Investigation of oxidative protein folding in protist mitochondria and elucidation of the catalytic mechanism of glutaredoxins

Protein import into the mitochondrial intermembrane space (IMS) of parasitic protists differs from other eukaryotes despite a general conservation of mitochondrial protein import signals. Imported proteins typically carry conserved cysteine-motifs that are oxidized in the IMS. In opisthokonta such as yeast and mammals, these motifs are recognized and oxidized by the oxidoreductase Mia40 leading to the formation of intramolecular disulfide bonds. Reduced Mia40 is subsequently re-oxidized by the sulfhydryl electron transferase Erv1. In kinetoplastida, such as Leishmania tarentolae, and in apicomplexan parasites, such as Plasmodium falciparum, no Mia40 homolog could be identified so far. However, conserved substrates and an Erv homolog in these parasites suggest the existence of a Mia40 replacement. Indeed, preliminary results revealed two disulfide-bonded interaction partners of PfErv as demonstrated by western blot analyses. The major objective of this thesis was the identification of a potential Mia40 replacement in the parasite L. tarentolae. Numerous experiments were performed in order to trap mixed disulfide intermediates between the model substrate LtsTim1 or LtErv and a potential interaction partner. A variety of protocols with alkylating, oxidizing or reducing agents did not reveal mixed disulfides between LtErv and the adapter replacement in western blot analyses. In addition, even with highly enriched LtErv protein levels after denaturing or native pulldown, no mixed disulfide intermediate could be identified. In contrast, staining against LtsTim1 in western blot analyses showed disulfide-bridged intermediates indicating potential heterodimers of LtsTim1 and Mia40 replacement candidates. However, an enrichment of these intermediates by affinity chromatography and further analysis failed because of systematic problems with the hydrophobicity of the substrate and the LtsTim1-antibody. To summarize, a potential replacement for Mia40 in parasitic protists remains to be identified and the oxidative folding machinery in the IMS of kinetoplastida and apicomplexa could not be unraveled during this PhD project. Four alternative cysteine-containing substrates of the oxidative protein folding pathway in the IMS were already designed during this project and might lead to a rather fast breakthrough in further experiments. Glutaredoxins (Grx) are highly conserved enzymes that play important roles in redox catalysis and iron metabolism and are found in almost all organisms. The traditional monothiol mechanism of Grx catalysis is divided into an oxidative half-reaction with the first glutathionylated substrate and a reductive half reaction with the reduced tripeptide glutathione (GSH) as the second substrate. However, this traditional model cannot explain how exactly the two different substrates of Grx are bound. Hence, two refined models of Grx catalysis namely the “glutathione scaffold model” and the “glutathione activator model” were previously proposed and experimentally confirmed for two residues of ScGrx7. This model can help to distinguish protein areas that either interact with the disulfide substrate (a scaffold site, including Glu170 in ScGrx7) or with the reducing agent (an activator site, including Lys105 in ScGrx7). The second objective of this PhD project was to test the general applicability of this model using the non-related enzyme PfGrx. Moreover, four additional residues that were previously suggested to contribute to the glutathione activator site in ScGrx7 were characterized in this thesis. Taken together, I confirmed the existence of two distinct glutathione interaction sites with the non-related model enzyme PfGrx. Moreover, I could identify Arg153 of ScGrx7 as another potential scaffold site residue. In addition, I could show that the two charge inversion mutants with a positively charged amino acid of the helix 3 residues Asp144 and Glu147 in ScGrx7 enhanced the interaction with the second substrate GSH. Hence, the helix 3 of these “gain-offunction” mutants indeed seems to affect the glutathione activator site. Furthermore, I could show that the introduced mutations influenced the pKa value of the active site cysteine of ScGrx7 only to a minor degree, except for ScGrx7K105E. Modeling of the transition states and analyses of the different mutants by roGFP assays may help to elucidate the structure-function relationships of Grx in further analyses

Data of oxygen- and pH-dependent oxidation of resveratrol

AbstractWe show here if under physiologically relevant conditions resveratrol (RSV) remains stable or not. We further show under which circumstances various oxidation products of RSV such as ROS can be produced. For example, in addition to the widely known effect of bicarbonate ions, high pH values promote the decay of RSV. Moreover, we analyse the impact of reduction of the oxygen partial pressure on the pH-dependent oxidation of RSV. For further interpretation and discussion of these focused data in a broader context we refer to the article “Hormetic shifting of redox environment by pro-oxidative resveratrol protects cells against stress” (Plauth et al., in press) [1]

