RESUMO
An enzyme catalyzed strategy for the synthesis of a chiral hydrazine from 3-cyclopentyl-3-oxopropanenitrile 5 and hydrazine hydrate 2 is presented. An imine reductase (IRED) from Streptosporangium roseum was identified to catalyze the reaction between 3-cyclopentyl-3-oxopropanenitrile 5 and hydrazine hydrate 2 to produce trace amounts of (R)-3-cyclopentyl-3-hydrazineylpropanenitrile 4. We employed a 2-fold approach to optimize the catalytic performance of this enzyme. First, a transition state analogue (TSA) model was constructed to illuminate the enzyme-substrate interactions. Subsequently, the Enzyme_design and Funclib methods were utilized to predict mutants for experimental evaluation. Through three rounds of site-directed mutagenesis, site saturation mutagenesis, and combinatorial mutagenesis, we obtained mutant M6 with a yield of 98% and an enantiomeric excess (ee) of 99%. This study presents an effective method for constructing a hydrazine derivative via IRED-catalyzed reductive amination of ketone and hydrazine. Furthermore, it provides a general approach for constructing suitable enzymes, starting from nonreactive enzymes and gradually enhancing their catalytic activity through active site modifications.
Assuntos
Biocatálise , Nitrilas , Oxirredutases , Pirazóis , Pirimidinas , Nitrilas/química , Nitrilas/metabolismo , Pirimidinas/química , Pirimidinas/biossíntese , Pirimidinas/metabolismo , Oxirredutases/metabolismo , Oxirredutases/genética , Pirazóis/química , Pirazóis/metabolismo , Iminas/química , Iminas/metabolismo , Estrutura Molecular , Hidrazinas/química , Engenharia de ProteínasRESUMO
Inhibition of sodium-glucose cotransporter 2 (SGLT2) by empagliflozin (EMPA) and other "flozins" can improve glycemic control under conditions of diabetes and kidney disease. Though they act on the kidney, they also offer cardiovascular and liver protection. Previously, we found that EMPA decreased circulating triglycerides and hepatic lipid and cholesterol esters in male TallyHo mice fed a high-milk-fat diet (HMFD). The goal of this study was to determine whether the liver protection is associated with a change in metabolic function by characterizing the hepatic and circulating metabolic and lipidomic profiles using targeted LC-MS. In both male and female mice, HMFD feeding significantly altered the circulating and hepatic metabolome compared with low-fat diet (LFD). Addition of EMPA resulted in the restoration of circulating orotate (intermediate in pyrimidine biosynthesis) and hepatic dihydrofolate (intermediate in the folate and methionine cycles) levels in males and acylcarnitines in females. These changes were partially explained by altered expression of rate-limiting enzymes in these pathways. This metabolic signature was not detected when EMPA was incorporated into an LFD, suggesting that the restoration requires the metabolic shift that accompanies the HMFD. Notably, the HMFD increased expression of 18 of 20 circulating amino acids in males and 11 of 20 in females, and this pattern was reversed by EMPA. Finally, we confirmed that SGLT2 inhibition upregulates ketone bodies including ß-hydroxybutyrate. Collectively, this study highlights the metabolic changes that occur with EMPA treatment, and sheds light on the possible mechanisms by which this drug offers liver and systemic protection.NEW & NOTEWORTHY Sodium-glucose cotransporter 2 (SGLT2) inhibitors, including empagliflozin, have emerged as a new treatment option for individuals with type 2 diabetes that have positive impacts on kidney and cardiovascular disease. However, less is known about their impact on other tissues, including the liver. Here, we report that empagliflozin reduces hepatic steatosis that is associated with restoring metabolic intermediates in the folate and pyrimidine biosynthesis pathways. These changes may lead to new approaches to treat nonalcoholic fatty liver disease.
