DDRDegronDiscovery · Substrate Selectivity in the Ubiquitin-Dependent Response to DNA Damage

Substrate Selectivity in the Ubiquitin-Dependent Response to DNA Damage

Our cells sense and adapt to external and internal changes in their environment by communication between proteins. A frequent way to transmit information is the interaction between proteins. In order to obtain a fast and dynamic network to transmit information these interactions need to be dynamic. One tool proteins use to store and transmit information as well as to change their behavior are modifications of their amino acids. The ubiquitin system uses such a modification to signal that some proteins are no longer needed, they can be degraded in order to recycle resources. One important task during the life of a cell is to keep the genomic information save, in order to perform its function as part of a multi-cellular organism. Failures to do so can result in human diseases like cancer. Cells have developed a sophisticated system to detect and repair damages to its DNA. An important aspect of DNA damage response (DDR) is the ubiquitin system. In the last years we have learned a lot about the protein networks that help keep the genome safe, but some aspects like the interaction of proteins necessary for ubiquitination and degradation are still incompletely understood because they are difficult to study. A better understanding of protein-protein interaction, ubiquitination and the DDR is important for finding novel ways to diagnose and treat human diseases like cancer. Furthermore, E3 ligases, part of the ubiquitin proteasome system, are being used by novel pharmaceutical drugs to artificially degrade and thereby inactivate proteins that are detrimental in a particular disease. In depth knowledge of E3 ligase will enable the development of these drugs for the benefit of patients´ health. In this project my main goal was to establish new technologies to study the importance of short amino acid stretches in proteins for their degradation during DNA repair pathways. For many E3 Ligases (the proteins responsible for tagging other proteins with ubiquitin) it is not known how they recognize their substrates. I wanted to use a technology called SPARK2 to screen thousands of peptide sequences simultaneously to test if they interact with the E3 ligase of interest and if they get degraded. The cells that contain peptide sequences that interact with the E3 ligase can be identified because they will produce a fluorescent protein and sequencing of the DNA of that cell allows me to identify the peptide sequence. In order to gain more information about the critical amino acids in that peptide sequence I planned to use MBRLE:pep, a technology that allows multiplexed measurements of protein-peptide interactions. The technology uses peptides presented on encoded beads. That means one particular peptide sequence is attached to beads with a unique code. We can then mix beads with tens to hundreds of different codes and still unambiguously identify the peptide sequence. The overall progress of the project is satisfactory. The infrastructure for both technologies has been established. The MRBLE:pep assay required to establish a collaboration to get access to a peptide synthesizer to be able to synthesize peptides on encoded beads as well as the setup of a new microscope. Initial experiments to show the feasibility of the approach have been performed. Team members can build on the established infrastructure to continue the project and use the technologies for other exciting discoveries.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз