage in colon cancer cells and fibroblasts from subjects with citrullinemia type I. Following acute induction of DNA damage with doxorubicin, ASS1 expression is elevated in the cytosol and the nucleus with at least a partial dependency on p53; ASS1 metabolically restrains cell cycle progression in the cytosol by restricting nucleotide synthesis. In the nucleus, ASS1 and ASL generate fumarate for the succination of SMARCC1, destabilizing the chromatin-remodeling complex SMARCC1-SNF5 to decrease gene transcription, specifically in a subset of the p53-regulated cell cycle genes. Thus, following DNA damage, ASS1 is part of the p53 network that pauses cell cycle progression, enabling genome maintenance and survival. Loss of ASS1 contributes to DNA damage and promotes cell cycle progression, likely contributing to cancer mutagenesis and, hence, adaptability potential.
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Argininosuccinato Sintasa , Núcleo Celular , Citosol , Daño del ADN , Proteína p53 Supresora de Tumor , Humanos , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Citosol/metabolismo , Argininosuccinato Sintasa/metabolismo , Argininosuccinato Sintasa/genética , Núcleo Celular/metabolismo , Ciclo Celular/genéticaRESUMEN
People with diabetes feature a life-risking susceptibility to respiratory viral infection, including influenza and SARS-CoV-2 (ref. 1), whose mechanism remains unknown. In acquired and genetic mouse models of diabetes, induced with an acute pulmonary viral infection, we demonstrate that hyperglycaemia leads to impaired costimulatory molecule expression, antigen transport and T cell priming in distinct lung dendritic cell (DC) subsets, driving a defective antiviral adaptive immune response, delayed viral clearance and enhanced mortality. Mechanistically, hyperglycaemia induces an altered metabolic DC circuitry characterized by increased glucose-to-acetyl-CoA shunting and downstream histone acetylation, leading to global chromatin alterations. These, in turn, drive impaired expression of key DC effectors including central antigen presentation-related genes. Either glucose-lowering treatment or pharmacological modulation of histone acetylation rescues DC function and antiviral immunity. Collectively, we highlight a hyperglycaemia-driven metabolic-immune axis orchestrating DC dysfunction during pulmonary viral infection and identify metabolic checkpoints that may be therapeutically exploited in mitigating exacerbated disease in infected diabetics.
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Células Dendríticas , Complicaciones de la Diabetes , Diabetes Mellitus , Susceptibilidad a Enfermedades , Hiperglucemia , Pulmón , Virosis , Animales , Ratones , Acetilcoenzima A/metabolismo , Acetilación , Cromatina/genética , Cromatina/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/patología , Complicaciones de la Diabetes/inmunología , Complicaciones de la Diabetes/metabolismo , Diabetes Mellitus/genética , Diabetes Mellitus/inmunología , Diabetes Mellitus/metabolismo , Glucosa/metabolismo , Histonas/metabolismo , Hiperglucemia/complicaciones , Hiperglucemia/inmunología , Hiperglucemia/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/virología , Linfocitos T/inmunología , Virosis/complicaciones , Virosis/inmunología , Virosis/mortalidad , Virus/inmunología , Modelos Animales de Enfermedad , HumanosRESUMEN
Multiple studies have identified metabolic changes within the tumor and its microenvironment during carcinogenesis. Yet, the mechanisms by which tumors affect the host metabolism are unclear. We find that systemic inflammation induced by cancer leads to liver infiltration of myeloid cells at early extrahepatic carcinogenesis. The infiltrating immune cells via IL6-pSTAT3 immune-hepatocyte cross-talk cause the depletion of a master metabolic regulator, HNF4α, consequently leading to systemic metabolic changes that promote breast and pancreatic cancer proliferation and a worse outcome. Preserving HNF4α levels maintains liver metabolism and restricts carcinogenesis. Standard liver biochemical tests can identify early metabolic changes and predict patients' outcomes and weight loss. Thus, the tumor induces early metabolic changes in its macroenvironment with diagnostic and potentially therapeutic implications for the host. SIGNIFICANCE: Cancer growth requires a permanent nutrient supply starting from early disease stages. We find that the tumor extends its effect to the host's liver to obtain nutrients and rewires the systemic and tissue-specific metabolism early during carcinogenesis. Preserving liver metabolism restricts tumor growth and improves cancer outcomes. This article is highlighted in the In This Issue feature, p. 1501.
