Oxidative posttranslational modifications in Plasmodium falciparum and coronaviruses

dc.contributor.advisorBecker, Katja
dc.contributor.advisorSträßer, Katja
dc.contributor.authorDillenberger, Melissa
dc.description.abstractThe protozoan parasite Plasmodium falciparum (P. falciparum) is the causative pathogen of the most severe form of malaria. For its survival and replication in human erythrocytes, P. falciparum depends on ATP as energy supply, which is almost entirely provided by the glycolytic pathway. Besides this strong dependence on glycolysis, the glycolytic enzymes of P. falciparum differ significantly from their human counterparts and are therefore considered promising drug targets. Moreover, many of those enzymes are shown to be targeted and potentially regulated by oxidative posttranslational modifications (oxPTMs). OxPTMs can occur at protein’s cysteine residues under both basal conditions and conditions that are associated with increased oxidative and/or nitrosative stress. Within this thesis, two glycolytic enzymes were enzymatically and structurally characterized: P. falciparum hexokinase (PfHK) and pyruvate kinase (PfPK). PfHK was confirmed to be a target for both S-glutathionylation and S-nitrosation. While both oxPTMs can inhibit the protein’s enzymatic activity, incubations with thioredoxin (PfTrx), glutaredoxin (PfGrx), and plasmoredoxin (PfPlrx) were shown to restore the activity after the modification. In addition, the first three-dimensional protein structure of PfHK was solved. The crystal structure reveals a Plasmodium-specific insertion in the small domain and confirms the tetrameric assembly already described for Plasmodium vivax hexokinase. This oligomerization is unique compared to hexokinases from other organisms. For PfPK, C49 and C343 were identified as important contributors to the functional and structural integrity of the enzyme. Respective cysteine-to-alanine mutants showed lowered enzymatic activities and substrate affinities. Furthermore, structural analysis revealed conformational changes within the mutant structures that are in accordance with the previous data. Overall, the present findings are an important contribution to understand the complex redox regulation of P. falciparum glycolytic enzymes and provide novel findings about their three-dimensional structure that will be advantageous for future structure-based drug design. Upon viral infections, both host cell and viral proteins are exposed to high levels of oxidative stress. However, viruses lack oxidative defense systems, and, in contrast to P. falciparum and many other organisms, there is a paucity of data about the effects of oxPTMs on viral proteins. The few studies available have shown conflicting effects: depending on the virus and protein studied, oxPTMs can induce either inhibitory or beneficial effects on the protein’s function or the whole replication. Coronaviruses are positive-stranded, enveloped RNA viruses causing various diseases in different mammals and birds. In humans, coronaviruses commonly cause mild respiratory tract infections. In this thesis, the methodological knowledge gained while studying oxPTMs of P. falciparum proteins was transferred to coronavirus nonstructural proteins (nsps). Among ~30 different nsps, several targets for S-glutathionylation could be identified. It was shown that the protease activity of nsp5 from two viral strains is inhibited upon S-glutathionylation. Cysteine-to-alanine mutants of severe acute respiratory syndrome coronavirus (SARS-CoV) nsp9 further identified single cysteines that may be more susceptible for modifications. In summary, the present data provides new insights into potential redox regulation of CoV proteins via oxPTMs. Finally, this thesis briefly summarizes results of a collaboration project in an excursus section. Using different experimental approaches, it could be confirmed that human spectrin is a target of S-glutathionylation.de_DE
dc.rightsIn Copyright*
dc.titleOxidative posttranslational modifications in Plasmodium falciparum and coronavirusesde_DE
local.affiliationFB 08 - Biologie und Chemiede_DE


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