ABSTRACT
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.
Subject(s)
Immunity , Neoplasms/immunology , Neoplasms/metabolism , Obesity/metabolism , Tumor Microenvironment , Adiposity , Animals , CD8-Positive T-Lymphocytes/immunology , Cell Line, Tumor , Cell Proliferation , Diet, High-Fat , Fatty Acids/metabolism , HEK293 Cells , Humans , Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism , Kinetics , Lymphocytes, Tumor-Infiltrating , Mice, Inbred C57BL , Mice, Knockout , Oxidation-Reduction , Principal Component Analysis , Procollagen-Proline Dioxygenase/metabolism , ProteomicsABSTRACT
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.
Subject(s)
Mitochondria/metabolism , Protein Interaction Maps , Sirtuin 3/metabolism , Acetylation , Adenosine Triphosphatases/metabolism , Animals , Carrier Proteins/metabolism , HeLa Cells , Humans , Immunoprecipitation , Membrane Proteins/metabolism , Mice , Mitochondrial Proteins/metabolism , Mitochondrial Proton-Translocating ATPases , Sirtuins/classification , Sirtuins/metabolismABSTRACT
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.
Subject(s)
Acylation/physiology , Mitochondria/metabolism , Mitochondria/physiology , Humans , Lysine/metabolism , Mitochondrial Proteins , Protein Processing, Post-Translational , Sirtuins/physiologyABSTRACT
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.
Subject(s)
Lymphocyte Activation , Proteomics , T-Lymphocytes , Phosphorylation , T-Lymphocytes/metabolism , Proteomics/methods , Phosphoproteins/metabolism , Animals , Humans , Protein Stability , Signal Transduction , Tandem Mass Spectrometry , MiceABSTRACT
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.
Subject(s)
Colorimetry/methods , Hypoxia-Inducible Factor-Proline Dioxygenases/analysis , Ketoglutaric Acids/analysis , Phenylhydrazines/chemistry , Enzyme Assays/methods , Humans , Hydroxylation , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism , Ketoglutaric Acids/chemistry , Kinetics , Substrate SpecificityABSTRACT
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.
Subject(s)
Histone Acetyltransferases/metabolism , Models, Biological , Nucleosomes/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Acetylation , Blotting, Western , Histone Acetyltransferases/genetics , Histones/metabolism , Kinetics , Lysine/metabolism , Methylation , Nucleosomes/genetics , Promoter Regions, Genetic/genetics , Protein Binding , Protein Subunits/genetics , Protein Subunits/metabolism , Schizosaccharomyces pombe Proteins/geneticsABSTRACT
In yeast, the conserved histone acetyltransferase (HAT) Gcn5 associates with Ada2 and Ada3 to form the catalytic module of the ADA and SAGA transcriptional coactivator complexes. Gcn5 also contains an acetyl-lysine binding bromodomain that has been implicated in regulating nucleosomal acetylation in vitro, as well as at gene promoters in cells. However, the contribution of the Gcn5 bromodomain in regulating site specificity of HAT activity remains unclear. Here, we used a combined acid-urea gel and quantitative mass spectrometry approach to compare the HAT activity of wild-type and Gcn5 bromodomain-mutant ADA subcomplexes (Gcn5-Ada2-Ada3). Wild-type ADA subcomplex acetylated H3 lysines with the following specificity; H3K14 > H3K23 > H3K9 ≈ H3K18 > H3K27 > H3K36. However, when the Gcn5 bromodomain was defective in acetyl-lysine binding, the ADA subcomplex demonstrated altered site-specific acetylation on free and nucleosomal H3, with H3K18ac being the most severely diminished. H3K18ac was also severely diminished on H3K14R, but not H3K23R, substrates in wild-type HAT reactions, further suggesting that Gcn5-catalyzed acetylation of H3K14 and bromodomain binding to H3K14ac are important steps preceding H3K18ac. In sum, this work details a previously uncharacterized cross-talk between the Gcn5 bromodomain "reader" function and enzymatic HAT activity that might ultimately affect gene expression. Future studies of how mutations in bromodomains or other histone post-translational modification readers can affect chromatin-templated enzymatic activities will yield unprecedented insight into a potential "histone/epigenetic code." MS data are available via ProteomeXchange with identifier PXD001167.
Subject(s)
Histone Acetyltransferases/chemistry , Histone Acetyltransferases/metabolism , Histones/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Acetylation , Animals , Binding Sites/genetics , Histone Acetyltransferases/genetics , Protein Binding/genetics , Protein Processing, Post-Translational , Protein Structure, Tertiary/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Transcription Factors/metabolism , Transcriptional ActivationABSTRACT
Sir2 is an NAD(+)-dependent histone deacetylase required to mediate transcriptional silencing and suppress rDNA recombination in budding yeast. We previously identified Tdh3, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as a high expression suppressor of the lethality caused by Sir2 overexpression in yeast cells. Here we show that Tdh3 interacts with Sir2, localizes to silent chromatin in a Sir2-dependent manner, and promotes normal silencing at the telomere and rDNA. Characterization of specific TDH3 alleles suggests that Tdh3's influence on silencing requires nuclear localization but does not correlate with its catalytic activity. Interestingly, a genetic assay suggests that Tdh3, an NAD(+)-binding protein, influences nuclear NAD(+) levels; we speculate that Tdh3 links nuclear Sir2 with NAD(+) from the cytoplasm.
