Cytidine Triphosphate Synthase Research Articles

Filamentation and inhibition of prokaryotic CTP synthase with ligands

AbstractCytidine triphosphate synthase (CTPS) plays a pivotal role in the de novo synthesis of cytidine triphosphate (CTP), a fundamental building block for RNA and DNA that is essential for life. CTPS is capable of directly binding to all four nucleotide triphosphates: adenine triphosphate, uridine triphosphate, CTP, and guanidine triphosphate. Furthermore, CTPS can form cytoophidia in vivo and metabolic filaments in vitro, undergoing regulation at multiple levels. CTPS is considered a potential therapeutic target for combating invasions or infections by viral or prokaryotic pathogens. Utilizing cryo‐electron microscopy, we determined the structure of Escherichia coli CTPS (ecCTPS) filament in complex with CTP, nicotinamide adenine dinucleotide (NADH), and the covalent inhibitor 6‐diazo‐5‐oxo‐ l‐norleucine (DON), achieving a resolution of 2.9 Å. We constructed a phylogenetic tree based on differences in filament‐forming interfaces and designed a variant to validate our hypothesis, providing an evolutionary perspective on CTPS filament formation. Our computational analysis revealed a solvent‐accessible ammonia tunnel upon DON binding. Through comparative structural analysis, we discern a distinct mode of CTP binding of ecCTPS that differs from eukaryotic counterparts. Combining biochemical assays and structural analysis, we determined and validated the synergistic inhibitory effects of CTP with NADH or adenine on CTPS. Our results expand our comprehension of the diverse regulatory aspects of CTPS and lay a foundation for the design of specific inhibitors targeting prokaryotic CTPS.

Abstract LB405: CTP synthase 1 is a synthetic vulnerability in prostate cancer treated with supraphysiological androgen

Abstract Bipolar androgen therapy (BAT) is the cyclical administration of supraphysiological androgen (SPA), which can be an effective treatment for 30-40% of patients with castration-resistant prostate cancer. We sought to improve the efficacy of BAT by better understanding its molecular consequences in prostate cancer. Given that the androgen receptor (AR) regulates cellular metabolism, we hypothesized that SPA induces dependencies on specific metabolic pathways that might be exploited therapeutically. A negative selection metabolism-focused CRISPR-KO screen in LNCaP cells treated with SPA suggested that the deletion of several enzymes involved in de novo nucleotide synthesis enhances growth suppression by SPA. A top hit was cytidine triphosphate synthase 1 (CTPS1). CTPS1 converts UTP to CTP in the final step of de novo pyrimidine synthesis. Exposure of SPA-treated prostate cancer cell lines to STP-B, a potent and highly selective inhibitor of CTPS1 (PMID 37008165), increased cell death in vitro and in vivo, indicating that CTPS1 constitutes a metabolic vulnerability in this context. Inhibition of CTPS1 with STP-B induced S phase cell cycle arrest and replication stress, as indicated by phosphorylation of RPA and CHEK proteins, which was augmented by treatment with SPA. CTPS1 likely becomes a vulnerability in SPA-treated prostate cancer due to the downregulation of MYC by SPA, which we found leads to a reduced abundance of enzymes required for nucleotide synthesis and nucleotides, particularly CTP. This rewires nucleotide synthesis and salvage pathway such that de novo synthesis of CTP is required to avoid replication stress and cell death. Altogether, this work suggests that inhibition of CTPS1 may enhance the efficacy of BAT through the induction of replication stress. Selective inhibitors of CTPS1 have entered clinical development (NCT05463263), increasing the feasibility of a combination therapy clinical trial design for patients with prostate cancer. Citation Format: Sheila Jonnatan, Rajendra Kumar, Varsha Vakkala, Karthik Vasan, Zachary R. Chalmers, Daniel R. Schmidt, Philip A. Beer, Matthew G. Vander Heiden, Samuel R. Denmeade, Navdeep S. Chandel, Laura A. Sena. CTP synthase 1 is a synthetic vulnerability in prostate cancer treated with supraphysiological androgen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB405.

