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1.
mBio ; 15(5): e0341423, 2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38572988

RESUMO

Acetyl-CoA carboxylases (ACCs) convert acetyl-CoA to malonyl-CoA, a key step in fatty acid biosynthesis and autotrophic carbon fixation pathways. Three functionally distinct components, biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT), are either separated or partially fused in different combinations, forming heteromeric ACCs. However, an ACC with fused BC-BCCP and separate CT has not been identified, leaving its catalytic mechanism unclear. Here, we identify two BC isoforms (BC1 and BC2) from Chloroflexus aurantiacus, a filamentous anoxygenic phototroph that employs 3-hydroxypropionate (3-HP) bi-cycle rather than Calvin cycle for autotrophic carbon fixation. We reveal that BC1 possesses fused BC and BCCP domains, where BCCP could be biotinylated by E. coli or C. aurantiacus BirA on Lys553 residue. Crystal structures of BC1 and BC2 at 3.2 Å and 3.0 Å resolutions, respectively, further reveal a tetramer of two BC1-BC homodimers, and a BC2 homodimer, all exhibiting similar BC architectures. The two BC1-BC homodimers are connected by an eight-stranded ß-barrel of the partially resolved BCCP domain. Disruption of ß-barrel results in dissociation of the tetramer into dimers in solution and decreased biotin carboxylase activity. Biotinylation of the BCCP domain further promotes BC1 and CTß-CTα interactions to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-HP via co-expression with a recombinant malonyl-CoA reductase in E. coli cells. This study revealed a heteromeric ACC that evolves fused BC-BCCP but separate CTα and CTß to complete ACC activity.IMPORTANCEAcetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step in fatty acid biosynthesis and autotrophic carbon fixation pathways across a wide range of organisms, making them attractive targets for drug discovery against various infections and diseases. Although structural studies on homomeric ACCs, which consist of a single protein with three subunits, have revealed the "swing domain model" where the biotin carboxyl carrier protein (BCCP) domain translocates between biotin carboxylase (BC) and carboxyltransferase (CT) active sites to facilitate the reaction, our understanding of the subunit composition and catalytic mechanism in heteromeric ACCs remains limited. Here, we identify a novel ACC from an ancient anoxygenic photosynthetic bacterium Chloroflexus aurantiacus, it evolves fused BC and BCCP domain, but separate CT components to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-hydroxypropionate (3-HP) via co-expression with recombinant malonyl-CoA reductase in E. coli cells. These findings expand the diversity and molecular evolution of heteromeric ACCs and provide a structural basis for potential applications in 3-HP biosynthesis.


Assuntos
Acetil-CoA Carboxilase , Carbono-Nitrogênio Ligases , Chloroflexus , Acetil-CoA Carboxilase/metabolismo , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/química , Carbono-Nitrogênio Ligases/metabolismo , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/química , Chloroflexus/genética , Chloroflexus/metabolismo , Chloroflexus/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Biotina/metabolismo , Biotina/biossíntese , Malonil Coenzima A/metabolismo , Acetilcoenzima A/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Ácido Graxo Sintase Tipo II
2.
PLoS One ; 19(4): e0297122, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38662671

RESUMO

Site specific biotinylation of AviTagged recombinant proteins using BirA enzyme is a widely used protein labeling technology. However, due to the incomplete biotinylation reactions and the lack of a purification method specific for the biotinylated proteins, it is challenging to purify the biotinylated sample when mixed with the non-biotinylated byproduct. Here, we have developed a monoclonal antibody that specifically recognizes the non-biotinylated AviTag but not the biotinylated sequence. After a ten-minute incubation with the resin that is conjugated with the antibody, the non-biotinylated AviTagged protein is trapped on the resin while the fully biotinylated material freely passes through. Therefore, our AviTrap (anti-AviTag antibody conjugated resin) provides an efficient solution for enriching biotinylated AviTagged proteins via a simple one-step purification.


