A major challenge is the lack of therapeutic approaches for numerous human diseases, which poses a societal challenge. The ubiquitin system is a major promising source for novel therapeutic approaches but its potential has not been fully exploited due to limited insight and lack of researchers trained in understanding and identifying avenues to target the ubiquitin system. Ubiquitin ligases and deubiquitinating enzymes (DUBs) comprises enzymes that actively add or remove ubiquitin from proteins to regulate cell physiology. How these enzymes selectively recognize their substrates is largely unknown but an emerging theme is that a globular domain in the enzyme binds a short linear interaction motif (SLiM) in the substrate. SLiMs are short peptide motifs that constitute ideal entry points for understanding the ubiquitin system as they provide detailed insight into substrate selection and how this regulates the underlying biology. Further, SLiMs provide direct routes to therapeutic exploitation as they serve as peptide scaffolds for drug development and tools to highjack the ubiquitin system. The potential of SLiMs has not been capitalized upon because a method for efficient identification was missing, interactions between relevant partners were lacking and this has hampered training of young researchers. UBIMOTIF overcomes these barriers by exploiting novel technological advances to identify SLiMs and uniting outstanding complementary researchers and industrial partners with a common vision to provide outstanding training of early career scientist in SLiMs and the ubiquitin system. The UBIMOTIF topic provides ideal training of young researchers, as it requires a multidisciplinary and intersectorial approach providing training in multiple cutting-edge technologies. The impact of UBIMOTIF will be unprecedented insight into the ubiquitin system, novel therapeutic opportunities and a skilled workforce that can fill the exhausted pipelines of European companies.

Aberrant cell cycle and redox regulation are hallmarks of cancer. While cell cycle and redox signaling are extensively studied, it remains poorly understood how both communicate in physiological conditions. One reason is the emphasis on oxidative stress as a signature of cancer cells. Only recently, emerging evidence indicates that reactive oxygen species (ROS) also function as signaling molecules in physiological conditions, and that some of their key targets are cysteine residues on cell cycle proteins. This indicates that more subtle changes in redox signaling can affect proliferation and have the potential to promote cancer. I propose to investigate how the cell cycle and redox homeostasis are coupled in a spatial-temporal manner and reveal the differences that distinguish physiological from pathological redox signaling. I will use genetic engineering to label endogenous cell cycle and redox proteins with fluorescent markers. I will measure relative and absolute molecule numbers of cell cycle and redox proteins, relate this to cell cycle and redox states, and determine the cysteines modified on cell cycle proteins. To functionally investigate the pathological potential of identified modifications I will use mammary 3D cell culture – a model that recapitulates many aspects of mammary architecture in vivo and is used to study early steps of breast tumorigenesis. I expect our work to provide us with a quantitative description of the interface between cell cycle and redox regulation. Although intracellular cell behavior is never completely deterministic, a reasonable quantitative model should be predictive to some degree and reveal how different levels of ROS can affect cell cycle decisions. Shedding light on the cell cycle targets of ROS will indicate the nodes that can be hijacked by cancer cells. Together, this work will provide a significantly improved basis for our understanding of redox signaling in tumorigenesis and indicate new strategies for treatment.

In higher eukaryotes, the RNA polymerase III (Pol III) participates in the transcription of small RNAs such as the tRNAs. RNA polymerase recruitment to their specific promoter relies on the activity of several transcription factors. Brf2 is a transcription factor that exclusively recruits RNA Pol III at the selenocysteine tRNA (tRNASec). Unpublished work from our group has unravelled an unanticipated central role of Brf2 in the oxidative stress response pathway, by acting as a cellular blockade during prolonged oxidative stress. We are interested in understanding the molecular determinants that govern RNA Pol III recruitment at tRNASec promoter and its interaction with Brf2-bound promoters. In general, RNA Pol III subunit’s size is conserved amongst the eukaryotic kingdom. However, an exception is the human Rpc5 subunit, whose C terminus has 450 residues that are not present in its yeast counterpart C37. Similarly to Brf2, the Rpc5 C-terminal extension is only present in higher metazoans, which suggests a phylogenetic link between these two proteins. The recruiting mechanism of RNA Pol III to Brf2-dependent promoters has not been described to date. Preliminary results in our lab provide evidences that indeed Rpc5 C terminus is responsible for the accurate recruitment of RNA Pol III at TBP/Brf2/DNA complex. Interestingly, structural homology predictions indicated that the human Rpc5 C-terminal extension is a eukaryotic homologue of the prokaryotic protein SelB, a factor that interacts with the tRNASec and with a specific region of mRNAs, the SECIS-element, during translation of SeCys containing proteins. This similarity suggests a regulatory role for Rpc5 C terminus in the interaction with the SECIS-element and/or the tRNASec. Our main objectives are to determine the structure of the Rpc5 C terminus in isolation and in complex with Brf2/TBP/DNA by X-ray crystallography and to characterise the role of Rpc5 C terminus in the context of tRNASec transcription.

Cancer is a major cause of death and suffering, rendering it a huge concern to the general public. Consequently, it has been targeted by application of the molecular techniques developed during the last 20-25 years, thereby improving diagnosis and treatment. Nevertheless, although current biomarker and treatment concepts often are successful initially, they subsequently frequently fail to achieve durable drug response and long-term survival for cancer patients. The study of somatic evolution in cancer is a very promising approach to rectify this situation. Fortunately, single cell genomic sequencing has recently begun to provide opportunity to unprecedented detailed insights into tumour evolution and new techniques are emerging for assaying the spatial distribution of tumour heterogeneity. Analysing and developing methods for these emerging data sets are under-researched areas that lie at the intersection of medical, evolutionary biology, and computational research. In CONTRA, European researchers with complementary expertise have joined forces in order to collectively facilitate training of future European computational cancer researchers. We will by applying modern recruitment and selection methods in combination with offering excellent training and employments conditions, including social security, facilitate recruitment of 15 excellent ESRs, across genders and geographical areas. We will train these 15 ESRs in the mathematical, computational, and applied skills required to tackle the complex analysis problems posed by somatic evolution in cancer. We will also teach them entrepreneurship and the requirements of pharmaceutical and biotech industry. Using a novel scheme, this substantial academic cancer expertise will be complemented with industrial expertise in software development, especially for biotech and pharmaceutical industry, which will unleash key components of the innovative force of the European commercial sector in the struggle against cancer.