Identification and functional characterization of genes involved in replicative senescence, stress resistance and apoptosis

This project part addresses the identification and functional characterization of candidate genes that derive from high-throughput screening systems of the individual project partners. Special emphasis is put on their role in regulation of life span, stress resistance and apoptotic response in human endothelial cells. Therefore we will test the influence of overexpression and knock-down of these candidates on cellular behaviour as already done for one of our previously isolated candidates, SNEV. We have shown that SNEV overexpression extends the replicative life span of human endothelial cells, that SNEV is a splicing factor and interacts with the proteasome. Additionally, it is known that SNEV has ubiquitin E3 ligase activity and is involved in DNA repair. This multifaceted nature prompts us to study the molecular mechanisms of life span expansion by SNEV. We will overexpress various deletion mutants of SNEV trying to determine the function of its single protein domains and to find dominant negative mutants. In addition, we aim to identify the substrate of SNEV’s E3 ubiquitin ligase activity, which might elucidate the pathways SNEV is involved in. Since more substrate is ubiquitinated by SNEV overexpression and concurrent proteasome inhibition, we will purify ubiquitinated proteins from cells with increased and decreased SNEV levels, compare the pattern by a novel 2-dimensional electrophoresis technique using fluorescence labels (DIGE) and thus identify candidate substrates. By the same technique, we will do the first systematic screening to spot overall differences in the selection of proteins for degradation, the “ubiquitinome”, during cellular aging. In summary, this project will identify novel targets from the highly interesting subproteome of ubiquitinated proteins, and will elucidate the influence of candidate genes selected from the gene lists of all partners of this project on replicative life span, stress resistance and apoptotic response of human endothelial cells.