CTP synthase forms cytoophidia in archaea

CTP synthase(CTPS) is an important metabolic enzyme that catalyzes the rate-Iimiting reaction of nucleotide CrP de novo synthesis.Since 2010,a series of studies have demonstrated that CTPS can form filamentous structures in bacteria and eukaryotes,which are termed cytoophidia.However,it is unknown whether cytoophidia exist in the third domain of life,archaea.Using Haloarcula hispanica as a model system,here we demonstrate that CTPS forms distinct intracellular compartments in archaea.Under stimulated emission depletion microscopy,we find that the structures of H.hispanica CTPS are elongated,similar to cytoophidia in bacteria and eukaryotes.When Haloarcula cells are cultured in lowsalt medium,the occurrence of cytoophidia increases dramatically.In addition,treatment of H.hispanica with a glutamine analog or overexpression of CTPS can promote cytoophidium assembly.Our study reveals that CTPS can fo rm cytoophidia in all three domains of life,suggesting that forming cytoophidia is an ancient property of CTPS.     

mTOR-S6K1 pathway mediates cytoophidium assembly

CTP synthase (CTPS), the rate-limiting enzyme in de novo CTP biosynthesis, has been demonstrated to assemble into evolutionarily conserved filamentous structures, termed cytoophidia, in Drosophila, bacteria, yeast and mammalian cells. However, the regulation and function of the cytoophidium remain elusive. Here, we provide evidence that the mechanistic target of rapamycin (mTOR) pathway controls cytoophidium assembly in mammalian and Drosophila cells. In mammalian cells, we find that inhibition of mTOR pathway attenuates cytoophidium formation. Moreover, CTPS cytoophidium assembly appears to be dependent on the mTOR complex 1 (mTORC1) mainly. In addition, knockdown of the mTORC1 downstream target S6K1 can inhibit cytoophidium formation, while overexpression of the constitutively active S6K1 reverses mTOR knockdown-induced cytoophidium disassembly. Finally, reducing mTOR protein expression results in a decrease of the length of cytoophidium in Drosophila follicle cells. Therefore, our study connects CTPS cytoophidium formation with the mTOR signaling pathway.     

Intracellular compartmentation of CTP synthase in Drosophila

Compartmentation is essential for the localization of biological processes within a eukaryotic cell. ATP synthase localizes to organelles such as mitochondria and chloroplasts. By contrast, little is known about the subcellular distribution of CTP synthase, the critical enzyme in the production of CTP, a high-energy molecule similar to ATP. Here I describe the identification of a novel intracellular structure con-taining CTP synthase, termed the cytoophidium, in Drosophila cells. I find that cytoophidia are present in all major cell types in the ovary and exist in a wide range of tissues such as brain, gut, trachea, testis, accessory gland, salivary gland and lymph gland. In addition, I find CTP synthase-containing cytoophidia in other fruit fly species. The observation of compartmentation of CTP synthase now permits a broad range of questions to be addressed concerning not only the structure and function of cytoophidia but also the organization and regulation of CTP synthesis.     

Temperature-sensitive cytoophidium assembly in Schizosaccharomyces pombe

The metabolic enzyme CTP synthase(CTPS) is able to compartmentalize into filaments,termed cytoophidia,in a variety of organisms including bacteria,budding yeast,fission yeast,fruit flies and mammals.A previous study in budding yeast shows that the filament-forming process of CTPS is not sensitive to temperature shift.Here we study CTPS filamentation in the fission yeast Schizosaccharomyces pombe.To our surprise,we find that both the length and the occurrence of cytoophidia in S.pombe decrease upon cold shock or heat shock.The temperature-dependent changes of cytoophidia are fast and reversible.Taking advantage of yeast genetics,we demonstrate that heat-shock proteins are required for cytoophidium assembly in S.pombe.Temperature sensitivity of cytoophidia makes S.pombe an attractive model system for future investigations of this novel membraneless organelle.     

Intracellular compartmentation of CTP synthase in Drosophila

Compartmentation is essential for the localization of biological processes within a eukaryotic cell.ATP synthase localizes to organelles such as mitochondria and chloroplasts.By contrast,little is known about the subcellular distribution of CTP synthase,the critical enzyme in the production of CTP,a high-energy molecule similar to ATP.Here I describe the identification of a novel intracellular structure con-taining CTP synthase,termed the cytoophidium,in Drosophila cells.I find that cytoophidia are present ...     

