Proteome-wide degron mapping by Global Protein Stability profiling

Abstract

The ubiquitin-proteasome system plays crucial roles in nearly every aspect of cell biology by regulating the degradation of specific proteins. There are approximately 600 E3 ubiquitin ligases that mediate specific protein degradation, often through the recognition of short linear peptide motifs known as degrons on their protein substrates. Despite the crucial role of degrons in selective protein degradation, our understanding of degrons is still incomplete and the number of known degrons remains limited. To accelerate our understanding of degrons, this dissertation presents a system for systematic identification of degrons at a proteome-wide level. The first part of this dissertation describes a pipeline that combines experimental Global Protein Stability profiling, machine learning, and a computational degron motif clustering algorithm for systematic degron identification. First, we leveraged Global Protein stability profiling and employed machine learning to identify 15,800 putative sequence-dependent degron peptides. We then performed scanning mutagenesis to define degron motifs for over 5,000 of these predicted degron peptides. Empowered by access to a broad-spectrum ubiquitin ligase inhibitor that inhibits all Cullin-RING ligases (CRLs), we focused on CRL-degrons and performed saturation mutagenesis to precisely define degron motifs for 219 CRL-degrons. To enable unbiased computational clustering of saturation mutagenesis footprints, we developed DegronID, a clustering algorithm that groups degron motifs based on their footprint similarity. Lastly, we mapped a subset of CRL-degron peptides to their cognate ubiquitin ligases via CRISPR screening and revealed 15 classes of CRL-degron pairs, of which many were previously unknown. The second part of this dissertation describes the application of Global Protein stability profiling to study regulated protein degradations by leveraging post-translational modification inhibitors. By measuring human ORFeome stability through Global Protein Stability profiling in the presence and absence of an inhibitor of OGT, the only writer for O-GlcNAcylation, we identified a large set of GFP-fusion proteins degraded by ubiquitin ligase CUL3KLHL9/13 in an O-GlcNAcylation regulated manner. While preliminary results suggested that many of the identified KLHL9/13 substrates are only degraded by KLHL9/13 when in the form of GFP-fusions, the molecular details and the physiologically relevant substrates of this nutrient regulated degradation pathway will continue to stimulate future research.