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1.
Int J Mol Sci ; 24(2)2023 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-36675157

RESUMO

4-coumarate: CoA ligase (4CL) is not only involved in the biosynthetic processes of flavonoids and lignin in plants but is also closely related to plant tolerance to abiotic stress. UV irradiation can activate the expression of 4CL genes in plants, and the expression of 4CL genes changed significantly in response to different phytohormone treatments. Although the 4CL gene has been cloned in potatoes, there have been fewer related studies of the 4CL gene family on the potato genome-wide scale. In this study, a total of 10 potato 4CL genes were identified in the potato whole genome. Through multiple sequence alignment, phylogenetic analysis as well as gene structure analysis indicated that the potato 4CL gene family could be divided into two subgroups. Combined with promoter cis-acting element analysis, transcriptome data, and RT-qPCR results indicated that potato 4CL gene family was involved in potato response to white light, UV irradiation, ABA treatment, MeJA treatment, and PEG simulated drought stress. Abiotic stresses such as UV, ABA, MeJA, and PEG could promote the up-regulated expression of St4CL6 and St4CL8 but inhibits the expression of St4CL5. The above results will increase our understanding of the evolution and expression regulation of the potato 4CL gene family and provide reference value for further research on the molecular biological mechanism of 4CL participating in response to diverse environmental signals in potatoes.


Assuntos
Solanum tuberosum , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Ligases/metabolismo , Filogenia , Plantas/metabolismo , Regiões Promotoras Genéticas , Regulação da Expressão Gênica de Plantas , Estresse Fisiológico/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
2.
Clin Transl Med ; 13(1): e1007, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36627765

RESUMO

BACKGROUND: SMC5/6 complex plays a vital role in maintaining genome stability, yet the relationship with human diseases has not been described. METHODS: SMC5 variation was identified through whole-exome sequencing (WES) and verified by Sanger sequencing. Immunoprecipitation, cytogenetic analysis, fluorescence activated cell sorting (FACS) and electron microscopy were used to elucidate the cellular consequences of patient's cells. smc5 knockout (KO) zebrafish and Smc5K371del knock-in mouse models were generated by CRISPR-Cas9. RNA-seq, quantitative real-time PCR (qPCR), western blot, microquantitative computed tomography (microCT) and histology were used to explore phenotypic characteristics and potential mechanisms of the animal models. The effects of Smc5 knockdown on mitotic clonal expansion (MCE) during adipogenesis were investigated through Oil Red O staining, proliferation and apoptosis assays in vitro. RESULTS: We identified a homozygous in-frame deletion of Arg372 in SMC5, one of the core subunits of the SMC5/6 complex, from an adult patient with microcephalic primordial dwarfism, chromosomal instability and insulin resistance. SMC5 mutation disrupted its interaction with its interacting protein NSMCE2, leading to defects in DNA repair and chromosomal instability in patient fibroblasts. Smc5 KO zebrafish showed microcephaly, short length and disturbed glucose metabolism. Smc5 depletion triggers a p53-related apoptosis, as concomitant deletion of the p53 rescued growth defects phenotype in zebrafish. An smc5K371del knock-in mouse model exhibited high mortality, severe growth restriction and fat loss. In 3T3-L1 cells, the knockdown of smc5 results in impaired MCE, a crucial step in adipogenesis. This finding implies that defective cell survival and differentiation is an important mechanism linking growth disorders and metabolic homeostasis imbalance.


Assuntos
Nanismo , Resistência à Insulina , Animais , Camundongos , Adulto , Humanos , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Resistência à Insulina/genética , Proteína Supressora de Tumor p53/genética , Nanismo/genética , Fenótipo , Instabilidade Cromossômica , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ligases/genética , Ligases/metabolismo
3.
Nat Chem Biol ; 19(1): 55-63, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36577875

RESUMO

Engineered destruction of target proteins by recruitment to the cell's degradation machinery has emerged as a promising strategy in drug discovery. The majority of molecules that facilitate targeted degradation do so via a select number of ubiquitin ligases, restricting this therapeutic approach to tissue types that express the requisite ligase. Here, we describe a new strategy of targeted protein degradation through direct substrate recruitment to the 26S proteasome. The proteolytic complex is essential and abundantly expressed in all cells; however, proteasomal ligands remain scarce. We identify potent peptidic macrocycles that bind directly to the 26S proteasome subunit PSMD2, with a 2.5-Å-resolution cryo-electron microscopy complex structure revealing a binding site near the 26S pore. Conjugation of this macrocycle to a potent BRD4 ligand enabled generation of chimeric molecules that effectively degrade BRD4 in cells, thus demonstrating that degradation via direct proteasomal recruitment is a viable strategy for targeted protein degradation.


