Analysis of the function of SUMO in the brain with a particular emphasis on synaptic transmission
Zusammenfassung der Projektergebnisse
SUMO has been implicated in the regulation of many neuronal proteins as well as in many neurodegenerative processes. The aim of my study was to shed some light on the molecular mechanisms of SUMO function in the brain and more precisely at the synapse. However, identification of SUMO substrates is not easy and SUMOylation is mainly studied in cell lines. Therefore, I developed new mouse models in order to facilitate purification of SUMOylated protein in vivo. At first, I developed three transgenic mouse line expressing Hise-SUMO under a neuron specific promoter. We could enrich SUMO substrates from transgenic mice brain as observed by Western blotting. However, the purification still carries a strong background that prevents analysis of the samples by proteomics. I am currently troubleshooting for a second purification step, e.g immunoprecipitation using anti-SUMO antibodies or antibodies against a putative substrate. Importantly, transgenic mouse do not show altered brain cytoarchitecture or changes in the localization of synaptic markers. Result obtained in the transgenic mice showed that it is necessary to develop tandem affinity purification in order to isolate SUMO substrates from mice brain. Therefore, I developed a second mouse model, knock-In mice carrying a double His6-HA tag in front of SUMO1. Preliminary results already showed an evident enrichment of SUMO1 substrates as compared to wild-type in Western blotting experiment. I am currently improving the purification methods and up-scaling the experiment in order to analyze the SUMO1 substrates using proteomics. In summary, the development of the transgenic mouse model did not lead to the expected result which is the identification of SUMO substrates in vivo and more precisely in the brain. Therefore, I had to establish a new mouse model, the Hise-HA-SUMO1 knock-In mice.