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Hígado , Neoplasias Pancreáticas , Humanos , Hígado/metabolismo , Carcinogénesis/patología , Hepatocitos , Neoplasias Pancreáticas/patología , Inmunidad Innata , Microambiente TumoralRESUMEN
The non-classical Major Histocompatibility Complex class II (MHCII) protein, H2-M, edits peptides bound to conventional MHCII in favor of stable peptide/MHCII (p/MHCII) complexes. Here, we show that H2-M deficiency affects B-1 cell survival, reduces cell renewal capacity, and alters immunoglobulin repertoire, allowing for the selection of cells specific for highly abundant epitopes, but not low-frequency epitopes. H2-M-deficient B-1 cells have shorter CDR3 length, higher content of positively charged amino acids, shorter junctional regions, less mutation frequency, and a skewed clonal distribution. Mechanistically, H2-M loss reduces plasma membrane p/MHCII association with B cell receptors (BCR) on B-1 cells and diminishes integrated BCR signal strength, a key determinant of B-1 cell selection, maturation, and maintenance. Thus, H2-M:MHCII interaction serves as a cell-intrinsic regulator of BCR signaling and influences the selection of the B-1 cell clonal repertoire.
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Linfocitos B/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Animales , Activación de Linfocitos/inmunología , RatonesRESUMEN
Major histocompatibility complex class II (MHC-II) antigen presentation underlies a wide range of immune responses in health and disease. However, how MHC-II antigen presentation is regulated by the peptide-loading catalyst HLA-DM (DM), its associated modulator, HLA-DO (DO), is incompletely understood. This is due largely to technical limitations: model antigen-presenting cell (APC) systems that express these MHC-II peptidome regulators at physiologically variable levels have not been described. Likewise, computational prediction tools that account for DO and DM activities are not presently available. To address these gaps, we created a panel of single MHC-II allele, HLA-DR4-expressing APC lines that cover a wide range of DO:DM ratio states. Using a combined immunopeptidomic and proteomic discovery strategy, we measured the effects DO:DM ratios have on peptide presentation by surveying over 10,000 unique DR4-presented peptides. The resulting data provide insight into peptide characteristics that influence their presentation with increasing DO:DM ratios. These include DM sensitivity, peptide abundance, binding affinity and motif, peptide length, and choice of binding register along the source protein. These findings have implications for designing improved HLA-II prediction algorithms and research strategies for dissecting the variety of functions that different APCs serve in the body.
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Presentación de Antígeno , Antígenos HLA-D , Antígenos de Histocompatibilidad Clase II , Proteómica , Células Presentadoras de Antígenos , Línea Celular , Antígenos HLA-DR , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Péptidos/metabolismoRESUMEN
Argininosuccinate lyase (ASL) is essential for the NO-dependent regulation of tyrosine hydroxylase (TH) and thus for catecholamine production. Using a conditional mouse model with loss of ASL in catecholamine neurons, we demonstrate that ASL is expressed in dopaminergic neurons in the substantia nigra pars compacta, including the ALDH1A1 + subpopulation that is pivotal for the pathogenesis of Parkinson disease (PD). Neuronal loss of ASL results in catecholamine deficiency, in accumulation and formation of tyrosine aggregates, in elevation of α-synuclein, and phenotypically in motor and cognitive deficits. NO supplementation rescues the formation of aggregates as well as the motor deficiencies. Our data point to a potential metabolic link between accumulations of tyrosine and seeding of pathological aggregates in neurons as initiators for the pathological processes involved in neurodegeneration. Hence, interventions in tyrosine metabolism via regulation of NO levels may be therapeutic beneficial for the treatment of catecholamine-related neurodegenerative disorders.
