Mentor: Andrea Kravats, Ph.D.

Endoplasmic reticulum (ER) resident molecular chaperones are essential for maintaining the integrity of the proteome during times of ER stress. Failure of these chaperone systems results in protein misfolding and subsequent aggregation, which have been linked to neurodegenerative diseases such as Alzheimer's, ALS, and Parkinson's disease. The ER Hsp70, BiP, has a well characterized chaperone cycle that is facilitated by co-chaperones to transition between high and low affinity states for misfolded client proteins; J-domain proteins stimulate BiP’s ATP hydrolysis rates and deliver misfolded clients, while nucleotide exchange factors facilitate BiP’s exchange of ADP to enable ATP rebinding. Additionally, it is well established that the BiP chaperone system collaborates with the ER Hsp90 chaperone, Grp94, in protein remodeling. The canonical client remodeling mechanism for BiP and Grp94 is conserved with other Hsp70-Hsp90 chaperones, where Hsp70 and cochaperones initiate client remodeling and recruit Hsp90 for enhanced remodeling. Previous studies from our lab investigating chaperone complexes formed during client remodeling showed correlations between the amount of BiP and J-protein in complex with Grp94. Binary complexes between BiP and Grp94 have been reported in numerous studies from our lab and others. Studies with cytosolic chaperones have indicated that Hsp70, J-proteins, and Hsp90 form ternary complexes during protein remodeling. Furthermore, J-proteins have been shown to interact with Hsp90s in the absence of Hsp70. These previous findings support the basis for our prediction that this interaction is further conserved in human ER chaperones. Pulldown assays were performed to confirm the interactions between Grp94 and a J-protein. Chemical crosslinking was carried out to further clarify the characteristics of the complex and its stoichiometry. Analysis of the crosslinked gels by mass spectrometry will identify direct points of physical interaction between the two chaperones. Together, these results will allow for improved understanding of these chaperones and their collaboration in protein remodeling. This information is helpful in rational drug design targeting protein-protein interactions.

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