Abstract 2842 Comparative Transcriptomics Reveal Insights into Vertebrate Brain Development Through a Goldfish Telencephalon Atlas

Ubiquitin-mediated protein degradation is a vital cellular pathway in eukaryotes, facilitating the selective degradation of proteins and thereby allowing cells to function efficiently and adapt to stress.In this study, we established a quantitative proteomics approach to uncover changes in the ubiquitinated proteome in cultured human cells.We utilized TR-TUBE (Trypsin Resistant-Tandem Ubiquitin Binding Entities) recombinant protein as a probe to enrich poly-ubiquitinated proteins from cell lysates.TR-TUBE is a previously developed synthetic protein that binds poly-ubiquitin chains with high affinity and has trypsin resistance due to the lack of lysine and arginine residues.We conjugated TR-TUBE to NHS-activated magnetic beads, which successfully isolated poly-ubiquitinated proteins from HEK293 cell lysates.Subsequent mass spectrometry analyses revealed the identities of these isolated proteins.Next, we applied this method to investigate the roles of CAND1 and CAND2 in regulating the ubiquitinated proteome.CAND1 and CAND2 are regulators of cullin-RING ligase (CRL), a major family of E3 ubiquitin ligases responsible for ubiquitinating about 20% of total cellular proteins.Through directly binding to the cullin scaffold of CRLs, CAND1/2 regulates the assembly and activity of almost all CRLs, promoting CRL1-, CRL3-, and CRL4-mediated ubiquitination, while conditionally inhibiting CRL2-mediated ubiquitination.Using the TR-TUBE based proteomics approach we optimized, we compared the ubiquitinated proteome in HEK293 cells with or without CAND1 and CAND2 double knockout (DKO).This analysis identified around 6000 ubiquitinated proteins from the HEK293 cells, about 20% of which displayed two-fold or greater changes in ubiquitination levels in DKO cells.Upon confirming the changes in ubiquitination, we will investigate the mechanisms by which CAND1 and CAND2 regulate these proteins.In summary, we have established a quantitative proteomics method that efficiently identifies altered ubiquitination within the human cell proteome.This method serves as a useful tool for uncovering cellular, chemical, and environmental factors that regulate protein ubiquitination.