RESUMO
Macrophages are critical to turn noninflamed "cold tumors" into inflamed "hot tumors". Emerging evidence indicates abnormal cholesterol metabolites in the tumor microenvironment (TME) with unclear function. Here, we uncovered the inducible expression of cholesterol-25-hydroxylase (Ch25h) by interleukin-4 (IL-4) and interleukin-13 (IL-13) via the transcription factor STAT6, causing 25-hydroxycholesterol (25HC) accumulation. scRNA-seq analysis confirmed that CH25Hhi subsets were enriched in immunosuppressive macrophage subsets and correlated to lower survival rates in pan-cancers. Targeting CH25H abrogated macrophage immunosuppressive function to enhance infiltrating T cell numbers and activation, which synergized with anti-PD-1 to improve anti-tumor efficacy. Mechanically, lysosome-accumulated 25HC competed with cholesterol for GPR155 binding to inhibit the kinase mTORC1, leading to AMPKα activation and metabolic reprogramming. AMPKα also phosphorylated STAT6 Ser564 to enhance STAT6 activation and ARG1 production. Together, we propose CH25H as an immunometabolic checkpoint, which manipulates macrophage fate to reshape CD8+ T cell surveillance and anti-tumor response.
Assuntos
Hidroxicolesteróis , Lisossomos , Macrófagos , Microambiente Tumoral , Animais , Hidroxicolesteróis/metabolismo , Camundongos , Macrófagos/imunologia , Macrófagos/metabolismo , Humanos , Lisossomos/metabolismo , Microambiente Tumoral/imunologia , Fator de Transcrição STAT6/metabolismo , Adenilato Quinase/metabolismo , Camundongos Endogâmicos C57BL , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Transdução de Sinais , Reprogramação MetabólicaRESUMO
Survival in the wild requires organismal adaptations to the availability of nutrients. Endosomes and lysosomes are key intracellular organelles that couple nutrition and metabolic status to cellular responses, but how they detect cytosolic ATP levels is not well understood. Here, we identify an endolysosomal ATP-sensitive Na(+) channel (lysoNa(ATP)). The channel is a complex formed by two-pore channels (TPC1 and TPC2), ion channels previously thought to be gated by nicotinic acid adenine dinucleotide phosphate (NAADP), and the mammalian target of rapamycin (mTOR). The channel complex detects nutrient status, becomes constitutively open upon nutrient removal and mTOR translocation off the lysosomal membrane, and controls the lysosome's membrane potential, pH stability, and amino acid homeostasis. Mutant mice lacking lysoNa(ATP) have much reduced exercise endurance after fasting. Thus, TPCs make up an ion channel family that couples the cell's metabolic state to endolysosomal function and are crucial for physical endurance during food restriction.
Assuntos
Trifosfato de Adenosina/metabolismo , Canais de Cálcio/metabolismo , Lisossomos/metabolismo , Canais de Sódio/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Adenilato Quinase/metabolismo , Aminoácidos/metabolismo , Animais , Canais de Cálcio/química , Canais de Cálcio/genética , Jejum , Técnicas de Inativação de Genes , Homeostase , Humanos , Concentração de Íons de Hidrogênio , Potenciais da Membrana , Camundongos , Resistência FísicaRESUMO
The biguanide drug metformin is widely prescribed to treat type 2 diabetes and metabolic syndrome, but its mode of action remains uncertain. Metformin also increases lifespan in Caenorhabditis elegans cocultured with Escherichia coli. This bacterium exerts complex nutritional and pathogenic effects on its nematode predator/host that impact health and aging. We report that metformin increases lifespan by altering microbial folate and methionine metabolism. Alterations in metformin-induced longevity by mutation of worm methionine synthase (metr-1) and S-adenosylmethionine synthase (sams-1) imply metformin-induced methionine restriction in the host, consistent with action of this drug as a dietary restriction mimetic. Metformin increases or decreases worm lifespan, depending on E. coli strain metformin sensitivity and glucose concentration. In mammals, the intestinal microbiome influences host metabolism, including development of metabolic disease. Thus, metformin-induced alteration of microbial metabolism could contribute to therapeutic efficacy-and also to its side effects, which include folate deficiency and gastrointestinal upset.
Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/microbiologia , Ácido Fólico/metabolismo , Hipoglicemiantes/farmacologia , Longevidade/efeitos dos fármacos , Metformina/farmacologia , Metionina/metabolismo , Adenilato Quinase/metabolismo , Envelhecimento/efeitos dos fármacos , Animais , Biguanidas/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Restrição Calórica , Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Escherichia coli/metabolismo , Humanos , Hipoglicemiantes/metabolismo , Metagenoma , Metformina/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Macromolecular function frequently requires that proteins change conformation into high-energy states1-4. However, methods for solving the structures of these functionally essential, lowly populated states are lacking. Here we develop a method for high-resolution structure determination of minorly populated states by coupling NMR spectroscopy-derived pseudocontact shifts5 (PCSs) with Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion6 (PCS-CPMG). Our approach additionally defines the corresponding kinetics and thermodynamics of high-energy excursions, thereby characterizing the entire free-energy landscape. Using a large set of simulated data for adenylate kinase (Adk), calmodulin and Src kinase, we find that high-energy PCSs accurately determine high-energy structures (with a root mean squared deviation of less than 3.5 angström). Applying our methodology to Adk during catalysis, we find that the high-energy excursion involves surprisingly small openings of the AMP and ATP lids. This previously unresolved high-energy structure solves a longstanding controversy about conformational interconversions that are rate-limiting for catalysis. Primed for either substrate binding or product release, the high-energy structure of Adk suggests a two-step mechanism combining conformational selection to this state, followed by an induced-fit step into a fully closed state for catalysis of the phosphoryl-transfer reaction. Unlike other methods for resolving high-energy states, such as cryo-electron microscopy and X-ray crystallography, our solution PCS-CPMG approach excels in cases involving domain rearrangements of smaller systems (less than 60 kDa) and populations as low as 0.5%, and enables the simultaneous determination of protein structure, kinetics and thermodynamics while proteins perform their function.
Assuntos
Adenilato Quinase , Adenilato Quinase/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , TermodinâmicaRESUMO
Assembly factors (AFs) prevent premature translation initiation on small (40S) ribosomal subunit assembly intermediates by blocking ligand binding. However, it is unclear how AFs are displaced from maturing 40S ribosomes, if or how maturing subunits are assessed for fidelity, and what prevents premature translation initiation once AFs dissociate. Here we show that maturation involves a translation-like cycle whereby the translation factor eIF5B, a GTPase, promotes joining of large (60S) subunits with pre-40S subunits to give 80S-like complexes, which are subsequently disassembled by the termination factor Rli1, an ATPase. The AFs Tsr1 and Rio2 block the mRNA channel and initiator tRNA binding site, and therefore 80S-like ribosomes lack mRNA or initiator tRNA. After Tsr1 and Rio2 dissociate from 80S-like complexes Rli1-directed displacement of 60S subunits allows for translation initiation. This cycle thus provides a functional test of 60S subunit binding and the GTPase site before ribosomes enter the translating pool.
Assuntos
Biossíntese de Proteínas , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Saccharomyces cerevisiae/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenilato Quinase/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/metabolismo , Endorribonucleases/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas Nucleares/metabolismo , Nucleosídeo-Trifosfatase/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
ABSTRACT: Chromosomal translocation (4;14), an adverse prognostic factor in multiple myeloma (MM), drives overexpression of the histone methyltransferase nuclear receptor binding SET domain protein 2 (NSD2). A genome-wide CRISPR screen in MM cells identified adenylate kinase 2 (AK2), an enzyme critical for high-energy phosphate transfer from the mitochondria, as an NSD2-driven vulnerability. AK2 suppression in t(4;14) MM cells decreased nicotinamide adenine dinucleotide phosphate (NADP[H]) critical for conversion of ribonucleotides to deoxyribonucleosides, leading to replication stress, DNA damage, and apoptosis. Driving a large genome-wide increase in chromatin methylation, NSD2 overexpression depletes S-adenosylmethionine, compromising the synthesis of creatine from its precursor, guanidinoacetate. Creatine supplementation restored NADP(H) levels, reduced DNA damage, and rescued AK2-deficient t(4;14) MM cells. As the creatine phosphate shuttle constitutes an alternative means for mitochondrial high-energy phosphate transport, these results indicate that NSD2-driven creatine depletion underlies the hypersensitivity of t(4;14) MM cells to AK2 loss. Furthermore, AK2 depletion in t(4;14) cells impaired protein folding in the endoplasmic reticulum, consistent with impaired use of mitochondrial adenosine triphosphate (ATP). Accordingly, AK2 suppression increased the sensitivity of MM cells to proteasome inhibition. These findings delineate a novel mechanism in which aberrant transfer of carbon to the epigenome creates a metabolic vulnerability, with direct therapeutic implications for t(4;14) MM.
