RESUMO
NLRs constitute a large, highly conserved family of cytosolic pattern recognition receptors that are central to health and disease, making them key therapeutic targets. NLRC5 is an enigmatic NLR with mutations associated with inflammatory and infectious diseases, but little is known about its function as an innate immune sensor and cell death regulator. Therefore, we screened for NLRC5's role in response to infections, PAMPs, DAMPs, and cytokines. We identified that NLRC5 acts as an innate immune sensor to drive inflammatory cell death, PANoptosis, in response to specific ligands, including PAMP/heme and heme/cytokine combinations. NLRC5 interacted with NLRP12 and PANoptosome components to form a cell death complex, suggesting an NLR network forms similar to those in plants. Mechanistically, TLR signaling and NAD+ levels regulated NLRC5 expression and ROS production to control cell death. Furthermore, NLRC5-deficient mice were protected in hemolytic and inflammatory models, suggesting that NLRC5 could be a potential therapeutic target.
Assuntos
Inflamação , Peptídeos e Proteínas de Sinalização Intracelular , NAD , Animais , Camundongos , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , NAD/metabolismo , Humanos , Imunidade Inata , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismo , Camundongos Knockout , Transdução de Sinais , Células HEK293 , Inflamassomos/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genética , Receptores Toll-Like/metabolismo , Masculino , Citocinas/metabolismo , Proteínas de Ligação ao CálcioRESUMO
Defects in translation lead to changes in the expression of proteins that can serve as drivers of cancer formation. Here, we show that cytosolic NAD+ synthesis plays an essential role in ovarian cancer by regulating translation and maintaining protein homeostasis. Expression of NMNAT-2, a cytosolic NAD+ synthase, is highly upregulated in ovarian cancers. NMNAT-2 supports the catalytic activity of the mono(ADP-ribosyl) transferase (MART) PARP-16, which mono(ADP-ribosyl)ates (MARylates) ribosomal proteins. Depletion of NMNAT-2 or PARP-16 leads to inhibition of MARylation, increased polysome association and enhanced translation of specific mRNAs, aggregation of their translated protein products, and reduced growth of ovarian cancer cells. Furthermore, MARylation of the ribosomal proteins, such as RPL24 and RPS6, inhibits polysome assembly by stabilizing eIF6 binding to ribosomes. Collectively, our results demonstrate that ribosome MARylation promotes protein homeostasis in cancers by fine-tuning the levels of protein synthesis and preventing toxic protein aggregation.
Assuntos
ADP-Ribosilação , Neoplasias Ovarianas/metabolismo , Biossíntese de Proteínas , Proteostase , Ribossomos/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Estresse do Retículo Endoplasmático , Tubas Uterinas/metabolismo , Feminino , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , NAD/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase , Conformação de Ácido Nucleico , Neoplasias Ovarianas/patologia , Poli(ADP-Ribose) Polimerases/metabolismo , Polirribossomos/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas Ribossômicas/metabolismoRESUMO
The malate shuttle is traditionally understood to maintain NAD+/NADH balance between the cytosol and mitochondria. Whether the malate shuttle has additional functions is unclear. Here we show that chronic viral infections induce CD8+ T cell expression of GOT1, a central enzyme in the malate shuttle. Got1 deficiency decreased the NAD+/NADH ratio and limited antiviral CD8+ T cell responses to chronic infection; however, increasing the NAD+/NADH ratio did not restore T cell responses. Got1 deficiency reduced the production of the ammonia scavenger 2-ketoglutarate (2-KG) from glutaminolysis and led to a toxic accumulation of ammonia in CD8+ T cells. Supplementation with 2-KG assimilated and detoxified ammonia in Got1-deficient T cells and restored antiviral responses. These data indicate that the major function of the malate shuttle in CD8+ T cells is not to maintain the NAD+/NADH balance but rather to detoxify ammonia and enable sustainable ammonia-neutral glutamine catabolism in CD8+ T cells during chronic infection.
Assuntos
Ácidos Cetoglutáricos , NAD , Humanos , Oxirredução , NAD/metabolismo , Ácidos Cetoglutáricos/metabolismo , Amônia , Malatos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Infecção Persistente , AntiviraisRESUMO
Mitochondrial diseases are clinically heterogeneous disorders caused by a wide spectrum of mutations in genes encoded by either the nuclear or the mitochondrial genome. Treatments for mitochondrial diseases are currently focused on symptomatic management rather than improving the biochemical defect caused by a particular mutation. This review focuses on the latest advances in the development of treatments for mitochondrial disease, both small molecules and gene therapies, as well as methods to prevent transmission of mitochondrial disease through the germline.