Assuntos
Compostos Benzidrílicos , Ácido Fólico , Glucosídeos , Fígado , Inibidores do Transportador 2 de Sódio-Glicose , Animais , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Fígado/metabolismo , Fígado/efeitos dos fármacos , Masculino , Feminino , Camundongos , Compostos Benzidrílicos/farmacologia , Glucosídeos/farmacologia , Ácido Fólico/sangue , Ácido Fólico/metabolismo , Pirimidinas/farmacologia , Pirimidinas/biossíntese , Transportador 2 de Glucose-Sódio/metabolismo , Dieta HiperlipídicaRESUMO
Cell proliferation requires metabolic reprogramming to accommodate biosynthesis of new cell components, and similar alterations occur in cancer cells. However, the mechanisms linking the cell cycle machinery to metabolism are not well defined. Cyclin D1, along with its main partner cyclin-dependent kinase 4 (Cdk4), is a pivotal cell cycle regulator and driver oncogene that is overexpressed in many cancers. Here, we examine hepatocyte proliferation to define novel effects of cyclin D1 on biosynthetic metabolism. Metabolomic studies reveal that cyclin D1 broadly promotes biosynthetic pathways including glycolysis, the pentose phosphate pathway, and the purine and pyrimidine nucleotide synthesis in hepatocytes. Proteomic analyses demonstrate that overexpressed cyclin D1 binds to numerous metabolic enzymes including those involved in glycolysis and pyrimidine synthesis. In the glycolysis pathway, cyclin D1 activates aldolase and GAPDH, and these proteins are phosphorylated by cyclin D1/Cdk4 in vitro. De novo pyrimidine synthesis is particularly dependent on cyclin D1. Cyclin D1/Cdk4 phosphorylates the initial enzyme of this pathway, carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and metabolomic analysis indicates that cyclin D1 depletion markedly reduces the activity of this enzyme. Pharmacologic inhibition of Cdk4 along with the downstream pyrimidine synthesis enzyme dihydroorotate dehydrogenase synergistically inhibits proliferation and survival of hepatocellular carcinoma cells. These studies demonstrate that cyclin D1 promotes a broad network of biosynthetic pathways in hepatocytes, and this model may provide insights into potential metabolic vulnerabilities in cancer cells.
Assuntos
Vias Biossintéticas , Ciclina D1 , Hepatócitos , Ciclina D1/genética , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Hepatócitos/metabolismo , Proteômica , Pirimidinas/biossíntese , Humanos , Animais , Camundongos , Linhagem CelularRESUMO
The internal tandem duplication of the FMS-like tyrosine kinase 3 (FLT3-ITD) is one of the most frequent genetic alterations in acute myeloid leukemia (AML). Limited and transient clinical benefit of FLT3 kinase inhibitors (FLT3i) emphasizes the need for alternative therapeutic options for this subset of myeloid malignancies. Herein, we showed that FLT3-ITD mutant (FLT3-ITD+) AML cells were susceptible toward inhibitors of DHODH, a rate-limiting enzyme of de novo pyrimidine biosynthesis. Genetic and pharmacological blockade of DHODH triggered downregulation of FLT3-ITD protein, subsequently suppressed activation of downstream ERK and STAT5, and promoted cell death of FLT3-ITD+ AML cells. Mechanistically, DHODH blockade triggered autophagy-mediated FLT3-ITD degradation via inactivating mTOR, a potent autophagy repressor. Notably, blockade of DHODH synergized with an FDA-approved FLT3i quizartinib in significantly impairing the growth of FLT3-ITD+ AML cells and improving tumor-bearing mice survival. We further demonstrated that DHODH blockade exhibited profound anti-proliferation effect on quizartinib-resistant cells in vitro and in vivo. In summary, this study demonstrates that the induction of degradation of FLT3-ITD protein by DHODH blockade may offer a promising therapeutic strategy for AML patients harboring FLT3-ITD mutation.
Assuntos
Di-Hidro-Orotato Desidrogenase , Leucemia Mieloide Aguda , Animais , Humanos , Camundongos , Autofagia , Tirosina Quinase 3 Semelhante a fms/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Mutação , Proteínas Oncogênicas/genética , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/biossíntese , Pirimidinas/metabolismoRESUMO
Whether TMPRSS2-ERG fusion and TP53 gene alteration coordinately promote prostate cancer (PCa) remains unclear. Here we demonstrate that TMPRSS2-ERG fusion and TP53 mutation / deletion co-occur in PCa patient specimens and this co-occurrence accelerates prostatic oncogenesis. p53 gain-of-function (GOF) mutants are now shown to bind to a unique DNA sequence in the CTNNB1 gene promoter and transactivate its expression. ERG and ß-Catenin co-occupy sites at pyrimidine synthesis gene (PSG) loci and promote PSG expression, pyrimidine synthesis and PCa growth. ß-Catenin inhibition by small molecule inhibitors or oligonucleotide-based PROTAC suppresses TMPRSS2-ERG- and p53 mutant-positive PCa cell growth in vitro and in mice. Our study identifies a gene transactivation function of GOF mutant p53 and reveals ß-Catenin as a transcriptional target gene of p53 GOF mutants and a driver and therapeutic target of TMPRSS2-ERG- and p53 GOF mutant-positive PCa.
