CTP synthase 1 explained

CTP synthase 1 is an enzyme that is encoded by the CTPS1 gene in humans.^{[1] [2]} CTP synthase 1 is an enzyme in the de novo pyrimidine synthesis pathway that catalyses the conversion of uridine triphosphate (UTP) to cytidine triphosphate (CTP). CTP is a key building block for the production of DNA, RNA and some phospholipids.

Structure and function

CTPS1 is an asymmetrical homotetramer with only three of its four monomers contributing to the catalytic domain. The substrates required for enzymatic activity are adenosine triphosphate (ATP), UTP and the amino acid glutamine. The ATP and UTP binding domains are located at the tetramer interface, whereas the glutamine binding domain is located away from the tetramer interface.^[3]

Glutamine is hydrolysed by the glutamine amidotransferase domain on the outside of the CTPS1 enzyme. The ammonia produced is channelled through to the synthase domain in the interior of the enzyme, to the tetrameric interface. ATP-dependent phosphorylation of UTP produces 4-phosphoryl UTP, which reacts with the ammonia to produce CTP. The reaction can also take place using ammonia in solution in place of the glutamine-derived ammonia. Guanosine triphosphate (GTP) is an allosteric activator of enzyme activity which stimulates the hydrolysis of glutamine. CTP is an allosteric inhibitor of enzyme activity; the CTP binding site overlaps with and impedes the UTP binding site. Thus, CTPS1 enzymatic activity is sensitive to the levels of all four essential ribonucleotides.^{[4] [5]}

De novo pyrimidine synthesis pathway

The conversion of UTP to CTP is the final and rate limiting step in the de novo pyrimidine synthesis pathway. This step is unusual as it is catalysed by two homologous enzymes, CTPS1 and CTPS2, which share 74% homology at the protein level in humans. Human genetics suggest different cellular dependencies on CTPS1 and CTPS2 activity (see below).

Pyrimidines can also be generated by a salvage pathway that recycles DNA. Whilst the salvage pathway is sufficient for pyrimidine production in non-dividing cells, de novo pyrimidine synthesis is required for dividing cells.

Clinical significance

Inherited mutations

Inherited CTPS1 deficiency is associated with a severe immunodeficiency syndrome characterised by life-threatening varicella zoster virus (VZV) and Epstein–Barr virus (EBV) infection in the first decade of life. Several cases of Epstein–Barr virus–associated lymphoproliferative disease have also been observed, including in the central nervous system. Importantly, no phenotype has been observed outside of the blood system, suggesting that CTPS2 is able to compensate for the CTPS1 loss in other tissues.^{[6] [7] [8]}

All individuals described to date are homozygous for the same splicing mutation in CTPS1, which results in skipping of exon 18 resulting in a severely hypomorphic enzyme. All reported families have ancestry in the North West of England, indicating a founder effect for the causative mutation.

The blood systems of individuals with inherited CTPS1 deficiency are characterised by the following:

1. Normal numbers of T cells, normal T cell subsets
2. Near absence of invariant NKT cells and mucosal associated invariant T cell
3. Normal numbers of total B cells, reduced proportion of memory B cells
4. Reduced numbers of NK cells
5. Severely impaired T cell proliferation response and reduced IL-2 secretion following activation
6. Impaired B cell proliferation response following activation
7. Elevated IgG with selective deficiencies of specific antibodies
8. Normal numbers and subset distribution of myeloid cells and dendritic cells

Inherited CTPS1 deficiency can be cured by allogeneic bone marrow transplantation.^{[9] [10]}

Cancer

Increased expression of CTPS1 has been reported to play a role in several different cancer types.

