Stefan Weger

Topors acts as a SUMO-1 E3 ligase for p53 in vitro and in vivo.

Human Topors, which was originally identified as cellular binding partner of DNA topoisomerase I and of p53, has recently been shown to function as an ubiquitin E3 ligase for p53 in a manner dependent on its N′‐terminally located RING finger. Here, we demonstrate that Topors also enhances the conjugation of the small ubiquitin‐like modifier 1 (SUMO‐1) to p53 in vivo and in a reconstituted in vitro system. The Topors SUMO‐1 E3 ligase activity does not depend upon its RING finger motif. In HeLa cells, Topors induced p53 sumoylation was accompanied by an increase in endogenous p53 protein levels. Furthermore, Topors enhances the sumoylation of a variety of other, yet unidentified, cellular proteins.

Topors, a p53 and topoisomerase I binding protein, interacts with the adeno-associated virus (AAV-2) Rep78/68 proteins and enhances AAV-2 gene expression.

The adeno-associated virus type 2 (AAV-2) Rep proteins are essential for AAV DNA replication and regulation of AAV gene expression. We have identified a cellular protein interacting with Rep78 and Rep68 in yeast two-hybrid analysis and in GST pull-down assays. This protein has recently been described as both a p53 (p53BP3) and a topoisomerase I interacting protein (Topors). It contains an arginine/serine-rich domain, a RING finger domain and five PEST sequences. A minimal sequence sufficient for interaction with Rep was mapped to Topors amino acids 871 to 917. We show that the same region is also involved in the interaction with p53. Rep sequences involved in interaction with Topors were mapped to Rep amino acids 172 to 481. Overexpression of Topors stimulated AAV gene expression in the absence of helper virus, suggesting a function of Topors as a transcriptional regulator.

The E3 ligase Topors induces the accumulation of polysumoylated forms of DNA topoisomerase I in vitro and in vivo.

Human Topors has originally been identified as binding partner of p53 and DNA topoisomerase I (TOP1). It can function as both an ubiquitin and SUMO‐1 E3 ligase for p53. Here we demonstrate that Topors enhances the formation of high‐molecular weight SUMO‐1 conjugates of TOP1 in a reconstituted in vitro system and also in human osteosarcoma cells, similar to treatment with CPT. In contrast to the situation observed with p53, overall sumoylation levels were rather unaffected. Experiments with TOP1 point mutants strongly suggest that the high‐molecular weight conjugates represent SUMO‐1 chains formed on a limited number of SUMO‐1 acceptor sites.

Experiments to localize the site for the anxiogenic action of NPY mediated by Y2 receptors in the mouse brain

Neuropeptide Y (NPY) is abundant in the nervous system. It acts through Y1, Y2, Y4 and Y5 receptors and is involved in a variety of brain functions. When applied locally into the amygdala, NPY exerts an anxiolytic action, presumably mediated by Y1 receptors. Depletion of Y2 receptors induces an anxiolytic phenotype, possibly by abolishing the release-inhibiting action of presynaptic Y2 receptors. In the present study we aimed to find the exact site of the presumed anxiogenic action mediated by Y2 receptors. We conducted site-specific deletions of Y2 receptors in Y2 mice by local injection of an AAV-Cre vector into the hippocampus and the amygdala. As controls, an AAV-GFP vector was injected in Y2 littermates at the same sites. Expression of Cre and GFP was verified by in situ hybridization and immunohistochemistry. Deletion of Y2 receptors was visualized by receptor autoradiography and in situ hybridization. After bilateral injection of an AAV-Cre vector into the basolateral amygdala, mice revealed a tendency towards an anxiolytic phenotype in the light-dark test (LDT). When deletion of Y2 receptors was confined to the central nucleus of the amygdala, an anxiolytic phenotype was observed in the elevated plus maze and the LDT. Moreover, a better stress coping ability was demonstrated in the tail suspension test. In contrast, no anxiolytic effect was detected after intrahippocampal injections. The experiments indicate that the anxiolytic and antidepressant-like effects of Y2 receptor deletion may be generated in certain subnuclei of the amygdala.

Reduced fear conditioning after viral vector mediated neuropeptide Y administration into the basolateral amygdala.

Background Neuropeptide Y (NPY) is a 36-amino-acid peptide that is abundantly expressed in the central nervous system. It is involved in various physiological and pathophysiological processes, including energy homeostasis, pain and epilepsy, but also in anxiety and depression. Consistent findings have demonstrated an anxiolytic effect of NPY. The presence of different NPY receptors in the amygdala and the effects of NPY on anxiety raise the question, whether NPY and its receptors may influence acquisition and extinction of conditioned fear. Therefore, we investigated NPY and NPY receptor knockout mice in Pavlovian fear conditioning.

Neuropeptide Y Y2 receptors modulate trace fear conditioning and spatial memory in the dorsal hippocampus

Background Neuropeptide Y (NPY), a highly conserved 36 amino acid peptide is widely distributed in the central nervous system. Besides its functions in various metabolic processes NPY has attracted considerable attention in modulating emotional-affective behavior. NPY exerts a pronounced anxiolytic effect most likely mediated by Y1 receptors, whereas stimulation of predominantly pre-synaptic Y2 receptors results in increased anxiety. The role of NPY Y2 receptors in the processing of emotional learning, however, remains still elusive.