title: Molecular Mechanism for the Thermo-Sensitive Phenotype of CHO-MT58 Cell Line Harbouring a Mutant CTP:Phosphocholine Cytidylyltransferase.
abstract: Control and elimination of malaria still represents a major public health challenge. Emerging parasite resistance to current therapies urges development of antimalarials with novel mechanism of action. Phospholipid biosynthesis of the Plasmodium parasite has been validated as promising candidate antimalarial target. The most prevalent de novo pathway for synthesis of phosphatidylcholine is the Kennedy pathway. Its regulatory and often also rate limiting step is catalyzed by CTP:phosphocholine cytidylyltransferase (CCT). The CHO-MT58 cell line expresses a mutant variant of CCT, and displays a thermo-sensitive phenotype. At non-permissive temperature (40°C), the endogenous CCT activity decreases dramatically, blocking membrane synthesis and ultimately leading to apoptosis. In the present study we investigated the impact of the analogous mutation in a catalytic domain construct of Plasmodium falciparum CCT in order to explore the underlying molecular mechanism that explains this phenotype. We used temperature dependent enzyme activity measurements and modeling to investigate the functionality of the mutant enzyme. Furthermore, MS measurements were performed to determine the oligomerization state of the protein, and MD simulations to assess the inter-subunit interactions in the dimer. Our results demonstrate that the R681H mutation does not directly influence enzyme catalytic activity. Instead, it provokes increased heat-sensitivity by destabilizing the CCT dimer. This can possibly explain the significance of the PfCCT pseudoheterodimer organization in ensuring proper enzymatic function. This also provide an explanation for the observed thermo-sensitive phenotype of CHO-MT58 cell line.
authors.affiliation: Institute of Enzymology, Research Centre for National Sciences, HAS, Budapest Hungary; Doctoral School of Multidisciplinary Medical Science, University of Szeged, Szeged, Hungary.
authors.forename: Lívia
authors.initials: L
authors.lastname: Marton
authors.affiliation: Institute of Enzymology, Research Centre for National Sciences, HAS, Budapest Hungary; Department of Applied Biotechnology and Food Science, Budapest University of Technology and Economics, Budapest, Hungary.
authors.forename: Gergely N
authors.initials: GN
authors.lastname: Nagy
authors.affiliation: Institute of Organic Chemistry, Research Centre for National Sciences, HAS, Budapest, Hungary.
authors.forename: Olivér
authors.initials: O
authors.lastname: Ozohanics
authors.affiliation: Department of Inorganic and Analytical Chemistry, Budapest University of Technology and Economics, Budapest, Hungary.
authors.forename: Anikó
authors.initials: A
authors.lastname: Lábas
authors.affiliation: Department of Inorganic and Analytical Chemistry, Budapest University of Technology and Economics, Budapest, Hungary.
authors.forename: Balázs
authors.initials: B
authors.lastname: Krámos
authors.affiliation: Department of Inorganic and Analytical Chemistry, Budapest University of Technology and Economics, Budapest, Hungary.
authors.forename: Julianna
authors.initials: J
authors.lastname: Oláh
authors.affiliation: Institute of Organic Chemistry, Research Centre for National Sciences, HAS, Budapest, Hungary.
authors.forename: Károly
authors.initials: K
authors.lastname: Vékey
authors.affiliation: Institute of Enzymology, Research Centre for National Sciences, HAS, Budapest Hungary; Department of Applied Biotechnology and Food Science, Budapest University of Technology and Economics, Budapest, Hungary.
authors.forename: Beáta G
authors.initials: BG
authors.lastname: Vértessy
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D020134:Catalytic Domain; D019916:Choline-Phosphate Cytidylyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017124:Conserved Sequence; D006224:Cricetinae; D003412:Cricetulus; D004795:Enzyme Stability; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010641:Phenotype; D010963:Plasmodium falciparum / Q000201:enzymology; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D013696:Temperature
chemical_list: D019916:Choline-Phosphate Cytidylyltransferase