RESUMEN
We designed metabolically engineered non-pathogenic strains of Escherichia coli to produce unsulfated chondroitin with and without chondroitin lyase to produce the chondroitin polymer or its related oligosaccharides. Chondroitin was synthesized using chondroitin synthase KfoC and chondroitin was degraded using Pl35, a chondroitin lyase from Pedobacter heparinus. Pl35 behaved as a true endo-enzyme generating a large panel of oligosaccharides ranging from trimers to 18-mers instead of the di- and tetramers obtained with most chondroitin lyases. Two series of oligosaccharides were characterized, sharing an unsaturated uronic acid (4-deoxy-α-L-threo-hex-4-enepyranosyluronic acid, â³UA) residue at their non-reducing end. The major "even-numbered" series was characterized by a terminal reducing N-acetylgalactosaminyl residue. The minor "odd-numbered" series oligosaccharides carried a terminal reducing glucuronic acid residue instead. Cultures were conducted in fed-batch conditions, and led to the production of up to 10 g L-1 chondroitin or chondroitin oligosaccharides. All products were purified and fully characterized using NMR and mass spectrometry analyses. This is the first report of the microbial production of large chondro-oligosaccharides.
Asunto(s)
Condroitín , Escherichia coli , Oligosacáridos , Escherichia coli/metabolismo , Escherichia coli/genética , Condroitín/química , Condroitín/metabolismo , Oligosacáridos/química , Oligosacáridos/síntesis química , Pedobacter/enzimología , Pedobacter/metabolismo , Condroitín Liasas/metabolismo , Condroitín Liasas/química , Ligasas de Carbono-Nitrógeno/metabolismo , Ligasas de Carbono-Nitrógeno/química , Ligasas de Carbono-Nitrógeno/genética , Ingeniería Metabólica , N-AcetilgalactosaminiltransferasasRESUMEN
The final step in the de novo synthesis of cytidine 5'-triphosphate (CTP) is catalyzed by CTP synthase (CTPS), which can form cytoophidia in all three domains of life. Recently, we have discovered that CTPS binds to ribonucleotides (NTPs) to form filaments, and have successfully resolved the structures of Drosophila melanogaster CTPS bound with NTPs. Previous biochemical studies have shown that CTPS can bind to deoxyribonucleotides (dNTPs) to produce 2'-deoxycytidine-5'-triphosphate (dCTP). However, the structural basis of CTPS binding to dNTPs is still unclear. In this study, we find that Drosophila CTPS can also form filaments with dNTPs. Using cryo-electron microscopy, we are able to resolve the structure of Drosophila melanogaster CTPS bound to dNTPs with a resolution of up to 2.7 Å. By combining these structural findings with biochemical analysis, we compare the binding and reaction characteristics of NTPs and dNTPs with CTPS. Our results indicate that the same enzyme can act bifunctionally as CTP/dCTP synthase in vitro, and provide a structural basis for these activities.
Asunto(s)
Ligasas de Carbono-Nitrógeno , Microscopía por Crioelectrón , Citidina Trifosfato , Drosophila melanogaster , Animales , Ligasas de Carbono-Nitrógeno/química , Ligasas de Carbono-Nitrógeno/metabolismo , Ligasas de Carbono-Nitrógeno/genética , Citidina Trifosfato/metabolismo , Citidina Trifosfato/química , Drosophila melanogaster/enzimología , Modelos Moleculares , Unión Proteica , Conformación Proteica , Nucleótidos de Desoxicitosina/metabolismo , Nucleótidos de Desoxicitosina/químicaRESUMEN
Methods that identify protein-protein interactions are essential for understanding molecular mechanisms controlling biological systems. Proximity-dependent labeling has proven to be a valuable method for revealing protein-protein interaction networks in living cells. A mutant form of the biotin protein ligase enzyme from Aquifex aeolicus (BioID2) underpins this methodology by producing biotin that is attached to proteins that enter proximity to it. This labels proteins for capture, extraction, and identification. In this chapter, we present a toolkit for BioID2 specifically adapted for use in E. coli, exemplified by the chemotaxis protein CheA. We have created plasmids containing BioID2 as expression cassettes for proteins (e.g., CheA) fused to BioID2 at either the N or C terminus, optimized with an 8 × GGS linker. We provide a methodology for expression and verification of CheA-BioID2 fusion proteins in E. coli cells, the in vivo biotinylation of interactors by protein-BioID2 fusions, and extraction and analysis of interacting proteins that have been biotinylated.
