RESUMEN
Progressive decline of the adaptive immune system with increasing age coincides with a sharp increase in cancer incidence. In this study, we set out to understand whether deficits in antitumor immunity with advanced age promote tumor progression and/or drive resistance to immunotherapy. We found that multiple syngeneic cancers grew more rapidly in aged versus young adult mice, driven by dysfunctional CD8+ T-cell responses. By systematically mapping immune cell profiles within tumors, we identified loss of tumor antigen-specific CD8+ T cells as a primary feature accelerating the growth of tumors in aged mice and driving resistance to immunotherapy. When antigen-specific T cells from young adult mice were administered to aged mice, tumor outgrowth was delayed and the aged animals became sensitive to PD-1 blockade. These studies reveal how age-associated CD8+ T-cell dysfunction may license tumorigenesis in elderly patients and have important implications for the use of aged mice as pre-clinical models of aging and cancer.
RESUMEN
T cell activation is a complex biological process of naive cells maturing into effector cells. Proteomic and phospho-proteomic approaches have provided critical insights into this process, yet it is not always clear how changes in individual proteins or phosphorylation sites have functional significance. Here, we developed the Phosphorylation Integrated Thermal Shift Assay (PITSA) that combines the measurement of protein or phosphorylation site abundance and thermal stability into a single tandem mass tags experiment and apply this method to study T cell activation. We quantified the abundance and thermal stability of over 7500 proteins and 5000 phosphorylation sites and identified significant differences in chromatin-related, TCR signaling, DNA repair, and proliferative phosphoproteins. PITSA may be applied to a wide range of biological contexts to generate hypotheses as to which proteins or phosphorylation sites are functionally regulated in a given system as well as the mechanisms by which this regulation may occur.
Asunto(s)
Activación de Linfocitos , Proteómica , Linfocitos T , Fosforilación , Linfocitos T/metabolismo , Proteómica/métodos , Fosfoproteínas/metabolismo , Animales , Humanos , Estabilidad Proteica , Transducción de Señal , Espectrometría de Masas en Tándem , RatonesRESUMEN
Aging results in remodeling of T cell immunity and is associated with poor clinical outcomes in age-related diseases such as cancer. Among the hallmarks of aging, changes in host and cellular metabolism critically affect the development, maintenance, and function of T cells. Although metabolic perturbations impact anti-tumor T cell responses, the link between age-associated metabolic dysfunction and anti-tumor immunity remains unclear. In this review, we summarize recent advances in our understanding of aged T cell metabolism, with a focus on the bioenergetic and immunologic features of T cell subsets unique to the aging process. We also survey insights into mechanisms of metabolic T cell dysfunction in aging and discuss the impacts of aging on the efficacy of cancer immunotherapy. As the average life expectancy continues to increase, understanding the interplay between age-related metabolic reprogramming and maladaptive T cell immunity will be instrumental for the development of therapeutic strategies for older patients.
Asunto(s)
Neoplasias , Subgrupos de Linfocitos T , Humanos , Anciano , Subgrupos de Linfocitos T/metabolismo , Metabolismo Energético , Inmunoterapia/métodos , Neoplasias/patología , Microambiente TumoralRESUMEN
Gain-of-function mutations in isocitrate dehydrogenase (IDH) in human cancers result in the production of d-2-hydroxyglutarate (d-2HG), an oncometabolite that promotes tumorigenesis through epigenetic alterations. The cancer cell-intrinsic effects of d-2HG are well understood, but its tumor cell-nonautonomous roles remain poorly explored. We compared the oncometabolite d-2HG with its enantiomer, l-2HG, and found that tumor-derived d-2HG was taken up by CD8+ T cells and altered their metabolism and antitumor functions in an acute and reversible fashion. We identified the glycolytic enzyme lactate dehydrogenase (LDH) as a molecular target of d-2HG. d-2HG and inhibition of LDH drive a metabolic program and immune CD8+ T cell signature marked by decreased cytotoxicity and impaired interferon-γ signaling that was recapitulated in clinical samples from human patients with IDH1 mutant gliomas.
