Towards discovering novel aspects of nuclear biology in the malaria parasite "Plasmodium falciparum"

The apicomplexan parasite P. falciparum continues to cause morbidity and mortality imposing a significant health and economic burden on human society. In light of antimalarial drug resistance and the lack of an effective vaccine there is an urgent need to understand the basic biology of Plasmodium parasites in much greater detail. In particular, basic nuclear processes such as those remain surprisingly unsought despite their importance in parasite survival and life cycle progression. Thus, identification and localisation of novel parasite proteins to areas of the nucleus is an important first step towards giving new insights into nuclear architecture and function. The main aim of this thesis was to compile an inventory of the nuclear proteome across the intra-erythrocytic cell cycle using high accuracy mass spectrometry coupled with bioinformatics and in vivo localisation experiments. The dataset was analysed for accuracy and retention of true nuclear proteins revealing a final list of 802 potential nuclear proteins with an estimated precision of 76%. Interestingly, the informational pool of this study was able to identify a large number of novel nuclear components including novel protein domains possibly involved in gene regulation, members of the nuclear pores, the nucleolus and the proteasome (chapter 2). Several transgenic parasite lines used for the experimental validation part of the nuclear core proteome were further investigated in more detail. One of these transgenic cell lines expresses the C-terminally tagged bromo-domain protein PF10_0328 and was investigated by co-immuoprecipitation experiments followed by LC-MS/MS to identify interacting proteins. Bromodomain proteins bind specifically to acetylated lysine residues in histone tails and are important regulators of transcription. Our results suggest that PF10_0328 acts in concert with two additional bromo-domain proteins in regulating transcription in P. falciparum (chapter 3). Further characterisation on the functional level of these three important regulators is currently ongoing in a collaborative effort. Characterisation of bromo-domain proteins could establish new intervention strategies against malaria as the recognition of acetylated histone tails by bromo-domains can be selectively prevented by small molecules. Furthermore, several proteins residing in the nuclear pores and the nucleolus of P. falciparum were used to visualise these structures in relation to chromosome end clusters based on fluorescence microscopy. We show that both structures, involved in nuclear-cytoplasmic trafficking and ribosomal biogenesis, respectively, do not appear to ‘cross-talk’ with silenced chromosome ends at the nuclear periphery of P. falciparum (chapter 4). In conclusion, I believe that my work about several aspects of gene regulation and nuclear architecture increases the understanding of the biology of this medically important pathogen and could have potential to identify new avenues for interventions against malaria.