Inactivation of cytidine triphosphate synthase 1 prevents fatal auto-immunity in mice

De novo synthesis of the pyrimidine, cytidine triphosphate (CTP), is crucial for DNA/RNA metabolism and depends on the CTP synthetases, CTPS1 and −2. Partial CTPS1 deficiency in humans has previously been shown to lead to immunodeficiency, with impaired expansion of T and B cells. Here, we examine the effects of conditional and inducible inactivation of Ctps1 and/or Ctps2 on mouse embryonic development and immunity. We report that deletion of Ctps1, but not Ctps2, is embryonic-lethal. Tissue and cells with high proliferation and renewal rates, such as intestinal epithelium, erythroid and thymic lineages, activated B and T lymphocytes, and memory T cells strongly rely on CTPS1 for their maintenance and growth. However, both CTPS1 and CTPS2 are required for T cell proliferation following TCR stimulation. Deletion of Ctps1 in T cells or treatment with a CTPS1 inhibitor rescued Foxp3-deficient mice from fatal systemic autoimmunity and reduced the severity of experimental autoimmune encephalomyelitis. These findings support that CTPS1 may represent a target for immune suppression.

Cytoophidia: a conserved yet promising mode of enzyme regulation in nucleotide metabolism.

Nucleotide biosynthesis encompasses both de novo and salvage synthesis pathways, each characterized by significant material and procedural distinctions. Despite these differences, cells with elevated nucleotide demands exhibit a preference for the more intricate de novo synthesis pathway, intricately linked to modes of enzyme regulation. In this study, we primarily scrutinize the biological importance of a conserved yet promising mode of enzyme regulation in nucleotide metabolism-cytoophidia. Cytoophidia, comprising cytidine triphosphate synthase or inosine monophosphate dehydrogenase, is explored across diverse biological models, including yeasts, Drosophila, mice, and human cancer cell lines. Additionally, we delineate potential biomedical applications of cytoophidia. As our understanding of cytoophidia deepens, the roles of enzyme compartmentalization and polymerization in various biochemical processes will unveil, promising profound impacts on both research and the treatment of metabolism-related diseases.

A circuitous route for in vitro multi-enzyme cascade production of cytidine triphosphate to overcome the thermodynamic bottleneck

Cytidine triphosphate (CTP), as a substance involved in the metabolism of phospholipids, proteins and nucleic acids, has precise drug effects and is a direct precursor for the synthesis of drugs such as citicoline. In this study, we established an in vitro six-enzyme cascade system to generate CTP. To avoid thermodynamic bottlenecks, we employed a circuitous and two-stage reaction strategy. Using cytidine as the key substrate, the final product CTP is obtained via the deamination and uridine phosphorylation pathways, relying on the irreversible reaction of cytidine triphosphate synthase to catalyze the amination of uridine triphosphate. Several extremophilic microbial-derived deaminases were screened and characterized, and a suitable cytidine deaminase was selected to match the first-stage reaction conditions. In addition, directed evolution modification of the rate-limiting enzyme CTP synthetase in the pathway yielded a variant that successfully relieved the product feedback inhibition, along with a 1.7-fold increase in activity over the wild type. After optimizing the reaction conditions, we finally carried out the catalytic reaction at an initial cytidine concentration of 20 mM, and the yield of CTP exceeded 82% within 10.0 h.Graphical

Cytoophidia Influence Cell Cycle and Size in Schizosaccharomyces pombe.

Cytidine triphosphate synthase (CTPS) forms cytoophidia in all three domains of life. Here we focus on the function of cytoophidia in cell proliferation using Schizosaccharomyces pombe as a model system. We find that converting His359 of CTPS into Ala359 leads to cytoophidium disassembly. By reducing the level of CTPS protein or specific mutation, the loss of cytoophidia prolongs the G2 phase and expands cell size. In addition, the loss-filament mutant of CTPS leads to a decrease in the expression of genes related to G2/M transition and cell growth, including histone chaperone slm9. The overexpression of slm9 alleviates the G2 phase elongation and cell size enlargement induced by CTPS loss-filament mutants. Overall, our results connect cytoophidia with cell cycle and cell size control in Schizosaccharomyces pombe.

