UNDERSTANDING CHAPERONE INTERACTIONS IN DISEASE

The correct folding of proteins after synthesis and stress-promoted denaturation is critical for cell viability in all organisms. The Hsp70 molecular chaperone is a key player in proteostasis, deciding which proteins are foldable and which are too badly damaged and need to be targeted for degradation. Hsp70 plays an important role as a drive of cancer, stabilizing key mutated oncoproteins such as HER2, p53, RNR, SHR and MUC1. This importance of Hsp70 in basic cell functions as well as human illness prompted us to examine novel ways to characterize Hsp70 genetic and physical interactors. In this thesis, we decided to tackle three main roadblocks in studying chaperone interactions; 1) purification of chaperone complexes at native stoichiometry in mammalian cells, 2) understanding the roles of co-chaperones in cancer 3) teasing apart bridged vs direct chaperone interactions. To solve the issue of native stoichiometry purification, we have utilized CRISPR-Cas9 genome engineering to insert epitope tags into the N-terminus of Hsp70 in mammalian cells. This tagged chaperone is present as the only Hsp70 in cells, is stable without the use of any selectable marker and allows expression of Hsp70 at native levels. To understand co-chaperone function in cancer, we used a novel chemogenomic screening technology on WT and DNAJA1 knockout HAP1 cells. In doing so, we have uncovered a dependence of a large proportion of approved oncology drugs on DNAJA1 status. Finally, we have used cross-linking mass spectrometry to define for the first time the direct interactors of Hsp70 in yeast. Our data reveals a wealth of information of fundamental Hsp70 function including discovery of active Hsp70 dimers, client binding throughout Hsp70 and a huge number of novel PTM-associated Hsp70 interactions. Overall, aside from gaining fundamental insight into the workings of Hsp70, this thesis provides a roadmap and tools for the chaperone community to explore novel biologically relevant Hsp70 interactions.