Molecular mechanism of the interaction of Hsp90 with cochaperones and clients
Final Report Abstract
The highly conserved Hsp90 chaperones control stability and activity of many essential signalling and regulatory proteins including about 60% of all human protein kinases, 30% of all human E3 ubiquitin ligases and many transcription factors. In this study, we aimed to better understand the physiological role of the two Hsp90 isoforms, Hsp90α and Hsp90β, in the cytosol and nucleus of human cells and to systematically analyze their regulation by posttranslational modifications. We developed a doxycycline-inducible CRISPRi knock-down system in HeLa cells that allows to reduce the cellular Hsp90 levels of each isoform separately to less than 5% of the wildtype level and of both simultaneously to some 30% of total cytosolic Hsp90 levels when cells stop dividing. Key findings of this study are (1) both isoforms are able to replace each other largely, if the knockdown of an isoform happens slowly over the course of 5 days, but not if the knock-down happens within one day; (2) the use of isoform-specific inhibitors revealed that inhibition of Hsp90 causes a toxic gain-of-function not loss-of-function phenotype; (3) time resolved proteomics showed that the majority of proteins that are reduced in concentration by limiting amounts of Hsp90 are proteins that interact with Hsp90 in the newly synthesized state and not clients that interact constitutively with Hsp90. The first client that fails upon reduction of Hsp90 levels is Cdk1 leading to cell cycle arrest in S phase and G2/M transition. According to high-throughput phosphoproteomics, human Hsp90α and Hsp90β are phosphorylated in 74 and 66 sites, respectively, but very few sites have been studied in more detail. We explored the suitability of the yeast model system for the identification of key regulatory residues in human Hsp90α. Replacement of three tyrosine residues in the N-terminal nucleotide binding domain of human Hsp90α by phosphomimetic glutamate and by non-phosphorylatable phenylalanine individually and in combination influenced growth of a yeast strain that had both endogenous Hsp90 genes deleted and affected the activity of 7 different Hsp90 clients in distinct ways. Our findings suggest that posttranslational modifications could bias Hsp90’s client specificity. This hypothesis was substantiated in a second study, in which we investigated six tyrosine phosphorylation sites at the hinge region between middle domain and C-terminal dimerization domain. Surprisingly, one phosphomimetic glutamate variant complemented yeast growth on fermentable carbon sources but not after the diauxic shift or on non-fermentable carbon sources. In contrast, the non-phosphorylatable phenylalanine variant of the same residue did not complement yeast growth. To elucidate the molecular mechanism, we are currently using our HeLa Hsp90 knock-down cell line.
Publications
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Modification of regulatory tyrosines biases human Hsp90α for interaction with cochaperones and clients.
Huo, Yuantao; Karnawat, Rishabh; Liu, Lixia; Knieß, Robert A.; Gross, Maike; Chen, Xuemei & Mayer, Matthias P.