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Familia de Aldehído Deshidrogenasa 1/genética , Familia de Aldehído Deshidrogenasa 1/metabolismo , Argininosuccinatoliasa/genética , Argininosuccinatoliasa/metabolismo , Neuronas Dopaminérgicas/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Fenotipo , Retinal-Deshidrogenasa/genética , Retinal-Deshidrogenasa/metabolismoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Argininosuccinate synthase (ASS1) downregulation in different tumors has been shown to support cell proliferation and yet, in several common cancer subsets ASS1 expression associates with poor patient prognosis. Here we demonstrate that ASS1 expression under glucose deprivation is induced by c-MYC, providing survival benefit by increasing nitric oxide synthesis and activating the gluconeogenic enzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase by S-nitrosylation. The resulting increased flux through gluconeogenesis enhances serine, glycine and subsequently purine synthesis. Notably, high ASS1-expressing breast cancer mice do not respond to immune checkpoint inhibitors and patients with breast cancer with high ASS1 have more metastases. We further find that inhibiting purine synthesis increases pyrimidine to purine ratio, elevates expression of the immunoproteasome and significantly enhances the response of autologous primary CD8+ T cells to anti-PD-1. These results suggest that treating patients with high-ASS1 cancers with purine synthesis inhibition is beneficial and may also sensitize them to immune checkpoint inhibition therapy.
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Argininosuccinato Sintasa , Neoplasias de la Mama , Animales , Argininosuccinato Sintasa/metabolismo , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Inhibidores de Puntos de Control Inmunológico , Ratones , PurinasRESUMEN
Citrin, encoded by SLC25A13 gene, is an inner mitochondrial transporter that is part of the malate-aspartate shuttle, which regulates the NAD+/NADH ratio between the cytosol and mitochondria. Citrullinemia type II (CTLN-II) is an inherited disorder caused by germline mutations in SLC25A13, manifesting clinically in growth failure that can be alleviated by dietary restriction of carbohydrates. The association of citrin with glycolysis and NAD+/NADH ratio led us to hypothesize that it may play a role in carcinogenesis. Indeed, we find that citrin is upregulated in multiple cancer types and is essential for supplementing NAD+ for glycolysis and NADH for oxidative phosphorylation. Consequently, citrin deficiency associates with autophagy, whereas its overexpression in cancer cells increases energy production and cancer invasion. Furthermore, based on the human deleterious mutations in citrin, we found a potential inhibitor of citrin that restricts cancerous phenotypes in cells. Collectively, our findings suggest that targeting citrin may be of benefit for cancer therapy.
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Carcinogénesis/genética , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Neoplasias/genética , Carbohidratos/genética , Citrulinemia/genética , Citrulinemia/metabolismo , Citosol/metabolismo , Citosol/patología , Regulación Neoplásica de la Expresión Génica/genética , Mutación de Línea Germinal/genética , Glutamatos/farmacología , Ácido Glutámico/análogos & derivados , Ácido Glutámico/farmacología , Glucólisis/genética , Humanos , Mitocondrias/metabolismo , Mitocondrias/patología , Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Fosforilación Oxidativa/efectos de los fármacosRESUMEN
Individuals with narcolepsy suffer from abnormal sleep patterns due to loss of neurons that uniquely supply hypocretin (HCRT). Previous studies found associations of narcolepsy with the human leukocyte antigen (HLA)-DQ6 allele and T-cell receptor α (TRA) J24 gene segment and also suggested that in vitro-stimulated T cells can target HCRT. Here, we present evidence of in vivo expansion of DQ6-HCRT tetramer+/TRAJ24+/CD4+ T cells in DQ6+ individuals with and without narcolepsy. We identify related TRAJ24+ TCRαß clonotypes encoded by identical α/ß gene regions from two patients and two controls. TRAJ24-G allele+ clonotypes only expand in the two patients, whereas a TRAJ24-C allele+ clonotype expands in a control. A representative tetramer+/G-allele+ TCR shows signaling reactivity to the epitope HCRT87-97. Clonally expanded G-allele+ T cells exhibit an unconventional effector phenotype. Our analysis of in vivo expansion of HCRT-reactive TRAJ24+ cells opens an avenue for further investigation of the autoimmune contribution to narcolepsy development.
