@article{WagnerDrouetTeschnerWolschkeetal.2021, author = {Wagner-Drouet, Eva and Teschner, Daniel and Wolschke, Christine and Sch{\"a}fer-Eckart, Kerstin and G{\"a}rtner, Johannes and Mielke, Stephan and Schreder, Martin and Kobbe, Guido and Hilgendorf, Inken and Klein, Stefan and Verbeek, Mareike and Ditschkowski, Markus and Koch, Martina and Lindemann, Monika and Schmidt, Traudel and Rascle, Anne and Barabas, Sascha and Deml, Ludwig and Wagner, Ralf and Wolff, Daniel}, title = {Comparison of cytomegalovirus-specific immune cell response to proteins versus peptides using an IFN-γ ELISpot assay after hematopoietic stem cell transplantation}, series = {Diagnostics}, volume = {11}, journal = {Diagnostics}, number = {2}, issn = {2075-4418}, doi = {10.3390/diagnostics11020312}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-228843}, year = {2021}, abstract = {Cytomegalovirus (CMV) infection is a major cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). Measuring CMV-specific cellular immunity may improve the risk stratification and management of patients. IFN-γ ELISpot assays, based on the stimulation of peripheral blood mononuclear cells with CMV pp65 and IE-1 proteins or peptides, have been validated in clinical settings. However, it remains unclear to which extend the T-cell response to synthetic peptides reflect that mediated by full-length proteins processed by antigen-presenting cells. We compared the stimulating ability of pp65 and IE-1 proteins and corresponding overlapping peptides in 16 HSCT recipients using a standardized IFN-γ ELISpot assay. Paired qualitative test results showed an overall 74.4\% concordance. Discordant results were mainly due to low-response tests, with one exception. One patient with early CMV reactivation and graft-versus-host disease, sustained CMV DNAemia and high CD8\(^+\) counts showed successive negative protein-based ELISpot results but a high and sustained response to IE-1 peptides. Our results suggest that the response to exogenous proteins, which involves their uptake and processing by antigen-presenting cells, more closely reflects the physiological response to CMV infection, while the response to exogenous peptides may lead to artificial in vitro T-cell responses, especially in strongly immunosuppressed patients.}, language = {en} } @phdthesis{Orth2021, author = {Orth, Barbara}, title = {Identification of an atypical peptide binding mode of the BTB domain of the transcription factor MIZ1 with a HUWE1-derived peptide}, doi = {10.25972/OPUS-25044}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-250447}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Ubiquitination is a posttranslational modification with immense impact on a wide range of cellular processes, including proteasomal degradation, membrane dynamics, transcription, translation, cell cycle, apoptosis, DNA repair and immunity. These diverse functions stem from the various ubiquitin chain types, topologies, and attachment sites on substrate proteins. Substrate recruitment and modification on lysine, serine or threonine residues is catalyzed by ubiquitin ligases (E3s). An important E3 that decides about the fate of numerous substrates is the HECT-type ubiquitin ligase HUWE1. Depending on the substrate, HUWE1 is involved in different processes, such as cell proliferation and differentiation, DNA repair, and transcription. One of the transcription factors that is ubiquitinated by HUWE1 is the MYC interacting zinc finger protein 1 (MIZ1). MIZ1 is a BTB/POZ (Bric-{\`a}-brac, Tramtrack and Broad-Complex/Pox virus and zinc finger) zinc finger (ZF) protein that binds to DNA through its 13 C2H2-type zinc fingers and either activates or represses the transcription of target genes, including genes involved in cell cycle arrest, such as P21CIP1 (CDKN1A). The precise functions of MIZ1 depend on its interactions with the MYC-MAX heterodimer, but also its heterodimerization with other BTB-ZF proteins, such as BCL6 or NAC1. How MIZ1 interacts with HUWE1 has not been studied and, as a consequence, it has not been possible to rationally develop tools to manipulate this interaction with specificity in order to better understand the effects of the interaction on the transcriptional function of MIZ1 on target genes or processes downstream. One aspect of my research, therefore, aimed at characterizing the MIZ1-HUWE1 interaction at a structural level. I determined a crystal structure of the MIZ1-BTB-domain in complex with a peptide, referred to as ASC, derived from a C terminal region of HUWE1, previously named 'activation segment'. The binding mode observed in this crystal structure could be validated by binding and activity assays in vitro and by cell-based co-IP experiments in the context of N-terminally truncated HUWE1 constructs. I was not able to provide unambiguous evidence for the identified binding mode in the context of full-length HUWE1, indicating that MIZ1 recognition by HUWE1 requires yet unknown regions in the cell. While the structural details of the MIZ1-HUWE1 interaction remains to be elucidated in the context of the full-length proteins, the binding mode between MIZ1BTB and ASC revealed an interesting, atypical