Assuntos
Neoplasias da Próstata , Regulador Transcricional ERG , Proteína Supressora de Tumor p53 , Animais , Humanos , Masculino , Camundongos , beta Catenina/genética , beta Catenina/metabolismo , Mutação com Ganho de Função , Proteínas de Fusão Oncogênica/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Proto-Oncogenes , Pirimidinas/biossíntese , Regulador Transcricional ERG/genética , Regulador Transcricional ERG/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Human uridine 5'-monophosphate synthase (HsUMPS) is a bifunctional enzyme that catalyzes the final two steps in de novo pyrimidine biosynthesis. The individual orotate phosphoribosyl transferase and orotidine monophosphate domains have been well characterized, but little is known about the overall structure of the protein and how the organization of domains impacts function. Using a combination of chromatography, electron microscopy, and complementary biophysical methods, we report herein that HsUMPS can be observed in two structurally distinct states, an enzymatically active dimeric form and a nonactive multimeric form. These two states readily interconvert to reach an equilibrium that is sensitive to perturbations of the active site and the presence of substrate. We determined that the smaller molecular weight form of HsUMPS is an S-shaped dimer that can self-assemble into relatively well-ordered globular condensates. Our analysis suggests that the transition between dimer and multimer is driven primarily by oligomerization of the orotate phosphoribosyl transferase domain. While the cellular distribution of HsUMPS is unaffected, quantification by mass spectrometry revealed that de novo pyrimidine biosynthesis is dysregulated when this protein is unable to assemble into inactive condensates. Taken together, our data suggest that HsUMPS self-assembles into biomolecular condensates as a means to store metabolic potential for the regulation of metabolic rates.
Assuntos
Condensados Biomoleculares , Orotato Fosforribosiltransferase , Orotidina-5'-Fosfato Descarboxilase , Uridina Monofosfato , Humanos , Orotato Fosforribosiltransferase/metabolismo , Orotidina-5'-Fosfato Descarboxilase/metabolismo , Pirimidinas/biossíntese , Uridina , Uridina Monofosfato/metabolismoRESUMO
CTNNB1, encoding ß-catenin protein, is the most frequently altered proto-oncogene in hepatic neoplasms. In this study, we studied the significance and pathological mechanism of CTNNB1 gain-of-function mutations in hepatocarcinogenesis. Activated ß-catenin not only triggered hepatic tumorigenesis but also exacerbated Tp53 deletion or hepatitis B virus infection-mediated liver cancer development in mouse models. Using untargeted metabolomic profiling, we identified boosted de novo pyrimidine synthesis as the major metabolic aberration in ß-catenin mutant cell lines and livers. Oncogenic ß-catenin transcriptionally stimulated AKT2, which then phosphorylated the rate-limiting de novo pyrimidine synthesis enzyme CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, dihydroorotase) on S1406 and S1859 to potentiate nucleotide synthesis. Moreover, inhibition of ß-catenin/AKT2-stimulated pyrimidine synthesis axis preferentially repressed ß-catenin mutant cell proliferation and tumor formation. Therefore, ß-catenin active mutations are oncogenic in various preclinical liver cancer models. Stimulation of ß-catenin/AKT2/CAD signaling cascade on pyrimidine synthesis is an essential and druggable vulnerability for ß-catenin mutant liver cancer.