Asunto(s)
Linfocitos T CD8-positivos , Carcinogénesis , Glutaratos , Isocitrato Deshidrogenasa , Neoplasias , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Carcinogénesis/genética , Carcinogénesis/metabolismo , Mutación con Ganancia de Función , Glutaratos/metabolismo , Humanos , Interferón gamma/metabolismo , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , L-Lactato Deshidrogenasa/antagonistas & inhibidores , L-Lactato Deshidrogenasa/metabolismo , Ratones , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/metabolismoRESUMEN
Cells adapt their metabolism to physiological stimuli, and metabolic heterogeneity exists between cell types, within tissues, and subcellular compartments. The liver plays an essential role in maintaining whole-body metabolic homeostasis and is structurally defined by metabolic zones. These zones are well-understood on the transcriptomic level, but have not been comprehensively characterized on the metabolomic level. Mass spectrometry imaging (MSI) can be used to map hundreds of metabolites directly from a tissue section, offering an important advance to investigate metabolic heterogeneity in tissues compared to extraction-based metabolomics methods that analyze tissue metabolite profiles in bulk. We established a workflow for the preparation of tissue specimens for matrix-assisted laser desorption/ionization (MALDI) MSI that can be implemented to achieve broad coverage of central carbon, nucleotide, and lipid metabolism pathways. Herein, we used this approach to visualize the effect of nutrient stress and excess on liver metabolism. Our data revealed a highly organized metabolic tissue compartmentalization in livers, which becomes disrupted under high fat diet. Fasting caused changes in the abundance of several metabolites, including increased levels of fatty acids and TCA intermediates while fatty livers had higher levels of purine and pentose phosphate-related metabolites, which generate reducing equivalents to counteract oxidative stress. This spatially conserved approach allowed the visualization of liver metabolic compartmentalization at 30 µm pixel resolution and can be applied more broadly to yield new insights into metabolic heterogeneity in vivo.
Asunto(s)
Dieta Alta en Grasa , Ayuno , Hígado , Metabolómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
Since the discovery of the prolyl hydroxylases domain (PHD) proteins and their canonical hypoxia-inducible factor (HIF) substrate two decades ago, a number of in vitro hydroxylation (IVH) assays for PHD activity have been developed to measure the PHD-HIF interaction. However, most of these assays either require complex proteomics mass spectrometry methods that rely on the specific PHD-HIF interaction or require the handling of radioactive material, as seen in the most commonly used assay measuring [14C]O2 release from labeled [14C]α-ketoglutarate. Here, we report an alternative rapid, cost-effective assay in which the consumption of α-ketoglutarate is monitored by its derivatization with 2,4-dinitrophenylhydrazine (2,4-DNPH) followed by treatment with concentrated base. We extensively optimized this 2,4-DNPH α-ketoglutarate assay to maximize the signal-to-noise ratio and demonstrated that it is robust enough to obtain kinetic parameters of the well-characterized PHD2 isoform comparable with those in published literature. We further showed that it is also sensitive enough to detect and measure the IC50 values of pan-PHD inhibitors and several PHD2 inhibitors in clinical trials for chronic kidney disease (CKD)-induced anemia. Given the efficiency of this assay coupled with its multiwell format, the 2,4-DNPH α-KG assay may be adaptable to explore non-HIF substrates of PHDs and potentially to high-throughput assays.