Quantitative Proteomic Analysis Deciphers the Molecular Mechanism for Endosperm Nuclear Division in Early Rice Seed Development.

Understanding the molecular mechanisms underlying early seed development is important in improving the grain yield and quality of crop plants. We performed a comparative label-free quantitative proteomic analysis of developing rice seeds for the WT and osctps1-2 mutant, encoding a cytidine triphosphate synthase previously reported as the endospermless 2 (enl2) mutant in rice, harvested at 0 and 1 d after pollination (DAP) to understand the molecular mechanism of early seed development. In total, 5231 proteins were identified, of which 902 changed in abundance between 0 and 1 DAP seeds. Proteins that preferentially accumulated at 1 DAP were involved in DNA replication and pyrimidine biosynthetic pathways. Notably, an increased abundance of OsCTPS1 was observed at 1 DAP; however, no such changes were observed at the transcriptional level. We further observed that the inhibition of phosphorylation increased the stability of this protein. Furthermore, in osctps1-2, minichromosome maintenance (MCM) proteins were significantly reduced compared with those in the WT at 1 DAP, and mutations in OsMCM5 caused defects in seed development. These results highlight the molecular mechanisms underlying early seed development in rice at the post-transcriptional level.

CTPS1 acts as a prognostic biomarker and therapeutic target in mantle cell lymphoma

Introduction: Mantle cell lymphoma (MCL) represents 6 to 8% of non-Hodgkin’s lymphoma with generally poor prognosis. Cytidine triphosphate synthase 1 (CTPS1) is a critical regulatory enzyme in cytidine metabolism, catalyzing the rate-limiting step in de novo CTP synthesis. Herein, we aim to investigate the clinical significance and functional mechanisms of CTPS1 in MCL patients. Methods: Lymph node, bone marrow biopsy, peripheral blood specimens and MCL cell lines were utilized as models. Liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS)-based analysis was performed to measure the steady-state level of metabolites. GSE93291 dataset and immunohistochemistry (IHC) staining were utilized to determine the relationship between expression levels of CTPS1 and patient outcome. CRISPR/Cas9 editing was employed to engineer the loss-of-function models of CTPS1 and other key enzymes involved in CTP synthesis. Results: Metabolic Profiling revealed dramatically aberrant cellular abundance of metabolic intermediates in the CTP synthesis pathway (Figure 1A). For GSE93291 analysis, a nucleotide metabolism related prognostic model was established by bioinformatic analysis and CTPS1 was screened out by highest regression coefficient (Figure 1B). IHC staining of CTPS1 in 105 MCL tissues confirmed that elevated CTPS1 expression was significantly associated with poor PFS (P = 0.038) and OS (P = 0.001) in MCL patients (Figure 1C-D). Multivariate Cox regression analysis showed that high CTPS1 expression was an independent prognostic indicator of both PFS and OS. CTPS1 depletion significantly impaired outgrowth and increased apoptosis of MCL cells, suggesting that CTPS1 is the major isozyme important for MCL survival (Figure 1E-F). In primary tumor samples, MYC protein levels were significantly correlated with CTPS1 protein levels (P < 0.001, R = 0.638). MYC knockout blocked CTPS1 mRNA and protein expression prior to induction of cell death, suggesting a requisite role of MYC in CTPS1 transcription regulation (Figure 1G). Remarkably, TP53 aberrant and ibrutinib-resistant MCL cells rely on cytidine metabolism (Figure 1H). Genetic or chemical inhibition (3-deazauridine) of CTPS1 reversed poor responsiveness of MCL cell lines to ibrutinib (Figure 1I). Cytidine level was significantly increased in TP53-deficient MCL cells and the effect of CTPS1 inhibition was independent of TP53 status (Figure 1K). Gene set enrichment analysis of RNA-sequencing revealed that CTPS1 inhibition induced DNA damage and apoptosis. Moreover, CTPS1 inhibition caused cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) activation, which triggered innate immune pathway, leading to cells growth inhibition (Figure 1L). Keywords: Aggressive B-cell non-Hodgkin lymphoma, Diagnostic and Prognostic Biomarkers, Metabolism No conflicts of interests pertinent to the abstract.

