Jeroen Demmers   Jeroen Demmers, Ph.D. studied Chemistry at Utrecht University (the Netherlands). He earned his PhD degree from the same university in 2002, working on the development of tools to study transmembrane peptide and proteins interactions with phospholipid bilayers using hydrogen-deuterium exchange and electrospray ionization mass spectrometry in the groups of Albert Heck and Antoinette Killian. As a postdoctoral fellow in the lab of Brian Chait at The Rockefeller University (New York) he worked on a project involving the proteomic identification of plasmid DNA binding proteins in yeast. In 2005, Jeroen moved to Erasmus University Medical Center to set up a proteomics lab and core facility and to initiate a research program in the field of protein mass spectrometry. Currently, he is an associate professor and director of the Erasmus MC proteomics core facility ( Research in his lab focuses on the molecular mechanisms of the ubiquitin–proteasome system and the lab develops quantitative proteomics technologies for the analysis of protein posttranslational modifications. The Demmers lab collaborates with many research groups within the institute and abroad on various topics, such as protein-protein interactions, immunopeptidomics, targeted proteomics, etc. Jeroen’s mission is to advocate the superiority of targeted proteomics in terms of specificity, quantitative accuracy, and – arguably – sensitivity over immunoblotting for the quantitative analysis of target proteins in complex biological mixtures. Although one currently still needs a dedicated mass spectrometry facility with skilled personnel and therefore higher initial costs, with the pace of technology development it is likely that small, user-friendly bench-top mass spectrometry equipment will democratize targeted proteomics assays soon. In addition, to push this transformation, the proteomics community should actively show the life science community what advantages it can bring.

Targeted mass spectrometry reveals that USP7 regulates the ncPRC1 Polycomb axis

INTRODUCTION: Ubiquitin-specific protease 7 (USP7) is a deubiquitylating enzyme that is involved in the regulation of multiple key cellular processes, including tumor suppression, transcription, epigenetics, the DNA damage response, and DNA replication. Whereas the role of USP7 in the p53 pathway is well established, a full picture of the USP7 regulatory network is lacking. For example, USP7 has been connected to the Polycomb system, but the molecular mechanism through which USP7 regulates Polycomb functions remains unclear. For debiquitinating enzymes, the regulatory mode of action is at the posttranslational level and, thus, proteomics tools are indispensable to study this. Here, we took an unbiased multi-omics approach with a strong targeted quantitative proteomics component to define the core USP7 network. METHODS: Relevant knock-outs, including Dox inducible USP7, were generated in HAP-1, DLD1 and U2OS cell lines. We performed interactomics and in-depth global proteomics to identify the USP7 interaction network. Targeted proteomics using parallel reaction monitoring (PRM) was done to accurately quantify all relevant players of the Polycomb system in a label free manner. For targeted proteomics a PRM regime on an Orbitrap Eclipse Tribrid was used to select for sets of previously selected peptides. Targeted proteomics data were analyzed with the Skyline software suite. Read More
RESULTS: Using a targeted mass spectrometry assay focused on a subset of potential USP7 target proteins and defined PTMs, we found that USP7 modulates the ncPRC1 axis at the posttranslational level through stabilization of the non-canonical Polycomb-repressive complexes ncPRC1.6 and ncPRC1.1. At the transcriptional level, USP7 silences AUTS2, the subunit that suppresses H2A ubiquitylation by ncPRC1.3/5. Collectively, these USP7 activities increase the genomic deposition of H2AK119ub1. Contradicting prevalent paradigms of Polycomb function, our findings reveal that changes in H2AK119ub1 are generally uncoupled from H3K27me3 and thus argue against a hierarchical relationship between these two repressive histone marks. Importantly, the connection of USP7 to the Polycomb system suggests that its role in cancer extends beyond regulation of p53. Furthermore, our interactomics assay shows that USP7 has a remarkable range of interaction partners, of which only a portion appears to be stabilized by USP7. Current studies address the relevance of these other USP7 partners. CONCLUSION: This multi-angle analysis establishes USP7 as a regulatory hub in a multinodal network involved in tumor biology, protein (de)ubiquitylation, and genome regulation. Combined, our multi-omics results provide a resource for future studies on the role of USP7 in (neuro)development and cancer.