Asunto(s)
Colorimetría/métodos , Prolina Dioxigenasas del Factor Inducible por Hipoxia/análisis , Ácidos Cetoglutáricos/análisis , Fenilhidrazinas/química , Pruebas de Enzimas/métodos , Humanos , Hidroxilación , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Ácidos Cetoglutáricos/química , Cinética , Especificidad por SustratoRESUMEN
Metabolic constraints in the tumor microenvironment constitute a barrier to effective antitumor immunity and similarities in the metabolic properties of T cells and cancer cells impede the specific therapeutic targeting of metabolism in either population. To identify distinct metabolic vulnerabilities of CD8+ T cells and cancer cells, we developed a high-throughput in vitro pharmacologic screening platform and used it to measure the cell type-specific sensitivities of activated CD8+ T cells and B16 melanoma cells to a wide array of metabolic perturbations during antigen-specific killing of cancer cells by CD8+ T cells. We illustrated the applicability of this screening platform by showing that CD8+ T cells were more sensitive to ferroptosis induction by inhibitors of glutathione peroxidase 4 (GPX4) than B16 and MC38 cancer cells. Overexpression of ferroptosis suppressor protein 1 (FSP1) or cytosolic GPX4 yielded ferroptosis-resistant CD8+ T cells without compromising their function, while genetic deletion of the ferroptosis sensitivity-promoting enzyme acyl-CoA synthetase long-chain family member 4 (ACSL4) protected CD8+ T cells from ferroptosis but impaired antitumor CD8+ T-cell responses. Our screen also revealed high T cell-specific vulnerabilities for compounds targeting NAD+ metabolism or autophagy and endoplasmic reticulum (ER) stress pathways. We focused the current screening effort on metabolic agents. However, this in vitro screening platform may also be valuable for rapid testing of other types of compounds to identify regulators of antitumor CD8+ T-cell function and potential therapeutic targets.
Asunto(s)
Antineoplásicos/farmacología , Linfocitos T CD8-positivos/efectos de los fármacos , Ferroptosis/efectos de los fármacos , Células Tumorales Cultivadas/efectos de los fármacos , Animales , Autofagia/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Retículo Endoplásmico/efectos de los fármacos , Femenino , Ferroptosis/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Neoplasias/tratamiento farmacológicoRESUMEN
Obesity is a major cancer risk factor, but how differences in systemic metabolism change the tumor microenvironment (TME) and impact anti-tumor immunity is not understood. Here, we demonstrate that high-fat diet (HFD)-induced obesity impairs CD8+ T cell function in the murine TME, accelerating tumor growth. We generate a single-cell resolution atlas of cellular metabolism in the TME, detailing how it changes with diet-induced obesity. We find that tumor and CD8+ T cells display distinct metabolic adaptations to obesity. Tumor cells increase fat uptake with HFD, whereas tumor-infiltrating CD8+ T cells do not. These differential adaptations lead to altered fatty acid partitioning in HFD tumors, impairing CD8+ T cell infiltration and function. Blocking metabolic reprogramming by tumor cells in obese mice improves anti-tumor immunity. Analysis of human cancers reveals similar transcriptional changes in CD8+ T cell markers, suggesting interventions that exploit metabolism to improve cancer immunotherapy.
Asunto(s)
Inmunidad , Neoplasias/inmunología , Neoplasias/metabolismo , Obesidad/metabolismo , Microambiente Tumoral , Adiposidad , Animales , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Proliferación Celular , Dieta Alta en Grasa , Ácidos Grasos/metabolismo , Células HEK293 , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Cinética , Linfocitos Infiltrantes de Tumor , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidación-Reducción , Análisis de Componente Principal , Procolágeno-Prolina Dioxigenasa/metabolismo , ProteómicaRESUMEN
Familial hemiplegic migraine is an episodic neurological disorder characterized by transient sensory and motor symptoms and signs. Mutations of the ion pump α2-Na/K ATPase cause familial hemiplegic migraine, but the mechanisms by which α2-Na/K ATPase mutations lead to the migraine phenotype remain incompletely understood. Here, we show that mice in which α2-Na/K ATPase is conditionally deleted in astrocytes display episodic paralysis. Functional neuroimaging reveals that conditional α2-Na/K ATPase knockout triggers spontaneous cortical spreading depression events that are associated with EEG low voltage activity events, which correlate with transient motor impairment in these mice. Transcriptomic and metabolomic analyses show that α2-Na/K ATPase loss alters metabolic gene expression with consequent serine and glycine elevation in the brain. A serine- and glycine-free diet rescues the transient motor impairment in conditional α2-Na/K ATPase knockout mice. Together, our findings define a metabolic mechanism regulated by astrocytic α2-Na/K ATPase that triggers episodic motor paralysis in mice.