The Interplay between Myc and CTP Synthase in Drosophila

CTP synthase (CTPsyn) is essential for the biosynthesis of pyrimidine nucleotides. It has been shown that CTPsyn is incorporated into a novel cytoplasmic structure which has been termed the cytoophidium. Here, we report that Myc regulates cytoophidium formation during Drosophila oogenesis. We have found that Myc protein levels correlate with cytoophidium abundance in follicle epithelia. Reducing Myc levels results in cytoophidium loss and small nuclear size in follicle cells, while overexpression of Myc increases the length of cytoophidia and the nuclear size of follicle cells. Ectopic expression of Myc induces cytoophidium formation in late stage follicle cells. Furthermore, knock-down of CTPsyn is sufficient to suppress the overgrowth phenotype induced by Myc overexpression, suggesting CTPsyn acts downstream of Myc and is required for Myc-mediated cell size control. Taken together, our data suggest a functional link between Myc, a renowned oncogene, and the essential nucleotide biosynthetic enzyme CTPsyn.     

The atlas of cytoophidia in Drosophila larvae

In 2010, cytidine 50-triphosphate synthase(CTPS) was reported to form the filamentous or serpentine structure in Drosophila, which we termed the cytoophidium. In the last decade, CTPS filaments/cytoophidia have been found in bacteria, budding yeast, human cells, mice, fission yeast, plants, and archaea,indicating that this mechanism is highly conserved in evolution. In addition to CTPS, other metabolic enzymes have been identified to have the characteristics of forming cytoophidia or similar advanced structures, demonstrating that this is a basic strategy of cells. Nevertheless, our understanding of the physiological function of the cytoophidium remains incomplete and elusive. Here, we took the larva of Drosophila melanogaster as a model to systematically describe the localization and distribution of cytoophidia in different tissues during larval development. We found that the distribution pattern of CTPS cytoophidia is dynamic and heterogenic in larval tissues. Our study provides a road map for further understanding of the function and regulatory mechanism of cytoophidia.     

川楝素对人胃癌细胞MKN-45中CTPS细胞蛇形成的影响及其机制*

目的:本研究旨在对川楝素(TSN)与胃癌细胞MKN-45中CTPS细胞蛇形成的联系进行初步探究。方法:以人胃癌细胞MKN-45为实验材料,设置7个处理组分别为:0、20、40、60、80、100、120 nmol/L TSN。每组3次重复,分别作用24 h、48 h、72 h,利用CCK-8法检测川楝素对MKN-45细胞增殖抑制作用,使用免疫荧光检测之后再通过激光共聚焦显微镜观察细胞内CTPS细胞蛇形态,qRT-PCR检测川楝素对MYC基因表达的影响。另外设置2个处理组为1 mmol/L DON和1 mmol/L MPA,每组3次重复,作用6 h然后采用免疫荧光检测细胞蛇形态。结果:免疫荧光结果显示,分别利用1 mmol/L DON和1 mmol/L MPA 处理MKN-45细胞后CTPS形成丝状的细胞蛇结构,意味着该细胞具有形成CTPS细胞蛇的能力;川楝素处理组的细胞增殖率明显低于0 nmol/L TSN组(P＜ 0.01);免疫荧光结果显示80 nmol/L的川楝素作用72 h时MKN-45细胞中CTPS细胞蛇形成数量最多;qRT-PCR检测结果显示,80 nmol/L川楝素作用24 h细胞内MYC表达明显降低(P＜0.05),48 h后细胞内MYC的表达量明显增多(P＜0.01)随后表达降低。结论:川楝素可能通过调节MYC的表达影响细胞内细胞蛇的组装。     

The proline synthesis enzyme P5CS forms cytoophidia in Drosophila

Compartmentation of enzymes via filamentation has arisen as a mechanism for the regulation of metabolism.In 2010,three groups independently reported that CTP synthase (CTPS) can assemble into a filamentous structure termed the cytoophidium.In searching for CTPS-interacting proteins,here we perform a yeast two-hybrid screening of Drosophila proteins and identify a putative CTPS-interacting protein,△~1-pyrroline-5-carboxylate synthase (P5CS).Using the Drosophila follicle cell as the in vivo model,we confirm that P5CS forms cytoophidia,which are associated with CTPS cytoophidia.Overexpression of P5CS increases the length of CTPS cytoophidia.Conversely,filamentation of CTPS affects the morphology of P5CS cytoophid ia.Finally,in vitro analyses confirm the filament-fo rming property of P5CS.Our work links CTPS with P5CS,two enzymes involved in the rate-limiting steps in pyrimidine and proline biosynthesis,respectively.     