Asunto(s)
Biotinilación , Escherichia coli , Mapeo de Interacción de Proteínas , Escherichia coli/genética , Escherichia coli/metabolismo , Mapeo de Interacción de Proteínas/métodos , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Biotina/metabolismo , Mapas de Interacción de Proteínas , Coloración y Etiquetado/métodos , Plásmidos/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/genética , Ligasas de Carbono-Nitrógeno/metabolismo , Ligasas de Carbono-Nitrógeno/genéticaRESUMEN
Proteins serve as the primary executors of cellular activities in organisms, and thus investigating the subcellular localization and interactions of proteins is crucial for understanding protein functions and elucidating the molecular mechanisms in organisms. Proximity labeling is a recently developed effective method for detecting protein-protein interactions in live cells. Compared with the conventional methods for studying protein-protein interactions, proximity labeling demonstrates high sensitivity, strong specificity, and low background and is widely employed in the research of protein-protein interactions between pathogens and hosts. This article reviews the recent progress in the development and applications of the biotin ligase BirA and its mutants and elucidates the functioning principles of several classical biotin ligases. This review aims to clarify the role of proximity labeling based on BirA and its mutants in identifying protein-protein interactions between pathogens and hosts.
Asunto(s)
Ligasas de Carbono-Nitrógeno , Interacciones Huésped-Patógeno , Mutación , Ligasas de Carbono-Nitrógeno/metabolismo , Ligasas de Carbono-Nitrógeno/genética , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Biotina/metabolismo , Humanos , Mapeo de Interacción de Proteínas , Escherichia coli/genética , Escherichia coli/metabolismoRESUMEN
Protein-protein interaction studies using proximity labeling techniques, such as biotin ligase-based BioID, have become integral in understanding cellular processes. Most studies utilize conventional 2D cell culture systems, potentially missing important differences in protein behavior found in 3D tissues. In this study, we investigated the protein-protein interactions of a protein, Bcl-2 Agonist of cell death (BAD), and compared conventional 2D culture conditions to a 3D system, wherein cells were embedded within a 3D extracellular matrix (ECM) mimic. Using BAD fused to the engineered biotin ligase miniTurbo (BirA*), we identified both overlapping and distinct BAD interactomes under 2D and 3D conditions. The known BAD binding proteins 14-3-3 isoforms and Bcl-XL interacted with BAD in both 2D and 3D. Of the 131 BAD-interactors identified, 56% were specific to 2D, 14% were specific to 3D, and 30% were common to both conditions. Interaction network analysis demonstrated differential associations between 2D and 3D interactomes, emphasizing the impact of the culture conditions on protein interactions. The 2D-3D overlap interactome encapsulated the apoptotic program, which is a well-known role of BAD. The 3D unique pathways were enriched in ECM signaling, suggestive of hitherto unknown functions for BAD. Thus, exploring protein-protein interactions in 3D provides novel clues into cell behavior. This exciting approach has the potential to bridge the knowledge gap between tractable 2D cell culture and organoid-like 3D systems.