Assuntos
Anticorpos Monoclonais , Biotinilação , Anticorpos Monoclonais/química , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Humanos , Biotina/química , Animais , Carbono-Nitrogênio Ligases/química , Carbono-Nitrogênio Ligases/metabolismo
3.
Int J Biol Macromol ; 267(Pt 2): 131510, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38608989

RESUMO

Bacterial diseases caused substantial yield losses worldwide, with the rise of antibiotic resistance, there is a critical need for alternative antibacterial compounds. Natural products (NPs) from microorganisms have emerged as promising candidates due to their potential as cost-effective and environmentally friendly bactericides. However, the precise mechanisms underlying the antibacterial activity of many NPs, including Guvermectin (GV), remain poorly understood. Here, we sought to explore how GV interacts with Guanosine 5'-monophosphate synthetase (GMPs), an enzyme crucial in bacterial guanine synthesis. We employed a combination of biochemical and genetic approaches, enzyme activity assays, site-directed mutagenesis, bio-layer interferometry, and molecular docking assays to assess GV's antibacterial activity and its mechanism targeting GMPs. The results showed that GV effectively inhibits GMPs, disrupting bacterial guanine synthesis. This was confirmed through drug-resistant assays and direct enzyme inhibition studies. Bio-layer interferometry assays demonstrated specific binding of GV to GMPs, with dependency on Xanthosine 5'-monophosphate. Site-directed mutagenesis identified key residues crucial for the GV-GMP interaction. This study elucidates the antibacterial mechanism of GV, highlighting its potential as a biocontrol agent in agriculture. These findings contribute to the development of novel antibacterial agents and underscore the importance of exploring natural products for agricultural disease management.


Assuntos
Adenosina/análogos & derivados , Antibacterianos , Ivermectina , Antibacterianos/farmacologia , Antibacterianos/química , Ivermectina/farmacologia , Ivermectina/análogos & derivados , Ivermectina/química , Simulação de Acoplamento Molecular , Produtos Biológicos/farmacologia , Produtos Biológicos/química , Testes de Sensibilidade Microbiana , Carbono-Nitrogênio Ligases/metabolismo , Carbono-Nitrogênio Ligases/química , Carbono-Nitrogênio Ligases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Mutagênese Sítio-Dirigida
4.
Nucleic Acids Res ; 52(7): e37, 2024 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-38452210

RESUMO

G-quadruplexes (G4s) are noncanonical nucleic acid structures pivotal to cellular processes and disease pathways. Deciphering G4-interacting proteins is imperative for unraveling G4's biological significance. In this study, we developed a G4-targeting biotin ligase named G4PID, meticulously assessing its binding affinity and specificity both in vitro and in vivo. Capitalizing on G4PID, we devised a tailored approach termed G-quadruplex-interacting proteins specific biotin-ligation procedure (PLGPB) to precisely profile G4-interacting proteins. Implementing this innovative strategy in live cells, we unveiled a cohort of 149 potential G4-interacting proteins, which exhibiting multifaceted functionalities. We then substantiate the directly binding affinity of 7 candidate G4-interacting-proteins (SF3B4, FBL, PP1G, BCL7C, NDUV1, ILF3, GAR1) in vitro. Remarkably, we verified that splicing factor 3B subunit 4 (SF3B4) binds preferentially to the G4-rich 3' splice site and the corresponding splicing sites are modulated by the G4 stabilizer PDS, indicating the regulating role of G4s in mRNA splicing procedure. The PLGPB strategy could biotinylate multiple proteins simultaneously, which providing an opportunity to map G4-interacting proteins network in living cells.


