Open Access

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.

Cellular protein homeostasis (proteostasis) maintains a functional proteome and thus proper cell function. Proteostasis is facilitated by the ubiquitin-proteasome system (UPS), an intracellular protein turnover machinery ensuring clearance of damaged, misfolded, old and/or unneeded regulatory proteins. This is particularly important in the central nervous system (CNS), where it is linked to neurodegeneration. Disruptions of the proteostasis systems cause the accumulation of misfolded proteins which are commonly seen in progressive neurodegenerative diseases also linked to neuroinflammation. Proper UPS function can protect cells from the accumulation of defective proteins, neurodegeneration and neuroinflammation. Furthermore, it has been found that loss of function mutations in the genes encoding UPS components are linked to systemic inflammation including neuroinflammation and/or neurodevelopmental disorders. Proteasome defects in patients suffering from these disorders cause decreased proteasome activity, accumulation of proteins, activation of proteotoxic stress responses and systemic inflammation. However, the molecular link between proteotoxic stress and the initiation of inflammatory signalling remained unclear. In Article 2, we summarized the importance of the UPS in immune cell proteostasis and function including activation of innate and adaptive immune responses. Although UPS function is notably important in innate immune signalling, the current understanding of the role of UPS in myeloid cell function in the CNS is limited. We also indicated the involvement of impaired UPS function in sterile systemic inflammation including neuroinflammation as well as tumour diseases and pathogen manipulation of immune cells. To investigate the molecular link behind proteasome impairment and systemic inflammation in the brain, we focused on microglia cells as the only immune residents of the CNS. In Article 1, we used a pharmacological inhibitor called bortezomib which targets β5 and β5i/LMP7 subunit activities in standard proteasome (SP) and immunoproteasome (IP), respectively. We showed for the first time on the molecular level that inhibition of proteasome activity by bortezomib triggers the accumulation of ubiquitylated proteins, proteotoxic stress responses and innate immune signalling activation depending on the induced proteotoxic stress response called unfolded protein response (UPR) in murine microglia. In particular, activation of the inositol-requiring protein 1α arm of UPR upon bortezomib treatment leads to systemic inflammation as indicated by type I interferon (IFN) response. IP enhance the proteolytic capacity of UPS by rapid clearance of proteins upon immune signalling activation. Microglia, like other immune cells, exhibit constitutive expression of IP as well as SP to maintain their cellular proteostasis. In Manuscript 3, we studied the particular impact of IP impairment on microglial cellular function. We showed accumulation of ubiquitin-modified proteins and activation of proteotoxic stress responses in IP-impaired mouse and human microglia models. Moreover, we identified possible IP substrates in microglia using β5i/LMP7 knockout mice as an IP deficiency model and, examined how IP deficiency affects microglia function. IP deficient microglia affected the ubiquitylation levels of proteins involved in multiple pathways such as immune responses, energy metabolism, cytoskeleton organisation, cell cycle and ribosome function. Based on the molecular analysis, we confirmed sterile activation of innate immune signalling mechanisms in IP impaired microglia. This is driven by the proteotoxic stress sensor protein kinase R (PKR). In addition, we were able to show that IP impairment altered levels of the microglial activation markers, which are also involved in motility, adhesion and phagocytosis of microglia. In this thesis, we highlight that UPS function is necessary to maintain microglial proteostasis and, that impairment of proteasome activities triggers sterile inflammation in microglia via activation of proteotoxic stress responses. The described activation of innate immune signalling mechanisms in microglia upon proteasome impairment may be considered as new therapeutic targets for patients suffering from rare protesomapathies or other disorders linked to dysregulated immune signalling.