Asunto(s)
Técnicas de Cultivo de Célula , Proteína Letal Asociada a bcl , Humanos , Proteína Letal Asociada a bcl/metabolismo , Técnicas de Cultivo de Célula/métodos , Mapas de Interacción de Proteínas , Matriz Extracelular/metabolismo , Mapeo de Interacción de Proteínas/métodos , Proteínas 14-3-3/metabolismo , Ligasas de Carbono-Nitrógeno/metabolismo , Ligasas de Carbono-Nitrógeno/genética , Unión Proteica , Proteína bcl-X/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas RepresorasRESUMEN
A host of metabolic enzymes reversibly self-assemble to form membrane-less, intracellular filaments under normal physiological conditions and in response to stress. Often, these enzymes reside at metabolic control points, suggesting that filament formation affords an additional regulatory mechanism. Examples include cytidine-5'-triphosphate (CTP) synthase (CTPS), which catalyzes the rate-limiting step for the de novo biosynthesis of CTP; inosine-5'-monophosphate dehydrogenase (IMPDH), which controls biosynthetic access to guanosine-5'-triphosphate (GTP); and ∆1-pyrroline-5-carboxylate (P5C) synthase (P5CS) that catalyzes the formation of P5C, which links the Krebs cycle, urea cycle, and proline metabolism. Intriguingly, CTPS can exist in co-assemblies with IMPDH or P5CS. Since GTP is an allosteric activator of CTPS, the association of CTPS and IMPDH filaments accords with the need to coordinate pyrimidine and purine biosynthesis. Herein, a hypothesis is presented furnishing a biochemical connection underlying co-assembly of CTPS and P5CS filaments - potent inhibition of CTPS by glutamate γ-semialdehyde, the open-chain form of P5C.
Asunto(s)
Ligasas de Carbono-Nitrógeno , IMP Deshidrogenasa , Animales , Humanos , Ligasas de Carbono-Nitrógeno/metabolismo , Ligasas de Carbono-Nitrógeno/genética , Citidina Trifosfato/metabolismo , Guanosina Trifosfato/metabolismo , IMP Deshidrogenasa/metabolismoRESUMEN
Phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) is a de novo purine biosynthetic enzyme. It has been found to be overexpressed in various types of cancer and is related to cell proliferation, invasion, the epithelial-mesenchymal transition, and efficient tumor growth. In this study, we describe a rat monoclonal antibody (mAb) 6A10, which was generated as an antigen of human PAICS. This mAb was generated to interact with the N-terminal region of human PAICS and was found to recognize endogenous PAICS enzymes in several cancer cells. Our results also indicated that it can recognize monkey and dog PAICS, which possess the same amino acid sequence in the antigenic region as human PAICS, but it does not recognize rat and mouse PAICS. Furthermore, our data indicated that this mAb is suitable for immunoprecipitation and immunoblotting use for several cancer cell lines. We, therefore, anticipate that mAb 6A10 will be useful for functional analyses of human PAICS in several cancers and for diagnosis of malignant transformation.
Asunto(s)
Anticuerpos Monoclonales , Humanos , Animales , Ratas , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/biosíntesis , Ratones , Perros , Purinas/inmunología , Línea Celular Tumoral , Ligasas de Carbono-Nitrógeno/inmunología , Ligasas de Carbono-Nitrógeno/genética , Especificidad de Anticuerpos/inmunología , Neoplasias/inmunología , Neoplasias/patología , Péptido SintasasRESUMEN
Staphylococcus aureus (S. aureus) presents a significant challenge in both nosocomial and community settings due to its pathogenicity. The emergence of drug-resistant strains exacerbates S. aureus infections, leading to increased mortality rates. PyrG, a member of the cytidine triphosphate (CTP) synthase family, serves as a crucial therapeutic target against S. aureus due to the pivotal role of CTP in cellular metabolism. However, the structural and mechanistic details of S. aureus PyrG remains unknown. Here, we successfully expressed and purified monomeric PyrG. Mutational experiments were conducted based on the results of molecular docking. Based on the results of the molecular docking, we carried out mutation experiments and found that Q386A dramatically decreased the CTP synthase activity compared to the wild-type protein, while Y54A almost completely abolished the activity. Exposure of S. aureus to the kinase inhibitor crizotinib increased expression of gene pyrG. Our results identify the two key sites on PyrG for the CTP synthase activity, and present PyrG gene expression increased during the treatment of crizotinib, which may eventually provide valuable guidance for the development of new drugs against S. aureus infections.