An Atypical Presentation of X-linked Lymphoproliferative Disease type 1 Presenting with Seronegative Coccidioidomycosis

X-linked lymphoproliferative disease (XLP-1), an inborn error of immunity (IEI) caused by pathogenic variants in SH2D1A encoding signaling lymphocyte activation molecule-associated protein (SAP), is characterized by hemophagocytic lymphohistiocytosis (HLH), lymphoma, and hypogammaglobulinemia. We report the first known case of XLP-1 presenting with seronegative coccidioidomycosis. CaseA 13-year-old boy with persistent asthma, recurrent otitis media requiring four sets of tympanostomy tubes, chronic sinusitis, and Common Variable Immunodeficiency (CVID) presented with acute-on-chronic hypoxic respiratory failure. He had been off subcutaneous immunoglobulin (SCIg) replacement for 2 years. Sputum culture was positive for H. influenzae, treated with cefepime and azithromycin and supplemental oxygen for acute hypoxic respiratory failure. Fever persisted despite 48 hours of antibiotics. CT chest showed bronchiectasis with mediastinal, subcarinal and hilar adenopathy. Coccidioides immitis IgG and IgM were negative, but urine antigen was positive. There was no radiologic or cerebrospinal fluid evidence of disseminated coccidioidomycosis.Immunologic workup revealed undetectable IgG, IgA, IgM, and IgE (table 1). Lymphocyte subsets showed severe T and NK cell lymphopenia. Total B cell count was normal, but he had decreased CD19+27+ and transitional B cells, with absent switched memory B cells and no plasmablasts (table 1). Lymphocyte proliferation to mitogens/antigens was decreased. Serum ferritin was normal, and EBV PCR was negative. A targeted 429-gene panel identified a hemizygous variant of uncertain significance (VUS) in SH2D1A, c.82_102dup (p.Ser28_Ser34dup). SAP expression was absent in CD4+ T cells and NK cells with a bimodal expression in CD8+ T cells, suggesting possible mosaicism in this subset. He was diagnosed with X-linked lymphoproliferative disease 1 (XLP-1) and treated with fluconazole, trimethoprim-sulfamethoxazole prophylaxis, and IgG replacement (1 g/kg intravenous initially, then transitioned to weekly SCIg targeting an IgG >1000 mg/dL), with clinical improvement, resolution of fevers and acute hypoxia. He was referred for hematopoietic cell transplant evaluation.Coccidioidomycosis has not been described in a new diagnosis of XLP-1 and should be considered in this IEI. Coccidioidomycosis susceptibility has been reported in other IEIs including impaired interleukin-12/interferon-gamma axis signaling, STAT3 loss of function, chronic granulomatous disease, and recently cytidine triphosphate synthase 1 (CTPS1) deficiency.Table 1Patient’s immunologic variables compared to reference range. Absolute values are shownImmunologic VariablePatient ValuesReference RangeIgG<70 (L)<70 mg/dLIgA<15 (L)<15 mg/dLIgM<8 (L)<8 mg/dLIgE<1.5 (L)<1.5 IU/uLTotal CD4+ T Cells11 (L)570–2400 cells/uLTotal CD8+ T Cells63 (L)210–1200 cells/uLTotal CD19+ B cells173165–1154 cells/uLCD19+ CD24++ CD38++ (Transitional B Cells)0 (L)8–392 cells/uLCD19+CD24+CD38+ (Naïve B Cells)12159–1146 cells/uLCD19+CD27-(Naïve B cells)169140–2269 cells/uLCD19+ CD27+ CD38+ IgM- (Plasmablasts)0<30 cells/uLCD19+CD27+IgD-IgM- (Switched-memory B cells)0 (L)6–190 cells/uLCD16+ CD56+ (NK Cells)32 (L)78–470 cells/uLL – low