The relevance of cold atmospheric plasmas (CAPs) in biomedicine has recently grown. The potential of CAPs has been discussed in multiple scientific works, highlighting its effectiveness in promoting wound healing, limiting cancer progression, and for sterilization of surfaces. Main bioactive molecules, such as reactive oxygen and nitrogen species (RONS), are proposed as key candidates in these processes. Indeed, the generation of cold plasma induces noble gas ionization which, reacting with atmospheric air molecules, generates species such as singlet oxygen, atomic oxygen radicals, nitric oxide radicals. Although molecular simulations have been conducted, the mechanism of action on biological molecules, as well as the possibility to tune plasmas to produce specific species cocktails (e.g., with different degree of oxidation power) has been not fully unleashed. In this dissertation, presented in form of 5 published scientific articles, focus has been placed on the interaction of plasmas with peptides and proteins, which are main biological effectors in cellular compartments. Precisely, through the development of liquid chromatography coupled mass spectrometry (LC-MS) methods, the effects of plasmas on peptides and proteins in form of oxidative post-translational modifications (oxPTMs) has been investigated. The characterization of these oxPTMs has been performed by treating peptide or protein aqueous solutions and on porcine skin tissues. It has been found that, introducing small amounts of different gases (oxygen, nitrogen, or both) or even water molecules, can made CAPs tunable tools to produce oxygen-species dominating effects versus nitrogen-species dominating effects. In addition to this, it was found that the amino acid position in a peptide or protein influences the quality and quantity of the resulting oxPTMs. Besides this, other important parameters like driven gases, admixture gases or treatment duration were identified as relevant factors for the modification of amino acids in the peptide structure. By comparing the effects between peptide solutions and complex matrices such as porcine skin, water has been identified as a valid vehicle to transport and amplify the plasma chemistry. In an experimental study, the inactivation of a protein (PLA2) was observed after CAP treatment and together with simulation studies, the specific dioxidation of tryptophane W128 was detected as a potential explanation for this inactivation, indicating the strong impact of plasma on biological targets. In summary, oxidative modifications found in peptide solutions were observed also in complex protein structures and sample matrices. In conclusion, this work provides a starting point for future studies of oxidative modifications in complex models and may thus be helpful for further investigations in the fields of plasma medicine and redox chemistry.

This thesis deals with the characterisation and engineering of new thermophilic PET hydrolases as potential candidates for an eco-friendly biocatalytic recycling approach for the upcycling or downcycling of polyethylene terephthalate (PET) on industrial scale. Furthermore, high-throughput screening methods are described that detect the products of PET hydrolysis. The high demand of PET in the packaging and textile industries with a global production of 82 million metric tons per year has significantly contributed to the global solid waste stream and environmental plastic pollution after its end-of-life. Although PET hydrolases have been identified in various microorganisms, only a handful of benchmark enzymes have been engineered for industrial applications. Therefore, the identification of new PET hydrolases from metagenomes or via protein engineering approaches, especially thermophilic PET hydrolases with optimal operating temperatures (i.e., increased thermostability and activity) near the glass transition temperature of the polymer PET, is a crucial step towards a bio-based circular plastic economy. Article I demonstrates that metagenome-derived thermophilic PET hydrolases can be significantly improved using different engineering approaches to achieve a similar activity level as the well-established leaf-branch-compost cutinase (LCC) F243I/D238C/S283C/Y127G variant (LCC ICCG). In Article II, thermostable variants of a mesophilic enzyme (PETase from Ideonella sakaiensis) were identified from a mutant library and characterised against PET substrates in various forms. Articles III and IV describe the application of high-throughput methods for the identification of novel PET hydrolases by directly assaying terephthalic acid (TPA), one of the monomeric building blocks of PET. Furthermore, Article IV describes the possibility of a one-pot conversion of the TPA-based aldehydes produced to their diamines as example for an open-loop upcycling method.

Die Dissertation beschreibt die Synthese verschiedener Nukleosidanaloga mit den notwendigen Modifizierungen und Funktionalitäten für einen Einsatz in der Phosphoramidit-basierten chemischen Oligonukleotidsynthese an fester Phase. Im Rahmen der Arbeit wurde ein nicht-kanonisches Desoxyadenosinderivat ausgehend von Allopurinol hergestellt. Außerdem wurden verschiedene Azid-modifizierte Nukleoside synthetisiert und Untersuchungen zur Herstellung eines Borono-modifizierten Adenosinderivats durchgeführt. Des Weiteren wurde ein Verfahren zur Bestimmung der Stabilität der Azidogruppe unter Standardbedingungen der Phosphoramidit-basierten chemischen Oligonukleotidsynthese demonstriert.