Proteomics of peroxisomes

Peroxisomes are single membrane bounded organelles performing essential biochemical pathways. The importance of this organelle is underscored by the fact that impairments of peroxisomes lead to several severe disorders, most of them with neurological involvement. This project is intended to investigate the protein composition of peroxisomes from different cell types and tissues using a proteomic approach. In the first step, we intend to generate a transgenic mouse that expresses a fusion protein consisting of a truncated peroxin-3 (pex3) and enhanced green fluorescent protein (EGFP) under the control of the ubiquitin-C promoter. The resulting transgenic mouse should have green auto-fluorescent peroxisomes in all different cell types. Thus, the dynamics of the peroxisome compartment can be followed by fluorescence or confocal laser microscopy during e.g. development or aging. Peroxisomes will be isolated from tissues of the transgenic mouse by affinity chromatography using antibody-coated magnetic beads and by density gradient centrifugation. Peroxisomal proteins will be separated by high-resolution 2D gel-electrophoresis and identification of all detected proteins will be done by MALDI-TOF MS, nano-ESI MS or microsequencing. Using these techniques we will initially establish a catalogue of peroxisomal proteins found in liver. Next, the same isolation method will be applied to mouse brain, as well as primary neuronal, astrocytic, microglial and oligodendroglial cell cultures. The individual peroxisomal protein spot pattern, obtained by 2D-PAGE, will be compared in order to elucidate cell type-specific differences in peroxisomal proteins and thereby in peroxisomal functions.

Peroxisomes are single membrane bounded organelles present in all cell types and tissues. A variety of important biochemical pathways are either completely or partially performed in peroxisomes as for example the synthesis of some fatty acids, plasmalogens or bile acid. General defects of peroxisomes or single peroxisomal enzyme defects leading to a multitude of inherited disorders such as Zelweger Syndrom or x-linked adrenoleukodystrophy. In addition, information is accumulating about peroxisomes as modulators or modifiers in diseases of complex inheritance, such as arteriosclerosis, cancer and Alzheimer`s disease. To enable us to analyse these essential organelles in more detail we have engineered a transgenic peroxisomal mouse model in which all peroxisomes are green fluorescent. Using this mouse model, which has been constructed in cooperation with Klaus-Armin Nave from the Max Planck Institute for Experimental Medicine, Göttingen, we have started to investigate the dynamic movements of the peroxisomes in living cells. As the fluorescent tag is located on the outer site of the peroxisomal membrane we can use this mouse model for the affinity purification of ultra pure peroxisomes for advanced proteom analysis of peroxisomes. In parallel we have performed conventional purify techniques to isolate peroxisomes form mouse liver and kidney. These peroxisomes have than been analysed on their protein composition using molecular biology methods in cooperation with Helmut E. Meyer and Bettina Warscheid from the Medical Proteom Center, Ruhr-University Bochum. In this approach we have identified 120 peroxisomal matrix proteins and isoforms thereof. Interestingly we were able to elucidate clear differences between the peroxisomes isolated form liver compared to the peroxisomes isolated form kidney. This gives us the first demonstration on the different peroxiosme protein composition and additional evidences that peroxisomes perform different biological pathways in different organs. Moreover, using these proteom approach we were able to identify a novel peroxisomal protein, which we currently further investigate.