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.

Heterologous expression of CTP:phosphocholine cytidylyltransferase from Plasmodium falciparum rescues Chinese Hamster Ovary cells deficient in the Kennedy phosphatidylcholine biosynthesis pathway

“…Since the catalytic domain is duplicated in Plasmodium CCT 22 , the Arg/His mutation was introduced at the respective positions of residue numbers 96 and 681 in the N-terminal and C-terminal halves of the protein, respectively. We have previously characterized this Arg/His point mutation within the second catalytic domain in vitro and demonstrated that the fold and the function of the protein is not damaged, whereas, thermal stability and dimer formation are heavily impaired 15 . To further prove that the rescue is linked to the enzymatic function of CCT, we engineered a Pf CCT variant harboring a mutation in the conserved HxGH motif, a unifying feature of the cytidylyltransferase superfamily which has been shown to function in substrate binding and catalysis 28 .…”

“…CHO-MT58 is a Chinese Hamster Ovarian (CHO) cell line generated by chemical mutagenesis from CHO-K1 14 . In CHO-MT58, the cct gene contains a point mutation resulting in an amino acid change (R140H) that deteriorates catalytic domain dimer formation 15 . While at 37 °C (permissive temperature) functional protein is formed albeit in smaller amount, at 40 °C (non-permissive temperature) an accelerated rate of CCT degradation is observed as a result of the point mutation.…”

CTP:phosphocholine cytidylyltransferase: Function, regulation, and structure of an amphitropic enzyme required for membrane biogenesis

“…2, instead of the homodimer arrangement common to most CCTs [224]. The dimerization interface and tertiary structure are likely very similar to the homodimeric CCTs, as shown by recent modeling and MD simulations of the P. falciparum CCT [224,225]. The CCT catalytic domain is highly conserved, and the structure of the rat catalytic domain is a very good model for other CCT catalytic domains [224].…”

Structural determinants of the catalytic mechanism of Plasmodium CCT, a key enzyme of malaria lipid biosynthesis

“…Enzyme activity measurements were performed as described previously, using a continuous coupled colorimetric assay with pyrophosphatase (PPase), purine nucleoside phosphorylase (PNP) auxiliary enzymes and the auxiliary substrate 7-methyl-6-thioguanosine (MESG) 16 , 30 , 39 . For CTP substrate titrations, CTP concentration was varied between 100 μM and 2 mM while ChoP concentration was kept constant at 5 mM.…”