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