Subject(s)
Gene Silencing , Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/genetics , Recombination, Genetic , Saccharomyces cerevisiae Proteins/genetics , Silent Information Regulator Proteins, Saccharomyces cerevisiae/genetics , Sirtuin 2/genetics , Cell Nucleus/metabolism , Chromatin/genetics , Cytoplasm/metabolism , DNA, Ribosomal/genetics , Gene Expression Regulation, Fungal , Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/metabolism , NAD/metabolism , Protein Interaction Maps/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/metabolism , Silent Information Regulator Proteins, Saccharomyces cerevisiae/metabolism , Sirtuin 2/metabolism , Transcription, GeneticABSTRACT
It is widely accepted that ubiquitin-conjugating enzymes contain an active site asparagine that serves as an oxyanion hole, thereby stabilizing a negatively charged transition state intermediate and promoting ubiquitin transfer. Using structural and biochemical approaches to study the role of the conserved asparagine to ubiquitin conjugation by Ubc13-Mms2, we conclude that the importance of this residue stems primarily from its structural role in stabilizing an active site loop.
Subject(s)
Asparagine/metabolism , Conserved Sequence , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin-Conjugating Enzymes/metabolism , Asparagine/chemistry , Catalytic Domain , Models, Molecular , Protein Conformation , Ubiquitin/metabolismABSTRACT
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.
ABSTRACT
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.
Subject(s)
Neoplasms , T-Lymphocyte Subsets , Humans , Aged , T-Lymphocyte Subsets/metabolism , Energy Metabolism , Immunotherapy/methods , Neoplasms/pathology , Tumor MicroenvironmentABSTRACT
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.
Subject(s)
Diet, High-Fat , Fasting , Liver , Metabolomics/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methodsABSTRACT
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.
Subject(s)
CD8-Positive T-Lymphocytes , Carcinogenesis , Glutarates , Isocitrate Dehydrogenase , Neoplasms , Animals , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Carcinogenesis/genetics , Carcinogenesis/metabolism , Gain of Function Mutation , Glutarates/metabolism , Humans , Interferon-gamma/metabolism , Isocitrate Dehydrogenase/genetics , Isocitrate Dehydrogenase/metabolism , L-Lactate Dehydrogenase/antagonists & inhibitors , L-Lactate Dehydrogenase/metabolism , Mice , Neoplasms/genetics , Neoplasms/immunology , Neoplasms/metabolismABSTRACT
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.
Subject(s)
Antineoplastic Agents/pharmacology , CD8-Positive T-Lymphocytes/drug effects , Ferroptosis/drug effects , Tumor Cells, Cultured/drug effects , Animals , Autophagy/drug effects , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Endoplasmic Reticulum/drug effects , Female , Ferroptosis/genetics , Humans , Mice , Mice, Inbred C57BL , Neoplasms/drug therapyABSTRACT
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.
Subject(s)
Astrocytes/metabolism , Ataxia/genetics , Metabolome/genetics , Migraine with Aura/genetics , Sodium-Potassium-Exchanging ATPase/genetics , Transcriptome , Animals , Astrocytes/pathology , Ataxia/metabolism , Ataxia/pathology , Brain/metabolism , Brain/pathology , Disease Models, Animal , Electroencephalography , Female , Functional Neuroimaging , Glycine/metabolism , Male , Mice , Mice, Knockout , Migraine with Aura/metabolism , Migraine with Aura/pathology , Rotarod Performance Test , Serine/metabolism , Sodium-Potassium-Exchanging ATPase/deficiencyABSTRACT
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.
Subject(s)
Induced Pluripotent Stem Cells/drug effects , Myocytes, Cardiac/metabolism , Protein Kinase Inhibitors/pharmacology , Acclimatization , Antineoplastic Agents/pharmacology , Cardiotoxicity/metabolism , Cell Differentiation/drug effects , Cells, Cultured , Erlotinib Hydrochloride/pharmacology , Gene Expression Profiling/methods , Humans , Induced Pluripotent Stem Cells/metabolism , Lapatinib/pharmacology , Protein-Tyrosine Kinases/metabolism , Signal Transduction/drug effects , Sorafenib/pharmacology , Sunitinib/pharmacologyABSTRACT
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.
Subject(s)
Chromatin/metabolism , Histone Demethylases/metabolism , Nuclear Proteins/metabolism , Oxygen/metabolism , Animals , Cell Hypoxia , HEK293 Cells , Histone Demethylases/genetics , Histones/metabolism , Humans , Hypoxia-Inducible Factor 1/metabolism , Jumonji Domain-Containing Histone Demethylases/genetics , Jumonji Domain-Containing Histone Demethylases/metabolism , MCF-7 Cells , Methylation , Mice , Nuclear Proteins/geneticsABSTRACT
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.
Subject(s)
Mutation/genetics , NAD/metabolism , Sequence Homology, Amino Acid , Sirtuin 2/chemistry , Sirtuin 2/genetics , Acetylation , Humans , Kinetics , Models, Molecular , Protein Stability , Saccharomyces cerevisiae/metabolism , Substrate SpecificityABSTRACT
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.
Subject(s)
Ammonia/metabolism , Breast Neoplasms/enzymology , Breast Neoplasms/pathology , Glutamate Dehydrogenase/metabolism , Amination , Animals , Aspartic Acid/metabolism , Biocatalysis , Cell Proliferation , Female , Glutamate Dehydrogenase/genetics , Humans , MCF-7 Cells , Mice , Proline/metabolism , RNA, Small Interfering/metabolism , Tumor MicroenvironmentABSTRACT
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.