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Autoinmunidad/inmunología , Linfocitos T CD4-Positivos/inmunología , Narcolepsia/inmunología , Orexinas/inmunología , Animales , Autoinmunidad/genética , Estudios de Casos y Controles , Proliferación Celular , Cristalografía por Rayos X , Drosophila , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/inmunología , Humanos , Región de Unión de la Inmunoglobulina/genética , Narcolepsia/genética , Tolerancia Periférica , Fenotipo , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunologíaRESUMEN
B cell receptors and surface-displayed peptide/MHCII complexes constitute two key components of the B-cell machinery to sense signals and communicate with other cell types during antigen-triggered activation. However, critical pathways synergizing antigen-BCR interaction and antigenic peptide-MHCII presentation remain elusive. Here, we report the discovery of factors involved in establishing such synergy. We applied a single-cell measure coupled with super-resolution microscopy to investigate the integrated function of two lysosomal regulators for peptide loading, HLA-DM and HLA-DO. In model cell lines and human tonsillar B cells, we found that tunable DM/DO stoichiometry governs DMfree activity for exchange of placeholder CLIP peptides with high affinity MHCII ligands. Compared to their naïve counterparts, memory B cells with less DMfree concentrate a higher proportion of CLIP/MHCII in lysosomal compartments. Upon activation mediated by high affinity BCR, DO tuning is synchronized with antigen internalization and rapidly potentiates DMfree activity to optimize antigen presentation for T-cell recruitment.
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Antígenos/inmunología , Linfocitos B/inmunología , Antígenos HLA-D/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Presentación de Antígeno/inmunología , Línea Celular , Humanos , Memoria Inmunológica/inmunología , Lisosomas/inmunologíaRESUMEN
Downregulation of the urea cycle enzyme argininosuccinate synthase (ASS1) by either promoter methylation or by HIF1α is associated with increased metastasis and poor prognosis in multiple cancers. We have previously shown that in normoxic conditions, ASS1 downregulation facilitates cancer cell proliferation by increasing aspartate availability for pyrimidine synthesis by the enzyme complex CAD. Here we report that in hypoxia, ASS1 expression in cancerous cells is downregulated further by HIF1α-mediated induction of miR-224-5p, making the cells more invasive and dependent on upstream substrates of ASS1 for survival. ASS1 was downregulated under acidic conditions, and ASS1-depleted cancer cells maintained a higher intracellular pH (pHi), depended less on extracellular glutamine, and displayed higher glutathione levels. Depletion of substrates of urea cycle enzymes in ASS1-deficient cancers decreased cancer cell survival. Thus, ASS1 levels in cancer are differentially regulated in various environmental conditions to metabolically benefit cancer progression. Understanding these alterations may help uncover specific context-dependent cancer vulnerabilities that may be targeted for therapeutic purposes. SIGNIFICANCE: Cancer cells in an acidic or hypoxic environment downregulate the expression of the urea cycle enzyme ASS1, which provides them with a redox and pH advantage, resulting in better survival.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/3/518/F1.large.jpg.
