Identifizierung und funktionelle Charakterisierung der SUMOylierung des Tight Junction-Proteins Zona occludens-2 (ZO-2)

Die Aufrechterhaltung und Integrität von Tight Junctions (TJs) ist von zentraler Bedeutung für die Zellpolarität in epithelialen und endothelialen Geweben. Als TJ-assoziiertes Adapter-Protein verbindet Zona occludens-2 (ZO-2) die integralen Tight Junction-Proteine mit dem Aktin-Cytoskelett und trägt dort zur Ausbildung des cytoplasmatischen Plaques bei. ZO-2 ist ein dual-lokalisiertes Nacos (nuclear and adhesion complexes)-Protein, das in konfluenten Zellen an der Plasmamembran und in dünn ausgesäten Zellen vorrangig im Kern zu finden ist. Die intrazelluläre Lokalisation von ZO-2 wird durch mehrere konservierte Kernimport- und Kernexport-Signale sowie posttranslationale Modifikationen u.a. innerhalb dieser Motive reguliert. Im Rahmen dieser Dissertation wurden im humanen ZO-2 mehrere evolutionär hochkonservierte potentielle SUMOylierungsstellen identifiziert und die Interaktion von ZO-2 mit Komponenten der SUMOylierungsmaschinerie nachgewiesen. Die SUMOylierung von ZO-2 konnte in vivo und in vitro bestätigt werden. Mit Hilfe von Mutationsanalysen wurde Lysin 730 als mögliche SUMO-Akzeptorstelle identifiziert. Die Mutation dieses Lysins führt zu einer vorrangig nukleären Lokalisation, wohingegen ein SUMO-ZO-2-Fusionsprotein, das ein konstitutiv SUMOyliertes ZO-2-Protein nachahmt, vermehrt im Cytoplasma zu finden ist. ZO-2 wurde in früheren Studien als Repressor des Wnt/β-Catenin-Signalwegs beschrieben. Das SUMO-ZO-2-Fusionsprotein besitzt keine transkriptionsreprimierende Aktivität, während die SUMOylierungs-defiziente ZO-2-K730R-Mutante die β-Catenin/TCF-4-vermittelte Transkriptionsaktivität sogar etwas stärker reprimiert. In diesem Zusammenhang wurde eine Interaktion von ZO-2 mit β-Catenin und GSK3β nachgewiesen. Aufgrund dieser Beobachtungen kann festgestellt werden, dass die intrazelluläre Lokalisation und die Signalfunktion von ZO-2 durch posttranslationale SUMOylierung reguliert wird

The Angular Momentum Problem in Cosmological Simulations of Disk Galaxy Formation

We conduct a systematic study of the angular momentum problem in numerical simulations of disk galaxy formation. We investigate the role of numerical resolution using a semi-cosmological setup which combines an efficient use of the number of particles in an isolated halo while preserving the hierarchical build-up of the disk through the merging of clumps. We perform the same simulation varying the resolution over 4 orders of magnitude. Independent on the level of resolution, the loss of angular momentum stays the same and can be tied to dynamical friction during the build-up phase. This is confirmed in a cosmological simulation. We also perform simulations including star formation and star formation and supernova feedback. While the former has no influence on the angular momentum problem, the latter reduces the loss to a level potentially in agreement with observations. This is achieved through a suppression of early star formation and therefore the formation of a large, slowly rotating bulge. We conclude that feedback is a critical component to achieve realistic disk galaxies in cosmological simulations. Numerical resolution is important, but by itself not capable of solving the angular momentum problem.Comment: 13 pages, 13 figures, submitted to MNRA

Impact of Partial Volume Correction on [18F]GE-180 PET Quantification in Subcortical Brain Regions of Patients with Corticobasal Syndrome.

Corticobasal syndrome (CBS) is a rare neurodegenerative condition characterized by four-repeat tau aggregation in the cortical and subcortical brain regions and accompanied by severe atrophy. The aim of this study was to evaluate partial volume effect correction (PVEC) in patients with CBS compared to a control cohort imaged with the 18-kDa translocator protein (TSPO) positron emission tomography (PET) tracer [18F]GE-180. Eighteen patients with CBS and 12 age- and sex-matched healthy controls underwent [18F]GE-180 PET. The cortical and subcortical regions were delineated by deep nuclei parcellation (DNP) of a 3D-T1 MRI. Region-specific subcortical volumes and standardized uptake values and ratios (SUV and SUVr) were extracted before and after region-based voxel-wise PVEC. Regional volumes were compared between patients with CBS and controls. The % group differences and effect sizes (CBS vs. controls) of uncorrected and PVE-corrected SUVr data were compared. Single-region positivity in patients with CBS was assessed by a >2 SD threshold vs. controls and compared between uncorrected and PVE-corrected data. Smaller regional volumes were detected in patients with CBS compared to controls in the right ventral striatum (p = 0.041), the left putamen (p = 0.005), the right putamen (p = 0.038) and the left pallidum (p = 0.015). After applying PVEC, the % group differences were distinctly higher, but the effect sizes of TSPO uptake were only slightly stronger due to the higher variance after PVEC. The single-region positivity of TSPO PET increased in patients with CBS after PVEC (100 vs. 83 regions). PVEC in the cortical and subcortical regions is valuable for TSPO imaging of patients with CBS, leading to the improved detection of elevated [18F]GE-180 uptake, although the effect sizes in the comparison against the controls did not improve strongly

TSPO acts as an immune resistance gene involved in the T cell mediated immune control of glioblastoma

Glioblastoma (GB) IDH-wildtype is the most malignant primary brain tumor. It is particularly resistant to current immunotherapies. Translocator protein 18 kDa (TSPO) is upregulated in GB and correlates with malignancy and poor prognosis, but also with increased immune infiltration. Here, we studied the role of TSPO in the regulation of immune resistance of human GB cells. The role of TSPO in tumor immune resistance was experimentally determined in primary brain tumor initiating cells (BTICs) and cell lines through genetic manipulation of TSPO expression and subsequent cocultures with antigen specific cytotoxic T cells and autologous tumor-infiltrating T cells. Death inducing intrinsic and extrinsic apoptotic pathways affected by TSPO were investigated. TSPO-regulated genes mediating apoptosis resistance in BTICs were identified through gene expression analysis and subsequent functional analyses. TSPO transcription in primary GB cells correlated with CD8+ T cell infiltration, cytotoxic activity of T cell infiltrate, expression of TNFR and IFNGR and with the activity of their downstream signalling pathways, as well as with the expression of TRAIL receptors. Coculture of BTICs with tumor reactive cytotoxic T cells or with T cell-derived factors induced TSPO up-regulation through T cell derived TNFα and IFNγ. Silencing of TSPO sensitized BTICs against T cell-mediated cytotoxicity. TSPO selectively protected BTICs against TRAIL-induced apoptosis by regulating apoptosis pathways. TSPO also regulated the expression of multiple genes associated with resistance against apoptosis. We conclude that TSPO expression in GB is induced through T cell-derived cytokines TNFα and IFNγ and that TSPO expression protects GB cells against cytotoxic T cell attack through TRAIL. Our data thereby provide an indication that therapeutic targeting of TSPO may be a suitable approach to sensitize GB to immune cell-mediated cytotoxicity by circumventing tumor intrinsic TRAIL resistance