structural feature of the BTB domain of MIZ1 that, to my knowledge, has not been described for other BTB-ZF proteins: The B3 region in MIZ1BTB is conformationally malleable, which allows for a HUWE1-ASC-peptide-mediated β-sheet extension of the upper B1/B2-strands, resulting in a mixed, 3 stranded β-sheet. Such β-sheet extension does not appear to occur in other homo- or heterodimeric BTB-ZF proteins, including MIZ1-heterodimers, since these proteins typically possess a pre-formed B3-strand in at least one subunit. Instead, BCL6 co repressor-derived peptides (SMRT and BCOR) were found to extend the lower β-sheet in BCL6BTB by binding to an adjacent 'lateral groove'. This interaction follows a 1:1 stoichiometry, whereas the MIZ1BTB-ASC-complex shows a 2:1 stoichiometry. The crystal structure of the MIZ1BTB-ASC-complex I determined, along with comparative binding studies of ASC with monomeric, homodimeric, and heterodimeric MIZ1BTB variants, respectively, suggests that ASC selects for MIZ1BTB homodimers. The structural data I generated may serve as an entry point for the prediction of additional interaction partners of MIZ1 that also have the ability to extend the upper β-sheet of MIZ1BTB. If successful, such interaction partners and structures thereof might aid the design of peptidomimetics or small-molecule inhibitors of MIZ1 signaling. Proof-of-principle for such a structure-guided approach targeting BTB domains has been provided by small-molecule inhibitors of BCL6BTB co-repressors interactions. If a similar approach led to molecules that interfere with specific interactions of MIZ1, they would provide intriguing probes to study MIZ1 biology and may eventually allow for the development of MIZ1-directed cancer therapeutics.}, subject = {Ubiquitin}, language = {en} } @article{AzzamiRitterTautzetal.2012, author = {Azzami, Klara and Ritter, Wolfgang and Tautz, J{\"u}rgen and Beier, Hildburg}, title = {Infection of honey bees with acute bee paralysis virus does not trigger humoral or cellular immune responses}, series = {Archives of Virology}, volume = {157}, journal = {Archives of Virology}, number = {4}, doi = {10.1007/s00705-012-1223-0}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-126863}, pages = {689-702}, year = {2012}, abstract = {We have studied the responses of honey bees at different life stages (Apis mellifera) to controlled infection with acute bee paralysis virus and have identified the haemolymph of infected larvae and adult worker bees as the compartment where massive propagation of ABPV occurs. Insects respond with a broad spectrum of induced innate immune reactions to bacterial infections, whereas defence mechanisms based on RNA interference play a major role in antiviral immunity. In this study, we have determined that honey bee larvae and adult workers do not produce a humoral immune reaction upon artificial infection with ABPV, in contrast to control individuals challenged with Escherichia coli. ABPV-infected bees produced neither elevated levels of specific antimicrobial peptides (AMPs), such as hymenoptaecin and defensin, nor any general antimicrobial activity, as revealed by inhibition-zone assays. Additionally, adult bees did not generate melanised nodules upon ABPV infection, an important cellular immune function activated by bacteria and viruses in some insects. Challenge of bees with both ABPV and E. coli showed that innate humoral and cellular immune reactions are induced in mixed infections, albeit at a reduced level.}, language = {en} } @article{MeyerzuHoersteCordesMausbergetal.2014, author = {Meyer zu H{\"o}rste, Gerd and Cordes, Steffen and Mausberg, Anne K. and Zozulya, Alla L. and Wessig, Carsten and Sparwasser, Tim and Mathys, Christian and Wiendl, Heinz and Hartung, Hans-Peter and Kieseier, Bernd C.}, title = {FoxP3+Regulatory T Cells Determine Disease Severity in Rodent Models of Inflammatory Neuropathies}, series = {PLOS ONE}, volume = {9}, journal = {PLOS ONE}, number = {10}, doi = {10.1371/journal.pone.0108756}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-115239}, pages = {e108756}, year = {2014}, abstract = {Inflammatory neuropathies represent disabling human autoimmune disorders with considerable disease variability. Animal models provide insights into defined aspects of their disease pathogenesis. Forkhead box P3 (FoxP3)+ regulatory T lymphocytes (Treg) are anti-inflammatory cells that maintain immune tolerance and counteract tissue damage in a variety of immune-mediated disorders. Dysfunction or a reduced frequency of Tregs have been associated with different human autoimmune disorders. We here analyzed the functional relevance of Tregs in determining disease manifestation and severity in murine models of autoimmune neuropathies. We took advantage of the DEREG mouse system allowing depletion of Treg with high specificity as well as anti-CD25 directed antibodies to deplete Tregs in mice in actively induced experimental autoimmune neuritis (EAN). Furthermore antibody-depletion was performed in an adoptive transfer model of chronic neuritis. Early Treg depletion increased clinical EAN severity both in active and adoptive transfer chronic neuritis. This was accompanied by increased proliferation of myelin specific T cells and histological signs of peripheral nerve inflammation. Late stage Treg depletion after initial disease manifestation however did not exacerbate inflammatory neuropathy symptoms further. We conclude that Tregs determine disease severity in experimental autoimmune neuropathies during the initial priming phase, but have no major disease modifying function after disease manifestation. Potential future therapeutic approaches targeting Tregs should thus be performed early in inflammatory neuropathies.