Assuntos
Biotina , Quadruplex G , Humanos , Biotina/metabolismo , Ligação Proteica , Fatores de Processamento de RNA/metabolismo , Carbono-Nitrogênio Ligases/metabolismo , Carbono-Nitrogênio Ligases/genética , Splicing de RNA , Células HEK293 , Proteínas de Ligação a RNA/metabolismo , Células HeLa
5.
Nat Chem Biol ; 20(5): 555-565, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38233583

RESUMO

Drug-ID is a novel method applying proximity biotinylation to identify drug-protein interactions inside living cells. The covalent conjugation of a drug with a biotin ligase enables targeted biotinylation and identification of the drug-bound proteome. We established Drug-ID for two small-molecule drugs, JQ1 and SAHA, and applied it for RNaseH-recruiting antisense oligonucleotides (ASOs). Drug-ID profiles the drug-protein interactome de novo under native conditions, directly inside living cells and at pharmacologically effective drug concentrations. It requires minimal amounts of cell material and might even become applicable in vivo. We studied the dose-dependent aggregation of ASOs and the effect of different wing chemistries (locked nucleic acid, 2'-methoxyethyl and 2'-Fluoro) and ASO lengths on the interactome. Finally, we demonstrate the detection of stress-induced, intracellular interactome changes (actinomycin D treatment) with an in situ variant of the approach, which uses a recombinant biotin ligase and does not require genetic manipulation of the target cell.


Assuntos
Biotinilação , Humanos , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Antissenso/química , Ribonuclease H/metabolismo , Carbono-Nitrogênio Ligases/metabolismo , Biotina/metabolismo , Biotina/química , Ligação Proteica
6.
Proteins ; 92(4): 435-448, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-37997490

RESUMO

Biotin (vitamin H or B7) is a coenzyme essential for all forms of life. Biotin has biological activity only when covalently attached to a few key metabolic enzyme proteins. Most organisms have only one attachment enzyme, biotin protein ligase (BPL), which attaches biotin to all target proteins. The sequences of these proteins and their substrate proteins are strongly conserved throughout biology. Structures of both the biotin ligase- and biotin-acceptor domains of mammals, plants, several bacterial species, and archaea have been determined. These, together with mutational analyses of ligases and their protein substrates, illustrate the exceptional specificity of this protein modification. For example, the Escherichia coli BPL biotinylates only one of the >4000 cellular proteins. Several bifunctional bacterial biotin ligases transcriptionally regulate biotin synthesis and/or transport in concert with biotinylation. The human BPL has been demonstrated to play an important role in that mutations in the BPL encoding gene cause one form of the disease, biotin-responsive multiple carboxylase deficiency. Promiscuous mutant versions of several BPL enzymes release biotinoyl-AMP, the active intermediate of the ligase reaction, to solvent. The released biotinoyl-AMP acts as a chemical biotinylation reagent that modifies lysine residues of neighboring proteins in vivo. This proximity-dependent biotinylation (called BioID) approach has been heavily utilized in cell biology.


Assuntos
Carbono-Nitrogênio Ligases , Proteínas de Escherichia coli , Animais , Humanos , Biotinilação , Biotina/química , Biotina/metabolismo , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/química , Carbono-Nitrogênio Ligases/metabolismo , Proteínas/metabolismo , Escherichia coli/metabolismo , Ligases/genética , Ligases/metabolismo , Bactérias/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mamíferos/metabolismo
7.
Adv Sci (Weinh) ; 10(25): e2301264, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37439412

RESUMO

Tumor stemness is associated with the recurrence and incurability of colorectal cancer (CRC), which lacks effective therapeutic targets and drugs. Glycinamide ribonucleotide transformylase (GART) fulfills an important role in numerous types of malignancies. The present study aims to identify the underlying mechanism through which GART may promote CRC stemness, as to developing novel therapeutic methods. An elevated level of GART is associated with poor outcomes in CRC patients and promotes the proliferation and migration of CRC cells. CD133+ cells with increased GART expression possess higher tumorigenic and proliferative capabilities both in vitro and in vivo. GART is identified to have a novel methyltransferase function, whose enzymatic activity center is located at the E948 site. GART also enhances the stability of RuvB-like AAA ATPase 1 (RUVBL1) through methylating its K7 site, which consequently aberrantly activates the Wnt/ß-catenin signaling pathway to induce tumor stemness. Pemetrexed (PEM), a compound targeting GART, combined with other chemotherapy drugs greatly suppresses tumor growth both in a PDX model and in CRC patients. The present study demonstrates a novel methyltransferase function of GART and the role of the GART/RUVBL1/ß-catenin signaling axis in promoting CRC stemness. PEM may be a promising therapeutic agent for the treatment of CRC.


Assuntos
Carbono-Nitrogênio Ligases , Neoplasias Colorretais , Humanos , Linhagem Celular Tumoral , Fosforribosilglicinamido Formiltransferase/metabolismo , Metiltransferases/metabolismo , beta Catenina/metabolismo , Neoplasias Colorretais/patologia , Via de Sinalização Wnt , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , DNA Helicases/farmacologia , Carbono-Nitrogênio Ligases/metabolismo
8.
Molecules ; 28(11)2023 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-37298742

RESUMO

(S)-Norcoclaurine is synthesized in vivo through a metabolic pathway that ends with (S)-norcoclaurine synthase (NCS). The former constitutes the scaffold for the biosynthesis of all benzylisoquinoline alkaloids (BIAs), including many drugs such as the opiates morphine and codeine and the semi-synthetic opioids oxycodone, hydrocodone, and hydromorphone. Unfortunately, the only source of complex BIAs is the opium poppy, leaving the drug supply dependent on poppy crops. Therefore, the bioproduction of (S)-norcoclaurine in heterologous hosts, such as bacteria or yeast, is an intense area of research nowadays. The efficiency of (S)-norcoclaurine biosynthesis is strongly dependent on the catalytic efficiency of NCS. Therefore, we identified vital NCS rate-enhancing mutations through the rational transition-state macrodipole stabilization method at the Quantum Mechanics/Molecular Mechanics (QM/MM) level. The results are a step forward for obtaining NCS variants able to biosynthesize (S)-norcoclaurine on a large scale.


Assuntos
Alcaloides , Benzilisoquinolinas , Carbono-Nitrogênio Ligases , Papaver , Alcaloides/metabolismo , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Codeína , Papaver/genética , Papaver/metabolismo
9.
Life Sci Alliance ; 6(9)2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37348953

RESUMO

The CTP nucleotide is a key precursor of nucleic acids metabolism essential for DNA replication. De novo CTP production relies on CTP synthetases 1 and 2 (CTPS1 and CTPS2) that catalyze the conversion of UTP into CTP. CTP synthetase activity is high in proliferating cells including cancer cells; however, the respective roles of CTPS1 and CTPS2 in cell proliferation are not known. By inactivation of CTPS1 and/or CTPS2 and complementation experiments, we showed that both CTPS1 and CTPS2 are differentially required for cell proliferation. CTPS1 was more efficient in promoting proliferation than CTPS2, in association with a higher intrinsic enzymatic activity that was more resistant to inhibition by 3-deaza-uridine, an UTP analog. The contribution of CTPS2 to cell proliferation was modest when CTPS1 was expressed but essential in absence of CTPS1. Public databases analysis of more than 1,000 inactivated cancer cell lines for CTPS1 or CTPS2 confirmed that cell growth is highly dependent of CTPS1 but less or not of CTPS2. Therefore, our results demonstrate that CTPS1 is the main contributor to cell proliferation.


Assuntos
Carbono-Nitrogênio Ligases , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Uridina Trifosfato/metabolismo , Proliferação de Células , Ciclo Celular , Linhagem Celular
10.
Front Endocrinol (Lausanne) ; 14: 1129162, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37143728