Assuntos
Adenilato Quinase , Histona-Lisina N-Metiltransferase , Mieloma Múltiplo , Translocação Genética , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Adenilato Quinase/metabolismo , Adenilato Quinase/genética , Cromossomos Humanos Par 14/genética , Epigenoma , Cromossomos Humanos Par 4/genética , Carbono/metabolismo , Linhagem Celular Tumoral , Proteínas RepressorasRESUMO
Enzymes play a vital role in life processes; they control chemical reactions and allow functional cycles to be synchronized. Many enzymes harness large-scale motions of their domains to achieve tremendous catalytic prowess and high selectivity for specific substrates. One outstanding example is provided by the three-domain enzyme adenylate kinase (AK), which catalyzes phosphotransfer between ATP to AMP. Here we study the phenomenon of substrate inhibition by AMP and its correlation with domain motions. Using single-molecule FRET spectroscopy, we show that AMP does not block access to the ATP binding site, neither by competitive binding to the ATP cognate site nor by directly closing the LID domain. Instead, inhibitory concentrations of AMP lead to a faster and more cooperative domain closure by ATP, leading in turn to an increased population of the closed state. The effect of AMP binding can be modulated through mutations throughout the structure of the enzyme, as shown by the screening of an extensive AK mutant library. The mutation of multiple conserved residues reduces substrate inhibition, suggesting that substrate inhibition is an evolutionary well conserved feature in AK. Combining these insights, we developed a model that explains the complex activity of AK, particularly substrate inhibition, based on the experimentally observed opening and closing rates. Notably, the model indicates that the catalytic power is affected by the microsecond balance between the open and closed states of the enzyme. Our findings highlight the crucial role of protein motions in enzymatic activity.
Assuntos
Trifosfato de Adenosina , Adenilato Quinase , Adenilato Quinase/metabolismo , Ligantes , Sítios de Ligação , Domínios Proteicos , Trifosfato de Adenosina/metabolismoRESUMO
Despite passing routine laboratory tests for semen quality, bulls used in artificial insemination exhibit significant variation in fertility. Routine analysis of fertility data identified a dairy bull with extreme subfertility (10% pregnancy rate). To characterize the subfertility phenotype, a range of in vitro, in vivo, and molecular assays were carried out. Sperm from the subfertile bull exhibited reduced motility and severely reduced caffeine-induced hyperactivation compared to controls. Ability to penetrate the zona pellucida, cleavage rate, cleavage kinetics, and blastocyst yield after IVF or AI were significantly lower than in control bulls. Whole-genome sequencing from semen and RNA sequencing of testis tissue revealed a critical mutation in adenylate kinase 9 (AK9) that impaired splicing, leading to a premature termination codon and a severely truncated protein. Mice deficient in AK9 were generated to further investigate the function of the gene; knockout males were phenotypically indistinguishable from their wild-type littermates but produced immotile sperm that were incapable of normal fertilization. These sperm exhibited numerous abnormalities, including a low ATP concentration and reduced motility. RNA-seq analysis of their testis revealed differential gene expression of components of the axoneme and sperm flagellum as well as steroid metabolic processes. Sperm ultrastructural analysis showed a high percentage of sperm with abnormal flagella. Combined bovine and murine data indicate the essential metabolic role of AK9 in sperm motility and/or hyperactivation, which in turn affects sperm binding and penetration of the zona pellucida. Thus, AK9 has been found to be directly implicated in impaired male fertility in mammals.