Asunto(s)
Astrocitos/metabolismo , Ataxia/genética , Metaboloma/genética , Migraña con Aura/genética , ATPasa Intercambiadora de Sodio-Potasio/genética , Transcriptoma , Animales , Astrocitos/patología , Ataxia/metabolismo , Ataxia/patología , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Electroencefalografía , Femenino , Neuroimagen Funcional , Glicina/metabolismo , Masculino , Ratones , Ratones Noqueados , Migraña con Aura/metabolismo , Migraña con Aura/patología , Prueba de Desempeño de Rotación con Aceleración Constante , Serina/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/deficienciaRESUMEN
Tyrosine kinase inhibitors (TKIs) are widely used to treat solid tumors but can be cardiotoxic. The molecular basis for this toxicity and its relationship to therapeutic mechanisms remain unclear; we therefore undertook a systems-level analysis of human cardiomyocytes (CMs) exposed to four TKIs. CMs differentiated from human induced pluripotent stem cells (hiPSCs) were exposed to sunitinib, sorafenib, lapatinib, or erlotinib, and responses were assessed by functional assays, microscopy, RNA sequencing, and mass spectrometry (GEO: GSE114686; PRIDE: PXD012043). TKIs have diverse effects on hiPSC-CMs distinct from inhibition of tyrosine-kinase-mediated signal transduction; cardiac metabolism is particularly sensitive. Following sorafenib treatment, oxidative phosphorylation is downregulated, resulting in a profound defect in mitochondrial energetics. Cells adapt by upregulating aerobic glycolysis. Adaptation makes cells less acutely sensitive to sorafenib but may have long-term negative consequences. Thus, CMs exhibit adaptive responses to anti-cancer drugs conceptually similar to those previously shown in tumors to mediate drug resistance.
Asunto(s)
Células Madre Pluripotentes Inducidas/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Aclimatación , Antineoplásicos/farmacología , Cardiotoxicidad/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Clorhidrato de Erlotinib/farmacología , Perfilación de la Expresión Génica/métodos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Lapatinib/farmacología , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Sorafenib/farmacología , Sunitinib/farmacologíaRESUMEN
Oxygen sensing is central to metazoan biology and has implications for human disease. Mammalian cells express multiple oxygen-dependent enzymes called 2-oxoglutarate (OG)-dependent dioxygenases (2-OGDDs), but they vary in their oxygen affinities and hence their ability to sense oxygen. The 2-OGDD histone demethylases control histone methylation. Hypoxia increases histone methylation, but whether this reflects direct effects on histone demethylases or indirect effects caused by the hypoxic induction of the HIF (hypoxia-inducible factor) transcription factor or the 2-OG antagonist 2-hydroxyglutarate (2-HG) is unclear. Here, we report that hypoxia promotes histone methylation in a HIF- and 2-HG-independent manner. We found that the H3K27 histone demethylase KDM6A/UTX, but not its paralog KDM6B, is oxygen sensitive. KDM6A loss, like hypoxia, prevented H3K27 demethylation and blocked cellular differentiation. Restoring H3K27 methylation homeostasis in hypoxic cells reversed these effects. Thus, oxygen directly affects chromatin regulators to control cell fate.
Asunto(s)
Cromatina/metabolismo , Histona Demetilasas/metabolismo , Proteínas Nucleares/metabolismo , Oxígeno/metabolismo , Animales , Hipoxia de la Célula , Células HEK293 , Histona Demetilasas/genética , Histonas/metabolismo , Humanos , Factor 1 Inducible por Hipoxia/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Células MCF-7 , Metilación , Ratones , Proteínas Nucleares/genéticaRESUMEN
Growing appreciation of the diversity of post-translational modifications (PTMs) in the mitochondria necessitates reevaluation of the roles these modifications play in both health and disease. Compared to the cytosol and nucleus, the mitochondrial proteome is highly acylated, and remodeling of the mitochondrial "acylome" is a key adaptive mechanism that regulates fundamental aspects of mitochondrial biology. It is clear that we need to understand the underlying chemistry that regulates mitochondrial acylation, as well as how chemical properties of the acyl chain impact biological functions. Here, we dissect the sources of PTMs in the mitochondria, review major mitochondrial pathways that control levels of PTMs, and highlight how sirtuin enzymes respond to the bioenergetic state of the cell via NAD+ availability to regulate mitochondrial biology. By providing a framework connecting the chemistry of these modifications, their biochemical consequences, and the pathways that regulate the levels of acyl PTMs, we will gain a deeper understanding of the physiological significance of mitochondrial acylation and its role in mitochondrial adaptation.