RESUMO

Targeting tumor cell metabolism is a new frontier in cancer management. Thus, metabolic pathway inhibitors could be used as anti-estrogen receptor α (ERα) breast cancer (BC) drugs. Here, the interplay among metabolic enzyme(s), the ERα levels and cell proliferation was studied. siRNA-based screen directed against different metabolic proteins in MCF10a, MCF-7 and MCF-7 cells genetically resistant to endocrine therapy (ET) drugs and metabolomic analyses in numerous BC cell lines unveil that the inhibition of GART, a key enzyme in the purine de novo biosynthetic pathway, induces ERα degradation and prevent BC cell proliferation. We report here that a reduced GART expression correlates with a longer relapse-free-survival (RFS) in women with ERα-positive BCs. ERα-expressing luminal A invasive ductal carcinomas (IDCs) are sensitive to GART inhibition and GART expression is increased in receptor-positive IDCs of high grade and stage and plays a role in the development of ET resistance. Accordingly, GART inhibition reduces ERα stability and cell proliferation in IDC luminal A cells where it deregulates 17ß-estradiol (E2):ERα signaling to cell proliferation. Moreover, the GART inhibitor lometrexol (LMX) and drugs approved for clinical treatment of primary and metastatic BC (4OH-tamoxifen and the CDK4/CDK6 inhibitors) exert synergic antiproliferative effects in BC cells. In conclusion, GART inhibition by LMX or other inhibitors of the de novo purine biosynthetic pathway could be a novel effective strategy for the treatment of primary and metastatic BCs.


Assuntos
Neoplasias da Mama , Carbono-Nitrogênio Ligases , Carcinoma Ductal de Mama , Feminino , Humanos , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Vias Biossintéticas , Recidiva Local de Neoplasia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Purinas , Carbono-Nitrogênio Ligases/metabolismo , Fosforribosilglicinamido Formiltransferase/metabolismo
11.
Protein Sci ; 32(6): e4648, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37106216

RESUMO

CTP synthases (CTPS) catalyze the de novo production of CTP using UTP, ATP, and l-glutamine with the anticancer drug metabolite gemcitabine-5'-triphosphate (dF-dCTP) being one of its most potent nucleotide inhibitors. To delineate the structural origins of this inhibition, we solved the structures of Escherichia coli CTPS (ecCTPS) in complex with CTP (2.0 Å), 2'-ribo-F-dCTP (2.0 Å), 2'-arabino-F-CTP (2.4 Å), dF-dCTP (2.3 Å), dF-dCTP and ADP (2.1 Å), and dF-dCTP and ATP (2.1 Å). These structures revealed that the increased binding affinities observed for inhibitors bearing the 2'-F-arabino group (dF-dCTP and F-araCTP), relative to CTP and F-dCTP, arise from interactions between the inhibitor's fluorine atom exploiting a conserved hydrophobic pocket formed by F227 and an interdigitating loop from an adjacent subunit (Q114-V115-I116). Intriguingly, crystal structures of ecCTPS•dF-dCTP complexes in the presence of select monovalent and divalent cations demonstrated that the in crystallo tetrameric assembly of wild-type ecCTPS was induced into a conformation similar to inhibitory ecCTPS filaments solely through the binding of Na+ -, Mg2+ -, or Mn2+ •dF-dCTP. However, in the presence of potassium, the dF-dCTP-bound structure is demetalated and in the low-affinity, non-filamentous conformation, like the conformation seen when bound to CTP and the other nucleotide analogues. Additionally, CTP can also induce the filament-competent conformation linked to high-affinity dF-dCTP binding in the presence of high concentrations of Mg2+ . This metal-dependent, compacted CTP pocket conformation therefore furnishes the binding environment responsible for the tight binding of dF-dCTP and provides insights for further inhibitor design.


Assuntos
Carbono-Nitrogênio Ligases , Gencitabina , Carbono-Nitrogênio Ligases/metabolismo , Escherichia coli/metabolismo , Nucleotídeos , Trifosfato de Adenosina , Cinética
12.
Methods Mol Biol ; 2626: 365-379, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36715916

RESUMO

We describe a tool, Spatio-Temporal Association Mapping of Proteins (STAMP), for identifying protein interactomes via proximity labeling. For a proof-of-principle study, we use cytidine 5'-triphosphate synthase (CTPS) as an example. CTPS, a metabolic enzyme, forms filamentous structures termed cytoophidia in various tissues. We apply STAMP to a variety of developmental stages and tissues in Drosophila including adult ovaries. Using a cell-specific GAL4 driver, we verify that TurboID can biotinylate the bait protein CTPS, making possible the identification of protein-protein interactions (PPIs) in individual cells. Using the wild-type and mutant CTPS as bait proteins, STAMP results in two distinct sets of proximate proteomes. Our results suggest that STAMP is a feasible tool to catch in vivo PPIs in situ at a defined spatiotemporal resolution.