Competition between ammonia derived from internal glutamine hydrolysis and hydroxylamine present in the solution for incorporation into UTP as catalysed by Lactococcus lactis CTP synthase

CTP synthase catalyses the reaction: glutamine+UTP+ATP --> glutamate+CTP+ADP+P(i). The reaction is greatly stimulated by the allosteric binding of GTP. In addition to glutamine that is hydrolysed by the enzyme to ammonia and glutamate, CTP synthase will also utilise external sources of amino donors such as NH(4)Cl. This reaction is no longer dependent on allosteric activation by GTP. Hydroxylamine is also a substrate for Lactococcus lactis CTP synthase and results in the formation of N4-OH CTP. This product has the feature that it absorbs at 300nm where CTP absorption was shown to be greatly reduced and enabled the determination of N4-OH CTP formation in the presence of CTP synthesis derived from glutamine hydrolysis. Differences in initial rates determined for the hydroxylamine dependent reaction at 291nm in the presence and absence of glutamine and GTP were ascribed to simultaneous CTP and N4-OH CTP synthesis in the presence of these compounds. A characterisation of the apparent inhibition by GTP and glutamine of N4-OH CTP synthesis determined at 300nm showed that glutamine dependent CTP synthesis occurs at a rate of about 60% of that in the absence of hydroxylamine. GTP dependent inhibition of the ammonium chloride dependent reaction of L. lactis CTP synthase by the glutamine analog glutamate gamma-semialdehyde showed a partial inhibition with a maximum inhibition of about 60%. These results are interpreted in terms of a "half of the sites" mechanism for glutamine hydrolysis on CTP synthase.     

CTP Synthase Is Required for Optic Lobe Homeostasis in Drosophila

CTP synthase(CTPsyn) is a metabolic enzyme responsible for the de novo synthesis of the nucleotide CTP. Several recent studies have shown that CTPsyn forms filamentous subcellular structures known as cytoophidia in bacteria, yeast, fruit flies and humans. However, it remains elusive whether and how CTPsyn and cytoophidia play a role during development. Here, we show that cytoophidia are abundant in the neuroepithelial stem cells in Drosophila optic lobes. Optic lobes are underdeveloped in CTPsyn mutants as well as in CTPsyn RNAi. Moreover, overexpressing CTPsyn impairs the development of optic lobes, specifically by blocking the transition from neuroepithelium to neuroblast. Taken together, our results indicate that CTPsyn is critical for optic lobe homeostasis in Drosophila.     

Steady-state kinetics of the glutaminase reaction of CTP synthase from Lactococcus lactis. The role of the allosteric activator GTP incoupling between glutamine hydrolysis and CTP synthesis.

CTP synthase catalyzes the reaction glutamine + UTP + ATP --> glutamate + CTP + ADP + Pi. The rate of the reaction is greatly enhanced by the allosteric activator GTP. We have studied the glutaminase half-reaction of CTP synthase from Lactococcus lactis and its response to the allosteric activator GTP and nucleotides that bind to the active site. In contrast to what has been found for the Escherichia coli enzyme, GTP activation of the L. lactis enzyme did not result in similar kcat values for the glutaminase activity and glutamine hydrolysis coupled to CTP synthesis. GTP activation of the glutaminase reaction never reached the levels of GTP-activated CTP synthesis, not even when the active site was saturated with UTP and the nonhydrolyzeable ATP-binding analog adenosine 5'-[gamma-thio]triphosphate. Furthermore, under conditions where the rate of glutamine hydrolysis exceeded that of CTP synthesis, GTP would stimulate CTP synthesis. These results indicate that the L. lactis enzyme differs significantly from the E. coli enzyme. For the E. coli enzyme, activation by GTP was found to stimulate glutamine hydrolysis and CTP synthesis to the same extent, suggesting that the major function of GTP binding is to activate the chemical steps of glutamine hydrolysis. An alternative mechanism for the action of GTP on L. lactis CTP synthase is suggested. Here the binding of GTP to the allosteric site promotes coordination of the phosphorylation of UTP and hydrolysis of glutamine for optimal efficiency in CTP synthesis rather than just acting to increase the rate of glutamine hydrolysis itself.     