Assuntos
Mitocôndrias/genética , Doenças Mitocondriais/terapia , Animais , DNA Mitocondrial/genética , Terapia Genética , Genoma Mitocondrial/genética , Humanos , Mitocôndrias/patologia , Doenças Mitocondriais/genética , Mutação , NAD/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Metabolic reprogramming is a key feature of many cancers, but how and when it contributes to tumorigenesis remains unclear. Here we demonstrate that metabolic reprogramming induced by mitochondrial fusion can be rate-limiting for immortalization of tumor-initiating cells (TICs) and trigger their irreversible dedication to tumorigenesis. Using single-cell transcriptomics, we find that Drosophila brain tumors contain a rapidly dividing stem cell population defined by upregulation of oxidative phosphorylation (OxPhos). We combine targeted metabolomics and in vivo genetic screening to demonstrate that OxPhos is required for tumor cell immortalization but dispensable in neural stem cells (NSCs) giving rise to tumors. Employing an in vivo NADH/NAD+ sensor, we show that NSCs precisely increase OxPhos during immortalization. Blocking OxPhos or mitochondrial fusion stalls TICs in quiescence and prevents tumorigenesis through impaired NAD+ regeneration. Our work establishes a unique connection between cellular metabolism and immortalization of tumor-initiating cells.
Assuntos
Neoplasias Encefálicas/metabolismo , Carcinogênese/metabolismo , Transformação Celular Neoplásica/metabolismo , Dinâmica Mitocondrial , NAD/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neurais/metabolismo , Fosforilação Oxidativa , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Carcinogênese/genética , Carcinogênese/patologia , Transformação Celular Neoplásica/patologia , Ciclo do Ácido Cítrico/genética , Biologia Computacional , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Glicólise/genética , Espectrometria de Massas , Metabolômica , Microscopia Eletrônica de Transmissão , Família Multigênica , Células-Tronco Neurais/patologia , Consumo de Oxigênio/genética , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Análise de Célula Única , Transcriptoma/genéticaRESUMO
Nicotinamide adenine dinucleotide (NAD+) is a coenzyme for redox reactions, making it central to energy metabolism. NAD+ is also an essential cofactor for non-redox NAD+-dependent enzymes, including sirtuins, CD38 and poly(ADP-ribose) polymerases. NAD+ can directly and indirectly influence many key cellular functions, including metabolic pathways, DNA repair, chromatin remodelling, cellular senescence and immune cell function. These cellular processes and functions are critical for maintaining tissue and metabolic homeostasis and for healthy ageing. Remarkably, ageing is accompanied by a gradual decline in tissue and cellular NAD+ levels in multiple model organisms, including rodents and humans. This decline in NAD+ levels is linked causally to numerous ageing-associated diseases, including cognitive decline, cancer, metabolic disease, sarcopenia and frailty. Many of these ageing-associated diseases can be slowed down and even reversed by restoring NAD+ levels. Therefore, targeting NAD+ metabolism has emerged as a potential therapeutic approach to ameliorate ageing-related disease, and extend the human healthspan and lifespan. However, much remains to be learnt about how NAD+ influences human health and ageing biology. This includes a deeper understanding of the molecular mechanisms that regulate NAD+ levels, how to effectively restore NAD+ levels during ageing, whether doing so is safe and whether NAD+ repletion will have beneficial effects in ageing humans.
Assuntos
Envelhecimento , Fenômenos Fisiológicos Celulares , Redes e Vias Metabólicas , Mitocôndrias/metabolismo , NAD/metabolismo , Animais , Metabolismo Energético , HumanosRESUMO
A decline in capillary density and blood flow with age is a major cause of mortality and morbidity. Understanding why this occurs is key to future gains in human health. NAD precursors reverse aspects of aging, in part, by activating sirtuin deacylases (SIRT1-SIRT7) that mediate the benefits of exercise and dietary restriction (DR). We show that SIRT1 in endothelial cells is a key mediator of pro-angiogenic signals secreted from myocytes. Treatment of mice with the NAD+ booster nicotinamide mononucleotide (NMN) improves blood flow and increases endurance in elderly mice by promoting SIRT1-dependent increases in capillary density, an effect augmented by exercise or increasing the levels of hydrogen sulfide (H2S), a DR mimetic and regulator of endothelial NAD+ levels. These findings have implications for improving blood flow to organs and tissues, increasing human performance, and reestablishing a virtuous cycle of mobility in the elderly.