Assuntos
Carbono-Nitrogênio Ligases , Animais , Feminino , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Citoesqueleto/metabolismo , Drosophila/metabolismo , Ovário/metabolismo , Proteoma/metabolismo
13.
Exp Cell Res ; 422(1): 113433, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-36423659

RESUMO

Although most cells are mononuclear, the nucleus can exist in the form of binucleate or even multinucleate to respond to different physiological processes. The male accessory gland of Drosophila is the organ that produces semen, and its main cells are binucleate. Here we observe that CTP synthase (CTPS) forms filamentous cytoophidia in binuclear main cells, primarily located at the cell boundary. In CTPSH355A, a point mutation that destroys the formation of cytoophidia, we find that the nucleation mode of the main cells changes, including mononucleates and vertical distribution of binucleates. Although the overexpression of CTPSH355A can restore the level of CTPS protein, it will neither form cytoophidia nor eliminate the abnormal nucleation pattern. Therefore, our data indicate that there is an unexpected functional link between the formation of cytoophidia and the maintenance of binucleation in Drosophila main cells.


Assuntos
Carbono-Nitrogênio Ligases , Drosophila , Animais , Masculino , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Drosophila/metabolismo
14.
J Med Chem ; 65(24): 16640-16650, 2022 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-36449304

RESUMO

Herein, we report the discovery of a first-in-class chemotype 2-(alkylsulfonamido)thiazol-4-yl)acetamides that act as pan-selective inhibitors of cytidine 5'-triphosphate synthetase (CTPS1/2), critical enzymes in the de novo pyrimidine synthesis pathway. Weak inhibitors identified from a high-throughput screening of 240K compounds have been optimized to a potent, orally active agent, compound 27, which has shown significant pharmacological responses at 10 mg/kg dose BID in a well-established animal model of inflammation.


Assuntos
Carbono-Nitrogênio Ligases , Inibidores Enzimáticos , Animais , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Carbono-Nitrogênio Ligases/metabolismo , Proliferação de Células , Ensaios de Triagem em Larga Escala
15.
Bioessays ; 44(12): e2200128, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36209393

RESUMO

Two enzymes involved in the synthesis of pyrimidine and purine nucleotides, CTP synthase (CTPS) and IMP dehydrogenase (IMPDH), can assemble into a single or very few large filaments called rods and rings (RR) or cytoophidia. Most recently, asymmetric cytoplasmic distribution of organelles during cell division has been described as a decisive event in hematopoietic stem cell fate. We propose that cytoophidia, which could be considered as membrane-less organelles, may also be distributed asymmetrically during mammalian cell division as previously described for Schizosaccharomyces pombe. Furthermore, because each type of nucleotide intervenes in distinct processes (e.g., membrane synthesis, glycosylation, and G protein-signaling), alterations in the rate of synthesis of specific nucleotide types could influence cell differentiation in multiple ways. Therefore, we hypothesize that whether a daughter cell inherits or not CTPS or IMPDH filaments determines its fate and that this asymmetric inheritance, together with the dynamic nature of these structures enables plasticity in a cell population.


Assuntos
Carbono-Nitrogênio Ligases , Schizosaccharomyces , Animais , IMP Desidrogenase/metabolismo , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Diferenciação Celular , Schizosaccharomyces/genética , Nucleotídeos/metabolismo , Mamíferos/metabolismo
16.
Int J Mol Sci ; 23(20)2022 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-36293272

