Project Details
Projekt
Identifying and characterizing readers of serine- and threonine-linked ubiquitin chains using non-hydrolyzable diubiquitins and chemical proteomics
Applicant
Tim Aguirre
Subject Area
Biochemistry
Term
since 2023
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 532187979
Ubiquitylation is a versatile post-translational modification involved in a myriad of cellular processes, including protein degradation, epigenetic regulation, and DNA damage responses. The reversible modification of proteins with ubiquitin (Ub) is tightly regulated by a sophisticated enzymatic machinery. It is common understanding in the ubiquitin field that ubiquitylation involves the formation of an isopeptide bond between the C-terminus of ubiquitin and the lysine side chain of another ubiquitin or the protein substrate. However, recent studies have provided proof that ubiquitin can also be attached to serine and threonine side chains via the formation of oxyester linkages. Intriguingly, these linkages were also found to occur between two successive Ub molecules in ubiquitin chains at residues Thr12, Thr14, Ser20, and Thr22. This revelation increases the already daunting complexity of the 'ubiquitin code' and requires the identification of cellular binding partners of oxyester-linked ubiquitin chains to comprehend their biological implications. Another challenge is to disclose the identity of the deubiquitinases (DUBs) involved in disassembling endogenous serine- and threonine-linked ubiquitin chains. The main aim of the proposed project is to identify interactors and DUBs of serine- and threonine-linked ubiquitin chains by employing synthetic diubiquitins as baits in pulldown proteomic experiments. Subsequently, putative interaction partners will be thoroughly characterized in vitro. Therefore, I will develop a methodology to synthesize non-hydrolyzable diubiquitin probes linked at Thr12, Thr14, Ser20, and Thr22 using solid phase peptide synthesis and copper-catalyzed azide-alkyne cycloadditions. I will furthermore establish a route to access the corresponding activity-based probes bearing an electrophilic warhead at the linkage site to capture ligases and DUBs. These probes will subsequently be used in pulldown experiments to identify interaction partners of ubiquitin chains linked via serine or threonine. The obtained list of enriched proteins will be vigilantly analyzed with regards to the enrichment score and potential clustering of certain protein families or classes and binding domains. Gene ontology analysis can provide valuable insights into the cellular processes that oxyester-linked ubiquitin chains might regulate. Selected putative binders will be recombinantly expressed and subjected to detailed biochemical and biophysical analysis in vitro. In addition, crystallization efforts or cryo-EM analysis will allow me to gain insights into specific binding interactions on a molecular level. Eventually, depending on which cellular pathway the identified interactors or DUBs play a role in, I will induce certain cell stresses like starvation, DANN damage, oxidation, or proteasome inhibition and observe the changes in interaction between the diubiquitin baits and the studied protein in cellula.
DFG Programme
WBP Fellowship
International Connection
Netherlands