Hormetic shifting of redox environment by pro-oxidative resveratrol protects cells against stress

AbstractResveratrol has gained tremendous interest owing to multiple reported health-beneficial effects. However, the underlying key mechanism of action of this natural product remained largely controversial. Here, we demonstrate that under physiologically relevant conditions major biological effects of resveratrol can be attributed to its generation of oxidation products such as reactive oxygen species (ROS). At low nontoxic concentrations (in general <50µM), treatment with resveratrol increased viability in a set of representative cell models, whereas application of quenchers of ROS completely truncated these beneficial effects. Notably, resveratrol treatment led to mild, Nrf2-specific gene expression reprogramming. For example, in primary epidermal keratinocytes derived from human skin this coordinated process resulted in a 1.3-fold increase of endogenously generated glutathione (GSH) and subsequently in a quantitative reduction of the cellular redox environment by 2.61mVmmol GSH per g protein. After induction of oxidative stress by using 0.78% (v/v) ethanol, endogenous generation of ROS was consequently reduced by 24% in resveratrol pre-treated cells. In contrast to the common perception that resveratrol acts mainly as a chemical antioxidant or as a target protein-specific ligand, we propose that the cellular response to resveratrol treatment is essentially based on oxidative triggering. In physiological microenvironments this molecular training can lead to hormetic shifting of cellular defense towards a more reductive state to improve physiological resilience to oxidative stress

Untersuchungen zur Rolle der RNA-Transportproteine Rec von HERV-K und REV von HIV-1 bei der Virusassemblierung

Diese Arbeit beschäftigt sich mit den RNA-Transportproteinen Rec des humanen endogenen Retrovirus K (HERV-K) und Rev des humanen Immundefizienz-Virus 1 (HIV-1) und ihrer Rolle während des nukleozytoplasmatischen RNA-Transports und der Virusassemblierung. Um ihrer Rolle als RNA-Transportproteine gerecht zu werden, müssen HERV-K Rec und HIV-1 Rev mit einer Vielzahl von Wirtsproteinen interagieren. Es wurde vor allem das Zusammenspiel mit möglichen Interaktionspartnern wie dem RNA-Transportprotein Staufen-1 oder dem antiviralen hochmolekularen APOBEC3G (apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1-like) untersucht. Co-Immunpräzipitationsversuche konnten eine bereits gezeigte Interaktion zwischen HERV-K Rec und Staufen-1 auch für das HIV-1 Rev nachweisen. Die spezifische Interaktion, die hierbei zwischen Staufen-1 und HIV-1 Rev nachgewiesen wurde, konnte durch Immunfluoreszenzversuche bestätigt werden. Es soll untersucht werden, ob die Interaktion von Staufen-1 und den RNA-Transportproteinen Rec und Rev möglicherweise einen antiviralen Kontrollmechanismus der Wirtszelle darstellt, indem HIV-1 Rev und HERV-K Rec über die Interaktion mit dem Staufen-1 in Stressgranula und antivirale HMM APOBEC3G-Komplexe rekrutiert und dort regulatorischen Mechanismen unterworfen werden. Die räumliche Nähe von HERV-K Rec zu A3G konnte bereits gezeigt werden. Allerdings konnte dies für das HIV-Rev bei Versuchen im Rahmen dieser Bachelorarbeit nicht bestätigt werden. Ebenso konnte keine Colokalisation mit TIA-1, einem Stressgranula-Markerprotein gezeigt werden. Weiteres Ziel dieser Arbeit ist die Aufreinigung von Staufen-1- und HERV-K Rec-Protein. Dies soll die Vorarbeit für eine entsprechende Antikörpergenerierung darstellen und konnte für das Staufen-1 erfolgreich realisiert werden. Eine Aufreinigung des HERV-K Rec war trotz Versuchsoptimierung aufgrund der Bildung sogenannter „Inclusion Bodies“ nicht möglich

The cytosolic glyoxalases of Plasmodium falciparum are dispensable during asexual blood-stage development