RESUMO

The reconfiguration of the primary metabolism is essential in plant-pathogen interactions. We compared the local metabolic responses of cucumber leaves inoculated with Pseudomonas syringae pv lachrymans (Psl) with those in non-inoculated systemic leaves, by examining the changes in the nicotinamide adenine dinucleotides pools, the concentration of soluble carbohydrates and activities/gene expression of carbohydrate metabolism-related enzymes, the expression of photosynthesis-related genes, and the tricarboxylic acid cycle-linked metabolite contents and enzyme activities. In the infected leaves, Psl induced a metabolic signature with an altered [NAD(P)H]/[NAD(P)+] ratio; decreased glucose and sucrose contents, along with a changed invertase gene expression; and increased glucose turnover and accumulation of raffinose, trehalose, and myo-inositol. The accumulation of oxaloacetic and malic acids, enhanced activities, and gene expression of fumarase and l-malate dehydrogenase, as well as the increased respiration rate in the infected leaves, indicated that Psl induced the tricarboxylic acid cycle. The changes in gene expression of ribulose-l,5-bis-phosphate carboxylase/oxygenase large unit, phosphoenolpyruvate carboxylase and chloroplast glyceraldehyde-3-phosphate dehydrogenase were compatible with a net photosynthesis decline described earlier. Psl triggered metabolic changes common to the infected and non-infected leaves, the dynamics of which differed quantitatively (e.g., malic acid content and metabolism, glucose-6-phosphate accumulation, and glucose-6-phosphate dehydrogenase activity) and those specifically related to the local or systemic response (e.g., changes in the sugar content and turnover). Therefore, metabolic changes in the systemic leaves may be part of the global effects of local infection on the whole-plant metabolism and also represent a specific acclimation response contributing to balancing growth and defense.


Assuntos
Carbono-Nitrogênio Ligases , Cucumis sativus , Pseudomonas syringae/fisiologia , Cucumis sativus/genética , Cucumis sativus/metabolismo , Carbono/metabolismo , Fosfoenolpiruvato Carboxilase/genética , beta-Frutofuranosidase/metabolismo , Malato Desidrogenase/metabolismo , Rafinose/metabolismo , Trealose/metabolismo , NAD/metabolismo , Fumarato Hidratase , Glucose-6-Fosfato/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Folhas de Planta/metabolismo , Fotossíntese/fisiologia , Metabolismo dos Carboidratos , Sacarose/metabolismo , Fosfatos/metabolismo , Oxigenases/metabolismo , Inositol/metabolismo , Carbono-Nitrogênio Ligases/metabolismo , Niacinamida/metabolismo , Adenina/metabolismo , Glucose/metabolismo
17.
Exp Cell Res ; 420(1): 113337, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36087798

RESUMO

CTP synthase (CTPS), a metabolic enzyme responsible for the de novo synthesis of CTP, can form filamentous structures termed cytoophidia, which are evolutionarily conserved from bacteria to humans. Here we used Schizosaccharomyces pombe to study the cytoophidium assembly regulation by ubiquitination. We tested the CTP synthase's capacity to be post-translationally modified by ubiquitin or be affected by the ubiquitination state of the cell and showed that ubiquitination is important for the maintenance of the CTPS filamentous structure in fission yeast. We have identified proteins which are in complex with CTPS, including specific ubiquitination regulators which significantly affect CTPS filamentation, and mapped probable ubiquitination targets on CTPS. Furthermore, we discovered that a cohort of deubiquitinating enzymes is important for the regulation of cytoophidium's filamentous morphology. Our study provides a framework for the analysis of the effects that ubiquitination and deubiquitination have on the formation of cytoophidia.


Assuntos
Carbono-Nitrogênio Ligases , Schizosaccharomyces , Humanos , Carbono-Nitrogênio Ligases/metabolismo , Citidina Trifosfato/metabolismo , Enzimas Desubiquitinantes/metabolismo , Schizosaccharomyces/metabolismo , Ubiquitinação , Ubiquitinas/metabolismo
18.
Cell Mol Life Sci ; 79(10): 534, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-36180607

RESUMO

Tissue architecture determines its unique physiology and function. How these properties are intertwined has remained unclear. Here we show that the metabolic enzyme CTP synthase (CTPS) form filamentous structures termed cytoophidia along the adipocyte cortex in Drosophila adipose tissue. Loss of cytoophidia, whether due to reduced CTPS expression or a point mutation that specifically abrogates its polymerization ability, causes impaired adipocyte adhesion and defective adipose tissue architecture. Moreover, CTPS influences integrin distribution and dot-like deposition of type IV collagen (Col IV). Col IV-integrin signaling reciprocally regulates the assembly of cytoophidia in adipocytes. Our results demonstrate that a positive feedback signaling loop containing both cytoophidia and integrin adhesion complex couple tissue architecture and metabolism in Drosophila adipose tissue.