Asunto(s)
Proteínas Bacterianas , Ligasas de Carbono-Nitrógeno , Staphylococcus aureus , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/química , Ligasas de Carbono-Nitrógeno/metabolismo , Ligasas de Carbono-Nitrógeno/aislamiento & purificación , Staphylococcus aureus/enzimología , Staphylococcus aureus/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/biosíntesis , Expresión Génica , Simulación del Acoplamiento Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/biosíntesisRESUMEN
Biotin (vitamin B7, or vitamin H) is a water-soluble B-vitamin that functions as a cofactor for carboxylases, i.e., enzymes involved in the cellular metabolism of fatty acids and amino acids and in gluconeogenesis; moreover, as reported, biotin may be involved in gene regulation. Biotin is not synthesized by human cells, but it is found in food and is also produced by intestinal bacteria. Biotin status/homeostasis in human individuals depends on several factors, including efficiency/deficiency of the enzymes involved in biotin recycling within the human organism (biotinidase, holocarboxylase synthetase), and/or effectiveness of intestinal uptake, which is mainly accomplished through the sodium-dependent multivitamin transporter. In the last years, administration of biotin at high/"pharmacological" doses has been proposed to treat specific defects/deficiencies and human disorders, exhibiting mainly neurological and/or dermatological symptoms and including biotinidase deficiency, holocarboxylase synthetase deficiency, and biotin-thiamine-responsive basal ganglia disease. On the other hand, according to warnings of the Food and Drug Administration, USA, high biotin levels can affect clinical biotin-(strept)avidin assays and thus lead to false results during quantification of critical biomarkers. In this review article, recent findings/advancements that may offer new insight in the abovementioned research fields concerning biotin will be presented and briefly discussed.
Asunto(s)
Biotina , Deficiencia de Biotinidasa , Biotinidasa , Homeostasis , Humanos , Biotina/metabolismo , Deficiencia de Biotinidasa/metabolismo , Deficiencia de Biotinidasa/diagnóstico , Deficiencia de Biotinidasa/genética , Deficiencia de Biotinidasa/tratamiento farmacológico , Biotinidasa/metabolismo , Biotinidasa/genética , Deficiencia de Holocarboxilasa Sintetasa/metabolismo , Ligasas de Carbono-Nitrógeno/metabolismo , Ligasas de Carbono-Nitrógeno/genética , Animales , Ataxia/metabolismo , Ataxia/genética , Enfermedades de los Ganglios BasalesRESUMEN
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.
Asunto(s)
Acetil-CoA Carboxilasa , Ligasas de Carbono-Nitrógeno , Chloroflexus , Acetil-CoA Carboxilasa/metabolismo , Acetil-CoA Carboxilasa/genética , Acetil-CoA Carboxilasa/química , Ligasas de Carbono-Nitrógeno/metabolismo , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/química , Chloroflexus/genética , Chloroflexus/metabolismo , Chloroflexus/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/enzimología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Biotina/metabolismo , Biotina/biosíntesis , Malonil Coenzima A/metabolismo , Acetilcoenzima A/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Acido Graso Sintasa Tipo IIRESUMEN
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.
Asunto(s)
Biotina , G-Cuádruplex , Humanos , Biotina/metabolismo , Unión Proteica , Factores de Empalme de ARN/metabolismo , Ligasas de Carbono-Nitrógeno/metabolismo , Ligasas de Carbono-Nitrógeno/genética , Empalme del ARN , Células HEK293 , Proteínas de Unión al ARN/metabolismo , Células HeLaRESUMEN
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.
