Plasmodium falciparum is the causative agent of the most lethal form of human malaria. In the absence of a vaccine and the emergence of drug-resistant parasites, novel targets against malaria are required. The ubiquitin-proteasome system (UPS), which involves the attachment of the small protein ubiquitin to target proteins, is essential to all eukaryotes and represents a promising target for antimalarial therapies. However, the components of this system are largely uncharacterised.

The work undertaken in this thesis explores the ubiquitin landscape of P.falciparum with a focus on E3 ubiquitin ligases as the most numerous and diverse component of this pathway. Activity based probes (ABPs) were used to identify ubiquitin pathway enzymes expressed and active during the asexual stage of P.falciparum. Two ABPs Ub-Dha and Ub-PA were used co-operatively in concert with immunoprecipitation and mass spectrometry (IP-MS/MS) to identify several families of ubiquitin enzymes as well as proteins of unknown function. In vitro ubiquitination assays validated the activity of identified E2 ubiquitin conjugating enzymes, and a HECT E3 ligase, PfHEUL. The catalytic residue of PfHEUL was found to be C8558 by site-directed mutagenesis, however, attempts to genetically modify the PfHEUL locus in vivo were unsuccessful indicating gene immutability and emphasising the importance of the gene function to asexual stage parasites.

Two families of Cullin-RING ligases (CRLs) were identified in P.falciparum, and a transgenic approach was used to tag endogenous PfCullin1 and PfCullin2 to facilitate localisation and co-immunoprecipitation studies. Associated proteins identified through this approach indicated a role for PfCullin1 in DNA replication and PfCullin2 in protein homeostasis. PfFBXO6, an F-box domain containing protein found to be associated with PfCullin1, was shown to interact with PfSkp1 and to be involved in DNA replication through interactions with the MCM complex in the nucleus. Conditional knockdown of PfFBXO6, in conjunction with ubiquitin-derived diGly remnant enrichment, revealed potential ubiquitination substrates of PfFBXO6 containing CRL1 (PfSCF^FBXO6) involved in DNA replication and gametocytogenesis. The utilisation of data-independent acquisition (DIA) mass spectrometry generated the largest Plasmodium ubiquitome to date.

These findings contribute to a deeper understanding of the ubiquitin pathway in P.falciparum and provide potential targets for the development of antimalarial therapies.