Deubiquitinating enzymes in selective autophagy
Dynamic protein ubiquitination plays an essential regulatory role in selective autophagy. This project aims to provide a comprehensive view on the function of deubiquitinating enzymes (DUBs) in different selective autophagy pathways. To systematically analyze DUBs that regulate mitophagy, pexophagy, and ER-phagy, we will perform a multiplex CRISPR/Cas9-based genetic screen targeting individual human DUBs and all possible combinations of DUB pairs. To study the mechanistic and functional role of identified DUBs in selective autophagy pathways, we will employ quantitative mass spectrometry-based proteomics, imaging, and biochemical methods.
Characterizing ubiquitin signaling by MS-based proteomics. Ubiquitin remnant profiling allows proteome-wide identification of endogenous ubiquitination sites. If combined with SILAC, ubiquitin remnant profiling can be used for relative quantification of ubiquitination sites in different conditions. To gain insights into the topology of ubiquitin chains, binders such as antibodies or recombinant ubiquitin binding domains can be used to enrich proteins modified with a specific type of ubiquitin chains. Enriched proteins are then digested in gel into peptides and analyzed by LC-MS/MS (Heidelberger et al., 2016).