Asunto(s)
Ligasas de Carbono-Nitrógeno , Proteínas de Escherichia coli , Animales , Humanos , Biotinilación , Biotina/química , Biotina/metabolismo , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/química , Ligasas de Carbono-Nitrógeno/metabolismo , Proteínas/metabolismo , Escherichia coli/metabolismo , Ligasas/genética , Ligasas/metabolismo , Bacterias/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mamíferos/metabolismoRESUMEN
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.
Asunto(s)
Ligasas de Carbono-Nitrógeno , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/metabolismo , Uridina Trifosfato/metabolismo , Proliferación Celular , Ciclo Celular , Línea CelularRESUMEN
(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.
Asunto(s)
Alcaloides , Bencilisoquinolinas , Ligasas de Carbono-Nitrógeno , Papaver , Alcaloides/metabolismo , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/metabolismo , Codeína , Papaver/genética , Papaver/metabolismoRESUMEN
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.
Asunto(s)
Ligasas de Carbono-Nitrógeno , Animales , Femenino , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/metabolismo , Citoesqueleto/metabolismo , Drosophila/metabolismo , Ovario/metabolismo , Proteoma/metabolismoRESUMEN
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.
Asunto(s)
Ligasas de Carbono-Nitrógeno , Drosophila , Animales , Masculino , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/metabolismo , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Drosophila/metabolismoRESUMEN
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.
Asunto(s)
Ligasas de Carbono-Nitrógeno , Schizosaccharomyces , Animales , IMP Deshidrogenasa/metabolismo , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/metabolismo , Diferenciación Celular , Schizosaccharomyces/genética , Nucleótidos/metabolismo , Mamíferos/metabolismoRESUMEN
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.
Asunto(s)
Ligasas de Carbono-Nitrógeno , Colágeno Tipo IV , Animales , Tejido Adiposo/metabolismo , Ligasas de Carbono-Nitrógeno/química , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/metabolismo , Drosophila/metabolismo , IntegrinasRESUMEN
Proximity-dependent biotinylation (PDB) combined with mass spectrometry analysis has established itself as a key technology to study protein-protein interactions in living cells. A widespread approach, BioID, uses an abortive variant of the E. coli BirA biotin protein ligase, a quite bulky enzyme with slow labeling kinetics. To improve PDB versatility and speed, various enzymes have been developed by different approaches. Here we present a small-size engineered enzyme: ultraID. We show its practical use to probe the interactome of Argonaute-2 after a 10 min labeling pulse and expression at physiological levels. Moreover, using ultraID, we provide a membrane-associated interactome of coatomer, the coat protein complex of COPI vesicles. To date, ultraID is the smallest and most efficient biotin ligase available for PDB and offers the possibility of investigating interactomes at a high temporal resolution.
Asunto(s)
Ligasas de Carbono-Nitrógeno , Proteínas de Escherichia coli , Biotina , Biotinilación , Ligasas de Carbono-Nitrógeno/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Espectrometría de Masas/métodos , Proteínas RepresorasRESUMEN
CTP synthase (CTPS) catalyzes the final step of de novo synthesis of the nucleotide CTP. In 2010, CTPS has been found to form filamentous structures termed cytoophidia in Drosophila follicle cells and germline cells. Subsequently, cytoophidia have been reported in many species across three domains of life: bacteria, eukaryotes and archaea. Forming cytoophidia appears to be a highly conserved and ancient property of CTPS. To our surprise, here we find that polar cells and stalk cells, two specialized types of cells composing Drosophila interfollicular stalks, do not possess obvious cytoophidia. We show that Myc level is low in these two types of cells. Treatment with a glutamine analog, 6-diazo-5-oxo-l-norleucine (DON), increases cytoophidium assembly in main follicle cells, but not in polar cells or stalk cells. Moreover, overexpressing Myc induces cytoophidium formation in stalk cells. When CTPS is overexpressed, cytoophidia can be observed both in stalk cells and polar cells. Our findings provide an interesting paradigm for the in vivo study of cytoophidium assembly and disassembly among different populations of follicle cells.