Discovery and characterization of novel inhibitors of CTP synthase 1 (CTPS1) for the treatment of autoimmune and inflammatory disease

Abstract The de novo pyrimidine biosynthetic pathway is an inducible cellular program which allows the rapid synthesis of pyrimidine nucleotides in proliferating cells, and the final rate-limiting step is catalyzed by cytidine triphosphate synthase (CTPS1 or CTPS2). A CTPS1 loss-of-function mutation in humans has illustrated the essential and non-redundant role of CTPS1 in activated lymphocyte proliferation. Therefore, selective CTPS1 inhibition is an attractive approach to specifically target pathological immune activity in autoimmune and inflammatory disease. Using cryo-EM and structure-based drug design, we have identified a series of potent, orally bioavailable, CTPS1-specific inhibitors, exemplified by the compound NTX-871. In-vitro, NTX-871 significantly inhibits the proliferation of T-cell derived lines and activated primary T-cells. These inhibitory effects are reversed by the addition of exogenous cytidine, illustrating the specificity of NTX-871 for CTPS1 and the de novo pyrimidine synthesis program. NTX-871 was also tested in several mouse in-vivo PD and autoimmune disease models. Oral dosing of NTX-871 significantly reduced T-cell proliferation in an ovalbumin-induced T-cell proliferation model, and QD or BID dosing of NTX-871 significantly improved disease phenotypes in models of delayed-type hypersensitivity, collagen-induced arthritis, and experimental autoimmune encephalomyelitis.

CTP Synthase 1 Is a Novel Therapeutic Target in Lymphoma.

Lymphoma is the most common hematological malignancy and is among the 10 most prevalent cancers worldwide. Although survival has been improved by modern immunochemotherapeutic regimens, there remains a significant need for novel targeted agents to treat both B-cell and T-cell malignancies. Cytidine triphosphate synthase 1 (CTPS1), which catalyzes the rate-limiting step in pyrimidine synthesis, plays an essential and nonredundant role in B-cell and T-cell proliferation but is complemented by the homologous CTPS2 isoform outside the hemopoietic system. This report describes the identification and characterization of CTPS1 as a novel target in B- and T-cell cancers. A series of small molecules have been developed which show potent and highly selective inhibition of CTPS1. Site-directed mutagenesis studies identified the adenosine triphosphate pocket of CTPS1 as the binding site for this small molecule series. In preclinical studies, a potent and highly selective small molecule inhibitor of CTPS1 blocked the in vitro proliferation of human neoplastic cells, showing the highest potency against lymphoid neoplasms. Importantly, pharmacological CTPS1 inhibition induced cell death by apoptosis in the majority of lymphoid cell lines tested, thus demonstrating a cytotoxic mechanism of action. Selective CTPS1 inhibition also inhibited the growth of neoplastic human B- and T- cells in vivo. These findings identify CTPS1 as a novel therapeutic target in lymphoid malignancy. A compound from this series is in phase 1/2 clinical studies for the treatment of relapsed/refractory B- and T-cell lymphoma (NCT05463263).

Identification of Potential Druggable Targets and Structure-Based Virtual Screening for Drug-like Molecules against the Shrimp Pathogen Enterocytozoon hepatopenaei.