Assuntos
Adenilato Quinase , Infertilidade , Sêmen , Animais , Bovinos , Feminino , Masculino , Camundongos , Gravidez , Adenilato Quinase/genética , Adenilato Quinase/metabolismo , Fertilidade , Mamíferos , Sêmen/metabolismo , Análise do Sêmen , Motilidade dos Espermatozoides , Espermatozoides/metabolismoRESUMO
Late in their maturation, nascent small (40S) ribosomal subunits bind 60S subunits to produce 80S-like ribosomes. Because of the analogy of this translation-like cycle to actual translation, and because 80S-like ribosomes do not produce any protein, it has been suggested that this represents a quality control mechanism for subunit functionality. Here we use genetic and biochemical experiments to show that the essential ATPase Fap7 promotes formation of the rotated state, a key intermediate in translocation, thereby releasing the essential assembly factor Dim1 from pre-40S subunits. Bypassing this quality control step produces defects in reading frame maintenance. These results show how progress in the maturation cascade is linked to a test for a key functionality of 40S ribosomes: their ability to translocate the mRNAâ tRNA pair. Furthermore, our data demonstrate for the first time that the translation-like cycle is a quality control mechanism that ensures the fidelity of the cellular ribosome pool.
Assuntos
Adenilato Quinase/metabolismo , Mudança da Fase de Leitura do Gene Ribossômico , Metiltransferases/metabolismo , Proteínas Nucleares/metabolismo , Nucleosídeo-Trifosfatase/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Adenilato Quinase/química , Adenilato Quinase/genética , Genótipo , Metiltransferases/química , Metiltransferases/genética , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/genética , Nucleosídeo-Trifosfatase/química , Nucleosídeo-Trifosfatase/genética , Fenótipo , Ligação Proteica , Conformação Proteica , Proteólise , Subunidades Ribossômicas Menores de Eucariotos/química , Subunidades Ribossômicas Menores de Eucariotos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Relação Estrutura-Atividade , Fatores de TempoRESUMO
Plasticity of the proteome is critical to adapt to varying conditions. Control of mitochondrial protein import contributes to this plasticity. Here, we identified a pathway that regulates mitochondrial protein import by regulated N-terminal processing. We demonstrate that dipeptidyl peptidases 8/9 (DPP8/9) mediate the N-terminal processing of adenylate kinase 2 (AK2) en route to mitochondria. We show that AK2 is a substrate of the mitochondrial disulfide relay, thus lacking an N-terminal mitochondrial targeting sequence and undergoing comparatively slow import. DPP9-mediated processing of AK2 induces its rapid proteasomal degradation and prevents cytosolic accumulation of enzymatically active AK2. Besides AK2, we identify more than 100 mitochondrial proteins with putative DPP8/9 recognition sites and demonstrate that DPP8/9 influence the cellular levels of a number of these proteins. Collectively, we provide in this study a conceptual framework on how regulated cytosolic processing controls levels of mitochondrial proteins as well as their dual localization to mitochondria and other compartments.