High expression of CTPS1 has been reported to impart a worse prognosis in myeloma, pancreatic cancer and breast cancer.^{[11] [12] [13] [14] [15] [16]}

miR-125b-5p was identified as a tumour suppressor which is down regulated in squamous cell lung cancer; CTPS1 is a potential target of miR-125b-5p, and loss of expression of this miR is predicted to result in increased expression of CTPS1.^[17]

CTPS1 knock down by shRNA inhibited tumour cell growth in a breast cancer model.^[18] CTPS1 knock down by CRISPR showed synergy with inhibition of ATR in a model of MYC-driven cancer.^[19]

CTPS1 as a therapeutic target

Cancer therapy

The high proliferation rates and metabolic activity of cancer cells are likely to result in a critical dependency on the de novo pyrimidine synthesis pathway. This dependency is exploited therapeutically by several chemotherapy drugs that block de novo pyrimidine synthesis, including the nucleotide analogues cytosine arabinoside (ara-C) and gemcitabine.^[20]

Cyclopentenyl cytosine (CPEC) is an inhibitor of both CTPS1 and CTPS2, with activity thought to be mediated by its 5'-triphosphate metabolite CPEC-TP. In phase 1 clinical studies, CPEC administration resulted in unpredictable and refractory hypotension, including fatal events, resulting in discontinuation of clinical development.^{[21] [22]}

Recently, selective small molecule inhibitors have been described with a high degree of selectivity for CTPS1 over CTPS2. The binding mode and mechanism of CTPS1 selectivity has been resolved by cryo-EM which showed docking of the compounds to the CTP binding site of the enzyme.^[23] A lead clinical candidate from this chemical series has shown efficacy in preclinical models of B and T cell neoplasia.^[24]

A first in human clinical trial of a selective CTPS1 inhibitor will open to recruitment for patients with relapsed/refractory B cell lymphoma or T cell lymphoma late summer 2022 (NCT05463263).

Anti-viral therapy

Nucleoside analogues have a long history in the treatment of viral infection.^{[25] [26]}

Specific inhibition of CTP synthase has been identified as a target for anti-viral therapies.^{[27] [28]}

Epstein–Barr virus (EBV) upregulates the expression of both CTPS1 and CTPS2 in infected B cells, with the expression of CTPS1 increasing earlier than CTPS2. The EBV protein ENBA-LP binds to the CTPS1 promoter, along with MYC and NFκB, to enhance expression of CTPS1.^[29]

SARS-CoV-2, the virus that causes COVID-19, uses CTPS1 from infected cells to drive its proliferation; inhibition of CTPS1 has been highlighted as a potential anti-viral therapy.^[30]