Asunto(s)
Acilación/fisiología , Mitocondrias/metabolismo , Mitocondrias/fisiología , Humanos , Lisina/metabolismo , Proteínas Mitocondriales , Procesamiento Proteico-Postraduccional , Sirtuinas/fisiologíaRESUMEN
Sirtuin 3 (SIRT3) is a NAD+-dependent deacetylase downregulated in aging and age-associated diseases such as cancer and neurodegeneration and in high-fat diet (HFD)-induced metabolic disorders. Here, we performed a small-molecule screen and identified an unexpected metabolic vulnerability associated with SIRT3 loss. Azaserine, a glutamine analog, was the top compound that inhibited growth and proliferation of cells lacking SIRT3. Using stable isotope tracing of glutamine, we observed its increased incorporation into de novo nucleotide synthesis in SIRT3 knockout (KO) cells. Furthermore, we found that SIRT3 KO cells upregulated the diversion of glutamine into de novo nucleotide synthesis through hyperactive mTORC1 signaling. Overexpression of SIRT3 suppressed mTORC1 and growth in vivo in a xenograft tumor model of breast cancer. Thus, we have uncovered a metabolic vulnerability of cells with SIRT3 loss by using an unbiased small-molecule screen.
Asunto(s)
Nucleótidos/biosíntesis , Sirtuina 3/deficiencia , Bibliotecas de Moléculas Pequeñas/farmacología , Secuencia de Aminoácidos , Animales , Azaserina/farmacología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Glutamina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones Noqueados , Ratones Desnudos , Regiones Promotoras Genéticas/genética , Transducción de Señal/efectos de los fármacos , Sirtuina 3/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Ammonia is a ubiquitous by-product of cellular metabolism; however, the biological consequences of ammonia production are not fully understood, especially in cancer. We found that ammonia is not merely a toxic waste product but is recycled into central amino acid metabolism to maximize nitrogen utilization. In our experiments, human breast cancer cells primarily assimilated ammonia through reductive amination catalyzed by glutamate dehydrogenase (GDH); secondary reactions enabled other amino acids, such as proline and aspartate, to directly acquire this nitrogen. Metabolic recycling of ammonia accelerated proliferation of breast cancer. In mice, ammonia accumulated in the tumor microenvironment and was used directly to generate amino acids through GDH activity. These data show that ammonia is not only a secreted waste product but also a fundamental nitrogen source that can support tumor biomass.
Asunto(s)
Amoníaco/metabolismo , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Glutamato Deshidrogenasa/metabolismo , Aminación , Animales , Ácido Aspártico/metabolismo , Biocatálisis , Proliferación Celular , Femenino , Glutamato Deshidrogenasa/genética , Humanos , Células MCF-7 , Ratones , Prolina/metabolismo , ARN Interferente Pequeño/metabolismo , Microambiente TumoralRESUMEN
Sirtuin enzymes depend on NAD+ to catalyze protein deacetylation. Therefore, the lowering of NAD+ during aging leads to decreased sirtuin activity and may speed up aging processes in laboratory animals and humans. In this study, we used a genetic screen to identify two mutations in the catalytic domain of yeast Sir2 that allow the enzyme to function in an NAD+-depleted environment. These mutant enzymes give rise to a significant increase of yeast replicative lifespan and increase deacetylation by the Sir2 ortholog, SIRT1, in mammalian cells. Our data suggest that these mutations increase the stability of the conserved catalytic sirtuin domain, thereby increasing the catalytic efficiency of the mutant enzymes. Our approach to identifying sirtuin mutants that permit function in NAD+-limited environments may inform the design of small molecules that can maintain sirtuin activity in aging organisms.