Assuntos
Neoplasias Hepáticas , Pirimidinas , beta Catenina , Animais , Ácido Aspártico , Carcinogênese , Di-Hidro-Orotase/genética , Di-Hidro-Orotase/metabolismo , Sistemas de Liberação de Medicamentos , Ligases , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/fisiopatologia , Camundongos , Nucleotídeos , Fosfatos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/biossíntese , beta Catenina/metabolismoRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolves rapidly under the pressure of host immunity, as evidenced by waves of emerging variants despite effective vaccinations, highlighting the need for complementing antivirals. We report that targeting a pyrimidine synthesis enzyme restores inflammatory response and depletes the nucleotide pool to impede SARS-CoV-2 infection. SARS-CoV-2 deploys Nsp9 to activate carbamoyl-phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase (CAD) that catalyzes the rate-limiting steps of the de novo pyrimidine synthesis. Activated CAD not only fuels de novo nucleotide synthesis but also deamidates RelA. While RelA deamidation shuts down NF-κB activation and subsequent inflammatory response, it up-regulates key glycolytic enzymes to promote aerobic glycolysis that provides metabolites for de novo nucleotide synthesis. A newly synthesized small-molecule inhibitor of CAD restores antiviral inflammatory response and depletes the pyrimidine pool, thus effectively impeding SARS-CoV-2 replication. Targeting an essential cellular metabolic enzyme thus offers an antiviral strategy that would be more refractory to SARS-CoV-2 genetic changes.
Assuntos
Antivirais , Aspartato Carbamoiltransferase , Tratamento Farmacológico da COVID-19 , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante) , Di-Hidro-Orotase , Inibidores Enzimáticos , Pirimidinas , SARS-CoV-2 , Replicação Viral , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Aspartato Carbamoiltransferase/antagonistas & inibidores , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/antagonistas & inibidores , Di-Hidro-Orotase/antagonistas & inibidores , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Inflamação/tratamento farmacológico , Camundongos , Pirimidinas/antagonistas & inibidores , Pirimidinas/biossíntese , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Fator de Transcrição RelA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Shigella flexneri is the main causative agent of the communicable diarrheal disease, shigellosis. It is estimated that about 80-165 million cases and > 1 million deaths occur every year due to this disease. S. flexneri causes dysentery mostly in young children, elderly and immunocompromised patients, all over the globe. Recently, due to the emergence of S. flexneri antibiotic resistance strains, it is a dire need to predict novel therapeutic drug targets in the bacterium and screen natural products against it, which could eliminate the curse of antibiotic resistance. Therefore, in current study, available antibiotic-resistant genomes (n = 179) of S. flexneri were downloaded from PATRIC database and a pan-genome and resistome analysis was conducted. Around 5059 genes made up the accessory, 2469 genes made up the core, and 1558 genes made up the unique genome fraction, with 44, 34, and 13 antibiotic-resistant genes in each fraction, respectively. Core genome fraction (27% of the pan-genome), which was common to all strains, was used for subtractive genomics and resulted in 384 non-homologous, and 85 druggable targets. Dihydroorotase was chosen for further analysis and docked with natural product libraries (Ayurvedic and Streptomycin compounds), while the control was orotic acid or vitamin B13 (which is a natural binder of this protein). Dynamics simulation of 50 ns was carried out to validate findings for top-scored inhibitors. The current study proposed dihydroorotase as a significant drug target in S. flexneri and 4-tritriacontanone & patupilone compounds as potent drugs against shigellosis. Further experiments are required to ascertain validity of our findings.