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Argininosuccinato Sintasa/metabolismo , Neoplasias/metabolismo , Adolescente , Adulto , Animales , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Hipoxia de la Célula/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Niño , Regulación hacia Abajo , Perfilación de la Expresión Génica , Glutamina/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Neoplasias/enzimología , Neoplasias/patología , Osteosarcoma/metabolismo , Osteosarcoma/patología , Oxidación-Reducción , Adulto JovenRESUMEN
The urea cycle (UC) is the main pathway by which mammals dispose of waste nitrogen. We find that specific alterations in the expression of most UC enzymes occur in many tumors, leading to a general metabolic hallmark termed "UC dysregulation" (UCD). UCD elicits nitrogen diversion toward carbamoyl-phosphate synthetase2, aspartate transcarbamylase, and dihydrooratase (CAD) activation and enhances pyrimidine synthesis, resulting in detectable changes in nitrogen metabolites in both patient tumors and their bio-fluids. The accompanying excess of pyrimidine versus purine nucleotides results in a genomic signature consisting of transversion mutations at the DNA, RNA, and protein levels. This mutational bias is associated with increased numbers of hydrophobic tumor antigens and a better response to immune checkpoint inhibitors independent of mutational load. Taken together, our findings demonstrate that UCD is a common feature of tumors that profoundly affects carcinogenesis, mutagenesis, and immunotherapy response.
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Genómica , Metabolómica , Neoplasias/patología , Urea/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Animales , Aspartato Carbamoiltransferasa/genética , Aspartato Carbamoiltransferasa/metabolismo , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/genética , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/metabolismo , Línea Celular Tumoral , Dihidroorotasa/genética , Dihidroorotasa/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Proteínas de Transporte de Membrana Mitocondrial , Neoplasias/metabolismo , Ornitina Carbamoiltransferasa/antagonistas & inhibidores , Ornitina Carbamoiltransferasa/genética , Ornitina Carbamoiltransferasa/metabolismo , Fosforilación/efectos de los fármacos , Pirimidinas/biosíntesis , Pirimidinas/química , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
In Extended Data Fig. 1a of this Letter, the flow cytometry plot depicting the surface phenotype of AML sample DD08 was a duplicate of the plot for AML sample DD06. Supplementary Data 4 has been added to the Supplementary Information of the original Letter to clarify the proteome data acquisition and presentation. The original Letter has been corrected online.
RESUMEN
The branched-chain amino acid (BCAA) pathway and high levels of BCAA transaminase 1 (BCAT1) have recently been associated with aggressiveness in several cancer entities. However, the mechanistic role of BCAT1 in this process remains largely uncertain. Here, by performing high-resolution proteomic analysis of human acute myeloid leukaemia (AML) stem-cell and non-stem-cell populations, we find the BCAA pathway enriched and BCAT1 protein and transcripts overexpressed in leukaemia stem cells. We show that BCAT1, which transfers α-amino groups from BCAAs to α-ketoglutarate (αKG), is a critical regulator of intracellular αKG homeostasis. Further to its role in the tricarboxylic acid cycle, αKG is an essential cofactor for αKG-dependent dioxygenases such as Egl-9 family hypoxia inducible factor 1 (EGLN1) and the ten-eleven translocation (TET) family of DNA demethylases. Knockdown of BCAT1 in leukaemia cells caused accumulation of αKG, leading to EGLN1-mediated HIF1α protein degradation. This resulted in a growth and survival defect and abrogated leukaemia-initiating potential. By contrast, overexpression of BCAT1 in leukaemia cells decreased intracellular αKG levels and caused DNA hypermethylation through altered TET activity. AML with high levels of BCAT1 (BCAT1high) displayed a DNA hypermethylation phenotype similar to cases carrying a mutant isocitrate dehydrogenase (IDHmut), in which TET2 is inhibited by the oncometabolite 2-hydroxyglutarate. High levels of BCAT1 strongly correlate with shorter overall survival in IDHWTTET2WT, but not IDHmut or TET2mut AML. Gene sets characteristic for IDHmut AML were enriched in samples from patients with an IDHWTTET2WTBCAT1high status. BCAT1high AML showed robust enrichment for leukaemia stem-cell signatures, and paired sample analysis showed a significant increase in BCAT1 levels upon disease relapse. In summary, by limiting intracellular αKG, BCAT1 links BCAA catabolism to HIF1α stability and regulation of the epigenomic landscape, mimicking the effects of IDH mutations. Our results suggest the BCAA-BCAT1-αKG pathway as a therapeutic target to compromise leukaemia stem-cell function in patients with IDHWTTET2WT AML.