Assuntos
Envelhecimento , Sulfeto de Hidrogênio/metabolismo , NAD/metabolismo , Animais , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Camundongos , Camundongos Knockout , Microvasos/metabolismo , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Neovascularização Fisiológica , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Condicionamento Físico Animal , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/genética , Sirtuína 1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
H2S is an endogenous gasotransmitter that plays an important role in physiological conditions. In this issue, Das et al. provide evidence that SIRT1-dependent angiogenesis is augmented by H2S-findings reinforced by Longchamp et al., who demonstrate that H2S-dependent angiogenesis is triggered by amino acid deprivation.
Assuntos
NAD , Sirtuína 1RESUMO
Eukaryotic mRNAs generally possess a 5' end N7 methyl guanosine (m7G) cap that promotes their translation and stability. However, mammalian mRNAs can also carry a 5' end nicotinamide adenine dinucleotide (NAD+) cap that, in contrast to the m7G cap, does not support translation but instead promotes mRNA decay. The mammalian and fungal noncanonical DXO/Rai1 decapping enzymes efficiently remove NAD+ caps, and cocrystal structures of DXO/Rai1 with 3'-NADP+ illuminate the molecular mechanism for how the "deNADding" reaction produces NAD+ and 5' phosphate RNA. Removal of DXO from cells increases NAD+-capped mRNA levels and enables detection of NAD+-capped intronic small nucleolar RNAs (snoRNAs), suggesting NAD+ caps can be added to 5'-processed termini. Our findings establish NAD+ as an alternative mammalian RNA cap and DXO as a deNADding enzyme modulating cellular levels of NAD+-capped RNAs. Collectively, these data reveal that mammalian RNAs can harbor a 5' end modification distinct from the classical m7G cap that promotes rather than inhibits RNA decay.
Assuntos
Processamento Pós-Transcricional do RNA , Estabilidade de RNA , Animais , Endorribonucleases/metabolismo , Células HEK293 , Humanos , Camundongos , NAD/metabolismo , Proteínas Nucleares/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA não Traduzido/metabolismoRESUMO
The process of aging and circadian rhythms are intimately intertwined, but how peripheral clocks involved in metabolic homeostasis contribute to aging remains unknown. Importantly, caloric restriction (CR) extends lifespan in several organisms and rewires circadian metabolism. Using young versus old mice, fed ad libitum or under CR, we reveal reprogramming of the circadian transcriptome in the liver. These age-dependent changes occur in a highly tissue-specific manner, as demonstrated by comparing circadian gene expression in the liver versus epidermal and skeletal muscle stem cells. Moreover, de novo oscillating genes under CR show an enrichment in SIRT1 targets in the liver. This is accompanied by distinct circadian hepatic signatures in NAD+-related metabolites and cyclic global protein acetylation. Strikingly, this oscillation in acetylation is absent in old mice while CR robustly rescues global protein acetylation. Our findings indicate that the clock operates at the crossroad between protein acetylation, liver metabolism, and aging.
Assuntos
Envelhecimento/metabolismo , Ritmo Circadiano , Fígado/metabolismo , Redes e Vias Metabólicas , Acetilcoenzima A/metabolismo , Acetilação , Envelhecimento/patologia , Animais , Restrição Calórica , Histonas/metabolismo , Fígado/patologia , Camundongos , NAD/metabolismo , Proteínas/metabolismo , Sirtuína 1/metabolismo , Células-Tronco/metabolismo , TranscriptomaRESUMO
Serine metabolism is involved in the fate decisions of immune cells; however, whether and how de novo serine synthesis shapes innate immune cell function remain unknown. Here, we first demonstrated that inflammatory macrophages have high expression of phosphoglycerate dehydrogenase (PHGDH, the rate-limiting enzyme of de novo serine synthesis) via nuclear factor κB signaling. Notably, the pharmacological inhibition or genetic modulation of PHGDH limits macrophage interleukin (IL)-1ß production through NAD+ accumulation and subsequent NAD+-dependent SIRT1 and SIRT3 expression and activity. Mechanistically, PHGDH not only sustains IL-1ß expression through H3K9/27 acetylation-mediated transcriptional activation of Toll-like receptor 4 but also supports IL-1ß maturation via NLRP3-K21/22/24/ASC-K21/22/24 acetylation-mediated activation of the NLRP3 inflammasome. Moreover, mice with myeloid-specific depletion of Phgdh show alleviated inflammatory responses in lipopolysaccharide-induced systemic inflammation. This study reveals a network by which a metabolic enzyme, involved in de novo serine synthesis, mediates post-translational modifications and epigenetic regulation to orchestrate IL-1ß production, providing a potential inflammatory disease target.