Assuntos
Proteínas Nucleares , Fatores de Transcrição , Proteínas Nucleares/metabolismo , Microscopia Crioeletrônica , Fatores de Transcrição/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ligases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
4.
Biochemistry ; 62(2): 462-475, 2023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36577516

RESUMO

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are natural products with remarkable chemical and functional diversities. These peptides are often synthesized as signals or antibiotics and frequently associated with quorum sensing (QS) systems. With the increasing number of available genomes, many hitherto unseen RiPP biosynthetic pathways have been mined, providing new resources for novel bioactive compounds. Herein, we investigated the underexplored biosynthetic potential of Streptococci, prevalent bacteria in mammal-microbiomes that include pathogenic, mutualistic, and commensal members. Using the transcription factor-centric genome mining strategy, we discovered a new family of lanthipeptide biosynthetic loci under the control of potential QS. By in vitro studies, we investigated the reaction of one of these lanthipeptide synthetases and found that it installs only one lanthionine moiety onto its short precursor peptide by connecting a conserved TxxC region. Bioinformatics and in vitro studies revealed that these lanthipeptide synthetases (class VI) are novel lanthipeptide synthetases with a truncated lyase, a kinase, and a truncated cyclase domain. Our data provide important insights into the processing and evolution of lanthipeptide synthetase to tailor smaller substrates. The data are important for obtaining a mechanistic understanding of the post-translational biosynthesis machinery of the growing variety of lanthipeptides.


Assuntos
Produtos Biológicos , Ligases , Ligases/metabolismo , Produtos Biológicos/metabolismo , Peptídeos/química , Antibacterianos/metabolismo , Ribossomos/metabolismo , Processamento de Proteína Pós-Traducional
5.
Eur J Pharmacol ; 940: 175457, 2023 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-36529278

RESUMO

Accumulating evidence suggests that de novo lipogenesis is a typical characteristic facilitating nonalcoholic fatty liver disease (NAFLD) progression. Gallic acid (GA) is a naturally occurring phenolic acid with metabolic disease-related clinical significance and preclinical benefits. This study aimed to evaluate the anti-steatotic potentials of GA in a fructose-induced NAFLD mouse model featuring a hepatic lipogenic phenotype. The results revealed that GA alleviated hepatic steatosis, oxidative stress, and inflammatory response in fructose-fed mice. Mechanistically, GA treatment restored AMP-activated protein kinase α (AMPKα) phosphorylation, resulting in downregulations of pro-lipogenic factors, including sterol regulatory element binding protein-1 (SREBP-1), fatty acid synthetase (FASN), and acetyl-CoA carboxylase (ACC), in hepatocytes of mice and in vitro. Furthermore, computational docking analysis indicated that GA could directly interact with AMPKα/ß subunits to stabilize its activation. These results suggest that GA ameliorates fructose-induced hepatosteatosis by restraining hepatic lipogenesis via AMPK-dependent suppression of the SREBP-1/ACC/FASN cascade. Altogether, this study demonstrates that GA supplement may be a promising therapeutic strategy in NAFLD, especially in the subset with enhanced hepatic lipogenesis.


Assuntos
Hepatopatia Gordurosa não Alcoólica , Humanos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Lipogênese/genética , Acetil-CoA Carboxilase/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Ácidos Graxos/metabolismo , Ligases/metabolismo , Ácido Gálico/farmacologia , Ácido Gálico/uso terapêutico , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Frutose/efeitos adversos , Fígado/metabolismo , Ácido Graxo Sintase Tipo I/metabolismo
6.
Biol Chem ; 404(1): 29-39, 2023 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-36215729