Assuntos
Carbono-Nitrogênio Ligases , Colágeno Tipo IV , Animais , Tecido Adiposo/metabolismo , Carbono-Nitrogênio Ligases/química , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Drosophila/metabolismo , Integrinas
19.
Microbiol Res ; 263: 127142, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35940107

RESUMO

Urea is one of the major components of the human urine and its breakdown by the uropathogens occurs mainly through the activity of the enzyme urease. However, a few reports suggest the presence of an alternate enzyme system for urea breakdown namely urea carboxylase (UC) and allophanate hydrolase (AH). We have previously reported the UC and AH system in the genome of a urease-negative uropathogen Kalamiella piersonii YU22 of the novel genus Kalamiella (reclassified recently as Pantoea).To validate the UC and AH activity in the presence of urea, we investigated the growth and urea utilization patterns of this bacterium. Growth kinetics, variations in media pH, NH4-N generation and UC and AH gene expressions were probed using urea-containing media. YU22 was able to grow in M9 media containing urea and increase the pH of the media due to the urea breakdown. Further, significantly higher concentrations of extracellular NH4-N (p < 0.001) was also detected in the cultures along with over-expression of UC and AH genes. The bacterium formed biofilm, and displayed swimming and swarming motilities in presence of urea. Additional glucose supply to urea boosted the colonization but ameliorated the media alkalization and ammonification through suppression of gene expressions encoding UC and AH. These results show that the urease-negative strain YU22 can utilize the UC and AH system for urea metabolism. We propose to further investigate the UC and AH system in other urease-negative uropathogens and its implications for pathogenicity and urinary tract colonization.


Assuntos
Alofanato Hidrolase , Carbono-Nitrogênio Ligases , Gammaproteobacteria , Alofanato Hidrolase/genética , Alofanato Hidrolase/metabolismo , Carbono-Nitrogênio Ligases/metabolismo , Gammaproteobacteria/metabolismo , Humanos , Ureia/metabolismo , Urease/genética
20.
Biomolecules ; 12(7)2022 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-35883427

RESUMO

Glutamine amidotransferases, enzymes that transfer nitrogen from Gln to various cellular metabolites, are modular, with the amidotransferase (GATase) domain hydrolyzing Gln, generating ammonia and the acceptor domain catalyzing the addition of nitrogen onto its cognate substrate. GMP synthetase (GMPS), an enzyme in the de novo purine nucleotide biosynthetic pathway, is a glutamine amidotransferase that catalyzes the synthesis of GMP from XMP. The reaction involves activation of XMP though adenylation by ATP in the ATP pyrophosphatase (ATPPase) active site, followed by channeling and attack of NH3 generated in the GATase pocket. This complex chemistry entails co-ordination of activity across the active sites, allosteric activation of the GATase domain to modulate Gln hydrolysis and channeling of ammonia from the GATase to the acceptor active site. Functional GMPS dimers associate through the dimerization domain. The crystal structure of the Gln-bound complex of Plasmodium falciparum GMPS (PfGMPS) for the first time revealed large-scale domain rotation to be associated with catalysis and leading to the juxtaposition of two otherwise spatially distal cysteinyl (C113/C337) residues. In this manuscript, we report on an unusual structural variation in the crystal structure of the C89A/C113A PfGMPS double mutant, wherein a larger degree of domain rotation has led to the dissociation of the dimeric structure. Furthermore, we report a hitherto overlooked signature motif tightly related to catalysis.


Assuntos
Amônia , Carbono-Nitrogênio Ligases , Trifosfato de Adenosina/química , Amônia/metabolismo , Carbono-Nitrogênio Ligases/metabolismo , Catálise , Glutamina/metabolismo , Cinética , Nitrogênio , Conformação Proteica
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