Assuntos
NAD , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Camundongos , Acetilação , Epigênese Genética , Inflamassomos/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , NAD/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Processamento de Proteína Pós-Traducional , Serina/metabolismoRESUMO
Retrons are toxin-antitoxin systems protecting bacteria against bacteriophages via abortive infection. The Retron-Eco1 antitoxin is formed by a reverse transcriptase (RT) and a non-coding RNA (ncRNA)/multi-copy single-stranded DNA (msDNA) hybrid that neutralizes an uncharacterized toxic effector. Yet, the molecular mechanisms underlying phage defense remain unknown. Here, we show that the N-glycosidase effector, which belongs to the STIR superfamily, hydrolyzes NAD+ during infection. Cryoelectron microscopy (cryo-EM) analysis shows that the msDNA stabilizes a filament that cages the effector in a low-activity state in which ADPr, a NAD+ hydrolysis product, is covalently linked to the catalytic E106 residue. Mutations shortening the msDNA induce filament disassembly and the effector's toxicity, underscoring the msDNA role in immunity. Furthermore, we discovered a phage-encoded Retron-Eco1 inhibitor (U56) that binds ADPr, highlighting the intricate interplay between retron systems and phage evolution. Our work outlines the structural basis of Retron-Eco1 defense, uncovering ADPr's pivotal role in immunity.
Assuntos
Bacteriófagos , Microscopia Crioeletrônica , NAD , NAD/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Bacteriófagos/imunologia , Hidrólise , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/imunologia , Sistemas Toxina-Antitoxina/genética , Escherichia coli/virologia , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/metabolismoRESUMO
Sirtuins are NAD(+)-dependent enzymes universally present in all organisms, where they play central roles in regulating numerous biological processes. Although early studies showed that sirtuins deacetylated lysines in a reaction that consumes NAD(+), more recent studies have revealed that these enzymes can remove a variety of acyl-lysine modifications. The specificities for varied acyl modifications may thus underlie the distinct roles of the different sirtuins within a given organism. This review summarizes the structure, chemistry, and substrate specificity of sirtuins with a focus on how different sirtuins recognize distinct substrates and thus carry out specific functions.
Assuntos
Histonas/química , NAD/química , Processamento de Proteína Pós-Traducional , Sirtuínas/química , Acilação , Expressão Gênica , Histonas/genética , Histonas/metabolismo , Humanos , Hidrólise , Cinética , Lipoilação , Modelos Moleculares , Ácido Mirístico/química , Ácido Mirístico/metabolismo , NAD/metabolismo , Plasmodium falciparum/química , Plasmodium falciparum/enzimologia , Estrutura Secundária de Proteína , Sirtuínas/genética , Sirtuínas/metabolismo , Especificidade por Substrato , Ácido Succínico/química , Ácido Succínico/metabolismo , Thermotoga maritima/química , Thermotoga maritima/enzimologiaRESUMO
Recent advances highlight a pivotal role for cellular metabolism in programming immune responses. Here, we demonstrate that cell-autonomous generation of nicotinamide adenine dinucleotide (NAD+) via the kynurenine pathway (KP) regulates macrophage immune function in aging and inflammation. Isotope tracer studies revealed that macrophage NAD+ derives substantially from KP metabolism of tryptophan. Genetic or pharmacological blockade of de novo NAD+ synthesis depleted NAD+, suppressed mitochondrial NAD+-dependent signaling and respiration, and impaired phagocytosis and resolution of inflammation. Innate immune challenge triggered upstream KP activation but paradoxically suppressed cell-autonomous NAD+ synthesis by limiting the conversion of downstream quinolinate to NAD+, a profile recapitulated in aging macrophages. Increasing de novo NAD+ generation in immune-challenged or aged macrophages restored oxidative phosphorylation and homeostatic immune responses. Thus, KP-derived NAD+ operates as a metabolic switch to specify macrophage effector responses. Breakdown of de novo NAD+ synthesis may underlie declining NAD+ levels and rising innate immune dysfunction in aging and age-associated diseases.