Assembly of IMPDH2-Based,CTPS-Based,and Mixed Rod/Ring Structures Is Dependent on Cell Type and Conditions of Induction

Inhibition of guanosine triphosphate(GTP)and cytidine triphosphate(CTP)biosynthetic pathways induces cells to assemble rod/ring(RR)structures,also named cytoophidia,which consist of the enzymes cytidine triphosphate synthase(CTPS)and inosine-50-monophosphate dehydrogenase 2(IMPDH2).We aim to explore the interaction of CTPS and IMPDH2 in the generation of RR structures.He La and COS-7 cells were cultured in normal conditions or in the presence of 6-diazo-5-oxo-L-norleucine(DON),ribavirin,or mycophenolic acid(MPA).Over 90%of DON-treated cells presented RR structures.In He La cells,35%of the RR structures were positive for IMPDH2alone,26%were CTPS alone,and 31%were IMPDH2/CTPS mixed,while in COS-7 cells,42%of RR were IMPDH2 alone,41%were CTPS alone,and 10%were IMPDH2/CTPS mixed.Ribavirin and MPA treatments induced only IMPDH2-based RR.Cells were also transfected with an N-terminal hemagglutinin(NHA)-tagged CTPS1 construct.Over 95%of NHA-CTPS1 transfected cells with DON treatment presented IMPDH2-based RR and almost 100%presented CTPS1-based RR;when treated with ribavirin,over 94%of transfected cells presented IMPDH2-based RR and 37%presented CTPS1-based RR,whereas 2%of untreated transfected cells presented IMPDH2-based RR and 28%presented CTPS1-based RR.These results may help in understanding the relationship between CTP and GTP biosynthetic pathways,especially concerning the formation of filamentous RR structures.     

Metabolic regulation via enzyme filamentation

Determining the mechanisms of enzymatic regulation is central to the study of cellular metabolism. Regulation of enzyme activity via polymerization-mediated strategies has been shown to be widespread, and plays a vital role in mediating cellular homeostasis. In this review, we begin with an overview of the filamentation of CTP synthase, which forms filamentous structures termed cytoophidia. We then highlight other important examples of the phenomenon. Moreover, we discuss recent data relating to the regulation of enzyme activity by compartmentalization into cytoophidia. Finally, we hypothesize potential roles for enzyme filament formation in the regulation of metabolism, development and disease.     

Ubiquitination and filamentous structure of cytidine triphosphate synthase

Living organisms respond to nutrient availability by regulating the activity of metabolic enzymes. Therefore, the reversible post-translational modification of an enzyme is a common regulatory mechanism for energy conservation. Recently, cytidine-5′-triphosphate (CTP) synthase was discovered to form a filamentous structure that is evolutionarily conserved from flies to humans. Interestingly, induction of the formation of CTP synthase filament is responsive to starvation or glutamine depletion. However, the biological roles of this structure remain elusive. We have recently shown that ubiquitination regulates CTP synthase activity by promoting filament formation in Drosophila ovaries during endocycles. Intriguingly, although the ubiquitination process was required for filament formation induced by glutamine depletion, CTP synthase ubiquitination was found to be inversely correlated with filament formation in Drosophila and human cell lines. In this article, we discuss the putative dual roles of ubiquitination, as well as its physiological implications, in the regulation of CTP synthase structure.     

Inhibition of E. coli CTP synthase by the "positive" allosteric effector GTP

Cytidine 5'-triphosphate (CTP) synthase catalyzes the ATP-dependent formation of CTP from UTP using either ammonia or l-glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as a positive allosteric effector to promote catalysis of glutamine hydrolysis. We show that at concentrations exceeding approximately 0.15 mM, GTP actually behaves as a negative allosteric effector of E. coli CTP synthase, inhibiting glutamine-dependent CTP formation. In addition, GTP inhibits NH(3)-dependent CTP formation in a concentration-dependent manner. However, GTP does not inhibit the enzyme's intrinsic glutaminase activity. Although the activation of CTP synthase by GTP does not display cooperative behavior, inhibition of both CTP synthase-catalyzed ammonia- and glutamine-dependent CTP synthesis by GTP do exhibit positive cooperativity. These results suggest that GTP binding affects CTP synthase catalysis in two ways: it activates enzyme-catalyzed glutamine hydrolysis and it inhibits the utilization of NH(3) as a substrate by the synthase domain.     

Drosophila CTP synthase can form distinct substrate- and product-bound filaments

Intracellular compartmentation is a key strategy for the functioning of a cell. In 2010, several studies revealed that the metabolic enzyme CTP synthase (CTPS) can form filamentous structures termed cytoophidia in prokaryotic and eukaryotic cells. However, recent structural studies showed that CTPS only forms inactive product-bound filaments in bacteria while forming active substrate-bound filaments in eukaryotic cells. In this study, using negative staining and cryo-electron microscopy, we demonstrate that Drosophila CTPS, whether in substrate-bound or product-bound form, can form filaments. Our results challenge the previous model and indicate that substrate-bound and product-bound filaments can coexist in the same species. We speculate that the ability to switch between active and inactive cytoophidia in the same cells provides an additional layer of metabolic regulation.     

