Characterization of an A-kinase anchoring protein-like suggests an alternative way of PKA anchoring in Plasmodium falciparum

International audienceBackground: The asexual intra‑erythrocytic multiplication of the malaria parasite Plasmodium falciparum is regulated by various molecular mechanisms. In eukaryotic cells, protein kinases are known to play key roles in cell cycle regula‑ tion and signaling pathways. The activity of cAMP‑dependent protein kinase (PKA) depends on A‑kinase anchoring proteins (AKAPs) through protein interactions. While several components of the cAMP dependent pathway—includ‑ ing the PKA catalytic and regulatory subunits—have been characterized in P. falciparum, whether AKAPs are involved in this pathway remains unclear. Here, PfAKAL, an open reading frame of a potential AKAP‑like protein in the P. falcipa-rum genome was identified, and its protein partners and putative cellular functions characterized. Methods: The expression of PfAKAL throughout the erythrocytic cycle of the 3D7 strain was assessed by RT‑qPCR and the presence of the corresponding protein by immunofluorescence assays. In order to study physical interactions between PfAKAL and other proteins, pull down experiments were performed using a recombinant PfAKAL protein and parasite protein extracts, or with recombinant proteins. These interactions were also tested by combining bio‑ chemical and proteomic approaches. As phosphorylation could be involved in the regulation of protein complexes, both PfAKAL and Pf14‑3‑3I phosphorylation was studied using a radiolabel kinase activity assay. Finally, to identify a potential function of the protein, PfAKAL sequence was aligned and structurally modeled, revealing a conserved nucleotide‑binding pocket; confirmed by qualitative nucleotide binding experiments. Results: PfAKAL is the first AKAP‑like protein in P. falciparum to be identified, and shares 23 % sequence identity with the central domain of human AKAP18δ. PfAKAL is expressed in mature asexual stages, merozoites and gametocytes. In spite of homology to AKAP18, biochemical and immunochemical analyses demonstrated that PfAKAL does not interact directly with the P. falciparum PKA regulatory subunit (PfPKA‑R), but instead binds and colocalizes with Pf14‑ 3‑3I, which in turn interacts with PfPKA‑R. In vivo, these different interactions could be regulated by phosphorylation, as PfPKA‑R and Pf14‑3‑3I, but not PfAKAL, are phosphorylated in vitro by PKA. Interestingly, PfAKAL binds nucleotides such as AMP and cAMP, suggesting that this protein may be involved in the AMP‑activated protein kinase (AMPK) pathway, or associated with phosphodiesterase activities. Conclusion: PfAKAL is an atypical AKAP that shares common features with human AKAP18, such as nucleotides binding. The interaction of PfAKAL with PfPKA‑R could be indirectly mediated through a join interaction with Pf14‑ 3‑3I. Therefore, PfPKA localization could not depend on PfAKAL, but rather involves other partners..org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated

Impact of Hemoglobin S Trait on Cell Surface Antibody Recognition of Plasmodium falciparum-Infected Erythrocytes in Pregnancy-Associated Malaria

International audienceBackground. Sickle cell trait (HbAS) confers partial protection against malaria by reducing the adhesion of Plasmodium falciparum-infected erythrocytes to host receptors, but little is known about its potential protection against placental malaria. Methods. Using flow cytometry, we assessed the recognition of HbAA and HbAS VAR2CSA-expressing infected erythrocytes, by plasma from 159 Beninese pregnant women with either HbAA (normal) or HbAS. Using multivariate linear models adjusted for gravidity, parasite infection at delivery, glucose-6-phosphate dehydrogenase deficiency, and α-thalassemia carriage, we observed significantly reduced cell surface antibody binding of HbAS-infected erythrocytes by plasma from HbAS compared with HbAA women (P < 10-3). Results. The difference in cell surface antibody binding was only observed when infected erythrocytes and plasma were associated according to the same hemoglobin genotype. Similar levels of VAR2CSA-specific antibody were measured by enzyme-linked immunosorbent assay in the 2 groups, suggesting that the altered interaction between VAR2CSA and HbAS women's antibodies could reflect abnormal display of VAR2CSA on HbAS erythrocytes. Conclusions. Our data stress the need for assessments of erythrocyte disorders such as the sickle cell trait in a population group when studying immunological responses to P falciparum