Identification and dissection of N-degron pathways using quantitative proteomics

• Applicant: Dr. Tanja Bange
• Subject Area: Biochemistry
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 504140321

Regulated protein degradation controls levels of all short-lived proteins to ensure cellular homeostasis and protects cells from accumulating abnormal proteins. Dysfunctional degradation causes multiple pathological processes, spanning from neurodegenerative disorders to cancer. Therefore, the identification and understanding of degradation pathways is of pivotal importance. This proposal aims to investigate a subset of protein degradation pathways, so called N-degron pathways, in which N-termini of proteins are recognized as degradation signals. N-degrons are very short (1-4 amino acids (aa)) motifs mainly determined by the first N-terminal (Nt-) aa of a protein. The most important players are E3 ligases, so called N-recognins, which recognize N-degrons and target the proteins for proteasomal degradation. Four N-degron pathways have been described so far.We set up a peptide pull-down approach combined with quantitative mass spectrometry to identify N-degrons and their cognate N-recognins. With this approach we identified a new N-degron starting with alanine (A) harboring a 4 aa IBM (IAP binding motif, with high flexibility on aa 2-4) and its cognate N-recognins, the so-called IAPs (inhibitor of apoptosis proteins), previously involved in apoptosis Importantly, proteins with A are only recognised if they are also a substrate of the Nt-acetyltransferase NatA and their natural acetylation has been omitted. This means that the N-degron is masked through acetylation and only exposed when Nt-acetylation is disturbed. These findings made a major contribution to the Nt-processing and N-degron field by implicating Nt-acetylation in protein stabilization and suggesting that not properly acetylated proteins can be removed from cells by IAPs.Hypothesis: Based on recent literature and my preliminary data, I hypothesize that more E3 ligases act as N-recognins in N-degron pathways than currently known. Studies so far have mainly focused on the role of the starting aa on protein stability and less on the identification of the E3 ligases involved. I have demonstrated that our approach is able to identify those E3 ligases. Therefore, I propose to apply it systematically to enlarge our picture of N-degron pathways.Key Objective: This project aims to unravel the identity and function of N-degron pathways. We will reach our goal through two objectives:1) Identification of E3 ligases recognizing N-degrons in a medium-throughput manner including all 20 aa by using peptide pull-downs combined with quantitative mass spectrometry (MS). 2) Validation and characterization of the N-recognin candidates/ new pathways identified in objective 1 with biochemical and cellular assays.Impact: A systematic screening for N-recognins and their characterization has not been done so far. The results will be novel and of high impact for various fields (Nt- processing, Nt-acetylation, protein quality control and stability, assignment of new roles to E3 ligases).

• DFG Programme: Research Grants