Assuntos
Adenilato Quinase/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Proteínas Mitocondriais/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Células HEK293 , Células HeLa , Humanos , Transporte ProteicoRESUMO
N6 -methyladenosine (m6 A) modification has been identified as one of the most important epigenetic regulation mechanisms in the development of human cancers. However, the association between m6 A-associated single-nucleotide polymorphisms (m6 A-SNPs) and lung cancer risk remains largely unknown. Here, we identified m6 A-SNPs and examined the association of these m6 A-SNPs with lung cancer risk in 13,793 lung cancer cases and 14,027 controls. In silico functional annotation was used to identify causal m6 A-SNPs and target genes. Furthermore, methylated RNA immunoprecipitation and quantitative real-time polymerase chain reaction (MeRIP-qPCR) assay was performed to assess the m6 A modification level of different genotypes of the causal SNP. In vitro assays were performed to validate the potential role of the target gene in lung cancer. A total of 8794 m6 A-SNPs were detected, among which 397 SNPs in nine susceptibility loci were associated with lung cancer risk, including six novel loci. Bioinformatics analyses indicated that rs1321328 in 6q21 was located around the m6 A modification site of AK9 and significantly reduced AK9 expression (ß = -0.15, p = 2.78 × 10-8 ). Moreover, AK9 was significantly downregulated in lung cancer tissues than that in adjacent normal tissues of samples from the Cancer Genome Atlas and Nanjing Lung Cancer Cohort. MeRIP-qPCR assay suggested that C allele of rs1321328 could significantly decrease the m6 A modification level of AK9 compared with G allele. In vitro assays verified the tumor-suppressing role of AK9 in lung cancer. These findings shed light on the pathogenic mechanism of lung cancer susceptibility loci linked with m6 A modification.
Assuntos
Adenina , Neoplasias Pulmonares , Polimorfismo de Nucleotídeo Único , Humanos , Adenina/análogos & derivados , Epigênese Genética , Genes Supressores de Tumor , Neoplasias Pulmonares/genética , Adenilato Quinase/metabolismoRESUMO
Drying protein-based drugs, usually via lyophilization, can facilitate storage at ambient temperature and improve accessibility but many proteins cannot withstand drying and must be formulated with protective additives called excipients. However, mechanisms of protection are poorly understood, precluding rational formulation design. To better understand dry proteins and their protection, we examine Escherichia coli adenylate kinase (AdK) lyophilized alone and with the additives trehalose, maltose, bovine serum albumin, cytosolic abundant heat soluble protein D, histidine, and arginine. We apply liquid-observed vapor exchange NMR to interrogate the residue-level structure in the presence and absence of additives. We pair these observations with differential scanning calorimetry data of lyophilized samples and AdK activity assays with and without heating. We show that the amino acids do not preserve the native structure as well as sugars or proteins and that after heating the most stable additives protect activity best.
Assuntos
Adenilato Quinase , Escherichia coli , Liofilização , Trealose , Liofilização/métodos , Adenilato Quinase/metabolismo , Trealose/química , Soroalbumina Bovina/química , Excipientes/química , Varredura Diferencial de Calorimetria , Maltose/química , Histidina/química , Arginina/química , Espectroscopia de Ressonância MagnéticaRESUMO
Adenylate kinase (AK) plays a crucial role in the metabolic monitoring of cellular adenine nucleotide homeostasis by catalyzing the reversible transfer of a phosphate group between ATP and AMP, yielding two ADP molecules. By regulating the nucleotide levels and energy metabolism, the enzyme is considered a disease modifier and potential therapeutic target for various human diseases, including malignancies and inflammatory and neurodegenerative disorders. However, lacking approved drugs targeting AK hinders broad studies on this enzyme's pathological importance and therapeutic potential. In this work, we determined the effect of a series of dinucleoside polyphosphate derivatives, commercially available (11 compounds) and newly synthesized (8 compounds), on the catalytic activity of human adenylate kinase isoenzyme 1 (hAK1). The tested compounds belonged to the following groups: (1) diadenosine polyphosphates with different phosphate chain lengths, (2) base-modified derivatives, and (3) phosphate-modified derivatives. We found that all the investigated compounds inhibited the catalytic activity of hAK1, yet with different efficiencies. Three dinucleoside polyphosphates showed IC50 values below 1 µM, and the most significant inhibitory effect was observed for P1-(5'-adenosyl) P5-(5'-adenosyl) pentaphosphate (Ap5A). To understand the observed differences in the inhibition efficiency of the tested dinucleoside polyphosphates, the molecular docking of these compounds to hAK1 was performed. Finally, we conducted a quantitative structure-activity relationship (QSAR) analysis to establish a computational prediction model for hAK1 modulators. Two PLS-regression-based models were built using kinetic data obtained from the AK1 activity analysis performed in both directions of the enzymatic reaction. Model 1 (AMP and ATP synthesis) had a good prediction power (R2 = 0.931, Q2 = 0.854, and MAE = 0.286), while Model 2 (ADP synthesis) exhibited a moderate quality (R2 = 0.913, Q2 = 0.848, and MAE = 0.370). These studies can help better understand the interactions between dinucleoside polyphosphates and adenylate kinase to attain more effective and selective inhibitors in the future.