Assuntos
Antibacterianos/farmacologia , Produtos Biológicos/antagonistas & inibidores , Descoberta de Drogas/métodos , Pirimidinas/farmacologia , Shigella flexneri/enzimologia , Simulação por Computador , Farmacorresistência Bacteriana , Pirimidinas/biossíntese , Shigella flexneri/efeitos dos fármacosRESUMO
Various populations of cells are recruited to the heart after cardiac injury, but little is known about whether cardiomyocytes directly regulate heart repair. Using a murine model of ischemic cardiac injury, we demonstrate that cardiomyocytes play a pivotal role in heart repair by regulating nucleotide metabolism and fates of nonmyocytes. Cardiac injury induced the expression of the ectonucleotidase ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which hydrolyzes extracellular ATP to form AMP. In response to AMP, cardiomyocytes released adenine and specific ribonucleosides that disrupted pyrimidine biosynthesis at the orotidine monophosphate (OMP) synthesis step and induced genotoxic stress and p53-mediated cell death of cycling nonmyocytes. As nonmyocytes are critical for heart repair, we showed that rescue of pyrimidine biosynthesis by administration of uridine or by genetic targeting of the ENPP1/AMP pathway enhanced repair after cardiac injury. We identified ENPP1 inhibitors using small molecule screening and showed that systemic administration of an ENPP1 inhibitor after heart injury rescued pyrimidine biosynthesis in nonmyocyte cells and augmented cardiac repair and postinfarct heart function. These observations demonstrate that the cardiac muscle cell regulates pyrimidine metabolism in nonmuscle cells by releasing adenine and specific nucleosides after heart injury and provide insight into how intercellular regulation of pyrimidine biosynthesis can be targeted and monitored for augmenting tissue repair.
Assuntos
Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Pirimidinas/biossíntese , Pirofosfatases/metabolismo , Regeneração , Transdução de Sinais , Monofosfato de Adenosina/genética , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , Traumatismos Cardíacos/genética , Traumatismos Cardíacos/metabolismo , Camundongos , Diester Fosfórico Hidrolases/genética , Pirofosfatases/genéticaRESUMO
DksA is a conserved RNA polymerase-binding protein known to play a key role in the stringent response of proteobacteria species, including many gastrointestinal pathogens. Here, we used RNA-sequencing of Escherichia coli, Salmonella bongori and Salmonella enterica serovar Typhimurium, together with phenotypic comparison to study changes in the DksA regulon, during Salmonella evolution. Comparative RNA-sequencing showed that under non-starved conditions, DksA controls the expression of 25%, 15%, and 20% of the E. coli, S. bongori, and S. enterica genes, respectively, indicating that DksA is a pleiotropic regulator, expanding its role beyond the canonical stringent response. We demonstrate that DksA is required for the growth of these three enteric bacteria species in minimal medium and controls the expression of the TCA cycle, glycolysis, pyrimidine biosynthesis, and quorum sensing. Interestingly, at multiple steps during Salmonella evolution, the type I fimbriae and various virulence genes encoded within SPIs 1, 2, 4, 5, and 11 have been transcriptionally integrated under the ancestral DksA regulon. Consequently, we show that DksA is necessary for host cells invasion by S. Typhimurium and S. bongori and for intracellular survival of S. Typhimurium in bone marrow-derived macrophages (BMDM). Moreover, we demonstrate regulatory inversion of the conserved motility-chemotaxis regulon by DksA, which acts as a negative regulator in E. coli, but activates this pathway in S. bongori and S. enterica. Overall, this study demonstrates the regulatory assimilation of multiple horizontally acquired virulence genes under the DksA regulon and provides new insights into the evolution of virulence genes regulation in Salmonella spp.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Evolução Molecular , Salmonella typhimurium/genética , Salmonella/genética , Salmonella/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Ciclo do Ácido Cítrico , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/classificação , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Transferência Genética Horizontal , Glicólise , Humanos , Pirimidinas/biossíntese , Regulon , Salmonella/citologia , Salmonella/patogenicidade , Infecções por Salmonella/microbiologia , Salmonella typhimurium/citologia , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , VirulênciaRESUMO
T-cell activation upon antigen stimulation is essential for the continuation of the adaptive immune response. Impairment of mitochondrial oxidative phosphorylation is a well-known disruptor of T-cell activation. Dihydroorotate dehydrogenase (DHODH) is a component of the de novo synthesis of pyrimidines, the activity of which depends on functional oxidative phosphorylation. Under circumstances of an inhibited oxidative phosphorylation, DHODH becomes rate-limiting. Inhibition of DHODH is known to block clonal expansion and expression of effector molecules of activated T cells. However, this effect has been suggested to be caused by downstream impairment of oxidative phosphorylation rather than a lower rate of pyrimidine synthesis. In this study, we successfully inhibit the DHODH of T cells with no residual effect on oxidative phosphorylation and demonstrate a dose-dependent inhibition of proliferation of activated CD3+ T cells. This block is fully rescued when uridine is supplemented. Inhibition of DHODH does not alter expression of effector molecules but results in decreased intracellular levels of deoxypyrimidines without decreasing cell viability. Our results clearly demonstrate the DHODH and mitochondrial linked pyrimidine synthesis as an independent and important cytostatic regulator of activated T cells.