}, language = {en} } @phdthesis{Dunkel2013, author = {Dunkel, Nico}, title = {Regulation of virulence-associated traits of the human fungal pathogen Candida albicans by nitrogen availability}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-83076}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Nitrogen-regulated pathogenesis describes the expression of virulence attributes as direct response to the quantity and quality of an available nitrogen source. As consequence of nitrogen availability, the opportunistic human fungal pathogen Candida albicans changes its morphology and secretes aspartic proteases [SAPs], both well characterized virulence attributes. C. albicans, contrarily to its normally non-pathogenic relative Saccharomyces cerevisiae, is able to utilize proteins, which are considered as abundant and important nitrogen source within the human host. To assimilate complex proteinaceous matter, extracellular proteolysis is followed by uptake of the degradation products through dedicated peptide transporters (di-/tripeptide transporters [PTRs] and oligopeptide transporters [OPTs]). The expression of both traits is transcriptionally controlled by Stp1 - the global regulator of protein utilization - in C. albicans. The aim of the present study was to elucidate the regulation of virulence attributes of the pathogenic fungus C. albicans by nitrogen availability in more detail. Within a genome wide binding profile of Stp1, during growth with proteins, more than 600 Stp1 target genes were identified, thereby confirming its role in the usage of proteins, but also other nitrogenous compounds as nitrogen source. Moreover, the revealed targets suggest an involvement of Stp1 in the general adaption to nutrient availability as well as in the environmental stress response. With the focus on protein utilization and nitrogen-regulated pathogenesis, the regulation of the major secreted aspartic protease Sap2 - additionally one of the prime examples of allelic heterogeneity in C. albicans - was investigated in detail. Thereby, the heterogezygous SAP2 promoter helped to identify an unintended genomic alteration as the true cause of a growth defect of a C. albicans mutant. Additionally, the promoter region, which was responsible for the differential activation of the SAP2 alleles, was delimited. Furthermore, general Sap2 induction was demonstrated to be mediated by distinct cis-acting elements that are required for a high or a low activity of SAP2 expression. For the utilization of proteins as nitrogen source it is also crucial to take up the peptides that are produced by extracellular proteolysis. Therefore, the function and importance of specific peptide transporters was investigated in C. albicans mutants, unable to use peptides as nitrogen source (opt1Δ/Δ opt2Δ/Δ opt3Δ/Δ opt4Δ/Δ opt5Δ/Δ ptr2Δ/Δ ptr22Δ/Δ septuple null mutants). The overexpression of individual transporters in these mutants revealed differential substrate specificities and expanded the specificity of the OPTs to dipeptides, a completely new facet of these transporters. The peptide-uptake deficient mutants were further used to elucidate, whether indeed proteins and peptides are an important in vivo nitrogen source for C. albicans. It was found that during competitive colonization of the mouse intestine these mutants exhibited wild-type fitness, indicating that neither proteins nor peptides are primary nitrogen sources required to efficiently support growth of C. albicans in the mouse gut. Adequate availability of the preferred nitrogen source ammonium represses the utilization of proteins and other alternative nitrogen sources, but also the expression of virulence attributes, like Sap secretion and nitrogen-starvation induced filamentation. In order to discriminate, whether ammonium availability is externally sensed or determined inside the cell by C. albicans, the response to exterior ammonium concentrations of ammonium-uptake deficient mutants (mep1Δ/Δ mep2Δ/Δ null mutants) was investigated. This study showed that presence of an otherwise suppressing ammonium concentration did not inhibit Sap2 proteases secretion and arginine-induced filamentation in these mutants. Conclusively, ammonium availability is primarily determined inside the cell in order to control the expression of virulence traits. In sum, the present work contributes to the current understanding of how C. albicans regulates expression of virulence-associated traits in response to the presence of available nitrogen sources - especially proteins and peptides - in order to adapt its lifestyle within a human host.}, subject = {Candida albicans}, language = {en} }