Enterocytozoon hepatopenaei (EHP) causes slow growth syndrome in shrimp, resulting in huge economic losses for the global shrimp industry. Despite worldwide reports, there are no effective therapeutics for controlling EHP infections. In this study, five potential druggable targets of EHP, namely, aquaporin (AQP), cytidine triphosphate (CTP) synthase, thymidine kinase (TK), methionine aminopeptidase2 (MetAP2), and dihydrofolate reductase (DHFR), were identified via functional classification of the whole EHP proteome. The three-dimensional structures of the proteins were constructed using the artificial-intelligence-based program AlphaFold 2. Following the prediction of druggable sites, the ZINC15 and ChEMBL databases were screened against targets using docking-based virtual screening. Molecules with affinity scores ≥ 7.5 and numbers of interactions ≥ 9 were initially selected and subsequently enriched based on their ADMET properties and electrostatic complementarities. Five compounds were finally selected against each target based on their complex stabilities and binding energies. The compounds CHEMBL3703838, CHEMBL2132563, and CHEMBL133039 were selected against AQP; CHEMBL1091856, CHEMBL1162979, and CHEMBL525202 against CTP synthase; CHEMBL4078273, CHEMBL1683320, and CHEMBL3674540 against TK; CHEMBL340488, CHEMBL1966988, and ZINC000828645375 against DHFR; and CHEMBL3913373, ZINC000016682972, and CHEMBL3142997 against MetAP2.The compounds exhibited high stabilities and low binding free energies, indicating their abilities to suppress EHP infections; however, further validation is necessary for determining their efficacy.

CTPS cytoophidia formation affects cell cycle progression and promotes TSN‑induced apoptosis of MKN45 cells.

Cytidine triphosphate synthase (CTPS) forms filamentous structures termed cytoophidia in numerous types of cell. Toosendanin (TSN) is a tetracyclic triterpenoid and induces CTPS to form cytoophidia in MKN45 cells. However, the effects of CTPS cytoophidia on the proliferation and apoptosis of human gastric cancer cells remain poorly understood. In the present study, CTPS-overexpression and R294D-CTPS mutant vectors were generated to assess the effect of CTPS cytoophidia on the proliferation and apoptosis of gastric cancer MKN45 cells. Formation of CTPS cytoophidia significantly inhibited MKN45 cell proliferation (evaluated using EdU incorporation assay), significantly blocked the cell cycle in G1 phase (assessed using flow cytometry) and significantly decreased mRNA and protein expression levels of cyclin D1 (assessed by reverse transcription-quantitative PCR and western blotting, respectively). Furthermore, the number of apoptotic bodies and apoptosis rate were markedly elevated and mitochondrial membrane potential was markedly decreased. Moreover, mRNA and protein expression levels of Bax increased and Bcl-2 decreased markedly in MKN45 cells following transfection with the CTPS-overexpression vector. The proliferation rate increased, percentage of G1/G0-phase cells decreased and apoptosis was attenuated in cells transfected with the R294D-CTPS mutant vector and this mutation did not lead to formation of cytoophidia. The results of the present study suggested that formation of CTPS cytoophidia inhibited proliferation and promoted apoptosis in MKN45 cells. These results may provide insights into the role of CTPS cytoophidia in cancer cell proliferation and apoptosis.

Drosophila CTP synthase regulates collective cell migration by controlling the polarized endocytic cycle.

Phosphatidylinositol (PI) 4,5-bisphosphate (PIP2) is involved in many biological functions. However, the mechanisms of PIP2 in collective cell migration remain elusive. This study highlights the regulatory role of cytidine triphosphate synthase (CTPsyn) in collective border cell migration through regulating the asymmetrical distribution of PIP2. We demonstrated that border cell clusters containing mutant CTPsyn cells suppressed migration. CTPsyn was co-enriched with Actin at the leading edge of the Drosophila border cell cluster where PIP2 was enriched, and this enrichment depended on the CTPsyn activity. Genetic interactions of border cell migration were found between CTPsyn mutant and genes in PI biosynthesis. The CTPsyn reduction resulted in loss of the asymmetric activity of endocytosis recycling. Also, genetic interactions were revealed between components of the exocyst complex and CTPsyn mutant, indicating that CTPsyn activity regulates the PIP2-related asymmetrical exocytosis activity. Furthermore, CTPsyn activity is essential for RTK-polarized distribution in the border cell cluster. We propose a model in which CTPsyn activity is required for the asymmetrical generation of PIP2 to enrich RTK signaling through endocytic recycling in collective cell migration.