Assuntos
Ativação Linfocitária/imunologia , Mitocôndrias/metabolismo , Pirimidinas/biossíntese , Proliferação de Células/fisiologia , Di-Hidro-Orotato Desidrogenase/antagonistas & inibidores , Humanos , Mitocôndrias/efeitos dos fármacosRESUMO
CAD (Carbamoyl-phosphate synthetase 2, Aspartate transcarbamoylase, and Dihydroorotase) is a multifunctional protein that participates in the initial three speed-limiting steps of pyrimidine nucleotide synthesis. Over the past two decades, extensive investigations have been conducted to unmask CAD as a central player for the synthesis of nucleic acids, active intermediates, and cell membranes. Meanwhile, the important role of CAD in various physiopathological processes has also been emphasized. Deregulation of CAD-related pathways or CAD mutations cause cancer, neurological disorders, and inherited metabolic diseases. Here, we review the structure, function, and regulation of CAD in mammalian physiology as well as human diseases, and provide insights into the potential to target CAD in future clinical applications.
Assuntos
Aspartato Carbamoiltransferase/metabolismo , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Di-Hidro-Orotase/metabolismo , Pirimidinas/biossíntese , Animais , Humanos , Mamíferos/metabolismoRESUMO
Glycine decarboxylase (GLDC) is a key enzyme of glycine cleavage system that converts glycine into one-carbon units. GLDC is commonly up-regulated and plays important roles in many human cancers. Whether and how GLDC is regulated by post-translational modifications is unknown. Here we report that mechanistic target of rapamycin complex 1 (mTORC1) signal inhibits GLDC acetylation at lysine (K) 514 by inducing transcription of the deacetylase sirtuin 3 (SIRT3). Upon inhibition of mTORC1, the acetyltransferase acetyl-CoA acetyltransferase 1 (ACAT1) catalyzes GLDC K514 acetylation. This acetylation of GLDC impairs its enzymatic activity. In addition, this acetylation of GLDC primes for its K33-linked polyubiquitination at K544 by the ubiquitin ligase NF-X1, leading to its degradation by the proteasomal pathway. Finally, we find that GLDC K514 acetylation inhibits glycine catabolism, pyrimidines synthesis and glioma tumorigenesis. Our finding reveals critical roles of post-translational modifications of GLDC in regulation of its enzymatic activity, glycine metabolism and tumorigenesis, and provides potential targets for therapeutics of cancers such as glioma.
Assuntos
Carcinogênese/genética , Glioma/genética , Glicina Desidrogenase (Descarboxilante)/metabolismo , Glicina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Acetil-CoA C-Acetiltransferase/metabolismo , Acetilação , Animais , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , Glioma/patologia , Células HEK293 , Humanos , Masculino , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Pirimidinas/biossíntese , Proteínas Repressoras/metabolismo , Sirtuína 3/genética , Sirtuína 3/metabolismo , Ativação Transcricional , Ubiquitinação/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway for pyrimidine nucleotides, and an attractive target for potential anticancer chemotherapy. By screening plant extracts and performing GC-MS analysis, we identified and characterized that the potent anticancer drug plumbagin (PLU), isolated from the carnivorous plant Nepenthes miranda, was a competitive inhibitor of DHOase. We also solved the complexed crystal structure of yeast DHOase with PLU (PDB entry 7CA1), to determine the binding interactions and investigate the binding modes. Mutational and structural analyses indicated the binding of PLU to DHOase through loop-in mode, and this dynamic loop may serve as a drug target. PLU exhibited cytotoxicity on the survival, migration, and proliferation of 4T1 cells and induced apoptosis. These results provide structural insights that may facilitate the development of new inhibitors targeting DHOase, for further clinical anticancer chemotherapies.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Produtos Biológicos/farmacologia , Vias Biossintéticas/efeitos dos fármacos , Di-Hidro-Orotase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Naftoquinonas/farmacologia , Pirimidinas/biossíntese , Antineoplásicos Fitogênicos/química , Sítios de Ligação , Produtos Biológicos/química , Domínio Catalítico , Di-Hidro-Orotase/química , Di-Hidro-Orotase/genética , Inibidores Enzimáticos/química , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Mutação , Naftoquinonas/química , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Mitochondria are organelles present in most eukaryotic cells, where they play major and multifaceted roles. The classical notion of the main mitochondrial function as the powerhouse of the cell per se has been complemented by recent discoveries pointing to mitochondria as organelles affecting a number of other auxiliary processes. They go beyond the classical energy provision via acting as a relay point of many catabolic and anabolic processes, to signaling pathways critically affecting cell growth by their implication in de novo pyrimidine synthesis. These additional roles further underscore the importance of mitochondrial homeostasis in various tissues, where its deregulation promotes a number of pathologies. While it has long been known that mitochondria can move within a cell to sites where they are needed, recent research has uncovered that mitochondria can also move between cells. While this intriguing field of research is only emerging, it is clear that mobilization of mitochondria requires a complex apparatus that critically involves mitochondrial proteins of the Miro family, whose role goes beyond the mitochondrial transfer, as will be covered in this review.
Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Transporte Biológico Ativo/fisiologia , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Pirimidinas/biossíntese , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Argininosuccinate synthase (ASS1) is a ubiquitous enzyme in mammals that catalyzes the formation of argininosuccinate from citrulline and aspartate. ASS1 genetic deficiency in patients leads to an autosomal recessive urea cycle disorder citrullinemia, while its somatic silence or down-regulation is very common in various human cancers. Here, we show that ASS1 functions as a tumor suppressor in breast cancer, and the pesticide spinosyn A (SPA) and its derivative LM-2I suppress breast tumor cell proliferation and growth by binding to and activating ASS1. The C13-C14 double bond in SPA and LM-2I while the Cys97 (C97) site in ASS1 are critical for the interaction between ASS1 and SPA or LM-2I. SPA and LM-2I treatment results in significant enhancement of ASS1 enzymatic activity in breast cancer cells, particularly in those cancer cells with low ASS1 expression, leading to reduced pyrimidine synthesis and consequently the inhibition of cancer cell proliferation. Thus, our results establish spinosyn A and its derivative LM-2I as potent ASS1 enzymatic activator and tumor inhibitor, which provides a therapeutic avenue for tumors with low ASS1 expression and for those non-tumor diseases caused by down-regulation of ASS1.
Assuntos
Argininossuccinato Sintase/metabolismo , Neoplasias da Mama/tratamento farmacológico , Citrulinemia/tratamento farmacológico , Ativadores de Enzimas/farmacologia , Macrolídeos/farmacologia , Proteínas Supressoras de Tumor/agonistas , Adulto , Idoso , Animais , Argininossuccinato Sintase/genética , Argininossuccinato Sintase/isolamento & purificação , Ácido Aspártico/metabolismo , Mama/patologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citrulina/metabolismo , Citrulinemia/genética , Ativadores de Enzimas/uso terapêutico , Feminino , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Macrolídeos/uso terapêutico , Metabolômica , Camundongos , Pessoa de Meia-Idade , Simulação de Acoplamento Molecular , Mutação , Ligação Proteica , Pirimidinas/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
TARGET OF RAPAMYCIN (TOR) is a conserved eukaryotic Ser/Thr protein kinase that coordinates growth and metabolism with nutrient availability. We conducted a medium-throughput functional genetic screen to discover essential genes that promote TOR activity in plants, and identified a critical regulatory enzyme, cytosolic phosphoribosyl pyrophosphate (PRPP) synthetase (PRS4). PRS4 synthesizes cytosolic PRPP, a key upstream metabolite in nucleotide synthesis and salvage pathways. We found that prs4 knockouts are embryo-lethal in Arabidopsis thaliana, and that silencing PRS4 expression in Nicotiana benthamiana causes pleiotropic developmental phenotypes, including dwarfism, aberrant leaf shape, and delayed flowering. Transcriptomic analysis revealed that ribosome biogenesis is among the most strongly repressed processes in prs4 knockdowns. Building on these results, we discovered that TOR activity is inhibited by chemical or genetic disruption of nucleotide biosynthesis, but that this effect can be reversed by supplying plants with nucleobases. Finally, we show that TOR transcriptionally promotes nucleotide biosynthesis to support the demands of ribosomal RNA synthesis. We propose that TOR coordinates ribosome biogenesis with nucleotide availability in plants to maintain metabolic homeostasis and support growth.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Nucleotídeos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ribossomos/metabolismo , Arabidopsis/embriologia , Arabidopsis/genética , Citosol/metabolismo , Inativação Gênica , Genes de Plantas , Fósforo/metabolismo , Células Vegetais/metabolismo , Desenvolvimento Vegetal , Purinas/biossíntese , Pirimidinas/biossíntese , Nicotiana/metabolismo , Transcriptoma/genéticaRESUMO
