The substrate quality of CK2 target sites has a determinant role on their function and evolution.

Most biological processes are regulated by signaling modules that bind to short linear motifs. For protein kinases, substrates may have full or only partial matches to the kinase recognition motif, a property known as "substrate quality." However, it is not clear whether differences in substrate quality represent neutral variation or if they have functional consequences. We examine this question for the kinase CK2, which has many fundamental functions. We show that optimal CK2 sites are phosphorylated at maximal stoichiometries and found in many conditions, whereas minimal substrates are more weakly phosphorylated and have regulatory functions. Optimal CK2 sites tend to be more conserved, and substrate quality is often tuned by selection. For intermediate sites, increases or decreases in substrate quality may be deleterious, as we demonstrate for a CK2 substrate at the kinetochore. The results together suggest a strong role for substrate quality in phosphosite function and evolution. A record of this paper's transparent peer review process is included in the supplemental information.

A screen for Plasmodium falciparum sporozoite surface protein binding to human hepatocyte surface receptors identifies novel host-pathogen interactions.

BACKGROUND: Sporozoite invasion of hepatocytes is an essential step in the Plasmodium life-cycle and has similarities, at the cellular level, to merozoite invasion of erythrocytes. In the case of the Plasmodium blood-stage, efforts to identify host-pathogen protein-protein interactions have yielded important insights including vaccine candidates. In the case of sporozoite-hepatocyte invasion, the host-pathogen protein-protein interactions involved are poorly understood. METHODS: To gain a better understanding of the protein-protein interaction between the sporozoite ligands and host receptors, a systematic screen was performed. The previous Plasmodium falciparum and human surface protein ectodomain libraries were substantially extended, resulting in the creation of new libraries comprising 88 P. falciparum sporozoite protein coding sequences and 182 sequences encoding human hepatocyte surface proteins. Having expressed recombinant proteins from these sequences, a plate-based assay was used, capable of detecting low affinity interactions between recombinant proteins, modified for enhanced throughput, to screen the proteins for interactions. The novel interactions identified in the screen were characterized biochemically, and their essential role in parasite invasion was further elucidated using antibodies and genetically manipulated Plasmodium parasites. RESULTS: A total of 7540 sporozoite-hepatocyte protein pairs were tested under conditions capable of detecting interactions of at least 1.2 µM KD. An interaction between the human fibroblast growth factor receptor 4 (FGFR4) and the P. falciparum protein Pf34 is identified and reported here, characterizing its affinity and demonstrating the blockade of the interaction by reagents, including a monoclonal antibody. Furthermore, further interactions between Pf34 and a second P. falciparum rhoptry neck protein, PfRON6, and between human low-density lipoprotein receptor (LDLR) and the P. falciparum protein PIESP15 are identified. Conditional genetic deletion confirmed the essentiality of PfRON6 in the blood-stage, consistent with the important role of this protein in parasite lifecycle. Pf34 was refractory to attempted genetic modification. Antibodies to Pf34 abrogated the interaction and had a modest effect upon sporozoite invasion into primary human hepatocytes. CONCLUSION: Pf34 and PfRON6 may be members of a functionally important invasion complex which could be a target for future interventions. The modified interaction screening assay, protein expression libraries and P. falciparum mutant parasites reported here may be a useful tool for protein interaction discovery and antigen candidate screening which could be of wider value to the scientific community.

PerTurboID, a targeted in situ method reveals the impact of kinase deletion on its local protein environment in the cytoadhesion complex of malaria-causing parasites.

Reverse genetics is key to understanding protein function, but the mechanistic connection between a gene of interest and the observed phenotype is not always clear. Here we describe the use of proximity labeling using TurboID and site-specific quantification of biotinylated peptides to measure changes to the local protein environment of selected targets upon perturbation. We apply this technique, which we call PerTurboID, to understand how the Plasmodium falciparum-exported kinase, FIKK4.1, regulates the function of the major virulence factor of the malaria-causing parasite, PfEMP1. We generated independent TurboID fusions of two proteins that are predicted substrates of FIKK4.1 in a FIKK4.1 conditional KO parasite line. Comparing the abundance of site-specific biotinylated peptides between wildtype and kinase deletion lines reveals the differential accessibility of proteins to biotinylation, indicating changes to localization, protein-protein interactions, or protein structure which are mediated by FIKK4.1 activity. We further show that FIKK4.1 is likely the only FIKK kinase that controls surface levels of PfEMP1, but not other surface antigens, on the infected red blood cell under standard culture conditions. We believe PerTurboID is broadly applicable to study the impact of genetic or environmental perturbation on a selected cellular niche.