Assuntos
Adenilato Quinase , Fosfatos de Dinucleosídeos , Relação Quantitativa Estrutura-Atividade , Humanos , Fosfatos de Dinucleosídeos/química , Fosfatos de Dinucleosídeos/síntese química , Fosfatos de Dinucleosídeos/farmacologia , Fosfatos de Dinucleosídeos/metabolismo , Cinética , Estrutura Molecular , Adenilato Quinase/metabolismo , Adenilato Quinase/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/químicaRESUMO
Starvation poses a fundamental challenge to cell survival. Whereas the role of autophagy in promoting energy homeostasis in this setting has been extensively characterized1, other mechanisms are less well understood. Here we reveal that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) inhibits coat protein I (COPI) transport by targeting a GTPase-activating protein (GAP) towards ADP-ribosylation factor 1 (ARF1) to suppress COPI vesicle fission. GAPDH inhibits multiple other transport pathways, also by targeting ARF GAPs. Further characterization suggests that this broad inhibition is activated by the cell during starvation to reduce energy consumption. These findings reveal a remarkable level of coordination among the intracellular transport pathways that underlies a critical mechanism of cellular energy homeostasis.
Assuntos
Metabolismo Energético , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Homeostase , Adenilato Quinase/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/metabolismo , Animais , Autofagia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Linhagem Celular , Chlorocebus aethiops , Cricetulus , Fibroblastos , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Proteínas Ativadoras de GTPase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/química , Humanos , Camundongos , Fosforilação , Ribonucleotídeos/metabolismo , InaniçãoRESUMO
Adaptation of organisms to environmental niches is a hallmark of evolution. One prevalent example is that of thermal adaptation, in which two descendants evolve at different temperature extremes1,2. Underlying the physiological differences between such organisms are changes in enzymes that catalyse essential reactions 3 , with orthologues from each organism undergoing adaptive mutations that preserve similar catalytic rates at their respective physiological temperatures4,5. The sequence changes responsible for these adaptive differences, however, are often at surface-exposed sites distant from the substrate-binding site, leaving the active site of the enzyme structurally unperturbed6,7. How such changes are allosterically propagated to the active site, to modulate activity, is not known. Here we show that entropy-tuning changes can be engineered into distal sites of Escherichia coli adenylate kinase, allowing us to quantitatively assess the role of dynamics in determining affinity, turnover and the role in driving adaptation. The results not only reveal a dynamics-based allosteric tuning mechanism, but also uncover a spatial separation of the control of key enzymatic parameters. Fluctuations in one mobile domain (the LID) control substrate affinity, whereas dynamic attenuation in the other domain (the AMP-binding domain) affects rate-limiting conformational changes that govern enzyme turnover. Dynamics-based regulation may thus represent an elegant, widespread and previously unrealized evolutionary adaptation mechanism that fine-tunes biological function without altering the ground state structure. Furthermore, because rigid-body conformational changes in both domains were thought to be rate limiting for turnover8,9, these adaptation studies reveal a new model for understanding the relationship between dynamics and turnover in adenylate kinase.