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Metilación de ADN , Isocitrato Deshidrogenasa/genética , Ácidos Cetoglutáricos/metabolismo , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/metabolismo , Transaminasas/metabolismo , Aminoácidos de Cadena Ramificada/metabolismo , Animales , Proliferación Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dioxigenasas , Epistasis Genética , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Isocitrato Deshidrogenasa/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/metabolismo , Ratones , Terapia Molecular Dirigida , Mutación , Células Madre Neoplásicas/patología , Pronóstico , Proteolisis , Proteómica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Transaminasas/deficiencia , Transaminasas/genéticaRESUMEN
Monocytes play a critical role in the innate and adaptive immune systems, performing phagocytosis, presenting antigen, and producing cytokines. They are a heterogeneous population that has been divided in humans into classical, intermediate, and non-classical subsets, but the roles of these subsets are incompletely understood. In this study, we investigated the expression patterns of MHC class II (MHCII) and associated molecules and find that the intermediate monocytes express the highest levels of the MHC molecules, HLA-DR (tested in n = 30 samples), HLA-DP (n = 30), and HLA-DQ (n = 10). HLA-DM (n = 30), which catalyzes the peptide exchange on the MHC molecules, is also expressed at the highest levels in intermediate monocytes. To measure HLA-DM function, we measured levels of MHCII-bound CLIP (class II invariant chain peptide, n = 23), which is exchanged for other peptides by HLA-DM. We calculated CLIP:MHCII ratios to normalize CLIP levels to MHCII levels, and found that intermediate monocytes have the lowest CLIP:MHCII ratio. We isolated the different monocyte subsets (in a total of 7 samples) and analyzed their responses to selected cytokines as model of monocyte activation: two M1-polarizing cytokines (IFNγ, GM-CSF), an M2-polarizing cytokine (IL-4) and IL-10. Classical monocytes exhibit the largest increases in class II pathway expression in response to stimulatory cytokines (IFNγ, GM-CSF, IL-4). All three subsets decrease HLA-DR levels after IL-10 exposure. Our findings argue that intermediate monocytes are the most efficient constitutive antigen presenting subset, that classical monocytes are recruited into an antigen presentation role during inflammatory responses and that IL-10 negatively regulates this function across all subsets.
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Citocinas/fisiología , Antígenos de Histocompatibilidad Clase II/inmunología , Monocitos/inmunología , Adulto , Citometría de Flujo , Humanos , Subgrupos LinfocitariosRESUMEN
Mature B lymphocytes (B cells) recognize antigens using their B cell receptor (BCR) and are activated to become antibody-producing cells. In addition, and integral to the development of a high-affinity antibodies, B cells utilize the specialized major histocompatibility complex class II (MHCII) antigen presentation pathway to process BCR-bound and internalized protein antigens and present selected peptides in complex with MHCII to CD4+ T cells. This interaction influences the fate of both types of lymphocytes and shapes immune outcomes. Specific, effective, and optimally timed antigen presentation by B cells requires well-controlled intracellular machinery, often regulated by the combined effects of several molecular events. Here, we delineate and summarize these events in four steps along the antigen presentation pathway: (1) antigen capture and uptake by B cells; (2) intersection of internalized antigen/BCRs complexes with MHCII in peptide-loading compartments; (3) generation and regulation of MHCII/peptide complexes; and (4) exocytic transport for presentation of MHCII/peptide complexes at the surface of B cells. Finally, we discuss modulation of the MHCII presentation pathway across B cell development and maturation to effector cells, with an emphasis on the shaping of the MHCII/peptide repertoire by two key antigen presentation regulators in B cells: HLA-DM and HLA-DO.
