Nature has evolved an additional level of genetic regulation by-passing direct changes in genetic code through the means of posttranslational modifications (PTMs) of nucleobases and histone proteins. Acetylation, methylation, phosphorylation, O-GlcNAcylation, ubiquitination, sumoylation, and ADP ribosylation are few common examples of various histone modifications. Identification of these modifications and subsequent access to homogeneously modified histone proteins are key for understanding the functional consequence of these PTMs. In this doctoral thesis, the role of PTMs of histone proteins in epigenetics was investigated with emphasis on understanding the role of O-GlcNAcylation in particular. In the second chapter, the functional consequence of O-GlcNAcylation at histone protein, H2B-Ser112 was explored. Homogeneously GlcNAcylated histones and nucleosomes were synthesized using protein chemical reactions. Mass Spectrometry (MS) based quantitative interaction proteomics revealed a direct interaction between GlcNAcylated nucleosomes and the Facilitates Chromatin Transcription (FACT) complex. Preferential binding of FACT to GlcNAcylated nucleosomes provides a molecular mechanism for FACT-driven transcriptional control. In the third chapter, the physical effect of O-GlcNAcylation on the nucleosome structure is described. Homogeneously GlcNAcylated histone protein, H2A-Thr101 was synthesized. The modified protein was used to reconstitute histone sub-complexes and nucleosomes. Various biophysical studies involving circular dichroism and native mass spectrometry revealed that H2A-T101 GlcNAcylation regulates the stability of the nucleosome structure, suggesting a role in transcriptional activation. In the fourth chapter, we discuss an interesting scenario where two PTMs - O-GlcNAcylation and phosphorylation - can compete for the same modification site of histone protein, H2B-Ser36. The resulting outcome is possibly a competitive antagonism or cross-talk, which can modulate the overall control of chromatin regulation. Using a "Tag-and-modify" approach, modified histone proteins bearing both modifications was synthesized, and was later used for nucleosome reconstitution. Quantitative interaction proteomics experiments with the modified nucleosome revealed key interacting protein partners for both the modifications.

Hepatitis B virus (HBV) is a leading cause of liver disease. Its success as a human pathogen is related to the immense production of subviral envelope particles (SVPs) contributing to viral persistence by interfering with immune functions. To explore cellular pathways involved in SVP formation and egress, we investigated host–pathogen interactions. Yeast-based proteomics revealed Sec24A, a component of the coat protein complex II (COPII), as an interaction partner of the HBV envelope S domain. To understand how HBV co-opts COPII as a proviral machinery, we studied roles of key Sec proteins in HBV-expressing liver cells. Silencing of Sar1, Sec23, and Sec24, which promote COPII assembly concomitant with cargo loading, strongly diminished endoplasmic reticulum (ER) envelope export and SVP secretion. By analysing Sec paralog specificities, we unexpectedly found that the HBV envelope is a selective interaction partner of Sec24A and Sec23B whose functions could not be substituted by their related isoforms. In support, we found that HBV replication upregulated Sec24A and Sec23B transcription. Furthermore, HBV encountered the Sec24A/Sec23B complex via an interaction that involved the N-terminal half of Sec24A and a di-arginine motif of its S domain, mirroring a novel ER export code. Accordingly, an interference with the COPII/HBV cross-talk might display a tool to effectively inhibit SVP release.

Argulus foliaceus (Linnaeus, 1758) is a member of the branchiuran family Argulidae and has a worldwide distribution, causing major economic impacts for freshwater aquacultured fish species worldwide. In the UK, it has economic impacts for both aquaculture and sports fishing industries. Previous studies observed haemorrhagic and inflammatory responses after Argulus infection, which has been taken to support the idea that the parasite secretes chemicals during the feeding process to assist with the ingestion of blood and epithelial tissue. The present study suggests that the blood-feeding ectoparasite of fish, A. foliaceus, may use similar mechanisms for evading host immune responses to those used by sea lice and other haematophagous arthropods. No previous studies have directly investigated the nature of the bioactive compounds / proteins, assumed to be released from these ectoparasites, and which are considered to contribute to feeding processes and host-parasite interactions during infection. Thus, the work described in this thesis was undertaken with the objective of identifying, describing and characterising the secretory components that have previously been suggested to be secreted from glandular cells associated with the feeding appendages of Argulus foliaceus. The current study applied transcriptomic and proteomic techniques in conjunction with in situ methods to investigate known immunomodulatory genes that may serve a function in parasite-host interactions. Overall, the findings of this project have generated considerable additional knowledge concerning the biology of Argulus spp. and have provided a list of proteins that may be used by the parasite to facilitate feeding processes by secreting these active molecules into the host and hence modulating their immune defence mechanisms. This information can be used as a baseline for developing freshwater lice control strategies to help prevent the spread of Argulosis in aquaculture by applying vaccination as means of control using the candidate antigens described in this study to specifically target Argulus spp. Knowledge generated by the work described in this thesis can also contribute to the development of drugs for controlling Argulus or functional components of feed that may serve to protect fish against this parasite. Furthermore, data from this thesis enhances the knowledge of the distribution of toxin/venom or venom-like substances in crustaceans and arthropods in general.