RESUMO

Hepatocellular carcinoma (HCC) is the most common primary human liver malignancy with high mortality. Liver cancer stem cells (CSCs) have been demonstrated to contribute to the recurrence, metastasis and drug resistance of liver cancer. Human HCC cohort analysis indicated that the epigenetic regulator polycomb chromobox homologue 4 (CBX4) was overexpressed in human HCC. Moreover, we found that CBX4 expression was significantly higher in CD44+ CD133+ Hep3B CSCs. Functionally, we demonstrated that CBX4 regulated cell proliferation, self-renewal, and metastasis ability of Hep3B CSCs. Bioinformatics analysis predicted that CBX4 was a direct target of microRNA-6838-5p (miR-6838-5p), which was further confirmed by luciferase reporter assay. MiR-6838-6p was down-regulated in HCC tumors and overexpression of miR-6838-5p attenuated the malignant traits of human liver CSCs in vitro. In addition, we found that miR-6838-5p/CBX4 axis modulates the biological properties of human liver CSCs via regulating ERK signaling. Overexpression of miR-6838-5p suppressed Hep3B xenograft tumor growth in vivo, while CBX4 overexpression abrogated the suppression effect, restored the angiogenesis, epithelial-to-mesenchymal transition (EMT), and ERK signaling in Hep3B tumor. In summary, our findings suggest that miR-6838-5p/CBX4 axis regulates liver tumor development and metastasis, which could be utilized as potential therapeutic target for HCC treatment.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroRNAs , Humanos , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Células-Tronco Neoplásicas/patologia , Proliferação de Células , Ligases/metabolismo , Proteínas do Grupo Polycomb/metabolismo
7.
Nat Commun ; 13(1): 7833, 2022 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-36539424

RESUMO

During lagging strand synthesis, DNA Ligase 1 (Lig1) cooperates with the sliding clamp PCNA to seal the nicks between Okazaki fragments generated by Pol δ and Flap endonuclease 1 (FEN1). We present several cryo-EM structures combined with functional assays, showing that human Lig1 recruits PCNA to nicked DNA using two PCNA-interacting motifs (PIPs) located at its disordered N-terminus (PIPN-term) and DNA binding domain (PIPDBD). Once Lig1 and PCNA assemble as two-stack rings encircling DNA, PIPN-term is released from PCNA and only PIPDBD is required for ligation to facilitate the substrate handoff from FEN1. Consistently, we observed that PCNA forms a defined complex with FEN1 and nicked DNA, and it recruits Lig1 to an unoccupied monomer creating a toolbelt that drives the transfer of DNA to Lig1. Collectively, our results provide a structural model on how PCNA regulates FEN1 and Lig1 during Okazaki fragments maturation.


Assuntos
DNA Polimerase III , Replicação do DNA , Humanos , Antígeno Nuclear de Célula em Proliferação/metabolismo , DNA Polimerase III/metabolismo , Ligases/metabolismo , DNA/metabolismo , Endonucleases Flap/metabolismo , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo
8.
Int J Mol Sci ; 23(23)2022 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-36499241

RESUMO

IFN-I is the key regulatory component activating and modulating the response of innate and adaptive immune system to bacterial as well as viral pathogens. IFN-I promotes the expression of IFN-induced genes (ISG) and, consequently, the production of chemokines, e.g., CXCL10. Those chemokines control migration and localization of immune cells in tissues, and, thus, are critical to the function of the innate immune system during infection. Consequently, the regulation of IFN-I signaling is essential for the proper induction of an immune response. Our previous study has shown that E3 ubiquitin ligase Pellino3 positively regulates IFNß expression and secretion. Herein, we examined the role of Pellino3 ligase in regulating CXCL10 expression in response to IFNß stimulation. Our experiments were carried out on murine macrophage cell line (BMDM) and human monocytes cell line (THP-1) using IFNß as a IFNAR ligand. We demonstrate that Pellino3 is important for IFNß-induced phosphorylation and nuclear translocation of STAT1/STAT2/IRF9 complex which interacts with CXCL10 promoter and enhances its expression. In this study, we characterize a novel molecular mechanism allowing Pellino3-dependent modulation of the IFNß-induced response in BMDM and THP-1 cell lines.


Assuntos
Quimiocina CXCL10 , Interferon Tipo I , Animais , Humanos , Camundongos , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Interferon Tipo I/metabolismo , Ligases/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Transdução de Sinais , Interferon beta/farmacologia
9.
Int J Mol Sci ; 23(23)2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36499418

RESUMO

Mycothiol (MSH), the major cellular thiol in Mycobacterium tuberculosis (Mtb), plays an essential role in the resistance of Mtb to various antibiotics and oxidative stresses. MshC catalyzes the ATP-dependent ligation of 1-O-(2-amino-2-deoxy-α-d-glucopyranosyl)-d-myo-inositol (GlcN-Ins) with l-cysteine (l-Cys) to form l-Cys-GlcN-Ins, the penultimate step in MSH biosynthesis. The inhibition of MshC is lethal to Mtb. In the present study, five new cysteinyl-sulfonamides were synthesized, and their binding affinity with MshC was evaluated using a thermal shift assay. Two of them bind the target with EC50 values of 219 and 231 µM. Crystal structures of full-length MshC in complex with these two compounds showed that they were bound in the catalytic site of MshC, inducing dramatic conformational changes of the catalytic site compared to the apo form. In particular, the observed closure of the KMSKS loop was not detected in the published cysteinyl-sulfamoyl adenosine-bound structure, the latter likely due to trypsin treatment. Despite the confirmed binding to MshC, the compounds did not suppress Mtb culture growth, which might be explained by the lack of adequate cellular uptake. Taken together, these novel cysteinyl-sulfonamide MshC inhibitors and newly reported full-length apo and ligand-bound MshC structures provide a promising starting point for the further development of novel anti-tubercular drugs targeting MshC.


