A proteomic approach towards the Hsp90-dependent ubiquitinylated proteome.

Since many Hsp90 client proteins are key players in tumour pathways, the ubiquitylation and subsequent degradation of Hsp90-substrates as a consequence of pharmacologically inhibiting Hsp90 represents an innovative approach for cancer therapy. We therefore identified Hsp90-binding proteins which accumulated as ubiquityl-tagged aggregates in the detergent insoluble fraction of HeLa cells as a consequence of simultaneously inhibiting Hsp90 and the proteasome. 2-DE followed by nanoLC-MS/MS of trypsinised protein spots provided the Hsp90-dependent ubiquitylated proteome which was finally annotated and functionally classified. The overall picture thus obtained emphasised the well-established role of Hsp90 in stabilising proteins involved in gene transcription and signal transduction. It also provided a novel Hsp90-related link to metabolic pathways as the inhibition of Hsp90 caused the ubiquitylation of a significant amount of metabolic enzymes. These findings serve to support cumulating indications which attribute Hsp90 to diverse stabilising functions beyond signal transduction and gene transcription.