Cyclic nucleotide signalling in malaria parasites

Abstract

The cyclic nucleotides 3', 5'-cyclic adenosine monophosphate (cAMP) and 3', 5'-cyclic guanosine monophosphate (cGMP) are intracellular messengers found in most animal cell types. They usually mediate an extracellular stimulus to drive a change in cell function through activation of their respective cyclic nucleotide-dependent protein kinases, PKA and PKG. The enzymatic components of the malaria parasite cyclic nucleotide signalling pathways have been identified, and the genetic and biochemical studies of these enzymes carried out to date are reviewed herein. What has become very clear is that cyclic nucleotides play vital roles in controlling every stage of the complex malaria parasite life cycle. Our understanding of the involvement of cyclic nucleotide signalling in orchestrating the complex biology of malaria parasites is still in its infancy, but the recent advances in our genetic tools and the increasing interest in signalling will deliver more rapid progress in the coming years.

Keywords: Plasmodium; anopheles; cyclase; cyclic nucleotides; malaria parasites; phosphodiesterase.

Figure 1.

Stage-specific involvement of cyclic nucleotide signalling components and calcium-dependent protein kinases. A schematic depicting all the key stages of the complex P. falciparum life cycle. The inner circles depict which life-cycle stages have been associated with specific cyclic nucleotide signalling components and calcium-dependent protein kinases.

Figure 2.

Localization of the key Plasmodium cyclic nucleotide signalling components in a merozoite prior to egress. A schematic showing the key cyclic nucleotide signalling players in a merozoite within a P. falciparum blood stage schizont. Some of the depicted cellular locations of pathway components are speculative (such as PDEα, PDEβ and GCα). Evidence suggests that a subpopulation of PKG is localized in the ER membrane, but the means of attachment is unknown. A rhoptry localization is depicted for ACβ based on data obtained from Toxoplasma gondii, where the location of the ACβ orthologue in the rhoptries is known to be dependent on the presence of ARO [7]. PKA is depicted as being rhoptry localized based on P. falciparum epitope tagging studies [8]. Also speculative is the existence of an (as yet) unidentified IP₃-gated calcium channel (IP3R) for which there is pharmacological evidence.

Figure 3.

Cartoon of PKG and PKA domain organization and structure in mammals and Plasmodium. (a) Mammalian PKGs are homodimers encoded by single genes where dimerization is mediated by the dimerization domain. The inactive form of the enzyme adopts a conformation such that the autoinhibitory sequence interacts with the substrate-binding lobe, preventing access of substrates to the catalytic site. Binding of four cGMP molecules to the homodimer causes a conformational change, exposing the catalytic the domain to allow phosphorylation of substrates. (b) Plasmodium PKG is also encoded by a single gene; however, it lacks a dimerization domain and evidence suggests it forms a monomer. Plasmodium PKG contains four consensus cyclic nucleotide-binding domains (CNBs). One of these domains (CNB-C) is degenerate, therefore, the binding of only three cGMP molecules is required for activation of the enzyme. (c) Mammalian PKA is a heterotetramer consisting of two regulatory domains and two catalytic domains; however, unlike mammalian PKG, the regulatory and catalytic subunits are encoded by separate genes. The autoinhibitory sequence within the regulatory subunit binds to the substrate-binding lobe in the inactive form. The binding of four cAMP molecules results in the dissociation of the catalytic subunits from the regulatory subunits and activation of PKA. (d) Plasmodium PKA regulatory and catalytic subunits are also encoded by two separate genes; however, the regulatory subunit lacks a dimerization docking domain and the inactive enzyme is thought to form a heterodimer. The binding of two cAMP molecules results in dissociation of the subunits and allows binding of substrate proteins to the catalytic subunit.

Figure 4.

Mutation of the PKG gatekeeper residue confers inhibitor insensitivity. The ATP-binding pocket of PKG contains a small hydrophobic pocket. Inhibitors such as the imidazopyridine, Compound 2, compete with ATP for binding to PKG, with the fluorophenyl group of the inhibitor interacting with the hydrophobic pocket. Substitution of the threonine gatekeeper residue with a bulkier glutamine residue prevents binding of Compound 2 but the kinase remains functional with ATP still able to bind.