Plasmodium falciparum Cyclic GMP-Dependent Protein Kinase Interacts with a Subunit of the Parasite Proteasome

Abstract

Malaria is caused by the protozoan parasite Plasmodium, which undergoes a complex life cycle in a human host and a mosquito vector. The parasite’s cyclic GMP (cGMP)-dependent protein kinase (PKG) is essential at multiple steps of the life cycle. Phosphoproteomic studies in Plasmodium falciparum erythrocytic stages and Plasmodium berghei ookinetes have identified proteolysis as a major biological pathway dependent on PKG activity. To further understand PKG’s mechanism of action, we screened a yeast two-hybrid library for P. falciparum proteins that interact with P. falciparum PKG (PfPKG) and tested peptide libraries to identify its phosphorylation site preferences. Our data suggest that PfPKG has a distinct phosphorylation site and that PfPKG directly phosphorylates parasite RPT1, one of six AAA+ ATPases present in the 19S regulatory particle of the proteasome. PfPKG and RPT1 interact in vitro, and the interacting fragment of RPT1 carries a PfPKG consensus phosphorylation site; a peptide carrying this consensus site competes with the RPT1 fragment for binding to PfPKG and is efficiently phosphorylated by PfPKG. These data suggest that PfPKG’s phosphorylation of RPT1 could contribute to its regulation of parasite proteolysis. We demonstrate that proteolysis plays an important role in a biological process known to require Plasmodium PKG: invasion by sporozoites of hepatocytes. A small-molecule inhibitor of proteasomal activity blocks sporozoite invasion in an additive manner when combined with a Plasmodium PKG-specific inhibitor. Mining the previously described parasite PKG-dependent phosphoproteomes using the consensus phosphorylation motif identified additional proteins that are likely to be direct substrates of the enzyme.

Inhibition of Resistance-Refractory P. falciparum Kinase PKG Delivers Prophylactic, Blood Stage, and Transmission-Blocking Antiplasmodial Activity

“…URP likely regulates ubiquitination and degradation of proteins by the proteasome, and it is reported to have phosphorylation sites at position S37 and S140 ( Collins et al., 2014 , Kumar et al., 2017 ). A recent report found that PKG phosphorylates RPT1, an AAA + ATPase present in the 19S regulatory unit of the proteasome, and regulates parasite proteolysis ( Govindasamy et al., 2019 ). Interestingly, the same report highlighted the importance of proteolysis for invasion by sporozoites of hepatocytes.…”

Ca 2+ signals critical for egress and gametogenesis in malaria parasites depend on a multipass membrane protein that interacts with PKG

“…In contrast, little is known about proteins interacting with PKG in eukaryotes as few partners have been identified (13,(47)(48)(49). In Plasmodium, the only previous data available for a PKG-interacting protein came from an in vitro study using the kinase domain of PKG (50), while a putative interaction between PKG and ICM1 was detected in a very recent elegant study profiling a potent inhibitor of PKG. However, conditional knockdown of ICM1 in that work resulted only in a minor growth defect, perhaps due to the different conditional system used (51), the TetR-DOZI aptamer system, which may require more than one cycle to achieve efficient down-regulation (52).…”

Plasmodium sporozoites on the move: Switching from cell traversal to productive invasion of hepatocytes

“…Upstream of Ca 2+ , cyclic guanosine monophosphate (cGMP) activates protein kinase G (PKG), resulting in the release of intracellular Ca 2+ (Carey et al, 2014; Kebaier & Vanderberg, 2010; Ono et al, 2008), via the action of phosphoinositide phospholipase C (PI‐PLC) and the production of inositol triphosphate (IP3) (Carey et al, 2014). The use of PKG inhibitors and PKG mutagenesis showed that PKG is required in P. berghei for TRAP secretion, sporozoite gliding motility and host cell invasion (Govindasamy et al, 2016; Govindasamy et al, 2019; Panchal & Bhanot, 2010). Serum albumin, a natural agonist for PKG‐dependent microneme secretion (Brown et al, 2016), also activates Ca 2+ ‐dependent motility in sporozoites (Kebaier & Vanderberg, 2010).…”