Systematic Investigation of Protein Quality Control Mechanisms using human peptidome libraries

Protein quality control (PQC) pathways monitor the proteome to preserve protein homeostasis. Exposed hydrophobic residues in aberrant or mislocalized protein substrates are a key feature recognized by distinct PQC mechanisms. It is believed that chaperones together with E3 ligases are responsible for the recognition and clearance of the PQC substrates; however, very little is known about the client range, selectivity and specificity of each of the PQC mechanisms. Here, we are interested in analyzing the PQC components recognizing aberrant or mislocalized proteins and mapping the recognition “code”. To this end, we used CRISPR/Cas9 approach to generate knock out cells for various E3 ligases believed to play a role in protein homeostasis regulation in human cells. Then, we used the GPS-peptidome approach, a high throughput technology for substrate discovery to identify specific substrates for each E3 ligase. Mutagenesis approach was used to map the minimal elements of hydrophobic sequences among substrates, and CRISPR screens are utilized to identify additional PQC components recognizing hydrophobicity. A detailed understanding of the specific hydrophobic sites on proteins that confer instability, coupled with genetics to uncover the cognate E3 ligases involved, is critical to further our understanding of how signal transduction remodels the proteome by specifically recognizing substrates’ aberrant features.