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Proteomic investigations of ubiquitin signals in DNA repair and chromatin organization

Final Report Summary - PROTEUS (Proteomic investigations of ubiquitin signals in DNA repair and chromatin organization)

The integrity of the human genome is constantly challenged by endogenous and environmental factors that can induce different types of DNA lesions. Failure to repair DNA lesions can lead to genomic instability and contribute to cancer and premature ageing. To counteract these potentially devastating effects caused by DNA damage, eukaryotic cells have evolved complex response mechanisms that regulate DNA repair and cell cycle progression and thereby enable cells to respond to DNA damage. Posttranslational modifications of chromatin-associated proteins play an essential role in the regulation of the cellular response to DNA damage. Over the last years modification of proteins by the 76 amino acid protein Ubiquitin has emerged as an important regulatory mechanism of DNA damage repair and signaling. Ubiquitin is covalently attached to lysine residues in specific protein substrates in a highly regulated enzymatic process. A number of ubiquitin-modifying enzymes are recruited to sites of DNA damage and are important for the repair of DNA lesions and genome stability. However, for majority of these enzymes the protein substrates and the molecular functions in the cellular response to DNA damage remain obscure.
Recent advances in mass spectrometry technology and development of novel methods for the enrichment of ubiquitylated peptides now permit to perform proteome wide analysis of endogenous ubiquitylation sites. Importantly, quantitative mass spectrometry-based approaches, such as stable isotope labeling with amino acids in cell culture, can be employed to determine the relative abundance of ubiquitylation sites after cellular perturbations. In this research project we employed quantitative mass spectrometry-based proteomics to investigate the functions of ubiquitin-modifying enzymes and ubiquitin-dependent signaling in the cellular response to DNA damage and on chromatin in general. We found that DNA damage induces site-specific ubiquitylation of proteins involved in DNA double strand break repair. Protein ubiquitylation regulates the assembly and disassembly of repair complexes on the chromatin and thereby plays an essential role in the maintenance of genome stability. The results of our studies demonstrate that ubiquitin-dependent extraction of repair factors from chromatin after repair has taken place is an important mechanism that promotes genome stability and cellular survival after DNA damage. Furthermore, we uncovered the substrates of the Ubiquitin-dependent remodeler VCP and identified its function in the regulation of the transcription factor c-Myc. Proteasome inhibitors are used in clinics for the treatment of multiple myeloma. VCP inhibitors are currently explored as an alternative approach to target the Ubiquitin-proteasome system in different types of hematological and solid malignancies. We found that VCP inhibition increases ubiquitylation of a different subset of proteins compared to proteasome inhibition, thus providing information that might help to understand the clinical effects of these inhibitors. Taken together, studies derived from this project provided better understanding of the regulatory roles of Ubiquitin-dependent signaling in human cells.