RESUMEN
Cancer cells hijack and remodel existing metabolic pathways for their benefit. Argininosuccinate synthase (ASS1) is a urea cycle enzyme that is essential in the conversion of nitrogen from ammonia and aspartate to urea. A decrease in nitrogen flux through ASS1 in the liver causes the urea cycle disorder citrullinaemia. In contrast to the well-studied consequences of loss of ASS1 activity on ureagenesis, the purpose of its somatic silencing in multiple cancers is largely unknown. Here we show that decreased activity of ASS1 in cancers supports proliferation by facilitating pyrimidine synthesis via CAD (carbamoyl-phosphate synthase 2, aspartate transcarbamylase, and dihydroorotase complex) activation. Our studies were initiated by delineating the consequences of loss of ASS1 activity in humans with two types of citrullinaemia. We find that in citrullinaemia type I (CTLN I), which is caused by deficiency of ASS1, there is increased pyrimidine synthesis and proliferation compared with citrullinaemia type II (CTLN II), in which there is decreased substrate availability for ASS1 caused by deficiency of the aspartate transporter citrin. Building on these results, we demonstrate that ASS1 deficiency in cancer increases cytosolic aspartate levels, which increases CAD activation by upregulating its substrate availability and by increasing its phosphorylation by S6K1 through the mammalian target of rapamycin (mTOR) pathway. Decreasing CAD activity by blocking citrin, the mTOR signalling, or pyrimidine synthesis decreases proliferation and thus may serve as a therapeutic strategy in multiple cancers where ASS1 is downregulated. Our results demonstrate that ASS1 downregulation is a novel mechanism supporting cancerous proliferation, and they provide a metabolic link between the urea cycle enzymes and pyrimidine synthesis.
Asunto(s)
Argininosuccinato Sintasa/deficiencia , Ácido Aspártico/metabolismo , Neoplasias/metabolismo , Pirimidinas/biosíntesis , Animales , Argininosuccinato Sintasa/metabolismo , Aspartato Carbamoiltransferasa/metabolismo , Proteínas de Unión al Calcio/antagonistas & inhibidores , Proteínas de Unión al Calcio/metabolismo , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/metabolismo , Línea Celular Tumoral , Proliferación Celular , Citrulinemia/metabolismo , Citosol/metabolismo , Dihidroorotasa/metabolismo , Regulación hacia Abajo , Activación Enzimática , Humanos , Masculino , Ratones , Ratones SCID , Neoplasias/enzimología , Neoplasias/patología , Transportadores de Anión Orgánico/antagonistas & inhibidores , Transportadores de Anión Orgánico/metabolismo , Fosforilación , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
l-Ascorbate, commonly known as vitamin C, serves as an antioxidant and cofactor essential for many biological processes. Distinct ascorbate biosynthetic pathways have been established for animals and plants, but little is known about the presence or synthesis of this molecule in invertebrate species. We have investigated ascorbate metabolism in the nematode Caenorhabditis elegans, where this molecule would be expected to play roles in oxidative stress resistance and as cofactor in collagen and neurotransmitter synthesis. Using high-performance liquid chromatography and gas-chromatography mass spectrometry, we determined that ascorbate is present at low amounts in the egg stage, L1 larvae, and mixed animal populations, with the egg stage containing the highest concentrations. Incubating C. elegans with precursor molecules necessary for ascorbate synthesis in plants and animals did not significantly alter ascorbate levels. Furthermore, bioinformatic analyses did not support the presence in C. elegans of either the plant or the animal biosynthetic pathway. However, we observed the complete (13)C-labeling of ascorbate when C. elegans was grown with (13)C-labeled Escherichia coli as a food source. These results support the hypothesis that ascorbate biosynthesis in invertebrates may proceed by a novel pathway and lay the foundation for a broader understanding of its biological role.