Cytidine triphosphate synthase activity and mRNA expression in normal human blood cells

Cytidine triphosphate (CTP) synthase is one of the key enzymes in pyrimidine nucleotide anabolic pathways. The activity of this enzyme is elevated in various malignancies including acute lymphocytic leukemia (ALL). In this study we investigated the activity of CTP synthase in various human blood cells isolated from healthy volunteers by density centrifugation and elutriation centrifugation. We also investigated the mRNA expression of CTP synthase in lymphocytes and monocytes. The highest activity of CTP synthase was found in thrombocytes (6.48 nmol CTP x mg(-1) x h(-1)), followed by that of monocytes (2.23), lymphocytes (1.69), granulocytes (0.52) and erythrocytes (0.42). The activity of CTP synthase in whole blood samples was at an intermediate level (1.27). The mRNA expression of CTP synthase in monocytes was comparable to that observed in lymphocytes.     

Alternative substrates for wild-type and L109A E. coli CTP synthases: kinetic evidence for a constricted ammonia tunnel.

Cytidine 5'-triphosphate (CTP) synthase catalyses the ATP-dependent formation of CTP from uridine 5'-triphosphate using either NH(3) or l-glutamine as the nitrogen source. The hydrolysis of glutamine is catalysed in the C-terminal glutamine amide transfer domain and the nascent NH(3) that is generated is transferred via an NH(3) tunnel [Endrizzi, J.A., Kim, H., Anderson, P.M. & Baldwin, E.P. (2004) Biochemistry43, 6447-6463] to the active site of the N-terminal synthase domain where the amination reaction occurs. Replacement of Leu109 by alanine in Escherichia coli CTP synthase causes an uncoupling of glutamine hydrolysis and glutamine-dependent CTP formation [Iyengar, A. & Bearne, S.L. (2003) Biochem. J.369, 497-507]. To test our hypothesis that L109A CTP synthase has a constricted or a leaky NH(3) tunnel, we examined the ability of wild-type and L109A CTP synthases to utilize NH(3), NH(2)OH, and NH(2)NH(2) as exogenous substrates, and as nascent substrates generated via the hydrolysis of glutamine, gamma-glutamyl hydroxamate, and gamma-glutamyl hydrazide, respectively. We show that the uncoupling of the hydrolysis of gamma-glutamyl hydroxamate and nascent NH(2)OH production from N(4)-hydroxy-CTP formation is more pronounced with the L109A enzyme, relative to the wild-type CTP synthase. These results suggest that the NH(3) tunnel of L109A, in the presence of bound allosteric effector guanosine 5'-triphosphate, is not leaky but contains a constriction that discriminates between NH(3) and NH(2)OH on the basis of size.     

Limited proteolysis of Escherichia coli cytidine 5'-triphosphate synthase. Identification of residues required for CTP formation and GTP-dependent activation of glutamine hydrolysis.

Cytidine 5'-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP using either ammonia or l-glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Limited trypsin-catalysed proteolysis, Edman degradation, and site-directed mutagenesis were used to identify peptide bonds C-terminal to three basic residues (Lys187, Arg429, and Lys432) of Escherichia coli CTP synthase that were highly susceptible to proteolysis. Lys187 is located at the CTP/UTP-binding site within the synthase domain, and cleavage at this site destroyed all synthase activity. Nucleotides protected the enzyme against proteolysis at Lys187 (CTP > ATP > UTP > GTP). The K187A mutant was resistant to proteolysis at this site, could not catalyse CTP formation, and exhibited low glutaminase activity that was enhanced slightly by GTP. K187A was able to form tetramers in the presence of UTP and ATP. Arg429 and Lys432 appear to reside in an exposed loop in the glutamine amide transfer (GAT) domain. Trypsin-catalyzed proteolysis occurred at Arg429 and Lys432 with a ratio of 2.6 : 1, and nucleotides did not protect these sites from cleavage. The R429A and R429A/K432A mutants exhibited reduced rates of trypsin-catalyzed proteolysis in the GAT domain and wild-type ability to catalyse NH3-dependent CTP formation. For these mutants, the values of kcat/Km and kcat for glutamine-dependent CTP formation were reduced approximately 20-fold and approximately 10-fold, respectively, relative to wild-type enzyme; however, the value of Km for glutamine was not significantly altered. Activation of the glutaminase activity of R429A by GTP was reduced 6-fold at saturating concentrations of GTP and the GTP binding affinity was reduced 10-fold. This suggests that Arg429 plays a role in both GTP-dependent activation and GTP binding.