Harvard Medical School, Genetics (External organization)

Description

Postdoctoral fellowship: High throughput charachterization of degradation pathways - Harvard Medical School

Degrons are the minimal elements within proteins that are recognized by E3 ligases to promote proteolysis. Despite the central role of degrons in proteostasis, our knowledge about degron motifs is still minimal. To characterize degron motifs in human proteins in a high-throughput manner, we developed a genome-wide approach called GPS-peptidome profiling. This technology is a hybrid of the Global Protein Stability (GPS) reporter system combined with a synthetic human peptidome library synthesized on high density oligonucleotide microarrays. GPS is based on a lentiviral construct encoding two fluorescent proteins: DsRed, which serves as an internal reference, and a GFP fusion peptide that is translated from an internal ribosome entry site (IRES). As both DsRed and the GFP fusion peptide are expressed from the same transcript, the GFP/DsRed ratio can be used to readout the effect of the fusion peptide on the stability of GFP. This powerful approach has allowed us to identify a large number of terminal degrons lying at both the N- and C-termini of protein substrates. A series of CRISPR screens identified the cognate E3 ligases that recognize these degrons. The C-terminome screen uncovered the first examples of C-degrons, while the N-terminome screen revealed extended specificity for UBR E3 ligases and a novel N-degron pathway centered on N-terminal glycine. Interestingly, dedicated Cullin2 E3 ligase complexes target both N-terminal and C-terminal glycine degrons. All of these degron motifs are depleted from the human proteome, suggesting evolutionary pressure to avoid degradation by E3 ligases targeting terminal degrons that in turn appears to have shaped the composition of metazoan proteomes. In addition, we uncovered important roles of terminal degrons in degrading proteins that fail to localize properly to cellular membranes and in destroying proteolytic fragments generated by caspases during apoptosis.

Period	2013 → 2019
Held at	Harvard Medical School, Genetics