Characterisation of the eukaryotic initiation factor 2alpha kinases of Plasmodium falciparum.
PhD thesis, University of Glasgow.
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Malaria remains a devastating disease with respect to both mortality and the constraints it places on the economic development of the countries in which it is endemic. Our laboratory is seeking new antimalarial targets, by characterising the protein kinases of the most lethal human malaria parasite, Plasmodium falciparum. As central components of many diverse signalling pathways, protein kinases are crucial for the control of proliferation and differentiation in other eukaryotes; we hypothesise that they play similar roles in P. falciparum. The life cycle of P. falciparum is complex, consisting of a series of tightly controlled stages of division and differentiation. In the related apicomplexan parasite Toxoplasma gondii, stress stimuli have been implicated in an important differentiation step, from rapidly dividing tachyzoites, to quiescent bradyzoites (which enable immune evasion). Evidence suggests that stress may also contribute to an essential differentiation stage, gametocytogenesis, in P. falciparum. In yeast and metazoans, part of the stress response is mediated through phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha), which results in selective translation of mRNAs encoding stress response proteins. Post-transcriptional control of gene expression is suspected to play an important role in P. falciparum. Importantly, the Goldberg laboratory recently demonstrated that similarly, in P. falciparum the eIF2alpha orthologue is phosphorylated in response to starvation.
Here we identify the P. falciparum orthologue of the translation initiation factor eIF2alpha and provide bioinformatic evidence for the presence of three eIF2alpha kinases in P. falciparum; PfeIK1, PfeIK2 and PfPK4, only one of which (PfPK4) has been described previously (Mohrle et al., 1997). We show that one of the novel eIF2alpha kinases, PfeIK1, is able to phosphorylate P. falciparum eIF2alpha in vitro. In addition, initial experiments support previous observations that PfPK4 is indeed an active protein kinase (Mohrle et al., 1997). We present evidence that PfPK4 is essential for asexual growth, which precludes straightforward reverse genetics studies aiming to determine its possible role in gametocytogenesis. In contrast, transgenic parasites allowed us to show that neither PfeIK1 nor PfeIK2 are required for asexual growth, or sexual development of the parasite in the mosquito vector. However, preliminary evidence (requiring confirmation) may indicate that parasites lacking PfeIK1 over-express PfPK4, which would suggest that PfeIK1 may play an important function in the parasite. This study strongly suggests that a mechanism for versatile regulation of translation by several kinases with a similar catalytic domain, but distinct regulatory domains, is conserved in P. falciparum.
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