Assuntos
Ligases , Mycobacterium tuberculosis , Proteínas de Bactérias/metabolismo , Cisteína/metabolismo , Glicopeptídeos/química , Inositol/metabolismo , Ligases/metabolismo , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/metabolismo , Sulfonamidas/farmacologia
10.
World J Microbiol Biotechnol ; 38(12): 255, 2022 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-36319705

RESUMO

Phosphate (Pi) is essential for life as it is an integral part of the universal chemical energy adenosine triphosphate (ATP), and macromolecules such as, DNA, RNA proteins and lipids. Despite the core roles and the need of this nutrient in living cells, some bacteria can grow in environments that are poor in Pi. The metabolic mechanisms that enable bacteria to proliferate in a low phosphate environment are not fully understood. In this study, the soil microbe Pseudomonas (P.) fluorescens was cultured in a control and a low Pi (stress) medium in order to delineate how energy homeostasis is maintained. Although there was no significant variation in biomass yield in these cultures, metabolites like isocitrate, oxaloacetate, pyruvate and phosphoenolpyruvate (PEP) were markedly increased in the phosphate-starved condition. Components of the glycolytic, glyoxylate and tricarboxylic acid cycles operated in tandem to generate ATP by substrate level phosphorylation (SLP) as NADH-producing enzymes were impeded. The α-ketoglutarate (KG) produced when glutamine, the sole carbon nutrient was transformed into phosphoenol pyruvate (PEP) and succinyl-CoA (SC), two high energy moieties. The metabolic reprogramming orchestrated by isocitrate lyase (ICL), phosphoenolpyruvate synthase (PEPS), pyruvate phosphate dikinase (PPDK), and succinyl-CoA synthetase fulfilled the ATP budget. Cell free extract experiments confirmed ATP synthesis in the presence of such substrates as PEP, oxaloacetate and isocitrate respectively. Gene expression profiling revealed elevated transcripts associated with numerous enzymes including ICL, PEPS, and succinyl-CoA synthetase (SCS). This microbial adaptation will be critical in promoting biological activity in Pi-poor ecosystems.


Assuntos
Pseudomonas fluorescens , Pseudomonas fluorescens/metabolismo , Trifosfato de Adenosina/metabolismo , Isocitratos/metabolismo , Fosfatos/metabolismo , Ecossistema , Fosfoenolpiruvato/metabolismo , Homeostase , Ácido Pirúvico/metabolismo , Oxaloacetatos/metabolismo , Ligases/metabolismo
11.
Nat Commun ; 13(1): 6647, 2022 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-36333342

RESUMO

Endothelial nitric oxide synthase (eNOS) decreases following inflammatory stimulation. As a master regulator of endothelial homeostasis, maintaining optimal eNOS levels is important during cardiovascular events. However, little is known regarding the mechanism of eNOS protection. In this study, we demonstrate a regulatory role for endothelial expression of 2'-5' oligoadenylate synthetase-like 1 (OASL1) in maintaining eNOS mRNA stability during athero-prone conditions and consider its clinical implications. A lack of endothelial Oasl1 accelerated plaque progression, which was preceded by endothelial dysfunction, elevated vascular inflammation, and decreased NO bioavailability following impaired eNOS expression. Mechanistically, knockdown of PI3K/Akt signaling-dependent OASL expression increased Erk1/2 and NF-κB activation and decreased NOS3 (gene name for eNOS) mRNA expression through upregulation of the negative regulatory, miR-584, whereas a miR-584 inhibitor rescued the effects of OASL knockdown. These results suggest that OASL1/OASL regulates endothelial biology by protecting NOS3 mRNA and targeting miR-584 represents a rational therapeutic strategy for eNOS maintenance in vascular disease.