Assuntos
Envelhecimento/fisiologia , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Inflamação/imunologia , Macrófagos/fisiologia , Mitocôndrias/metabolismo , NAD/metabolismo , Animais , Células Cultivadas , Homeostase , Imunidade Inata , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Cinurenina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação Oxidativa , Pentosiltransferases/genética , Fagocitose , Transdução de Sinais , Triptofano/metabolismoRESUMO
The adoption of Warburg metabolism is critical for the activation of macrophages in response to lipopolysaccharide. Macrophages stimulated with lipopolysaccharide increase their expression of nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in NAD+ salvage, and loss of NAMPT activity alters their inflammatory potential. However, the events that lead to the cells' becoming dependent on NAD+ salvage remain poorly defined. We found that depletion of NAD+ and increased expression of NAMPT occurred rapidly after inflammatory activation and coincided with DNA damage caused by reactive oxygen species (ROS). ROS produced by complex III of the mitochondrial electron-transport chain were required for macrophage activation. DNA damage was associated with activation of poly(ADP-ribose) polymerase, which led to consumption of NAD+. In this setting, increased NAMPT expression allowed the maintenance of NAD+ pools sufficient for glyceraldehyde-3-phosphate dehydrogenase activity and Warburg metabolism. Our findings provide an integrated explanation for the dependence of inflammatory macrophages on the NAD+ salvage pathway.
Assuntos
Dano ao DNA , Macrófagos/metabolismo , NAD/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Acrilamidas/farmacologia , Animais , Células Cultivadas , Citocinas/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Células HEK293 , Humanos , Inflamação/metabolismo , Ativação de Macrófagos , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Piperidinas/farmacologiaRESUMO
Immunoregulatory B cells impede antitumor immunity through unknown features and mechanisms. We report the existence of leucine-tRNA-synthase-2 (LARS2)-expressing B cell (LARS B) subset with a transforming growth factor-ß1 (TGF-ß1)-dominant regulatory feature in both mouse and human progressive colorectal cancer (CRC). Of note, LARS B cells exhibited a leucine nutrient preference and displayed active mitochondrial aminoacyl-tRNA biosynthesis. They were located outside the tertiary lymphoid structure and correlated with colorectal hyperplasia and shortened survival in CRC patients. A leucine diet induced LARS B cell generation, whereas LARS B cell deletion by Lars2 gene ablation or leucine blockage repressed CRC immunoevasion. Mechanistically, LARS2 programmed mitochondrial nicotinamide adenine dinucleotide (NAD+) regeneration and oxidative metabolism, thus determining the regulatory feature of LARS B cells in which the NAD-dependent protein deacetylase sirtuin-1 (SIRT1) was involved. We propose a leucine-dieting scheme to inhibit LARS B cells, which is safe and useful for CRC therapy.
Assuntos
Aminoacil-tRNA Sintetases , Neoplasias Colorretais , Animais , Humanos , Leucina , Camundongos , Mitocôndrias/metabolismo , NAD/metabolismo , RNA de TransferênciaRESUMO
Longevity-promoting caloric restriction is thought to trigger downregulation of mammalian target of rapamycin complex 1 (mTORC1) signaling and upregulation of SIRT1 activity with associated health benefits. Here, we show that mTORC1 signaling in intestinal stem cells (ISCs) is instead upregulated during calorie restriction (CR). SIRT1 deacetylates S6K1, thereby enhancing its phosphorylation by mTORC1, which leads to an increase in protein synthesis and an increase in ISC number. Paneth cells in the ISC niche secrete cyclic ADP ribose that triggers SIRT1 activity and mTORC1 signaling in neighboring ISCs. Notably, the mTOR inhibitor rapamycin, previously reported to mimic effects of CR, abolishes this expansion of ISCs. We suggest that Paneth cell signaling overrides any direct nutrient sensing in ISCs to sculpt the observed response to CR. Moreover, drugs that modulate pathways important in CR may exert opposing effects on different cell types.