The enzymes glyoxalase 1 and 2 (Glo1 and Glo2) are found in most eukaryotes and catalyze the glutathione-dependent conversion of 2-oxoaldehydes to 2-hydroxycarboxylic acids. Four glyoxalases are encoded in the genome of the malaria parasite Plasmodium falciparum, the cytosolic enzymes PfGlo1 and PfcGlo2, the apicoplast enzyme PftGlo2, and an inactive Glo1-like protein that also carries an apicoplast-targeting sequence. Inhibition or knockout of the Plasmodium glyoxalases was hypothesized to lead to an accumulation of 2-oxoaldehydes and advanced glycation end-products (AGE) in the host-parasite unit and to result in parasite death. Here, we generated clonal P. falciparum strain 3D7 knockout lines for PFGLO1 and PFcGLO2 using the CRISPR-Cas9 system. Although 3D7Δglo1 knockout clones had an increased susceptibility to external glyoxal, all 3D7Δglo1 and 3D7Δcglo2 knockout lines were viable and showed no significant growth phenotype under standard growth conditions. Furthermore, the lack of PfcGlo2, but not PfGlo1, increased gametocyte commitment in the knockout lines. In summary, PfGlo1 and PfcGlo2 are dispensable during asexual blood-stage development while the loss of PfcGlo2 may induce the formation of transmissible gametocytes. These combined data show that PfGlo1 and PfcGlo2 are most likely not suited as targets for selective drug development

Quantitative assessment of the determinant structural differences between redox-active and inactive glutaredoxins

Class I glutaredoxins are enzymatically active, glutathione-dependent oxidoreductases, whilst class II glutaredoxins are typically enzymatically inactive, Fe-S cluster-binding proteins. Enzymatically active glutaredoxins harbor both a glutathione-scaffold site for reacting with glutathionylated disulfide substrates and a glutathione-activator site for reacting with reduced glutathione. Here, using yeast ScGrx7 as a model protein, we comprehensively identified and characterized key residues from four distinct protein regions, as well as the covalently bound glutathione moiety, and quantified their contribution to both interaction sites. Additionally, we developed a redox-sensitive GFP2-based assay, which allowed the real-time assessment of glutaredoxin structure-function relationships inside living cells. Finally, we employed this assay to rapidly screen multiple glutaredoxin mutants, ultimately enabling us to convert enzymatically active and inactive glutaredoxins into each other. In summary, we have gained a comprehensive understanding of the mechanistic underpinnings of glutaredoxin catalysis and have elucidated the determinant structural differences between the two main classes of glutaredoxins

A single-cysteine mutant and chimeras of essential Leishmania Erv can complement the loss of Erv1 but not of Mia40 in yeast

Mia40/CHCHD4 and Erv1/ALR are essential for oxidative protein folding in the mitochondrial intermembrane space of yeast and mammals. In contrast, many protists, including important apicomplexan and kinetoplastid parasites, lack Mia40. Furthermore, the Erv homolog of the model parasite Leishmania tarentolae (LtErv) was shown to be incompatible with Saccharomyces cerevisiae Mia40 (ScMia40). Here we addressed structure-function relationships of ScErv1 and LtErv as well as their compatibility with the oxidative protein folding system in yeast using chimeric, truncated, and mutant Erv constructs. Chimeras between the N-terminal arm of ScErv1 and a variety of truncated LtErv constructs were able to rescue yeast cells that lack ScErv1. Yeast cells were also viable when only a single cysteine residue was replaced in LtErvC17S. Thus, the presence and position of the C-terminal arm and the kinetoplastida-specific second (KISS) domain of LtErv did not interfere with its functionality in the yeast system, whereas a relatively conserved cysteine residue before the flavodomain rendered LtErv incompatible with ScMia40. The question whether parasite Erv homologs might also exert the function of Mia40 was addressed in another set of complementation assays. However, neither the KISS domain nor other truncated or mutant LtErv constructs were able to rescue yeast cells that lack ScMia40. The general relevance of Erv and its candidate substrate small Tim1 was analyzed for the related parasite L. infantum. Repeated unsuccessful knockout attempts suggest that both genes are essential in this human pathogen and underline the potential of mitochondrial protein import pathways for future intervention strategies