Studies of human HSP70 isoforms residing in the cytoplasm and mitochondria and their high molecular weight oligomers: interaction with co-chaperones and client proteins

Abstract

Among all molecular chaperones, Hsp70 family stands out due to its important role in cellular homeostasis, since they act as a pivot receiving and delivering client proteins from/to other chaperone molecules. Hsp70 act assisting the folding of nascent proteins, preventing protein aggregation, directing of proteins to organelles or for degradation and recovering proteins from aggregates, among others. Therefore, Hsp70s develop crucial functions in cellular proteostasis. This project aims at in-depth studies of the functional mechanism of human Hsp70 residing in cell cytoplasm (Hsp70-1A) and mitochondria (mtHsp70/GRP75/HspA9 /PBP74/mortalin). In mammals, mtHsp70 is also called mortalin due to its involvement with apoptosis, senescence and cancer, as there is a significant change in its level of expression in tumor cells, generating great interest in its study, including as a target for pharmacological inhibition. Although not known since the 1990s, the production of recombinant human mortalin was not possible due to a self-aggregation process, which was recently surpassed by the proponent group. However, Hsp70s undergo self oligomerization/aggregation process, leading to the hypothesis that these would be cellular deposits of Hsp70. Thus, not only the investigation of the structural-functional structure of the monomeric Hsp70, including their regulating factors and interaction with client proteins, is of great relevance as well as the study of Hsp70 oligomers formed in vivo. In this context, this project aims: i) to analyze, in vivo, a presence and functionality of the high molecular weight oligomers of mortalin and Hsp70-1A; ii) to evaluate the in vitro effect of hHep1, hGrpE#1 and hGrpE#2 co-chaperones on mortalin and its high molecular weight oligomers; iii) to obtain the structure and function relationship of human hDjA3 and hDja20 co-chaperones, mitochondrial J-proteins, and evaluate their effects on monomeric mortalin and high molecular weight oligomers and Hsp70-1A; iv) to evaluate the structure of high molecular weight oligomers of mortalin and Hsp70-1A by electron transmission microscopy; v) study the interaction of mortalin, Hsp70-1A and their respective oligomers of high molecular mass with a p53 and mutant client protein, which is involved with a formation of cancerous cells. Both Hsp70-1A and a mortalin (which is also found in the cytoplasm, nucleus and endoplasmic reticulum) act by sequestering the p53 in the cellular cytoplasm, justifying the inclusion of this objective in the proposal. (AU)