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Class I and class II glutaredoxins (Grxs) are glutathione (GSH)-dependent proteins, that function as oxidoreductases (class I) or mediate cellular iron trafficking (class II). Some members of class I Grxs like human Grx2 are able to complex a [2Fe-2S] cluster and form a dimeric holo complex, which renders them catalytically inactive and is the basis for their function as redox sensors. Class II Grxs like human Grx5 also complex [2Fe-2S] clusters, however these proteins transfer the clusters to other proteins. Both functionally distinct classes share a similar thioredoxin fold and conserved interaction sites for the non-covalently binding of GSH, which is required to complex the [2Fe-2S] cluster. Furthermore, the proteins from both classes contain a highly nucleophilic active site cysteine that would allow both classes to catalyze GSH-dependent oxidoreduction reactions. Despite of these similar features, only class I Grxs are able to form a mixed disulfide with GSH and to reversibly transfer it to protein thiols (de-/glutathionylation). Interestingly, neither class I Grxs nor class II Grxs can effectively compensate the loss of an essential member of the other class. Even though some structural differences were described earlier, the basis for their different functions remained unknown. In particular, the lack of catalytic activity of class II Grxs as oxidoreductases could not be explained. Here, we demonstrate that the different conformations of a conserved lysyl side chain are the molecular determinant of the oxidoreductase or Fe-S transfer activity of class I and II Grxs, respectively. A specific loop structure that is conserved in all class II Grxs determines one lysyl conformation that prevents the formation of a mixed disulfide of the active site cysteinyl thiol with GSH. Using engineered mutants of hGrx2 and hGrx5, we demonstrated that the exchange of the distinct loop between the classes results in a loss of oxidoreductase function of class I hGrx2 and the gain of oxidoreductase activity of class II hGrx5. The altered GSH binding mode also profoundly changes the [2Fe-2S] cluster binding of the engineered mutants and thereby also influences stability of the holo complexes, a pre-determinant for [Fe-S] cluster transfer activity. With the minor shift of 2 Å in a conserved lysyl side chain orientation we were not only able to modify the catalytic activity of two small human mitochondrial proteins, but on a much larger scale also provided evidence for the previously unknown structural basis that determines the function of all class I and class II Grxs.
The oxidoreductase activity of hGrx2 was also analyzed in vivo in a model of doxorubicin cell toxicity. Applying a mass spectrometrical approach, we identified various mitochondrial proteins as targets for redox regulation. Furthermore, our results gave reason to reconsider some common assumptions regarding doxorubicin-induced apoptosis and the protective function of mitochondrial Grx2.
This thesis is about the establishment and the application of novel methods and tools that are re-lated to the most widely used enzyme class: hydrolases. It covers all fields from the identification to the application of these valuable enzymes with particular focus on lactonases, acylases and proteases. The activity assay introduced in Article I substantially extends the method toolbox for studies on lactonases and acylases that interfere with the bacterial cell-cell communication system. Article II describes a fully automatized robotic platform that represents the next-level tool for the high-throughput enzyme screening in the microtiter plate format. It was used, for instance, for the screening for improved porcine aminoacylase I variants. Diverse aspects of the protease-mediated hydrolysis of non-resistant proteins for the purification of resistant target proteins are highlighted in Article III.
Understanding the fundamental mechanisms in the extracellular matrix of cells (ECM) is crucial for the development of drugs and biomaterials. Therefore, an atomistic model of the extracellular matrix is a cost-efficient way to observe influences of drugs, test the effect of mutations or misfolds in proteins or study the properties of fibril or network-forming peptides.
With this thesis, a refined molecular model of an adhesion complex is proposed that contains collagen, fibronectin and the cell receptor integrin. During the building of the model, major new insights are given for each of these proteins and a powerful protein-folding algorithm is
developed.
