Phosphorylation in thePlasmodium falciparumproteome: A meta-analysis of publicly available data sets

AbstractMalaria is a deadly disease caused by Apicomplexan parasites of thePlasmodiumgenus. Several species of thePlasmodiumgenus are known to be infectious to human, of whichP. falciparumis the most virulent. Post-translational modifications (PTMs) of proteins coordinate cell signalling and hence, regulate many biological processes inP. falciparumhomeostasis and host infection, of which the most highly studied is phosphorylation. Phosphosites on proteins can be identified by tandem mass spectrometry (MS) performed on enriched samples (phosphoproteomics), followed by downstream computational analyses. We have performed a large-scale meta-analysis of 11 publicly available phosphoproteomics datasets, to build a comprehensive atlas of phosphosites in theP. falciparumproteome, using robust pipelines aimed at strict control of false identifications. We identified a total of 28,495 phosphorylated sites onP. falciparumproteins at 5% false localisation rate (FLR) and, of those, 18,100 at 1% FLR. We identified significant sequence motifs, likely indicative of different groups of kinases, responsible for different groups of phosphosites. Conservation analysis identified clusters of phosphoproteins that are highly conserved, and others that are evolving faster within thePlasmodiumgenus, and implicated in different pathways. We were also able to identify over 180,000 phosphosites withinPlasmodiumspecies beyondfalciparum, based on orthologue mapping. We also explored the structural context of phosphosites, identifying a strong enrichment for phosphosites on fast evolving (low conservation) intrinsically disordered regions (IDRs) of proteins. In other species, IDRs have been shown to have an important role in modulating protein-protein interactions, particularly in signalling, and thus warranting further study for their roles in host- pathogen interactions. All data has made available via UniProtKB, PRIDE and PeptideAtlas, with visualisation interfaces for exploring phosphosites in the context of other data onPlasmodiumproteins.Author SummaryPlasmodiumparasites continue to pose a significant global health threat, with a high proportion of the world at risk of malaria. It is imperative to gain new insights into cell signalling and regulation of biological processes in these parasites to develop effective treatments. This study focused on post- translational modifications (PTMs) of proteins, specifically phosphorylation. We conducted a meta- analysis of 11 publicly available phosphoproteomics datasets, identifying over 28,000 phosphorylated sites onP. falciparumproteins, using very rigorous statistics to avoid reporting false positives, and mapping to over 180,000 phosphorylation sites on other species ofPlasmodium.The analysis revealed distinct sequence motifs associated with different groups of phosphosites (and likely indicative of different upstream kinases), and differences in the downstream pathways regulated. Conservation analysis highlighted clusters of phosphoproteins evolving at different rates within thePlasmodiumgenus. Notably, phosphorylation was enriched in regions of proteins lacking distinct structural elements, known as intrinsically disordered regions (IDRs), which are poorly conserved across the genus – we speculate that they are important for modulating protein interactions. The findings provide valuable insights into the molecular mechanisms ofP. falciparum, with potential implications for understanding host-pathogen interactions. The comprehensive dataset generated is now publicly accessible, serving as a valuable resource for the scientific community through UniProtKB, PRIDE, and PeptideAtlas.