Enzymes known as protein kinases regulate a huge variety of biological processes inside cells by attaching small tags known as phosphate groups onto specific locations on certain proteins. For example, the parasite that causes malaria infections in humans and great apes, injects a protein kinase called FIKK4.1 into certain cells in its host. This enzyme then adds phosphate groups to various parasite and host proteins that, in turn, causes them to form a large group of proteins (known as the cytoadhesion complex) to protect the parasite from being cleared by the hosts' immune defences. However, it remains unclear how and where the complex forms, and how the parasite regulates it. Proximity labelling is a well-established method that allows researchers to label and identify proteins that are near to a protein of interest. To investigate how the FIKK4.1 enzyme alters host cells to make the cytoadhesion complex, Davies et al. combined proximity labelling with methods that disturb the normal state of cells at a specific timepoint during development. The team used this new approach ­ named PerTurboID ­ to identify the proteins surrounding three components in the cytoadhesion complex. This made it possible to create a map of proteins that FIKK4.1 is likely to modify to build and control the cytoadhesion complex. Further experiments examined what happened to these surrounding proteins when FIKK4.1 was inactivated. This revealed that some protein targets of FIKK4.1 become either more or less accessible to other enzymes that attach a molecule known as biotin to proteins. This could be a result of structural changes in the cytoadhesion complex that are normally regulated by the FIKK4.1 kinase. In the future, PerTurboID may be useful to study how genetics or environmental changes affect other groups of proteins within specific environments inside cells, such as protein complexes required for DNA replication or cell division, or assembly of temporal structures required for cell movement.

An exported kinase family mediates species-specific erythrocyte remodelling and virulence in human malaria.

The most severe form of human malaria is caused by Plasmodium falciparum. Its virulence is closely linked to the increase in rigidity of infected erythrocytes and their adhesion to endothelial receptors, obstructing blood flow to vital organs. Unlike other human-infecting Plasmodium species, P. falciparum exports a family of 18 FIKK serine/threonine kinases into the host cell, suggesting that phosphorylation may modulate erythrocyte modifications. We reveal substantial species-specific phosphorylation of erythrocyte proteins by P. falciparum but not by Plasmodium knowlesi, which does not export FIKK kinases. By conditionally deleting all FIKK kinases combined with large-scale quantitative phosphoproteomics we identified unique phosphorylation fingerprints for each kinase, including phosphosites on parasite virulence factors and host erythrocyte proteins. Despite their non-overlapping target sites, a network analysis revealed that some FIKKs may act in the same pathways. Only the deletion of the non-exported kinase FIKK8 resulted in reduced parasite growth, suggesting the exported FIKKs may instead support functions important for survival in the host. We show that one kinase, FIKK4.1, mediates both rigidification of the erythrocyte cytoskeleton and trafficking of the adhesin and key virulence factor PfEMP1 to the host cell surface. This establishes the FIKK family as important drivers of parasite evolution and malaria pathology.

A Novel Tool for the Generation of Conditional Knockouts To Study Gene Function across the Plasmodium falciparum Life Cycle.

Plasmodium falciparum has a complex life cycle that involves interaction with multiple tissues inside the human and mosquito hosts. Identification of essential genes at all different stages of the P. falciparum life cycle is urgently required for clinical development of tools for malaria control and eradication. However, the study of P. falciparum is limited by the inability to genetically modify the parasite throughout its life cycle with the currently available genetic tools. Here, we describe the detailed characterization of a new marker-free P. falciparum parasite line that expresses rapamycin-inducible Cre recombinase across the full life cycle. Using this parasite line, we were able to conditionally delete the essential invasion ligand AMA1 in three different developmental stages for the first time. We further confirm efficient gene deletion by targeting the nonessential kinase FIKK7.1.IMPORTANCE One of the major limitations in studying P. falciparum is that so far only asexual stages are amenable to rapid conditional genetic modification. The most promising drug targets and vaccine candidates, however, have been refractory to genetic modification because they are essential during the blood stage or for transmission in the mosquito vector. This leaves a major gap in our understanding of parasite proteins in most life cycle stages and hinders genetic validation of drug and vaccine targets. Here, we describe a method that supports conditional gene deletion across the P. falciparum life cycle for the first time. We demonstrate its potential by deleting essential and nonessential genes at different parasite stages, which opens up completely new avenues for the study of malaria and drug development. It may also allow the realization of novel vaccination strategies using attenuated parasites.

A versatile strategy for rapid conditional genome engineering using loxP sites in a small synthetic intron in Plasmodium falciparum.

Conditional genome engineering in the human malaria pathogen Plasmodium falciparum remains highly challenging. Here we describe a strategy for facile and rapid functional analysis of genes using an approach based on the Cre/lox system and tailored for organisms with short and few introns. Our method allows the conditional, site-specific removal of genomic sequences of essential and non-essential genes by placing loxP sites into a short synthetic intron to produce a module (loxPint) can be placed anywhere in open reading frames without compromising protein expression. When duplicated, the loxPint module serves as an intragenic recombineering point that can be used for the fusion of gene elements to reporters or the conditional introduction of point mutations. We demonstrate the robustness and versatility of the system by targeting the P. falciparum merozoite surface protein 1 gene (msp1), which has previously proven refractory to genetic interrogation, and the parasite exported kinase FIKK10.1.