Malaria remains a detrimental tropical disease caused by the protozoan parasites known as Plasmodium and is threatening almost half the worlds population. Nitric oxide (NO) and NO-derived reactive nitrogen species are well-known in vivo effector molecules for controlling the malaria parasites in both mosquito transmitting vectors and humans. However, NO targets and the profound mechanism of NO actions in the malaria parasites remain largely unexplored. Protein S-nitrosylation (SNO), a protein posttranslational modification induced by NO and RNS, has been recognized to substantially mediate diverse biological effects of NO in vivo. In this thesis, we describe a comprehensive analysis of protein S-nitrosylation in the most deadly malaria parasite Plasmodium falciparum. By using a biotin-switch assay coupled with mass spectrometry, we have identified, for the first time, 319 potential S-nitrosylation targets in Plasmodium falciparum that are widespread in various cellular pathways. Interestingly, some metabolic pathways such as glycolysis show an accumulation of S-nitrosylated proteins, indicating a high susceptibility to NO-mediated regulation. A major redox protein, P. falciparum thioredoxin 1 (PfTrx1) was found to be able to transfer or remove NO to or from other proteins, thus possibly mediating the transnitrosylation and denitrosylation in the parasites. We thus propose a redox status-based model of the role of PfTrx1 in the regulation of SNO in P. falciparum. The model suggests that PfTrx1 may protect the parasite from nitrosative stress via protein denitrosylation, whereas upon intensive nitrosative stress PfTrx1 may switch its role to transduce nitrosative stress via protein transnitrosylation. We believe that this study contributes to our understanding of how central metabolic processes in malaria parasites are regulated by NO and suggests that P. falciparum employs the thioredoxin system to deal with the nitrosative challenge.The protein turnover via the ubiquitin-proteasome system (UPS) is well-acknowledged as an essential mechanism profoundly involved in many cellular events in eukaryotes. The classical UPS pathway in eukaryotes typically consists of polyubiquitination of protein substrates, polyubiquitinylated substrates transportation, and substrate recognition by the eukaryotic 26S proteasome, and substrate degradation by the proteasome. Although compelling data have indicated the presence of a functional 26S proteasome network in P. falciparum, the componential integrity and the functionality of the plasmodial 26S proteasome has not been systematically studied so far. In this thesis, we aimed to characterize the mechanism of substrate recognition by the 26S proteasome of P. falciparum. By using in silico analysis and biochemical investigations, we have identified three ubiquitin receptor domains inherited by two plasmodial proteasome subunits, the Rpn10 and Rpn13 subunits. These newly identified plasmodial ubiquitin receptor domains include two putative ubiquitin-interacting motif (UIM) domains and a putative Pleckstrin-like receptor for ubiquitin (Pru) domain. The P. falciparum UIM domains (PfUIMs) and Pru domain (PfPru) were demonstrated to bind ubiquitin-like domains (UBLs) from the plasmodial homologues of Rad23 and Dsk2, which are important UBL-containing proteins involved in the transportation of proteasomal substrates to the 26S proteasome. Strikingly, only PfUIM2 domain was found to be able to directly bind polyubiquitin chains, indicating that PfUIM2 is the site for the direct recognition of polyubiquitinylated substrates for proteasomal degradation. Based on the affinity of PfUIM2 domain to the UBL domain of the plasmodial Rad23, we successfully established a simple and efficient affinity purification method to isolate the P. falciparum 26S proteasome complex together with a number of putative proteasome-interacting proteins (PIPs) directly from the parasite extracts. With this method, we provide the first insight into the componential composition of the plasmodial 26S proteasome. More importantly, the co-purification of putative PIPs in P. falciparum has allowed us to take a first view of the protein metabolism network orchestrated by the plasmodial UPS, and may provide new targets for developing novel antimalarial agents targeting the plasmodial UPS or related pathways.
Verknüpfung zu Publikationen oder weiteren Datensätzen
