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Modification of transmembrane peptides to probe SNARE-induced membrane fusion and cross-presentation of membrane-buried epitopes

dc.contributor.advisorDiederichsen, Ulf Prof. Dr.
dc.contributor.authorSchirmacher, Anastasiya
dc.date.accessioned2021-02-19T08:23:02Z
dc.date.available2021-02-19T08:23:02Z
dc.date.issued2021-02-19
dc.identifier.urihttp://hdl.handle.net/21.11130/00-1735-0000-0005-1576-F
dc.identifier.urihttp://dx.doi.org/10.53846/goediss-8449
dc.language.isoengde
dc.publisherNiedersächsische Staats- und Universitätsbibliothek Göttingende
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subject.ddc540de
dc.titleModification of transmembrane peptides to probe SNARE-induced membrane fusion and cross-presentation of membrane-buried epitopesde
dc.typedoctoralThesisde
dc.contributor.refereeDiederichsen, Ulf Prof. Dr.
dc.date.examination2020-03-11
dc.description.abstractengMembrane proteins play an important role in cellular function as receptors, transporters, anchors, and catalysts. Despite constituting 20-30% of the human proteome only 3 % of all characterized proteins are membrane proteins so far. However, their number is growing fast recently and model peptides from synthetic origins contribute greatly to uncovering their function. For this thesis customized solid-phase peptide synthesis (SPPS) and high-performance liquid chromatography (HPLC) techniques have been applied as valuable tools to generate such compounds to study SNARE (soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor) protein-mediated membrane fusion and antigen presentation of membrane-buried epitopes. SNARE proteins promote the merger of neuronal vesicles with the presynaptic membrane as a key step of neuronal exocytosis. Localized in both membranes, they specifically recognize each other via the SNARE motif and thereby pull the membranes closely together. Building on a previously developed SNARE mimetics E3Syb and K3Sx, spatio-temporal control of membrane fusion was sought out to zoom in on the different stages of the latter and illuminate the role of the transmembrane domains. Attractive electrostatic interactions between glutamic acid and lysine residues are systematically canceled in a selection of photocleavably protected E3Syb and K3Sx derivatives. Analysis of the model fusogens via fluorescence spectroscopy in bulk lipid mixing assays revealed minor influence of commonly used photocleavable protecting groups NVOC and DEACM on the fusion efficiency. This led to the development of a novel photocleavable protection strategy for coiled coil peptides based on spanning of the hydrophobic core of the coiled coils with a DEACM based hydrocarbon linker inspired by stapled peptides. Studying antigen cross-presentation, typically two principal cross-presentation pathways are considered. The phagosome-to-cytosol (P2C) pathway and the vacuolar pathway are known to dominate the processing of epitopes in soluble protein domains. However, they have not yet been shown to include steps that would be required to extract, process, and transport epitopes from membrane proteins. Thus, artificial transmembrane peptides containing known tumor epitope NY-ESO1[157-165] have been designed and the epitope was equipped with a small bio-orthogonal linker for fluorescent labeling to enable intracellular tracking without interference with peptide processing. A variety of peptides with different epitope and linker positioning were synthesized scanned for being processed by monocyte-derived dendritic cells isolated from human donor blood. Cross-presentation was verified by quantification of cytokine interferon γ (INFγ) secreted by CD8+ T cells upon activation and suitable peptides were used to gradually dissect cross-presentation of membrane-buried epitopes using inhibitors of pathway-specific metabolic processes. The studies indicated independence from the transporter associated with antigen processing (TAP) which could hint at membrane proteins not following the P2C pathway.de
dc.contributor.coRefereeJahn, Reinhard Prof. Dr.
dc.subject.engHigh-performance liquid chromatography (HPLC)de
dc.subject.engSolid-phase peptide synthesis (SPPS)de
dc.subject.engSNARE-mimeticsde
dc.subject.engBulk vesicle fusion assaysde
dc.subject.engT cell activation assayde
dc.subject.engMembrane-buried antigende
dc.subject.engBio-orthogonal fluorescent labelingde
dc.subject.engPhotocleavable protection groupsde
dc.identifier.urnurn:nbn:de:gbv:7-21.11130/00-1735-0000-0005-1576-F-0
dc.affiliation.instituteFakultät für Chemiede
dc.subject.gokfullChemie  (PPN62138352X)de
dc.identifier.ppn1748734202
dc.creator.birthnameMyanovska


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