Asunto(s)
Mutación/genética , NAD/metabolismo , Homología de Secuencia de Aminoácido , Sirtuina 2/química , Sirtuina 2/genética , Acetilación , Humanos , Cinética , Modelos Moleculares , Estabilidad Proteica , Saccharomyces cerevisiae/metabolismo , Especificidad por SustratoRESUMEN
Mitochondrial sirtuins, SIRT3-5, are NAD+-dependent deacylases and ADP-ribosyltransferases that are critical for stress responses. However, a comprehensive understanding of sirtuin targets, regulation of sirtuin activity, and the relationships between sirtuins remains a key challenge in mitochondrial physiology. Here, we employ systematic interaction proteomics to elucidate the mitochondrial sirtuin protein interaction landscape. This work reveals sirtuin interactions with numerous functional modules within mitochondria, identifies candidate sirtuin substrates, and uncovers a fundamental role for sequestration of SIRT3 by ATP synthase in mitochondrial homeostasis. In healthy mitochondria, a pool of SIRT3 binds ATP synthase, but upon matrix pH reduction with concomitant loss of mitochondrial membrane potential, SIRT3 dissociates. This release correlates with rapid deacetylation of matrix proteins, and SIRT3 is required for recovery of membrane potential. In vitro reconstitution experiments, as well as analysis of CRISPR/Cas9-engineered cells, indicate that pH-dependent SIRT3 release requires H135 in the ATP5O subunit of ATP synthase. Our SIRT3-5 interaction network provides a framework for discovering novel biological functions regulated by mitochondrial sirtuins.
Asunto(s)
Mitocondrias/metabolismo , Mapas de Interacción de Proteínas , Sirtuina 3/metabolismo , Acetilación , Adenosina Trifosfatasas/metabolismo , Animales , Proteínas Portadoras/metabolismo , Células HeLa , Humanos , Inmunoprecipitación , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Mitocondriales/metabolismo , ATPasas de Translocación de Protón Mitocondriales , Sirtuinas/clasificación , Sirtuinas/metabolismoRESUMEN
Gcn5 is a conserved acetyltransferase that regulates transcription by acetylating the N-terminal tails of histones. Motivated by recent studies identifying a chemically diverse array of lysine acyl modifications in vivo, the acyl-chain specificity of the acetyltransferase human Gcn5 (Gcn5L2) was examined. Whereas Gcn5L2 robustly catalyzes lysine acetylation, the acyltransferase activity of Gcn5L2 becomes progressively weaker with increasing acyl-chain length. To understand how Gcn5 discriminates between different acyl-CoA molecules, structures of the catalytic domain of human Gcn5L2 bound to propionyl-CoA and butyryl-CoA were determined. Although the active site of Gcn5L2 can accommodate propionyl-CoA and butyryl-CoA without major structural rearrangements, butyryl-CoA adopts a conformation incompatible with catalysis that obstructs the path of the incoming lysine residue and acts as a competitive inhibitor of Gcn5L2 versus acetyl-CoA. These structures demonstrate how Gcn5L2 discriminates between acyl-chain donors and explain why Gcn5L2 has weak activity for acyl moieties that are larger than an acetyl group.
Asunto(s)
Factores de Transcripción p300-CBP/química , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Acilcoenzima A/metabolismo , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Conformación Proteica , Especificidad por SustratoRESUMEN
Monoubiquitinated histone H2B plays multiple roles in transcription activation. H2B is deubiquitinated by the Spt-Ada-Gcn5 acetyltransferase (SAGA) coactivator, which contains a four-protein subcomplex known as the deubiquitinating (DUB) module. The crystal structure of the Ubp8/Sgf11/Sus1/Sgf73 DUB module bound to a ubiquitinated nucleosome reveals that the DUB module primarily contacts H2A/H2B, with an arginine cluster on the Sgf11 zinc finger domain docking on the conserved H2A/H2B acidic patch. The Ubp8 catalytic domain mediates additional contacts with H2B, as well as with the conjugated ubiquitin. We find that the DUB module deubiquitinates H2B both in the context of the nucleosome and in H2A/H2B dimers complexed with the histone chaperone, FACT, suggesting that SAGA could target H2B at multiple stages of nucleosome disassembly and reassembly during transcription.