Computational assessment of Withania somnifera phytomolecules as putative inhibitors of Mycobacterium tuberculosis CTP synthase PyrG

Genome evolution of Mycobacterium tuberculosis (Mtb) produces new strains resistant to various pre-existing anti-tubercular drugs. Hence, there is an urgent need to explore potent compounds with the most negligible side effects and effective Mtb inhibition. Mtb PyrG (CTP synthase) is a crucial enzyme for the conversion of the uridine triphosphate (UTP) into cytidine triphosphate (CTP) and is essential for the growth of Mtb. Thus, in this study, phytochemicals of Withania somnifera (W. somnifera) were screened to find the potential inhibitors against Mtb PyrG. Molecular docking resulted in the identification of quercetin 3-rutinoside-7-glucoside, rutin, chlorogenic acid and isochlorogenic acid C with a substantial docking score (from −12.6 to −10.8 kcal/mol) contributed by significant intermolecular interactions. Furthermore, 100 ns molecular dynamics simulation, ADME analysis and free binding energy calculations support the stability of docked complexes and drug-likeness for selected compounds, respectively. Collectively, these findings suggest that phytochemicals present in W. somnifera can be considered for further evaluation against Mtb in a series of in vitro and in vivo models. Communicated by Ramaswamy H. Sarma

Cytidine Triphosphate Synthase Four From Arabidopsis thaliana Attenuates Drought Stress Effects.

Cytidine triphosphate synthase (CTPS) catalyzes the final step in pyrimidine de novo synthesis. In Arabidopsis, this protein family consists of five members (CTPS1–5), and all of them localize to the cytosol. Specifically, CTPS4 showed a massive upregulation of transcript levels during abiotic stress, in line with increased staining of CTPS4 promoter:GUS lines in hypocotyl, root and to lesser extend leaf tissues. In a setup to study progressive drought stress, CTPS4 knockout mutants accumulated less fresh and dry weight at days 5–7 and showed impaired ability to recover from this stress after 3 days of rewatering. Surprisingly, a thorough physiological characterization of corresponding plants only revealed alterations in assimilation and accumulation of soluble sugars including those related to drought stress in the mutant. Bimolecular fluorescence complementation (BiFC) studies indicated the interaction of CTPS4 with other isoforms, possibly affecting cytoophidia (filaments formed by CTPS formation. Although the function of these structures has not been thoroughly investigated in plants, altered enzyme activity and effects on cell structure are reported in other organisms. CTPS activity is required for cell cycle progression and growth. Furthermore, drought can lead to the accumulation of reactive oxygen species (ROS) and by this, to DNA damage. We hypothesize that effects on the cell cycle or DNA repair might be relevant for the observed impaired reduced drought stress tolerance of CTPS4 mutants.

Structural basis for ligand binding modes of CTP synthase

Cytidine triphosphate synthase (CTPS), which comprises an ammonia ligase domain and a glutamine amidotransferase domain, catalyzes the final step of de novo CTP biosynthesis. The activity of CTPS is regulated by the binding of four nucleotides and glutamine. While glutamine serves as an ammonia donor for the ATP-dependent conversion of UTP to CTP, the fourth nucleotide GTP acts as an allosteric activator. Models have been proposed to explain the mechanisms of action at the active site of the ammonia ligase domain and the conformational changes derived by GTP binding. However, actual GTP/ATP/UTP binding modes and relevant conformational changes have not been revealed fully. Here, we report the discovery of binding modes of four nucleotides and a glutamine analog 6-diazo-5-oxo-L-norleucine in Drosophila CTPS by cryo-electron microscopy with near-atomic resolution. Interactions between GTP and surrounding residues indicate that GTP acts to coordinate reactions at both domains by directly blocking ammonia leakage and stabilizing the ammonia tunnel. Additionally, we observe the ATP-dependent UTP phosphorylation intermediate and determine interacting residues at the ammonia ligase. A noncanonical CTP binding at the ATP binding site suggests another layer of feedback inhibition. Our findings not only delineate the structure of CTPS in the presence of all substrates but also complete our understanding of the underlying mechanisms of the allosteric regulation and CTP synthesis.