Assuntos
Aterosclerose , MicroRNAs , Humanos , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ligases/metabolismo , Células Endoteliais/metabolismo , MicroRNAs/genética , Aterosclerose/genética , Aterosclerose/prevenção & controle , Aterosclerose/metabolismo , RNA Mensageiro/metabolismo , Estabilidade de RNA , Óxido Nítrico/metabolismo , Células Cultivadas
12.
Int J Mol Sci ; 23(21)2022 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-36361943

RESUMO

Size is the most important quality attribute of giant pumpkin fruit. Different concentrations and application frequencies of α-naphthaleneacetic acid (NAA) and 24-epibrassinolide (EBR) were sprayed on the leaves and fruits of giant pumpkin at different growth stages to determine their effects and the mechanism responsible for fruit size increase. NAA+EBR application improved source strength, and further analysis indicated that NAA+EBR markedly boosted net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr) and the expression level and activity of galactitol synthetase (GolS), raffinose synthetase (RS), and stachyose synthetase (STS), resulting in an increase in the synthesis of photoassimilate, especially stachyose. Concomitantly, NAA+EBR spray increased stachyose and sucrose contents throughout pumpkin fruit growth and the concentrations of glucose and fructose at 0 and 20 days post-anthesis (DPA) in peduncle phloem sap, implying that such treatment improved the efficiency of assimilate transport from the peduncle to the fruit. Furthermore, it improved the expression and activity of alkaline α-galactosidase (AGA), facilitating assimilate unloading, providing carbon skeletons and energy for fruit growth, and increasing fruit weight by more than 44.1%. Therefore, exogenous NAA and EBR increased source capacity, transportation efficiency, and sink strength, overall promoting the synthesis and distribution of photoassimilate, ultimately increasing fruit size.


Assuntos
Cucurbita , Frutas , Frutas/metabolismo , Açúcares/metabolismo , Ligases/metabolismo
13.
Nutrients ; 14(19)2022 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-36235710

RESUMO

A maternal low-protein (LP) diet during gestation and/or lactation results in metabolic syndrome in their offspring. Here, we investigated the effect of maternal LP diet during puberty and adulthood on the metabolic homeostasis of glucose and lipids in offspring. Female mice were fed with normal-protein (NP) diet or a LP diet for 11 weeks. Male offspring were then fed with a high-fat diet (NP-HFD and LP-HFD groups) or standard chow diet (NP-Chow and LP-Chow groups) for 4 months. Results showed that maternal LP diet during puberty and adulthood did not alter the insulin sensitivity and hepatic lipid homeostasis of their offspring under chow diet, but aggravated insulin resistance, hepatic steatosis, and hypercholesterolemia of offspring in response to a post-weaning HFD. Accordingly, transcriptomics study with offspring's liver indicated that several genes related to glucose and lipid metabolism, including lipoprotein lipase (Lpl), long-chain acyl-CoA synthetase 1 (Acsl1), Apoprotein A1 (Apoa1), major urinary protein 19 (Mup19), cholesterol 7α hydroxylase (Cyp7a1) and fibroblast growth factor 1 (Fgf1), were changed by maternal LP diet. Taken together, maternal LP diet during puberty and adulthood could disarrange the expression of metabolic genes in the liver of offspring and aggravate insulin resistance and hepatic steatosis in offspring fed a HFD.


Assuntos
Fígado Gorduroso , Resistência à Insulina , Efeitos Tardios da Exposição Pré-Natal , Animais , Apoproteínas/metabolismo , Colesterol 7-alfa-Hidroxilase/metabolismo , Coenzima A/metabolismo , Dieta Hiperlipídica/efeitos adversos , Dieta com Restrição de Proteínas/efeitos adversos , Fígado Gorduroso/metabolismo , Feminino , Fator 1 de Crescimento de Fibroblastos/metabolismo , Glucose/metabolismo , Ligases/metabolismo , Metabolismo dos Lipídeos/fisiologia , Lipídeos , Lipase Lipoproteica/metabolismo , Fígado/metabolismo , Masculino , Fenômenos Fisiológicos da Nutrição Materna , Camundongos , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Maturidade Sexual
14.
Sci Rep ; 12(1): 17990, 2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36289272

