Molecular Mechanism for the Thermo-Sensitive Phenotype of CHO-MT58 Cell Line Harbouring a Mutant XXXX : @entity2945 Cytidylyltransferase.

@entity73130 @entity101 @entity93 @entity2434 @entity601 @entity3964 @entity311 @entity2945
UNASSIGNED: Control and elimination of @entity311 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 @entity3964 parasite has been validated as promising candidate antimalarial target. The most prevalent de novo pathway for synthesis of @entity2434 is the Kennedy pathway. Its regulatory and often also rate limiting step is catalyzed by @entity93 : @entity2945 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 @entity3964 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 @entity601 simulations to assess the inter-subunit interactions in the dimer. Our results demonstrate that the @entity73130 mutation does not directly influence enzyme catalytic activity. Instead, it provokes @entity101 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.