Assuntos
Adaptação Biológica , Adenilato Quinase/química , Adenilato Quinase/metabolismo , Regulação Alostérica , Temperatura Baixa , Escherichia coli/enzimologia , Adaptação Biológica/genética , Adenilato Quinase/genética , Regulação Alostérica/genética , Sítios de Ligação/genética , Domínio Catalítico/genética , Entropia , Escherichia coli/genética , Modelos Moleculares , Mutação , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Especificidade por SubstratoRESUMO
Diabetes is a complex metabolic syndrome that is characterized by prolonged high blood glucose levels and frequently associated with life-threatening complications1,2. Epidemiological studies have suggested that diabetes is also linked to an increased risk of cancer3-5. High glucose levels may be a prevailing factor that contributes to the link between diabetes and cancer, but little is known about the molecular basis of this link and how the high glucose state may drive genetic and/or epigenetic alterations that result in a cancer phenotype. Here we show that hyperglycaemic conditions have an adverse effect on the DNA 5-hydroxymethylome. We identify the tumour suppressor TET2 as a substrate of the AMP-activated kinase (AMPK), which phosphorylates TET2 at serine 99, thereby stabilizing the tumour suppressor. Increased glucose levels impede AMPK-mediated phosphorylation at serine 99, which results in the destabilization of TET2 followed by dysregulation of both 5-hydroxymethylcytosine (5hmC) and the tumour suppressive function of TET2 in vitro and in vivo. Treatment with the anti-diabetic drug metformin protects AMPK-mediated phosphorylation of serine 99, thereby increasing TET2 stability and 5hmC levels. These findings define a novel 'phospho-switch' that regulates TET2 stability and a regulatory pathway that links glucose and AMPK to TET2 and 5hmC, which connects diabetes to cancer. Our data also unravel an epigenetic pathway by which metformin mediates tumour suppression. Thus, this study presents a new model for how a pernicious environment can directly reprogram the epigenome towards an oncogenic state, offering a potential strategy for cancer prevention and treatment.
Assuntos
Adenilato Quinase/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus/metabolismo , Glucose/metabolismo , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , DNA/química , DNA/metabolismo , Metilação de DNA , Diabetes Mellitus/genética , Dioxigenases , Estabilidade Enzimática , Epigênese Genética , Hemoglobinas Glicadas/análise , Humanos , Hiperglicemia/metabolismo , Metformina/farmacologia , Metformina/uso terapêutico , Camundongos , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/genética , Fosforilação , Fosfosserina/metabolismo , Especificidade por Substrato , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cancer cells acquire metabolic reprogramming to satisfy their high biogenetic demands, but little is known about how metabolic remodeling enables cancer cells to survive stress associated with genomic instability. Here, we show that the mitochondrial methylenetetrahydrofolate dehydrogenase (MTHFD2) is transcriptionally suppressed by p53, and its up-regulation by p53 inactivation leads to increased folate metabolism, de novo purine synthesis, and tumor growth in vivo and in vitro. Moreover, MTHFD2 unexpectedly promotes nonhomologous end joining in response to DNA damage by forming a complex with PARP3 to enhance its ribosylation, and the introduction of a PARP3-binding but enzymatically inactive MTHFD2 mutant (e.g., D155A) sufficiently prevents DNA damage. Notably, MTHFD2 depletion strongly restrains p53-deficient cell proliferation and sensitizes cells to chemotherapeutic agents, indicating a potential role for MTHFD2 depletion in the treatment of p53-deficient tumors.