Asunto(s)
Endopeptidasas/química , Histona Acetiltransferasas/química , Histonas/química , Proteínas Nucleares/química , Proteínas de Unión al ARN/química , Proteínas de Saccharomyces cerevisiae/química , Transactivadores/química , Factores de Transcripción/química , Ubiquitinación , Animales , Dominio Catalítico , Cristalografía por Rayos X , Nucleosomas/enzimología , Multimerización de Proteína , Estructura Secundaria de Proteína , Activación Transcripcional , Ubiquitina/química , Xenopus laevis , Dedos de ZincRESUMEN
The Spt-Ada-Gcn5 acetyltransferase (SAGA) coactivator complex hyperacetylates histone tails in vivo in a manner that depends upon histone 3 lysine 4 trimethylation (H3K4me3), a histone mark enriched at promoters of actively transcribed genes. SAGA contains a separable subcomplex known as the histone acetyltransferase (HAT) module that contains the HAT, Gcn5, bound to Sgf29, Ada2, and Ada3. Sgf29 contains a tandem Tudor domain that recognizes H3K4me3-containing peptides and is required for histone hyperacetylation in vivo. However, the mechanism by which H3K4me3 recognition leads to lysine hyperacetylation is unknown, as in vitro studies show no effect of the H3K4me3 modification on histone peptide acetylation by Gcn5. To determine how H3K4me3 binding by Sgf29 leads to histone hyperacetylation by Gcn5, we used differential fluorescent labeling of histones to monitor acetylation of individual subpopulations of methylated and unmodified nucleosomes in a mixture. We find that the SAGA HAT module preferentially acetylates H3K4me3 nucleosomes in a mixture containing excess unmodified nucleosomes and that this effect requires the Tudor domain of Sgf29. The H3K4me3 mark promotes processive, multisite acetylation of histone H3 by Gcn5 that can account for the different acetylation patterns established by SAGA at promoters versus coding regions. Our results establish a model for Sgf29 function at gene promoters and define a mechanism governing crosstalk between histone modifications.
Asunto(s)
Histona Acetiltransferasas/metabolismo , Modelos Biológicos , Nucleosomas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Acetilación , Western Blotting , Histona Acetiltransferasas/genética , Histonas/metabolismo , Cinética , Lisina/metabolismo , Metilación , Nucleosomas/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
Sirtuins were originally shown to regulate a wide array of biological processes such as transcription, genomic stability, and metabolism by catalyzing the NAD(+) -dependent deacetylation of lysine residues. Recent proteomic studies have revealed a much wider array of lysine acyl modifications in vivo than was previously known, which has prompted a reevaluation of sirtuin substrate specificity. Several sirtuins have now been shown to preferentially remove propionyl, succinyl, and long-chain fatty acyl groups from lysines, which has changed our understanding of sirtuin biology. In light of these developments, we revisited the acyl specificity of several well-studied archaeal and bacterial sirtuins. We find that the Archaeoglobus fulgidus sirtuins, Sir2Af1 and Sir2Af2, preferentially remove succinyl and myristoyl groups, respectively. Crystal structures of Sir2Af1 bound to a succinylated peptide and Sir2Af2 bound to a myristoylated peptide show how the active site of each enzyme accommodates a noncanonical acyl chain. As compared to its structure in complex with an acetylated peptide, Sir2Af2 undergoes a conformational change that expands the active site to accommodate the myristoyl group. These findings point to both structural and biochemical plasticity in sirtuin active sites and provide further evidence that sirtuins from all three domains of life catalyze noncanonical deacylation.