Emerging zoonotic viruses are a constant threat to human and animal health. Therefore, knowledge about the host factors influencing viral pathogenicity is highly welcome as a basis for developing treatment or vaccine strategies. In order to identify host factors that potentially determine the
pathogenicity of three highly pathogenic (’high consequence’) zoonotic viruses, the interactomes of
selected viral proteins were analysed in parallel with the interactomes of the homologous proteins from closely related viruses which lack high pathogenicity. For this purpose, affinity purification mass spectrometry (AP-MS) was performed with the virus proteins as baits and lists of candidate proteins were generated that may determine the pathotype and warrant follow-up studies to characterise their function concerning the viral life cycles. In detail, the interactomes of virus pairs from the arenaviruses, filoviruses and henipaviruses were studied. The following protein homologues were selected: for filoviruses, the transcription factor VP30, the co-transcription factor VP35 and matrix protein VP40 of the non-pathogenic Reston virus
(RESTV, species Reston ebolavirus), the pathogenic Ebola virus (EBOV, species Zaire ebolavirus),
and, in addition, the Lloviu virus (LLOV, species Lloviu cuevavirus); in case of the arenaviruses
the nucleoprotein (NP), matrix protein (Z) and glycoprotein (GP) of the pathogenic Junín virus (JUNV, species Argentine mammarenavirus) and the non-pathogenic Tacaribe virus (TCRV, species Tacaribe mammarenavirus); and for the henipaviruses, the fusion protein F of the apathogenic Cedar virus (CedV, species Cedar henipavirus) and the pathogenic Nipah virus (NiV, species Nipah henipavirus). The experimental approach was to express the tagged bait proteins in human cells by transfection with appropriate constructs, purify the interactomes by affinity enrichment and analyse their protein content by MS. Quantitation was performed by labelling with stable isotopes or by label-free quantification (LFQ). High-confidence interactions for the LFQ approach were identified using the Mass Spectrometry interaction STatistics (MiST) scoring tool. Qualitative and quantitative data were used to identify a limited number of candidates for follow-up research. Additionally,
the interactomes were analysed with bioinformatical tools like term enrichment analysis and network analysis to identify cellular pathways which are possibly impacted by the expression of viral proteins. A novel specific interactor of EBOV VP30 was identified, ubiquitin carboxyl-terminal hydrolase7
(USP7, also known as HAUSP), and the interaction was partially characterised. The interaction was confirmed by reverse-pull-down experiments, and the Kd value (determined by Microscale Thermophoresis, MST) was found to be lower than for the interaction of USP7 with the RESTV VP30.
This work adds insight into virus protein interactomes, especially for the often neglected low pathogenic virus species. Furthermore, the pathogenicity of the viruses was refl ected to some degree
in the interactomes of their proteins. The generated interactome data for the different virus species
create a basis in the search for interactions that determine pathogenicity.
The central aim of this thesis was the investigation of protein/polyanion interaction using circular dichroism (CD) spectroscopy, enzyme immune assay (EIA), isothermal titration calorimetry (ITC) and flow cytometry (FC). A further aim was to understand why an endogenous protein becomes immuno-genic when forming a complex. The focus was on the protein platelet factor (PF4), which gained wide interest in the clinical field, due to its role in the life-threatening, immune-driven, adverse drug effect heparin-induced thrombocytopenia (HIT). PF4 is a small homotetrameric chemokine with several basic amino acids on its surface, forming a positively charged ring. The antibodies that are formed during HIT recognize an epitope exposed on PF4, when it is in a complex with heparin at a certain molar ratio at which, PF4 tetramers are aligned on the heparin and forced into close approximation. The main results and conclusions of the thesis are summarized below: 5.1 Evolutionary Conservation of PF4 (Paper I – PF4/Evolution) By carrying out an amino acid sequence survey we found that the positively charged amino acids contributing to the heparin binding site on the surface of PF4 and related proteins are highly conserved in all vertebrates, including fish species. PF4 interacts with the phospholipid lipid A, the innermost part of the lipopolysaccharide (LPS) of Gram negative bacteria. We showed that the shorter the sugar chain of the O antigen, outer and inner core of the LPS were the more PF4 was binding. The interaction of PF4 with lipid A is inhibited by heparin, suggesting that the amino acids known to contribute to heparin binding are also involved in binding to lipid A. 5.2 PF4 Interaction with Polyanions (PA) of varying Length and Degree of Sulfation (Paper II – PF4/PA) CD spectroscopy was found to be a powerful technique to monitor structural changes of PF4 caused by binding to various clinically relevant polyanions. Therefore PF4 was titrated with different PA to investigate the dependencies: i. impact of the PF4:PA molar ratio, ii. degree of polymerization of the PA and iii. degree of sulfation of the PA. In all cases, exposure of HIT-relevant epitope(s) was only observed for PA that also induced changes in secondary structure of PF4. A comparison of results of an