Assuntos
Aminoidrolases/genética , Dano ao DNA , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Enzimas Multifuncionais/genética , Transcrição Gênica , Proteína Supressora de Tumor p53/deficiência , Adenilato Quinase/metabolismo , Aminoidrolases/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Carbono/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Respiração Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Humanos , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Enzimas Multifuncionais/metabolismo , Mutação/genética , Neoplasias/genética , Neoplasias/patologia , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica/efeitos dos fármacos , Ribonucleotídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Proteína Supressora de Tumor p53/genéticaRESUMO
Differentiation-inducing factor 1 (DIF-1), found in Dictyostelium discoideum, has antiproliferative and glucose-uptake-promoting activities in mammalian cells. DIF-1 is a potential lead for the development of antitumor and/or antiobesity/antidiabetes drugs, but the mechanisms underlying its actions have not been fully elucidated. In this study, we searched for target molecules of DIF-1 that mediate the actions of DIF-1 in mammalian cells by identifying DIF-1-binding proteins in human cervical cancer HeLa cells and mouse 3T3-L1 fibroblast cells using affinity chromatography and liquid chromatography-tandem mass spectrometry and found mitochondrial malate dehydrogenase (MDH2) to be a DIF-1-binding protein in both cell lines. Since DIF-1 has been shown to directly inhibit MDH2 activity, we compared the effects of DIF-1 and the MDH2 inhibitor LW6 on the growth of HeLa and 3T3-L1 cells and on glucose uptake in confluent 3T3-L1 cells in vitro. In both HeLa and 3T3-L1 cells, DIF-1 at 10-40 µM dose-dependently suppressed growth, whereas LW6 at 20 µM, but not at 2-10 µM, significantly suppressed growth in these cells. In confluent 3T3-L1 cells, DIF-1 at 10-40 µM significantly promoted glucose uptake, with the strongest effect at 20 µM DIF-1, whereas LW6 at 2-20 µM significantly promoted glucose uptake, with the strongest effect at 10 µM LW6. Western blot analyses showed that LW6 (10 µM) and DIF-1 (20 µM) phosphorylated and, thus, activated AMP kinase in 3T3-L1 cells. Our results suggest that MDH2 inhibition can suppress cell growth and promote glucose uptake in the cells, but appears to promote glucose uptake more strongly than it suppresses cell growth. Thus, DIF-1 may promote glucose uptake, at least in part, via direct inhibition of MDH2 and a subsequent activation of AMP kinase in 3T3-L1 cells.
Assuntos
Glucose , Malato Desidrogenase , Animais , Humanos , Camundongos , Células 3T3-L1/efeitos dos fármacos , Células 3T3-L1/metabolismo , Adenilato Quinase/metabolismo , Dictyostelium/metabolismo , Glucose/metabolismo , Células HeLa/efeitos dos fármacos , Células HeLa/metabolismo , Malato Desidrogenase/antagonistas & inibidores , Malato Desidrogenase/metabolismo , Mamíferos/metabolismoRESUMO
GP-2250, a novel anticancer agent, severely limits the energy metabolism, as demonstrated by the inhibition of hexokinase 2 and glyceraldehyde-3-phosphate dehydrogenase and a decrease of ATP. Rescue experiments with supplementary pyruvate or oxaloacetate demonstrated that a TCA cycle deficit largely contributed to cytotoxicity. Activation of the energy-deficit sensor, AMP-dependent protein kinase, was associated with increased phosphorylation of acetyl-CoA carboxylase and Raptor, pointing to a possible deficit in the synthesis of fatty acids and proteins as essential cell components. Binding of p65 to DNA was dose-dependently reduced in nuclear lysates. A transcriptional deficit of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) was substantiated by the downregulation of cyclin D1 and of the anti-apoptotic Bcl2, in line with reduction in tumour cell proliferation and induction of apoptosis, respectively. The upregulation of p53 concomitant with an excess of ROS supported apoptosis. Thus, the anticancer activity of GP-2250 is a result of disruption of energy metabolism and inhibition of tumour promotion by NF-κB.
Assuntos
Antineoplásicos , Neoplasias Pancreáticas , Humanos , NF-kappa B/metabolismo , Adenilato Quinase/metabolismo , Quinase I-kappa B/metabolismo , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Apoptose , Fosforilação , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Metabolismo Energético , Neoplasias PancreáticasRESUMO
Reticular dysgenesis (RD) is a rare genetic disease caused by gene mutations in the ATP:AMP phosphotransferase, adenylate kinase 2 (AK2). Patients with RD suffer from severe combined immunodeficiency with neutrophil maturation arrest. Although hematopoietic stem cell transplantation can be a curative option, it is invasive, and complications of agranulocytosis-induced infection worsen the prognosis. Here, we report that the use of UK-5099, an inhibitor of the mitochondrial pyruvate carrier (MPC), on hemo-angiogenic progenitor cells (HAPCs) derived from AK2-deficient induced pluripotent stem cells improved neutrophil maturation. Reactive oxygen species (ROS) levels in AK2-deficient HAPCs remained unchanged throughout all experiments, implying that UK-5099 improved the phenotype without affecting ROS levels. Overall, our results suggest that the MPC is a potential therapeutic target for the treatment of neutrophil maturation defects in RD.