Charge preference of the Mammalian protein quality control machinery

Many elements of the protein quality control machinery (PQC) have a strong tendency to bind hydrophobic protein segments enriched in basic residues. Up until recently, a rationale for this charge preference was lacking. Over a decade ago, the Switch lab showed that stretches of hydrophobic amino acids with a tendency to aggregate (so-called Aggregation-Prone Regions or APRs) are systematically flanked by charged residues. The term “aggregation gatekeepers” (GKs) was coined to describe these charged residues, as they reduce the aggregation tendency of the APR they flank. Recent research from the Switch lab revealed an intricate link between GKs and chaperones: it was shown that the E. coli Hsp70 homolog DnaK specifically recognizes basic GKs because of their relatively poor aggregation-breaking capacity. Hence the charge preference for the PQC can be rationalized as a recognition of poorly gatekeeped aggregation-prone regions. The aim of this dissertation is to exploit recent advances in protein proximity labeling through biotin ligation in order to perform a proteome-wide screen for GK-specific APR interactors in Mammalian cells. In this way, we aim to expand our previous findings to a Mammalian context and definitively confirm the PQC charge bias. Furthermore, this screen is likely to yield previously unidentified GK-specific PQC elements, which will be thoroughly characterised both in vitro as well as in vivo.