immune ¬assay with CD spectroscopic data showed that the extent of complex anti¬genicity correlates well with the magnitude of changes in PF4 secondary structure, and that the structural changes of PF4 have to exceed a certain threshold to achieve PF4/PA complex antigenicity. These findings allowed us to calculate expectation intervals for complex antigenicity solely using CD spectroscopic data. To our knowledge, this was the first demonstration that the capability of drugs to induce antigenicity of PF4 can be assessed without the necessity of in vivo studies or the use of antibodies obtained from immunized patients specific for the antigens. The antigenicity of PF4 in complex is not restricted to negative charges originating from sulfate groups, PA with phosphate groups are also capable (binding to phospholipids). We investigated inorganic polyphosphates (polyP) with a chain length of 75 Pi and showed that the induced secondary structural changes are even higher compared to the changes induced by the different heparins and that the PF4/P75 complexes are antigenic as well. 5.3 PF4 Interaction with defined oligomeric Heparins (Paper III – PF4/defined Heparins) We tested highly purified, monodisperse heparins. In contrast to the clinically relevant but relatively undefined (high polydispersity index) glycosamino glycans reported in paper II (PF4/PA). The defined heparins induced higher secondary structural changes. Here we showed for the first time that strong conformational changes during PF4/PA complex formation are necessary but not sufficient for to the expression of the anti-PF4/heparin antibody binding site. Also, the size of the complexes is not the only prerequisite for anti-PF4/heparin antibody binding (tested by atomic force microscopy). By ITC we found that antigenicity is only induced if the PF4/PA complex has a high binding enthalpy and the complex formation leads to a negative change in entropy. 5.4 PF4/Polyphosphates (polyP) Complex Antigenicity and Interaction with Escherichia coli (E. coli, Paper IV – PF4/polyP) PolyP with chain lengths of 45 Pi and 75 Pi induced remarkable secondary structural changes in the PF4 molecule, thereby exposing the epitope recognized by anti-PF4/heparin antibodies. The induced conformational changes were similar to the changes induced by the defined heparins. Again a high binding enthalpy was observed but here in connection with a positive change in entropy. Further we showed that polyP (≥45 Pi) enhance PF4 binding to the surface of Gram negative E. coli at intermediate concentration and disrupt the binding at elevated polyP concentrations. The increased amounts of PF4 on the bacterial surface also improved the binding of anti-PF4/heparin antibodies and thereby the phagocytosis of the bacteria by poly¬morpho¬nuclear leucocytes. 5.5 Nucleic acid based Aptamers induce structural Changes in the PF4 Molecule (Paper V – PF4/Aptamer) Nucleic acids are another class of molecules containing phosphate groups. Especially after cell damage their extra¬cellular concentration can be locally quite high (>2 mg/ml). We found that certain aptamers form complexes with PF4 and thereby inducing anti-PF4/aptamer antibodies which cross-react with PF4/heparin complexes. Moreover by CD spectroscopy we showed that the protein C-aptamer caused similar secondary structural changes of PF4 like heparin, but already at much lower concentration. The maximally induced changes by the protein-C aptamer were even higher and persisted over a broader concentration range. 5.6 Protamine Interaction with Heparin (Paper VI – PS/Heparin) After the intensive investigation of the complex formation between PF4 and many different classes of PA we assessed another protein for structural changes upon complex formation with heparin. Protamine (PS) a protein in routinely used in post-cardiac surgery to reverse the anticoagulant effects of heparin was found to unfold but not to refold with increasing concentration of PA in solution. 5.7 Conclusion and Outlook When starting this thesis, it was believed that repetitive structures formed by PF4 on a heparin chain mold the epitope recognized by antibodies inducing HIT. These repetitive structures might exhibit similarities with viral capsids and are therefore recognized by the immune system of some patients. We found that induced by the close approximation PF4 changes its conformation, thereby exposing a neoepitope. The conserved positively charged amino acids of the heparin binding site and the involvement of these amino acids in the binding to lipid A confirm our hypothesis of PF4 as part of an ancient immune-mediated host defense mechanism. As possible consequence of the “primitive mechanism of defense” the highly variable O-antigens of LPS might have significantly contributed to an efficient escape mechanism by hiding the structures that made the bacteria vulnerable. In turn polyP might be an adaption of the host improve pathogen recognition by PF4 and further by antibodies inducing phagocytosis of the PF4-marked objects. Although shown only for PF4 and PS, our findings might be applicable to other proteins that also express epitopes upon changes in their secondary structure. Our physicochemical methods may further be applied: i. to drug development for the prediction of antigenicity induced by polyanionic drugs, ii. to guide the development of synthetic heparins and other polyanion based drugs, e.g. aptamers, that do not lead to HIT and iii. to provide relevant aspects for other biological functions of heparins.