Anne-Claire Boschat (Presenter)
Institut Imagine
Bio: I discovered mass spectrometry during my master's degree, while I was studying at the Ecole Supérieure de Biotechnologie de Strasbourg (France). I became especially interested in this technique, owing to the power of this detection and identification tool, as well as its large application domains. I had the opportunity to fulfill my wish to work in this field during my final internship in the private proteomics laboratory of a large biotech company: New England Biolabs (Ipswich, MA, U.S.A.). There, for nine months, I used mass spectrometry for assessing the quality of the enzymes produced by the company, as well as for developing a way to compare the proteome redox state of different bacterial strains. After I graduated with a engineering degree in 2011, New England Biolabs offered me to continue working with them for a while, and I spent one more year studying and practicing mass spectrometry at NEB, using multiple types of MS techniques such as multidimensional protein identification technology (MudPIT) analysis using nLC ESI MS/MS (Ion Trap Orbitrap XL), nanoESI MS/MS (Ion Trap with an integrated Chip/NanoESI interface) and MALDI-TOF. When I came back in France at the end of the year 2012, I wanted to apply my knowledge of mass spectrometry to the medical field. I was hired at the mass spectrometry platform shared between Necker Hospital-Sick Children and Imagine Institute (Paris, FR). As an engineer, I am in charge of research, developments and analytical validations of biomarkers assay using HPLC system coupled with tandem mass spectrometry (Triple Quad and Orbitrap), more especially on immunodeficiency diseases. I collaborate with doctors and researchers working for both Imagine Institute and Necker-Hospital, and I especially like the fact that my work can have a direct impact on the well-being of patients. One year ago, my supervisor offered me to start working on a PhD thesis in parallel of my daily responsibilities. The goal of my thesis is to employ large spectral metabolomics analyses as a way to gain a better insight into human immune deficiencies.
Authorship: Anne-Claire Boschat (1,2,3), Emmanuel Martin (3,4), Peter Arkwright (5), Robert Barouki (1,2,6), Sylvain Latour (3,4), Sylvia Sanquer (1,2)
(1) Plateforme de Métabolomique et Protéomique, Institut Imagine, Paris 75015, France (2) Laboratoire de Biochimie Métabolomique et Protéomique, Hôpital Necker Enfants-Malades, Paris 75015, France. (3) Université Paris Descartes Sorbonne Paris Cité, Institut Imagine, Paris 75015, France. (4) Laboratoire Activation Lymphocytaire et Susceptibilité à l’EBV, INSERM UMR 1163, Institut Imagine, Paris 75015, France. (5) Royal Manchester Children's Hospital, Manchester, United Kingdom.(6) INSERM UMR-S 1124, Université Paris Descartes, Centre Universitaire des Saints-Pères, Paris, France.
| Short Abstract Cytosine 5’-Triphosphate Synthase (CTPS) is known to be a central enzyme in the de novo synthesis of CTP. Recently, a CTPS loss-of-function homozygous mutation inducing combined immunodeficiency in human was identified. Here we present an analytical method for the measurement of CTPS activity by LC-MS/MS. CTPS activity was measured in peripheral blood mononuclear cells issued from healthy donors, as well as of an immunodeficient patient with a rare recessive homozygous mutation in CTPS1 gene. This assay is a useful tool to better characterize the enzyme and could also provide an effective way of screening new inhibitors of CTPS. |
Long Abstract
A severe metabolism adaptation is necessary in lymphocyte cell division, after the antigen recognition [1]. Cytosine 5’-triphosphate (CTP) synthetase (CTPS) enzyme (EC 6.3.4.2) is a key enzyme involved in pyrimidine biosynthesis that interconverts uridine 5'-triphosphate nucleotide (UTP) into CTP [2-4]. CTPS displays two coupled enzymatic activities: glutaminase activity with L-glutamine hydrolysis and amination of UTP with the NH3 group that has been liberated from glutamine. GTP and ATP are positive allosteric effectors of the glutaminase and amination activities, respectively. Two isoforms of the enzyme exist (CTPS 1 and 2), but to date their specific roles are unknown. CTPS catalyzes the final step in the de novo synthesis of CTP, and its [5] regulation is critical to normal mammalian cell growth [6]. Like all purine and pyrimidine nucleotides, CTP indeed plays an important role in the biosynthesis of nucleic acids and membrane phospholipids which make CTPS a well-known target for the development of anti-proliferation growth agents [7-11]. Moreover, a central role for CTPS in lymphocyte proliferation has been recently reported and linked to a novel and life-threatening immunodeficiency, characterized by an impaired capacity of antigen induced lymphocytes proliferation [12].
