Studies on the role of cholesterol and coronin 1 in antigen-presenting cells

Antigen recognition and presentation to subsequently induce an appropriate host response is dependent on the action of antigen-presenting cells such as dendritic cells and macrophages. In this thesis, the function of cholesterol and coroninin antigen-presenting cells was studied. In the first part of this thesis, the delivery of exogenous antigens into the MHC class I pathway, termed cross-presentation, was investigated. Cross-presentation is important for the establishment of an immune response against viruses or tumour cells in vivo. Antigens to be cross-presented are frequently internalized via macropinocytosis. Here it is shown, that by cholesterol-depletion of antigen-presenting cells macropinosome formation was abolished resulting in an impaired crosspresentation of exogenous antigens. In accordance with a role of cholesterol in cross-presentation, modification of antigens by palmitoylation, a modification known to increase the affinity to cholesterol, resulted in a strongly enhanced uptake and improved cross-presentation of the antigen. Together, these results indicate that cholesterol plays an important role in macropinocytosis and in the subsequent delivery of antigens into the MHC class I pathway. To explore palmitoylation as a modification that would enhance cross-presentation of antigens, we found that such modification often results in the insolubility of the modified antigen. For specific antigens however the use of palmitoylation to improve cross-presentation of soluble proteins could be explored for the development of new vaccines. The second part of this thesis focused on coronin 1, a member of the WD repeat protein family of actin-binding proteins termed coronins. In contrast to the other mammalian coronins, coroninis expressed predominantly in leukocytes arguing for a role in leukocyte specific processes. To understand a function for coronin 1, the structure of coroninwas analyzed. Coroninconsists of three structural domains: a N-terminal region containingWD40 repeats, which is connected by a linker region with a C-terminal coiled coil domain. Coroninoccurs in vivo as homotrimeric complexes, which associate with the plasma membrane and with the cytoskeleton via two distinct binding domains. It was found, that association of coroninwith the cytoskeleton was mediated by coiled coil induced trimerization of a stretch of positively charged residues within the linker region. In contrast, plasma membrane binding was independent of the oligomerization state of coroninand required the presence of the N-terminal, WD repeat-containing domain. By bridging the F-actin cytoskeleton with the plasma membrane coronin1 may serve as a linker integrating outside signals with the remodelling of the F-actin cytoskeleton.