Periodic Reporting for period 1 - DDRDegronDiscovery (Substrate Selectivity in the Ubiquitin-Dependent Response to DNA Damage)
Berichtszeitraum: 2021-11-01 bis 2023-10-31
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.
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.
The project required a novel microscope that we developed in collaboration with Sutter Instruments. We wanted to have that in place before the start of the project, but initial tests showed that some additional modifications where necessary. I installed additional filters to allow deep UV imaging, a different light source for the UV light and more narrow bandwidth filter sets for fluorescent imaging led to successful imaging of lanthanide encoded beads. We thereby established the first dedicated microscope for imaging of lanthanide encoded beads.
MRBLE:pep requires the direct synthesis of peptides on encoded beads. The peptide synthesizer I had access to at the beginning of the project did not fulfil the technical requirements to obtain satisfactory results. After testing different synthesizers at the University of Copenhagen and the Technical University of Denmark we gained access to a synthesizer that matched the requirements. I successfully synthesized peptides on encoded beads and visualized a protein-peptide interaction using the build for purpose microscope.
I also established the SPARK2 assay in the lab and showed that it is feasible to detect Protein-Protein interactions that are based on short linear motifs similar to many interactions seen between E3 ligases and its substrates. In addition, I established a similar assay called FLUOPPI capable of visualizing this interaction type as a contingency plan for possible difficulties with the SPARK2 assay.
At this point of the project the results were not exploited or disseminated.
MRBLE:pep requires the direct synthesis of peptides on encoded beads. The peptide synthesizer I had access to at the beginning of the project did not fulfil the technical requirements to obtain satisfactory results. After testing different synthesizers at the University of Copenhagen and the Technical University of Denmark we gained access to a synthesizer that matched the requirements. I successfully synthesized peptides on encoded beads and visualized a protein-peptide interaction using the build for purpose microscope.
I also established the SPARK2 assay in the lab and showed that it is feasible to detect Protein-Protein interactions that are based on short linear motifs similar to many interactions seen between E3 ligases and its substrates. In addition, I established a similar assay called FLUOPPI capable of visualizing this interaction type as a contingency plan for possible difficulties with the SPARK2 assay.
At this point of the project the results were not exploited or disseminated.
Protein-protein interactions were long considered undruggable, but many are interesting targets for pharmaceutical intervention to treat human diseases. In addition, in recent years the development of heterobifuctional molecules that artificially bridge an E3 ligase to a target protein has fueled an increased interest in E3 ligases and protein-protein interactions. MRBLE:pep can be used for multiplexed analysis of protein-protein interaction and thereby enables the research of these interactions in particular for difficult to produce proteins. This project helped develop a functional microscope that makes MRBLE:pep more accessible for other scientists. I could further show that SPARK2 assay can be used to investigate weak and transient protein-protein interactions and can likely be exploited to analyze this type of interactions in a high throughput pooled screening set up.