Current clinical trials of combined EGFR-tyrosine kinase inhibitors (TKI) and immune checkpoint blockade (ICB) therapies show no additional effect. This raises questions regarding whether EGFR-TKIs attenuate ICB-enhanced CD8+ T lymphocyte function. Here we show that the EGFR-TKI afatinib suppresses CD8+ T lymphocyte proliferation, and we identify CAD, a key enzyme of de novo pyrimidine biosynthesis, to be a novel afatinib target. Afatinib reduced tumor-infiltrating lymphocyte numbers in Lewis lung carcinoma (LLC)-bearing mice. Early afatinib treatment inhibited CD8+ T lymphocyte proliferation in patients with non-small cell lung cancer, but their proliferation unexpectedly rebounded following long-term treatment. This suggests a transient immunomodulatory effect of afatinib on CD8+ T lymphocytes. Sequential treatment of afatinib with anti-PD1 immunotherapy substantially enhanced therapeutic efficacy in MC38 and LLC-bearing mice, while simultaneous combination therapy showed only marginal improvement over each single treatment. These results suggest that afatinib can suppress CD8+ T lymphocyte proliferation by targeting CAD, proposing a timing window for combined therapy that may prevent the dampening of ICB efficacy by EGFR-TKIs. SIGNIFICANCE: This study elucidates a mechanism of afatinib-mediated immunosuppression and provides new insights into treatment timing for combined targeted therapy and immunotherapy. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3270/F1.large.jpg.
Assuntos
Afatinib/farmacologia , Antineoplásicos Imunológicos/farmacologia , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Desoxirribonucleases/antagonistas & inibidores , Agentes de Imunomodulação/farmacologia , Pirimidinas/biossíntese , Animais , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Quimioterapia Combinada , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/antagonistas & inibidoresRESUMO
Emergence of drug resistance and adverse effects often affect the efficacy of nucleoside analogues in the therapy of Herpes simplex type 1 (HSV-1) and type 2 (HSV-2) infections. Host-targeting antivirals could therefore be considered as an alternative or complementary strategy in the management of HSV infections. To contribute to this advancement, here we report on the ability of a new generation inhibitor of a key cellular enzyme of de novo pyrimidine biosynthesis, the dihydroorotate dehydrogenase (DHODH), to inhibit HSV-1 and HSV-2 in vitro replication, with a potency comparable to that of the reference drug acyclovir. Analysis of the HSV replication cycle in MEDS433-treated cells revealed that it prevented the accumulation of viral genomes and reduced late gene expression, thus suggesting an impairment at a stage prior to viral DNA replication consistent with the ability of MEDS433 to inhibit DHODH activity. In fact, the anti-HSV activity of MEDS433 was abrogated by the addition of exogenous uridine or of the product of DHODH, the orotate, thus confirming DHODH as the MEDS433 specific target in HSV-infected cells. A combination of MEDS433 with dipyridamole (DPY), an inhibitor of the pyrimidine salvage pathway, was then observed to be effective in inhibiting HSV replication even in the presence of exogenous uridine, thus mimicking in vivo conditions. Finally, when combined with acyclovir and DPY in checkerboard experiments, MEDS433 exhibited highly synergistic antiviral activity. Taken together, these findings suggest that MEDS433 is a promising candidate as either single agent or in combination regimens with existing direct-acting anti-HSV drugs to develop new strategies for treatment of HSV infections.