title: Systematic Survey of the Plasmodium falciparum Hemoglobin Catabolic Pathway in Intact Cells Using the Digestomics Approach
Creators: name: Desmonts De Lamache, Dimitri Jean Marcel
Publisher: name: Graduate Studies
Year: 2024
Subjects: subject: Biology
subject: Biochemistry
subject: Plasmodium falciparum
subject: Proteomics
subject: Hemoglobin
subject: Software development
subject: Parasitology
subject: Bioinformatics
subject: FOS: Computer and information sciences
subject: Pathology

description: Hemoglobin (Hb) catabolism is an essential component of Plasmodium parasites’ intraerythrocytic asexual reproduction cycle. This catabolic process has been extensively studied over the past six decades as a potential target for antimalarial drugs. The current model describes Hb proteolysis as a semi-ordered process involving the concerted action of various endo- and exopeptidases, including aspartic, cysteine, metalloproteases and aminopeptidases. Importantly, this model is largely based on in vitro experiments using purified Plasmodium and recombinant proteases, and the molecular underpinnings of this catabolic process in intact cells remain poorly understood. This gap in knowledge directly results from the non-linear nature of protein catabolism in living cells. Proteolytic cascades, such as the Hb proteolytic pathway in Plasmodium parasites, produce large collections of closely related peptides that do not have well defined cut sites that can be defined a priory, making them inherently difficult to analyze comprehensively. Existing software tools rely on a defined precursor-product relationship to identify peptides and are thereby not well suited for analyzing semi-ordered proteolytic cascades occurring in vivo. To address this, I developed DigestR, an open-source software tool specifically designed for the analysis of large LC-MS proteomics datasets. DigestR enables users to visualize naturally occurring peptides, align them to a reference proteome, and display them at either a proteome-wide or protein-specific level. Additionally, DigestR allows for the analysis and plotting of peptide size, cleavage sites, net charges, and hydrophobicity, facilitating the identification of proteolytic patterns in natural systems and placing them in a biological relevant context. I first demonstrated the functionality of digestR with in vitro digestion experiments using known proteins and proteases. I then used digestR to conduct the first systematic survey of naturally occurring peptides resulting in from Hb catabolism by P. falciparum. To establish a baseline phenotype for characterizing phenotypic changes upon protease inhibition, I performed a temporal (throughout the parasite intraerythrocytic life cycle) and spatial (intracellular vs. extracellular) analysis of Hb-derived peptides. The data showed significant differences in Hb catabolism over the course of the parasite’s life cycle, with greater peptide diversity observed at the ring stage and maximal peptide relative abundances at the trophozoite stage. This was associated with differences in peptide cleavage sites and peptide length indicative of changes in proteolytic activity. I also observed significant differences between the intracellular and extracellular Hb peptidomes, highlighting a bias in peptide export against positively charged peptides. Furthermore, I consistently observed an enrichment of intracellular peptides with tyrosine residues at position P2 across groups, suggesting for a potential protective role against oxidative stress during hemoglobin catabolism. Subsequently, I analyzed perturbations in proteolytic activity upon protease knockout or inhibition and found that the diversity of naturally occurring peptides cannot be solely explained by the currently documented cut sites. My data demonstrates that, at the trophozoite stage, plasmepsins play a targeted role, while falcipains contribute to the bulk of Hb digestion. More importantly, I provide the first evidence that uncharacterized serine protease(s) may be major contributors of the hemoglobin degradation pathway. Finally, the accumulation of peptides around the same loci in both wild-type, knockout and inhibited strains, combined with the nested digestion patterns, suggests that hemoglobin endoproteases generate discrete hemoglobin fragments at a relatively small number of loci for further processing by aminopeptidases. In summary, this body of work provides the first map of naturally occurring peptides resulting from hemoglobin digestion by Plasmodium falciparum, ties enzyme activity to specific products, and highlights the existence uncharacterized proteases, which may serve as targets for future antimalarial drugs.