Hsp90 molecular chaperone : the effect on breast cancer cell invasion and functional interactions with Aha1 co-chaperone

Abstract

One of the most important aspects of the molecular chaperone Hsp90’s activity is the ATP-ase cycle. The cycle of ATP hydrolysis is an important part of the recycling of Hsp90 and drives its conformational changes. Co-chaperones regulate this ATP-hydrolysis as well as the interactions with substrate proteins. The Aha1 co-chaperone has been described in human cells to activate the relatively weak intrinsic ATP-ase activity of Hsp90. This work describes our investigations into the mechanism of interaction between Aha1 and Hsp90. We demonstrate that the binding of Aha1 to the Hsp90 is dependent on adenosine nucleotide binding, and in physiological conditions is prone to inhibition with the specific inhibitor of Hsp90, 17-AAG. Using high throughput screening of random mutants of Hsp90 in yeast we isolated a novel mutant of Hsp90 with increased affinity to Aha1. Mapping the interaction, we for the first time confirm the hypothesis of a more elaborate mechanism of interaction between Aha1 and the N-domain of Hsp90, crucial for the efficient stimulation of Hsp90 ATP-ase activity. This thesis also describes a novel mechanism for the inhibitor resistance of Hsp90. The in vivo resistance is related to the increased in vitro ATP-ase activity of Hsp90 and dependent on Aha1 in case of the novel mutant we described. Due to its involvement in chaperoning of malignant states Hsp90 is a subject of intensive research. A prominent role in cancer progression has been attributed to extracellular secreted Hsp90. Studying the role of Hsp90 in breast cancer cell invasion we demonstrated that the inhibitory effect of anti Hsp90 drugs is not related to inhibition of the activation of extracellular gelatinases, but is rather linked to the deregulation of signaling pathways involved in formation of focal adhesions. We postulate an exosomal pathway as a mechanism of Hsp90 secretion. Using the inhibitor resistant allele of Hsp90 we study isoform specificity of Hsp90, and demonstrate that the Hsp90α activity alone is not sufficient to promote survival of the MDA-MB 231 cancer cell line. We also investigated the effect of the post-translational modification of reversible acetylation of Hsp90 on the interaction with a model substrate, the p53 tumor suppressor. Reconstitution of the system using purified recombinant proteins we establish a role for the acetylation status of Hsp90 on the rescue of binding of WT p53 to the specific promoter sequences.