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

Liedgens, Linda

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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.

Item Type: Dissertation
Supervisor: Lanzer, Prof. Dr. Michael
Date of thesis defense: 18 September 2018
Date Deposited: 22 Oct 2018 08:09
Date: 2019
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 570 Life sciences
Uncontrolled Keywords: mitochondrial, protein, import, glutaredoxins
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