An Integrated Approach to Unravelling Malaria Cell Signalling Pathways

In the current thesis we analyse protein phosphorylation pathways in P. falciparum, the protozoa responsible for the most virulent form of malaria in order to both understand the role and scope of this protein modification in the parasite, and to explore its feasibility as a new drug target. With the aim to map phosphorylation pathways controlled by P. falciparum Casein Kinase 2 (PfCK2), we developed a new chemical-biological approach based on γ-modified ATP analogues bearing reporting groups on the transferred phosphate in order to selectively tag CK2 substrates. Despite being able to efficiently synthesise a small set of analogues, the data presented here shows that the P-N linkage bond between the nucleotide and the tag is stable during the assay conditions but not during the product analysis due to its acidic liability (e.g. with HPLC, MALDI); suggesting that a different type of linkage should be chosen in the future. Detailed characterisation studies of the parasite PfCK2 presented here showed a number of important features differing from human CK2. Docking analyses with a CK2 inhibitor showed that the PfCK2 ATP binding pocket is smaller than human CK2 due to the presence of Val116 and Leu45 which in the human kinase are replaced by more bulky isoleucine residues: Ile120 and Ile49. The difference between the human and parasite CK2 orthologues extends further to mechanisms of activation and regulation. Shown here is the autophosphorylation of PfCK2 that, unlike the human orthologue, occurs within subdomain I at Thr63. This autophosphorylation is essential for full catalytic activity. In addition we also showed that Thr63 phosphorylation regulates the interaction between the calalytic α-subunit and the regulatory β2-subunit. Here, we also presented evidence for tyrosine phosphorylated proteins in parasite infected red blood cells. PfCK2 can act as a dual specificity kinase phosphorylating P. falciparum Minichromosome Maintenance protein 2 (PfMCM2) on Tyr16 in vitro. It is therefore possible that PfCK2 may contribute to tyrosine phosphorylation within the parasite. Finally, we also reported a study regarding MCM2-Ser13 phosphorylation which successfully identified PfCK1 as the kinase responsible for this event

PfCK2 auto-phosphorylates <i>in vitro</i> on threonine 63.

<p><b>A</b>: <i>In vitro</i> kinase assay for GST-PfCK2 autophosphorylation, top panel: autoradiograph, bottom panel: Coomassie stain. <b>B</b>: LC-MS/MS trace identifying phosphorylation of PfCK2 at T63; right: Also shown is the hypothetical fragmentation table where the b-ions and y-ions detected in the LC-MS/MS spectra are shown in red and bold, respectively. <b>C</b>: Sequence of PfCK2 showing the phosphopeptide identified in the LC-MS/MS analysis (underlined) and the threonine 63 phosphorylation site (in red).</p

Structural analysis of PfCK2α inhibition by quinalizarin.

<p><b>A</b>: <i>In vitro</i> inhibition assay showing the affect of various concentrations of quinalizarin on the activity of human protein kinase CK2α (red) and PfCK2α (blue). Date represents the mean ± S.E.M (n = 3) <b>B</b>: Superimposition of the calculate <i>in silico</i> homology model for PfCK2α (purple) with the <i>Zea mays</i> protein kinase CK2α crystal structure (green, PDB code: 3FL5, resolution 2.30 Å); <b>B</b>: Superimposition of the molecular docking of the PfCK2 homology model with quinalizarin (purple) and the co-crystal structure of <i>Z. mays</i> protein kinase CK2α and quinalizarin (green); non-conserved residues are indicated in bold.</p

Autophosphorylation of PfCK2 regulates kinase activity.

<p>The activity of PfCK2α and a mutant PfCK2α where threonine 63 was mutated to alanine (T63A) was tested in <i>in vitro</i> kinase assays using α-casein as a substrate. <b>A</b>: Example of the <i>in vitro</i> kinase assay with PfCK2α and the T63A mutant. Top panel: autoradiograph, bottom panel: Coomassie stain. <b>B</b>: kinase activity quantification. Date represents the mean ± S.E.M (n = 3) <b>C</b>: LC-MS/MS trace of PfCK2 identifying T63 phosphorylation from a shizont stage lysate of <i>P. falciparum</i>. Indicated are the b-ions and b-ions (−98daltons) that were identified in the LC-MS/MS spectra. Also shown is the hypothetical fragmentation table where the ions that were identified in the LC-MS/MS spectra are shown in red.</p

PfCK2 phosphorylates MCM2 on Ser13 and Tyr16 <i>in vitro</i>.

<p><b>A</b>: In vitro kinase assay using a GST fusion protein containing a N-terminal portion of MCM2 (GST-MCM2) or the same fusion protein but where residue Y16 is mutated to an phenylalanine (Y16F) or where residue S13 is mutated to an alanine (S13A) or where both S13 and Y16 are mutated to an alanine and phenylalanine respectively (S13A/Y16F). Top panel: autoradiograph, bottom panel: Coomassie stain. <b>B</b>: LC-MS/MS trace of the fusion protein GST-PfMCM2 containing the S13 to alanine mutation following phosphorylation with PfCK2 indicating the phosphorylation of residue Y16. Also shown is the fragmentation table (detected b-ions and y-ions are represented respectively in bold red and bold blue). <b>C</b>: N-terminal sequence of PfMCM2 protein showing the phospho-peptide identified in the LC-MS/MS analysis that contains the tyrosine phosphorylated residue (in red).</p