Chloroplast quality control pathways are dependent on plastid DNA synthesis and nucleotides provided by cytidine triphosphate synthase two.

Reactive oxygen species (ROS) produced in chloroplasts cause oxidative damage, but also signal to initiate chloroplast quality control pathways, cell death, and gene expression. The Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant produces the ROS singlet oxygen in chloroplasts that activates such signaling pathways, but the mechanisms are largely unknown. Here we characterize one fc2 suppressor mutation and map it to CYTIDINE TRIPHOSPHATE SYNTHASE TWO (CTPS2), which encodes one of five enzymes in Arabidopsis necessary for de novo cytoplasmic CTP (and dCTP) synthesis. The ctps2 mutation reduces chloroplast transcripts and DNA content without similarly affecting mitochondria. Chloroplast nucleic acid content and singlet oxygen signaling are restored by exogenous feeding of the dCTP precursor deoxycytidine, suggesting ctps2 blocks signaling by limiting nucleotides for chloroplast genome maintenance. An investigation of CTPS orthologs in Brassicaceae showed CTPS2 is a member of an ancient lineage distinct from CTPS3. Complementation studies confirmed this analysis; CTPS3 was unable to compensate for CTPS2 function in providing nucleotides for chloroplast DNA and signaling. Our studies link cytoplasmic nucleotide metabolism with chloroplast quality control pathways. Such a connection is achieved by a conserved clade of CTPS enzymes that provide nucleotides for chloroplast function, thereby allowing stress signaling to occur.

CTP synthase is essential for early endosperm development by regulating nuclei spacing.

SummaryCereal grain endosperms are an important source of human nutrition. Nuclear division in early endosperm development plays a major role in determining seed size; however, this development is not well understood. We identified the rice mutant endospermless 2 (enl2), which shows defects in the early stages of endosperm development. These phenotypes arise from mutations in OsCTPS1 that encodes a cytidine triphosphate synthase (CTPS). Both wild‐type and mutant endosperms were normal at 8 h after pollination (HAP). In contrast, at 24 HAP, enl2 endosperm had approximately 10–16 clumped nuclei while wild‐type nuclei had increased in number and migrated to the endosperm periphery. Staining of microtubules in endosperm at 24 HAP revealed that wild‐type nuclei were evenly distributed by microtubules while the enl2‐2 nuclei were tightly packed due to their reduction in microtubule association. In addition, OsCTPS1 interacts with tubulins; thus, these observations suggest that OsCTPS1 may be involved in microtubule formation. OsCTPS1 transiently formed macromolecular structures in the endosperm during early developmental stages, further supporting the idea that OsCTPS1 may function as a structural component during endosperm development. Finally, overexpression of OsCTPS1 increased seed weight by promoting endosperm nuclear division, suggesting that this trait could be used to increase grain yield.

CTPS forms the cytoophidium in zebrafish

Cytidine triphosphate synthase (CTPS) catalyzes the rate-limiting step of de novo CTP biosynthesis. An intracellular structure of CTPS, the cytoophidium, has been found in many organisms including prokaryotes and eukaryotes. Formation of the cytoophidium has been suggested to regulate the activity and stability of CTPS and may participate in certain physiological events. Herein, we demonstrate that both CTPS1a and CTPS1b in zebrafish are able to form the cytoophidium in cultured cells. A point mutation, H355A, abrogates cytoophidium assembly of zebrafish CTPS1a and CTPS1b. In addition, we show the presence of CTPS cytoophidia in multiple tissues of larval and adult fish under normal conditions, while treatment with a CTPS inhibitor 6-diazo-5-oxo-l-norleucine (DON) can induce more cytoophidia in some tissues. Our findings reveal that forming the CTPS cytoophidium is a natural phenomenon of zebrafish and provide valuable information for future research on the physiological importance of this intracellular structure in vertebrates.