RESUMO

Although tyrosine kinase inhibitors (TKIs) improve the prognosis of chronic myeloid leukemia (CML) patients, resistance to TKIs and residual leukemia stem cells (LSCs) inevitably become the bottleneck of cure. Therefore, we need to explore novel treatment strategies based on conventional treatment strategies. Our previous study found that CML cell senescence may be one of the main factors to achieve clinical cure of CML. Studies have shown that lipid metabolism plays a key role in cellular senescence. Here, we found that long-chain acyl-CoA synthetase 1 (ACSL1) was significantly up-regulated in senescent CML cells. Furthermore, we demonstrated that overexpression of ACSL1 induces senescence and inhibits cell growth in K562 cells by altering cell cycle progression, and enhances the proliferation-inhibiting effect of imatinib. Overexpression of ACSL1 enhances imatinib-induced tumorigenic decline in K562 cells in vivo. Knockdown of ACSL1 reverses imatinib-induced senescence in K562 cells. Mechanistically, overexpression of ACSL1 induced senescence in K562 cells via the SIRT1/p53/p21 axis. Collectively, our study showed that ACSL1 promotes imatinib-induced K562 cells senescence and tumor growth by regulating SIRT1/p53/p21 pathway. The ACSL1/SIRT1/p53 signal axis is a novel mechanism of cell senescence in CML and a new potential target for eradication of CML LSCs.


Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Sirtuína 1 , Humanos , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Sirtuína 1/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Células K562 , Senescência Celular , Inibidores de Proteínas Quinases/farmacologia , Ligases/metabolismo , Coenzima A/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo
15.
BMC Biol ; 20(1): 228, 2022 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-36209095

RESUMO

BACKGROUND: One-carbon metabolism, which includes the folate and methionine cycles, involves the transfer of methyl groups which are then utilised as a part of multiple physiological processes including redox defence. During the methionine cycle, the vitamin B12-dependent enzyme methionine synthetase converts homocysteine to methionine. The enzyme S-adenosylmethionine (SAM) synthetase then uses methionine in the production of the reactive methyl carrier SAM. SAM-binding methyltransferases then utilise SAM as a cofactor to methylate proteins, small molecules, lipids, and nucleic acids. RESULTS: We describe a novel SAM methyltransferase, RIPS-1, which was the single gene identified from forward genetic screens in Caenorhabditis elegans looking for resistance to lethal concentrations of the thiol-reducing agent dithiothreitol (DTT). As well as RIPS-1 mutation, we show that in wild-type worms, DTT toxicity can be overcome by modulating vitamin B12 levels, either by using growth media and/or bacterial food that provide higher levels of vitamin B12 or by vitamin B12 supplementation. We show that active methionine synthetase is required for vitamin B12-mediated DTT resistance in wild types but is not required for resistance resulting from RIPS-1 mutation and that susceptibility to DTT is partially suppressed by methionine supplementation. A targeted RNAi modifier screen identified the mitochondrial enzyme methylmalonyl-CoA epimerase as a strong genetic enhancer of DTT resistance in a RIPS-1 mutant. We show that RIPS-1 is expressed in the intestinal and hypodermal tissues of the nematode and that treating with DTT, ß-mercaptoethanol, or hydrogen sulfide induces RIPS-1 expression. We demonstrate that RIPS-1 expression is controlled by the hypoxia-inducible factor pathway and that homologues of RIPS-1 are found in a small subset of eukaryotes and bacteria, many of which can adapt to fluctuations in environmental oxygen levels. CONCLUSIONS: This work highlights the central importance of dietary vitamin B12 in normal metabolic processes in C. elegans, defines a new role for this vitamin in countering reductive stress, and identifies RIPS-1 as a novel methyltransferase in the methionine cycle.


Assuntos
Sulfeto de Hidrogênio , Ácidos Nucleicos , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Carbono/metabolismo , Ditiotreitol/metabolismo , Ácido Fólico/metabolismo , Homocisteína/metabolismo , Sulfeto de Hidrogênio/metabolismo , Ligases/metabolismo , Lipídeos , Mercaptoetanol/metabolismo , Metionina/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Oxigênio/metabolismo , Substâncias Redutoras/metabolismo , S-Adenosilmetionina/metabolismo , Compostos de Sulfidrila/metabolismo , Vitamina B 12/metabolismo , Vitamina B 12/farmacologia , Vitaminas/metabolismo
16.
J Microbiol ; 60(12): 1139-1152, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36279104

