Characterization of Adaptor proteins and their Posttranslational Modifications involved in ALT (alternative lengthening of telomeres) using Proteomics

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Telomerase-negative tumor cells use mechanisms collectively known as Alternative Lengthening of Telomeres (ALT) to counteract the replication-associated telomere shortening. It is generally accepted that most, if not all, ALT mechanisms use homologous recombination to maintain telomere length. However, the precise molecular mechanisms are unknown. Most ALT cells display characteristic features comprising telomere length heterogeneity, high levels of telomere-sister chromatid exchanges and nuclear structures called ALT-associated PML bodies (APBs). PML bodies are macromolecular structures ubiquitously present in most cell types. These bodies are involved in many different protein-protein and protein-nucleic acid interactions. However, the interaction of PML bodies with chromosome extremities is a characteristic feature of ALT cells, bringing telomeres into close proximity and promoting post-replicative recombination in S/G2 phase. It is currently unknown how this spatiotemporal control is achieved. We speculate that unique adaptor proteins provide a bridge between telomeres and the PML body or specific post-translational modifications of constitutive telomere binding proteins are important. In this application, we propose to investigate both possibilities. To identify adaptor proteins, we combine an innovative approach based on the PML-infiltrating Herpes virus protein ICP0 as a bait for purification and in depth characterization by state-of-the-art quantitative proteomics to define the telosome-PML complex in ALT cells. Furthermore, using high-resolution mass spectrometry, we will establish the first comprehensive chart of posttranslational modifications of telomere-associated proteins in ALT. While there have been several posttranslational modifications described in the literature, most of them have only been reported in telomerase-positive cell lines and currently it is unknown whether these modifications are also present in ALT and what their role might be in ALT. We expect that the results of this study will allow us a better understanding of the ALT mechanisms. In this proposal we unite the expertise in ALT telomere biology (Draskovic and Boussin), a new purification strategy (Draskovic), a diverse glioma stem cell line collection as a model (Boussin) and the experience in mass spectrometry based proteomics (Butter) to investigate what defines an APB and how its formation is regulated.