Chlamydien sind obligat intrazelluläre Bakterien, die sich in einem membranumschlossenen Kompartiment namens Inklusion vermehren. Nach Infektion fragmentiert der Golgi-Apparat der Wirtszelle in kleine Membranstapel. Dies verbessert die Aufnahme von Sphingolipiden und ist deshalb für die chlamydiale Vermehrung essentiell. Die infektionsinduzierte Golgi-Fragmentierung geschieht nach Spaltung des Golgi-Matrix-Proteins Golgin-84. In dieser Arbeit konnte, durch den Vergleich mit bekannten Substraten und Inhibitorstudien, die chlamydiale Protease CPAF (Chlamydia protease-like activity factor) als das Enzym identifiziert werden, das diese Spaltung induziert, abhängig von der Anwesenheit zweier Rab-Proteine, Rab6 und Rab11, die den zellulären Vesikeltransport kontrollieren und zur Inklusion rekrutiert werden. Die Fragmentierung des Golgi-Apparates verhinderte dessen Relokalisierung während der Zellpolarisierung nach Einbringen eines migratorischen Stimulus. Sowohl infizierte als auch Golgin-84-depletierte Zellen migrierten langsamer und randomisiert in einem Motilitätsassay. Die Relokalisierung des Golgi-Apparates konnte durch seine Stabilisierung mittels WEHD oder Rab-Depletion wieder gewonnen werden, was die Zellmotilität teilweise wieder herstellte. Darüber hinaus konnte gezeigt werden, dass die Infektion außer der Golgi-Reorientierung die Signaltransduktion durch GTPasen beeinflusst. Die Aktivität von Cdc42 in infizierten Zellen war erhöht und die Interaktionen mit vielen ihrer Effektoren laut quantitativer Massenspektrometrie stark verändert. Die Ergebnisse dieser Arbeit zeigen, dass CPAF die für Chlamydien lebenswichtige Golgin-84 Prozessierung und Fragmentierung des Golgi-Apparates auslöst. Dies verringert die Mobilität der Wirtszelle, vor allem da der Golgi-Apparat während der Polarisierung nicht mehr ausgerichtet werden kann, des Weiteren durch Modulierung der Protein-Protein-Interaktionen von Cdc42. / Chlamydia are obligate intracellular human pathogens that proliferate inside a membrane-bound compartment called the inclusion. In infected cells, the Golgi apparatus is fragmented into small ministacks that are aligned around the inclusion. This facilitates uptake of host cell sphingolipids and is essential for chlamydial development. Infection-induced Golgi fragmentation happens after processing of the Golgi matrix protein golgin-84. This work could, via comparison with well-known substrates and inhibitor studies, identify the chlamydial protease CPAF (Chlamydia protease-like activity factor) as the enzyme accountable for this cleavage. Golgi Fragmentation depended on two Rab proteins, Rab6 and Rab11, which control vesicle transport and are recruited to the Chlamydia inclusion. As a consequence of Golgi fragmentation, cells lost the capacity to reorient the Golgi apparatus during polarization after a migratory stimulus. Both infected and golgin-84 depleted cells with a permanently fragmented Golgi apparatus displayed decelerated and furthermore randomized migration in a motility assay. Relocalization of the Golgi apparatus could be restored via stabilizing WEHD treatment or Rab depletion which partly rescued cell motility. Moreover, it could be shown that migration signaling via small GTPases was influenced by Chlamydia infection. Infected cells exhibited activation of the small polarity GTPase Cdc42. Numerous interactions with downstream effectors were strongly altered in infected cells according to quantitative mass spectrometry. Particularly, the binding of Cdc42 to migration-associated effectors was decreased. The results of this work show that CPAF, by processing of golgin-84, induces Golgi fragmentation which is vitally important for Chlamydia. This disturbs host cell motility because the Golgi apparatus cannot be reoriented during polarization and, additionally, via the modulation of protein-protein-interactions of Cdc42.