In the present study, we describe a rapid, specific, radioactivity free, and convenient HPLC coupled with tandem mass spectrometry assay for the quantification of the 2 coupled enzyme activities of CTPS, using glutamine as a nitrogen source. Because of the novel implication of CTPS in the immune system, we developed and validated this assay in peripheral blood mononuclear cells (PBMC) lysates and determined the kinetic parameters Vmax and Km for CTPS in both resting and stimulated PBMCs. This assay uses a simple sample preparation, a stable isotope of CTP as internal standard and MRM mode. The linear range for CTP was 2-451 uM and the LOQ was 2µM. All relative standard deviation and relative error values obtained in intra- and inter-day analyses were below 11%. Ten ug of cytosolic proteins was found to be the lowest quantity of proteins that have to be used for satisfactorily determine CTPS activity. The time required for the complete conversion of UTP into CTP was found unusually long for an enzyme since the plateau of the enzymatic reaction was achieved 400 min after the incorporation of the substrates glutamine and UTP. Michaelis-Menten parameters for CTPS in resting lymphocytes was 0.2837 mM for Km and 83±20 fmol/min/mg of proteins for Vmax and in stimulated lymphocytes, a 5-fold increase was found for Vmax (379±90 fmol/min/mg of proteins increase) while Km was unchanged at 0.2309 mM. CTPS activity was measured in lymphocytes derived from normal patients, as well as in patients displaying others immunodeficiencies.
In conclusion, we developed and validated a robust, sensitive and specific LC-MS/MS assay method for measuring the two coupled enzyme activities of CTPS. This assay could be a useful tool to better characterize the 2 isoforms of CTPS. It could also provide an effective way to screen new inhibitors of CTPS. Coupled with quantification of endogenous nucleotides in cells, it could also provide insights for a better understanding of the overall nucleotides metabolism mechanisms, especially in case of dysregulation inducing immune disorders.
References & Acknowledgements:
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2.Iyengar, A. & Bearne, S. L. An assay for cytidine 5′-triphosphate synthetase glutaminase activity using high performance liquid chromatography. Anal. Biochem. 308, 396–400 (2002).
3.Long, C. W. & Pardee, A. B. Cytidine Triphosphate Synthetase of Escherichia coli B I. PURIFICATION AND KINETICS. J. Biol. Chem. 242, 4715–4721 (1967).
4.Van Kuilenburg, A. B. et al. Determination of CTP synthetase activity in crude cell homogenates by a fast and sensitive non-radiochemical assay using anion-exchange high-performance liquid chromatography. J. Chromatogr. B. Biomed. Sci. App. 693, 287–295 (1997).
5.Evans, D. R. & Guy, H. I. Mammalian Pyrimidine Biosynthesis: Fresh Insights into an Ancient Pathway. J. Biol. Chem. 279, 33035–33038 (2004).
6.Korte, D. de, Haverkort, W. A., Boer, M. de, Gennip, A. H. van & Roos, D. Imbalance in the Nucleotide Pools of Myeloid Leukemia Cells and HL-60 Cells: Correlation with Cell-Cycle Phase, Proliferation, Differentiation, and Transformation. Cancer Res. 47, 1841–1847 (1987).
7.Hatse, S., De Clercq, E. & Balzarini, J. Role of antimetabolites of purine and pyrimidine nucleotide metabolism in tumor cell differentiation. Biochem. Pharmacol. 58, 539–555 (1999).
8.Verschuur, A. C. et al. In vitro inhibition of cytidine triphosphate synthetase activity by cyclopentenyl cytosine in paediatric acute lymphocytic leukaemia. Br. J. Haematol. 110, 161–169 (2000).
9.Endrizzi, J. A., Kim, H., Anderson, P. M. & Baldwin, E. P. Crystal Structure of Escherichia coli Cytidine Triphosphate Synthetase, a Nucleotide-Regulated Glutamine Amidotransferase/ATP-Dependent Amidoligase Fusion Protein and Homologue of Anticancer and Antiparasitic Drug Targets†,‡. Biochemistry (Mosc.) 43, 6447–6463 (2004).
10.Nadkarni, A. K. et al. Differential Biochemical Regulation of the URA7- and URA8-encoded CTP Synthetases from Saccharomyces cerevisiae. J. Biol. Chem. 270, 24982–24988 (1995).
11.Gao, W. Y., Johns, D. G. & Mitsuya, H. Potentiation of the anti-HIV activity of zalcitabine and lamivudine by a CTP synthase inhibitor, 3-deazauridine. Nucleosides Nucleotides Nucleic Acids 19, 371–377 (2000).
12.Martin, E. et al. CTP synthase 1 deficiency in humans reveals its central role in lymphocyte proliferation. Nature 510, 288–292 (2014).
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