Molecular chaperones are essential proteins that assist in the folding of substrate ‘client’ proteins to adopt their functionally active three-dimensional structures. The process of protein folding in the cell occurs in a highly concentrated crowded cellular environment among various other macromolecules and amidst various cell stresses which result in issues of aberrant protein folding into toxic species and aggregates. Thus, to counteract these stressors, cells have evolved a complex network of chaperone proteins to maintain protein homeostasis, or proteostasis. Hsp70 is an essential molecular chaperone that acts on clients important for a wide variety of cellular functions. Hsp70 can facilitate refolding of clients to regain their function. However, it can also target client proteins to proteasomal degradation. Turnover of aberrantly folded or aggregation prone proteins such as tau implicates Hsp70 in various pathologies including neurodegenerative diseases.
Another class of protein chaperones, termed ‘holdases’, act to delay protein aggregation. The small heat shock proteins (sHSP) systems possess such activity, binding to non-native conformations of clients. sHsps such as Hsp27 and αB crystallin exist as distributions of large oligomeric species that respond dynamically to pH and temperature stresses. Recent studies have demonstrated oligomeric rearrangements occur for sHsps to protect client proteins. A major outstanding question is how do these oligomeric assemblies’ complex structures sense cell stress or protein unfolding or aggregation. In addition to sensing cell stress, sHsps and holdase chaperones are also capable of bridging with the activities of other classes of chaperones, including the Hsp70 chaperone system. Hsp70 functions in concert with a network of co-chaperone proteins which diversify its protein folding capabilities. BAG3 is a nucleotide exchange factor (NEF) that facilitates the exchange of ADP and ATP in Hsp70. In addition, interactions with sHsp family chaperones have emerged, making it a promising target in elucidating the link between these two functionally distinct chaperone systems.
The overall theme to my thesis work has been to characterize protein homeostasis achieved through pro-folding and pro-degradation pathways. A major focus of my thesis concerns the ability of Hsp70 to work in concert with the CHIP E3 ubiquitin ligase to target tau for polyubiquitination in a chaperone dependent manner, thus facilitating protein turnover. Another focus has been on a pro-folding function of chaperones, the so-called holdase function, where I have explored the structural rearrangements of the sHsp αB crystallin as well as another multifunctional chaperone, peroxiredoxin, and how these conformational changes and oligomeric rearrangements trigger with external stress and correlate with activation of chaperone activity. This thesis also explores the cooperation between sHsps and Hsp70 to
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facilitate protein refolding, where I characterize rearrangements that occur in the Hsp27 oligomer distribution modulated by BAG3, and its implications on Hsp70 binding. One of the major techniques utilized in my thesis work is electron microscopy, obtaining structural information of protein complexes, from obtaining low resolution size distributions of sHsp oligomers to pushing resolution of Hsp70 in complex with CHIP beyond quaternary structural information to sub-nanometer resolution of the peroxiredoxin in its active chaperone form in complex with substrate. These studies serve as a foundation for future work on obtaining the structural basis of the decision process where chaperone proteins decide the fate of their client substrates.PHDBiological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/144085/1/orvvdom_1.pd