RESUMO

Acetyl-CoA synthetase (ACS) is the enzyme that irreversibly catalyzes the synthesis of acetyl-CoA from acetate, CoA-SH, and ATP via acetyl-AMP as an intermediate. In this study, we demonstrated that AcsA1 (MSMEG_6179) is the predominantly expressed ACS among four ACSs (MSMEG_6179, MSMEG_0718, MSMEG_3986, and MSMEG_5650) found in Mycobacterium smegmatis and that a deletion mutation of acsA1 in M. smegmatis led to its compromised growth on acetate as the sole carbon source. Expression of acsA1 was demonstrated to be induced during growth on acetate as the sole carbon source. The acsA1 gene was shown to be negatively regulated by Crp1 (MSMEG_6189) that is the major cAMP receptor protein (CRP) in M. smegmatis. Using DNase I footprinting analysis and site-directed mutagenesis, a CRP-binding site (GGTGA-N6-TCACA) was identified in the upstream regulatory region of acsA1, which is important for repression of acsA1 expression. We also demonstrated that inhibition of the respiratory electron transport chain by inactivation of the major terminal oxidase, aa3 cytochrome c oxidase, led to a decrease in acsA1 expression probably through the activation of CRP. In conclusion, AcsA1 is the major ACS in M. smegmatis and its gene is under the negative regulation of Crp1, which contributes to some extent to the induction of acsA1 expression under acetate conditions. The growth of M. smegmatis is severely impaired on acetate as the sole carbon source under respiration-inhibitory conditions.


Assuntos
Proteína Receptora de AMP Cíclico , Mycobacterium smegmatis , Mycobacterium smegmatis/genética , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/metabolismo , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Bactérias/metabolismo , Acetatos/metabolismo , Carbono/metabolismo , Ligases/genética , Ligases/metabolismo
17.
Methods Enzymol ; 676: 369-384, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36280358

RESUMO

TurboID is a new and efficient proximity labeling system that was first developed in living mammalian cells. TurboID is a modified bacterial biotin ligase that can be fused to a bait protein, which can then modify proximal interacting proteins with biotin. Prey proteins subsequently labeled with biotin tags will be pulled down with streptavidin-coated beads and identified by mass spectrometry-based proteomics. TurboID has been recently applied to living plant cells and provided promising results in identification of interacting proteins. Mitogen-activated protein kinase 4 (MPK4) is important for plant growth, development, and defense; however, the molecular mechanisms underlying the range of MPK4 functions are not completely known. Here we use modern proteomics together with the TurboID in a proof-of-concept study to profile the MPK4 interactome and uncover the functions of MPK4 in plant signaling cascades.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteômica/métodos , Biotina/metabolismo , Estreptavidina/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Ligases/metabolismo , Mamíferos/metabolismo
18.
Nat Commun ; 13(1): 6238, 2022 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-36266306

RESUMO

5-Aminolevulinic acid-based photodynamic therapy heavily depends on the biological transformation efficiency of 5-aminolevulinic acid to protoporphyrin IX, while the lack of an effective delivery system and imaging navigation are major hurdles in improving the accumulation of protoporphyrin IX and optimizing therapeutic parameters. Herein, we leverage a synthetic biology approach to construct a transdermal theranostic microneedle patch integrated with 5-aminolevulinic acid and catalase co-loaded tumor acidity-responsive copper-doped calcium phosphate nanoparticles for efficient 5-aminolevulinic acid-based photodynamic therapy by maximizing the enrichment of intratumoral protoporphyrin IX. We show that continuous oxygen generation by catalase in vivo reverses tumor hypoxia, enhances protoporphyrin IX accumulation by blocking protoporphyrin IX efflux (downregulating hypoxia-inducible factor-1α and ferrochelatase) and upregulates protoporphyrin IX biosynthesis (providing exogenous 5-aminolevulinic acid and upregulating ALA-synthetase). In vivo fluorescence/photoacoustic duplex imaging can monitor intratumoral oxygen saturation and protoporphyrin IX metabolic kinetics simultaneously. This approach thus facilitates the optimization of therapeutic parameters for different cancers to realize Ca2+/Cu2+-interferences-enhanced repeatable photodynamic therapy, making this theranostic patch promising for clinical practice.


Assuntos
Neoplasias , Fotoquimioterapia , Humanos , Fotoquimioterapia/métodos , Ácido Aminolevulínico/farmacologia , Catalase/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fármacos Fotossensibilizantes , Ferroquelatase/metabolismo , Biologia Sintética , Cobre/metabolismo , Protoporfirinas/metabolismo , Neoplasias/tratamento farmacológico , Oxigênio/metabolismo , Ligases/metabolismo , Linhagem Celular Tumoral
19.
Endocrinology ; 163(12)2022 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-36256598

