Abstract:
The polyamines putrescine, spermidine and spermine play essential roles in the proliferation and differentiation of most eukaryotic cells. Inhibition of the polyamine pathway is known to have antitumour and antiparasitic effects and á-difluoromethylornithine (DFMO), a polyamine biosynthesis inhibitor, is clinically used in the treatment of African sleeping sickness caused by Trypanosoma brucei gambiense. Ornithine decarboxylase (ODC) and Sadenosylmethionine decarboxylase (AdoMetDC) are the rate-limiting enzymes in polyamine metabolism. Usually, these enzymes are individually regulated, however, in the malaria parasite, Plasmodium falciparum, these enzymes are part of a unique bifunctional PfAdoMetDC/ODC protein. In addition, compared to homologous proteins, this malarial protein contains six unique parasite-specific inserted regions, which can be targeted with novel drugs. A modified restriction enzyme-mediated inverse PCR method was developed to delete the largest parasite-specific insert (411 bp) from the large PfAdoMetDC/ODC gene (4257 bp). The method was compared to existing deletion mutagenesis PCR protocols and was shown to be the most effective method (80% mutagenesis efficiency) as opposed to the 40% positively mutated clones obtained with the overlapping primer method in deleting a >100 bp region. The independent removal of all three the PfAdoMetDC domain parasite-specific inserts and subsequent activity analysis thereof showed that these inserts are essential for the catalytic activities of both the decarboxylase domains. Plasmodia conserved secondary structures within these inserts were identified and were also shown to be very important for domain activities, possibly through protein-protein interactions across and within the domains of the bifunctional complex for the efficient regulation of intracellular polyamine levels. The N-terminally located O1 insert in the PfODC domain is a highly conserved and structurally distinct insert, which is essential for both domain activities. Previous studies showed that the deletion of this insert prevents dimerisation of the PfODC monomers and as a result influences association of PfODC with the PfAdoMetDC domain to form the bifunctional ~330 kDa complex. In addition, immobilisation of the insert via the mutagenesis of flanking Gly residues and the disruption of a single conserved α-helix within the insert severely affected both PfODC and PfAdoMetDC activities. It was thus hypothesised that the helix is involved in protein-protein interactions and the dimerisation of the PfODC domain. Size-exclusion chromatography of the monofunctional PfODC and bifunctional PfAdoMetDC/ODC proteins with disrupted helices resulted in the elution of only the monomeric (~85 kDa) and heterodimeric PfAdoMetDC/ODC (~160 kDa) proteins, respectively. The mono- and bifunctional wild type and immobile proteins eluted as both dimeric PfODC (~170 kDa) and heterotetrameric (~330 kDa) fractions as a result of intact protein-protein interactions. These results were subsequently exploited in the design and application of a parasite-specific, mechanistically novel, inhibitory peptide specific for this non-homologous insert in the bifunctional protein. A 1000x molar excess of a synthetic peptide, complementary to the α-helix within the O1 insert but opposite in charge, resulted in a ~40% inhibition of the PfODC enzyme. This study thus provides a proof-of-principle for the use of an inhibitory peptide targeting a parasite-specific insert in the dimerisation interface of a uniquely bifunctional malarial protein.