Assuntos
Células-Tronco Adultas/metabolismo , Restrição Calórica , Complexos Multiproteicos/metabolismo , Transdução de Sinais , Sirtuína 1/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Proliferação de Células , ADP-Ribose Cíclica/metabolismo , Dieta , Mucosa Intestinal/metabolismo , Intestinos/citologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , NAD/metabolismo , Organoides/metabolismo , Fosforilação , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Sirtuína 2/metabolismoRESUMO
The glycerol-3-phosphate shuttle (G3PS) is a major NADH shuttle that regenerates reducing equivalents in the cytosol and produces energy in the mitochondria. Here, we demonstrate that G3PS is uncoupled in kidney cancer cells where the cytosolic reaction is â¼4.5 times faster than the mitochondrial reaction. The high flux through cytosolic glycerol-3-phosphate dehydrogenase (GPD) is required to maintain redox balance and support lipid synthesis. Interestingly, inhibition of G3PS by knocking down mitochondrial GPD (GPD2) has no effect on mitochondrial respiration. Instead, loss of GPD2 upregulates cytosolic GPD on a transcriptional level and promotes cancer cell proliferation by increasing glycerol-3-phosphate supply. The proliferative advantage of GPD2 knockdown tumor can be abolished by pharmacologic inhibition of lipid synthesis. Taken together, our results suggest that G3PS is not required to run as an intact NADH shuttle but is instead truncated to support complex lipid synthesis in kidney cancer.
Assuntos
Glicerol-3-Fosfato Desidrogenase (NAD+) , Neoplasias Renais , Lipídeos , Humanos , Glicerol/metabolismo , Glicerol-3-Fosfato Desidrogenase (NAD+)/genética , Glicerol-3-Fosfato Desidrogenase (NAD+)/metabolismo , Glicerolfosfato Desidrogenase/genética , Glicerolfosfato Desidrogenase/metabolismo , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Lipídeos/biossíntese , NAD/metabolismo , Oxirredução , Fosfatos/metabolismoRESUMO
SIR2-HerA, a bacterial two-protein anti-phage defense system, induces bacterial death by depleting NAD+ upon phage infection. Biochemical reconstitution of SIR2, HerA, and the SIR2-HerA complex reveals a dynamic assembly process. Unlike other ATPases, HerA can form various oligomers, ranging from dimers to nonamers. When assembled with SIR2, HerA forms a hexamer and converts SIR2 from a nuclease to an NAD+ hydrolase, representing an unexpected regulatory mechanism mediated by protein assembly. Furthermore, high concentrations of ATP can inhibit NAD+ hydrolysis by the SIR2-HerA complex. Cryo-EM structures of the SIR2-HerA complex reveal a giant supramolecular assembly up to 1 MDa, with SIR2 as a dodecamer and HerA as a hexamer, crucial for anti-phage defense. Unexpectedly, the HerA hexamer resembles a spiral staircase and exhibits helicase activities toward dual-forked DNA. Together, we reveal the supramolecular assembly of SIR2-HerA as a unique mechanism for switching enzymatic activities and bolstering anti-phage defense strategies.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Sirtuínas , Fagos T , Adenosina Trifosfatases/genética , Proteínas de Bactérias/genética , NAD , Sirtuínas/metabolismo , Escherichia coli/enzimologia , Escherichia coli/virologia , Proteínas de Escherichia coli/metabolismoRESUMO
While antibiotics are intended to specifically target bacteria, most are known to affect host cell physiology. In addition, some antibiotic classes are reported as immunosuppressive for reasons that remain unclear. Here, we show that Linezolid, a ribosomal-targeting antibiotic (RAbo), effectively blocked the course of a T cell-mediated autoimmune disease. Linezolid and other RAbos were strong inhibitors of T helper-17 cell effector function in vitro, showing that this effect was independent of their antibiotic activity. Perturbing mitochondrial translation in differentiating T cells, either with RAbos or through the inhibition of mitochondrial elongation factor G1 (mEF-G1) progressively compromised the integrity of the electron transport chain. Ultimately, this led to deficient oxidative phosphorylation, diminishing nicotinamide adenine dinucleotide concentrations and impairing cytokine production in differentiating T cells. In accordance, mice lacking mEF-G1 in T cells were protected from experimental autoimmune encephalomyelitis, demonstrating that this pathway is crucial in maintaining T cell function and pathogenicity.