RESUMO

Two well-known protein complexes in mammalian cells, mTOR type 1 and type 2 (mTORC1/2) are involved in several cellular processes such as protein synthesis, cell proliferation, and commonly dysregulated in cancer. An acyl-CoA synthetase type 4 (ACSL4) is one of the most recently mTORC1/2 regulators described, in breast cancer cells. The expression of ACSL4 is hormone-regulated in adrenocortical cells and required for steroid biosynthesis. mTORC1/2 have been reported to be crucial in the proliferation of human adrenocortical tumor cells H295R and interestingly reported at several subcellular locations, which has brought cell biology to the vanguard of the mTOR signaling field. In the present work, we study the regulation of mTORC1/2 activation by angiotensin II (Ang II)-the trophic hormone for adrenocortical cells-the subcellular localization of mTORC1/2 signaling proteins and the role of ACSL4 in the regulation of this pathway, in H295R cells. Ang II promotes activation by phosphorylation of mTORC1/2 pathway proteins in a time-dependent manner. Mitochondrial pools of ribosomal protein S6, protein kinase B (Akt) in threonine 308, and serine 473 and Rictor are phosphorylated and activated. Glycogen synthase kinase type 3 (GSK3) is phosphorylated and inactivated in mitochondria, favoring mTORC1 activation. Epidermal growth factor, a classic mTORC1/2 activator, promoted unique activation kinetics of mTORC1/2 pathway, except for Akt phosphorylation. Here, we demonstrate that ACSL4 is necessary for mTORC1/2 effectors phosphorylation and H295R proliferation, triggered by Ang II. Ang II promotes activation of mitochondrial mTORC1/2 signaling proteins, through ACSL4, with a direct effect on adrenocortical cellular proliferation.


Assuntos
Angiotensina II , Proteínas Proto-Oncogênicas c-akt , Animais , Humanos , Angiotensina II/farmacologia , Angiotensina II/metabolismo , Coenzima A/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Ligases/metabolismo , Mamíferos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Mitocôndrias/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo
20.
Int J Mol Sci ; 23(19)2022 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-36233324

RESUMO

Feeding ramie cultivars (Boehmaria nivea L.) are an important feedstock for livestock. Increasing their biomass and improving their nutritional values are essential for animal feeding. Gibberellin (GA3) and ethylene (ETH) are two plant hormones that regulate the growth, development, and metabolism of plants. Herein, we report effects of the GA3 and ETH application on the growth and plant metabolism of feeding ramie in the field. A combination of GA3 and ETH was designed to spray new plants. The two hormones enhanced the growth of plants to produce more biomass. Meanwhile, the two hormones reduced the contents of lignin in leaves and stems, while increased the content of flavonoids in leaves. To understand the potential mechanisms behind these results, we used RNA-seq-based transcriptomics and UPLC-MS/MS-based metabolomics to characterize gene expression and metabolite profiles associated with the treatment of GA3 and ETH. 1562 and 2364 differentially expressed genes (DEGs) were obtained from leaves and stems (treated versus control), respectively. Meanwhile, 99 and 88 differentially accumulated metabolites (DAMs) were annotated from treated versus control leaves and treated versus control stems, respectively. Data mining revealed that both DEGs and DAMs were associated with multiple plant metabolisms, especially plant secondary metabolism. A specific focus on the plant phenylpropanoid pathway identified candidates of DEGs and DEMs that were associated with lignin and flavonoid biosynthesis. Shikimate hydroxycinnamoyl transferase (HCT) is a key enzyme that is involved in the lignin biosynthesis. The gene encoding B. nivea HCT was downregulated in the treated leaves and stems. In addition, genes encoding 4-coumaryl CoA ligase (4CL) and trans-cinnamate 4-monooxygenase (CYP73A), two lignin pathway enzymes, were downregulated in the treated stems. Meanwhile, the reduction in lignin in the treated leaves led to an increase in cinnamic acid and p-coumaryl CoA, two shared substrates of flavonoids that are enhanced in contents. Taken together, these findings indicated that an appropriate combination of GA3 and ETH is an effective strategy to enhance plant growth via altering gene expression and plant secondary metabolism for biomass-enhanced and value-improved feeding ramie.


Assuntos
Boehmeria , Giberelinas , Boehmeria/metabolismo , Cromatografia Líquida , Coenzima A/metabolismo , Etilenos , Flavonoides , Regulação da Expressão Gênica de Plantas , Giberelinas/farmacologia , Hormônios , Ligases/metabolismo , Lignina/metabolismo , Compostos Organofosforados , Reguladores de Crescimento de Plantas/farmacologia , Plantas/metabolismo , Espectrometria de Massas em Tandem , Transcinamato 4-Mono-Oxigenase/genética , Transcinamato 4-Mono-Oxigenase/metabolismo , Transferases/metabolismo
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