Further reading

• Thomas PE, Lamb BJ, Chu EH . Purification of cytidine-triphosphate synthetase from rat liver, and demonstration of monomer, dimer and tetramer . Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology . 953 . 3 . 334–344 . April 1988 . 3355843 . 10.1016/0167-4838(88)90042-8 . free . 2027.42/27539 .
• Jin J, Smith FD, Stark C, Wells CD, Fawcett JP, Kulkarni S, Metalnikov P, O'Donnell P, Taylor P, Taylor L, Zougman A, Woodgett JR, Langeberg LK, Scott JD, Pawson T . Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization . Current Biology . 14 . 16 . 1436–1450 . August 2004 . 15324660 . 10.1016/j.cub.2004.07.051 . free . 2004CBio...14.1436J .
• Verschuur AC, Van Gennip AH, Muller EJ, Voûte PA, Vreken P, Van Kuilenburg AB . Cytidine triphosphate synthase activity and mRNA expression in normal human blood cells . Biological Chemistry . 380 . 1 . 41–46 . January 1999 . 10064135 . 10.1515/BC.1999.005 . 43352567 .
• Yamauchi M, Yamauchi N, Meuth M . Molecular cloning of the human CTP synthetase gene by functional complementation with purified human metaphase chromosomes . The EMBO Journal . 9 . 7 . 2095–2099 . July 1990 . 2113467 . 551928 . 10.1002/j.1460-2075.1990.tb07377.x .
• Suzuki Y, Yoshitomo-Nakagawa K, Maruyama K, Suyama A, Sugano S . Construction and characterization of a full length-enriched and a 5'-end-enriched cDNA library . Gene . 200 . 1–2 . 149–156 . October 1997 . 9373149 . 10.1016/S0378-1119(97)00411-3 .
• Higgins MJ, Loiselle D, Haystead TA, Graves LM . Human cytidine triphosphate synthetase 1 interacting proteins . Nucleosides, Nucleotides & Nucleic Acids . 27 . 6 . 850–857 . June 2008 . 18600551 . 10.1080/15257770802146502 . 33594588 .
• Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P, Mann M . Global, in vivo, and site-specific phosphorylation dynamics in signaling networks . Cell . 127 . 3 . 635–648 . November 2006 . 17081983 . 10.1016/j.cell.2006.09.026 . free .
• Higgins MJ, Graves PR, Graves LM . Regulation of human cytidine triphosphate synthetase 1 by glycogen synthase kinase 3 . The Journal of Biological Chemistry . 282 . 40 . 29493–29503 . October 2007 . 17681942 . 10.1074/jbc.M703948200 . free .
• Takahashi E, Yamauchi M, Tsuji H, Hitomi A, Meuth M, Hori T . Chromosome mapping of the human cytidine-5'-triphosphate synthetase (CTPS) gene to band 1p34.1-p34.3 by fluorescence in situ hybridization . Human Genetics . 88 . 1 . 119–121 . November 1991 . 1959918 . 10.1007/BF00204942 . 21062598 .
• Whelan J, Phear G, Yamauchi M, Meuth M . Clustered base substitutions in CTP synthetase conferring drug resistance in Chinese hamster ovary cells . Nature Genetics . 3 . 4 . 317–322 . April 1993 . 7981751 . 10.1038/ng0493-317 . 34685308 .
• Gevaert K, Staes A, Van Damme J, De Groot S, Hugelier K, Demol H, Martens L, Goethals M, Vandekerckhove J . Global phosphoproteome analysis on human HepG2 hepatocytes using reversed-phase diagonal LC . Proteomics . 5 . 14 . 3589–3599 . September 2005 . 16097034 . 10.1002/pmic.200401217 . 895879 .
• Maruyama K, Sugano S . Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides . Gene . 138 . 1–2 . 171–174 . January 1994 . 8125298 . 10.1016/0378-1119(94)90802-8 .
• Han GS, Sreenivas A, Choi MG, Chang YF, Martin SS, Baldwin EP, Carman GM . Expression of Human CTP synthetase in Saccharomyces cerevisiae reveals phosphorylation by protein kinase A . The Journal of Biological Chemistry . 280 . 46 . 38328–38336 . November 2005 . 16179339 . 1400552 . 10.1074/jbc.M509622200 . free .
• Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Villén J, Li J, Cohn MA, Cantley LC, Gygi SP . Large-scale characterization of HeLa cell nuclear phosphoproteins . Proceedings of the National Academy of Sciences of the United States of America . 101 . 33 . 12130–12135 . August 2004 . 15302935 . 514446 . 10.1073/pnas.0404720101 . free . 2004PNAS..10112130B .
• Ewing RM, Chu P, Elisma F, Li H, Taylor P, Climie S, McBroom-Cerajewski L, Robinson MD, O'Connor L, Li M, Taylor R, Dharsee M, Ho Y, Heilbut A, Moore L, Zhang S, Ornatsky O, Bukhman YV, Ethier M, Sheng Y, Vasilescu J, Abu-Farha M, Lambert JP, Duewel HS, Stewart II, Kuehl B, Hogue K, Colwill K, Gladwish K, Muskat B, Kinach R, Adams SL, Moran MF, Morin GB, Topaloglou T, Figeys D . Large-scale mapping of human protein-protein interactions by mass spectrometry . Molecular Systems Biology . 3 . 1 . 89 . 2007 . 17353931 . 1847948 . 10.1038/msb4100134 .
• Chang YF, Martin SS, Baldwin EP, Carman GM . Phosphorylation of human CTP synthetase 1 by protein kinase C: identification of Ser(462) and Thr(455) as major sites of phosphorylation . The Journal of Biological Chemistry . 282 . 24 . 17613–17622 . June 2007 . 17463002 . 2081159 . 10.1074/jbc.M702799200 . free .

Notes and References

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2. Web site: Entrez Gene: CTP synthase.
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4. Lauritsen I, Willemoës M, Jensen KF, Johansson E, Harris P . Structure of the dimeric form of CTP synthase from Sulfolobus solfataricus . Acta Crystallographica. Section F, Structural Biology and Crystallization Communications . 67 . Pt 2 . 201–208 . February 2011 . 21301086 . 3034608 . 10.1107/S1744309110052334 .
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