Hsp90 inhibition induces both protein-specific and global changes in the ubiquitinome

J Proteomics. 2015 Apr 29:120:215-29. doi: 10.1016/j.jprot.2015.02.020. Epub 2015 Mar 14.


Inhibition of the essential chaperone Hsp90 with drugs causes a global perturbation of protein folding and the depletion of direct substrates of Hsp90, also called clients. Ubiquitination and proteasomal degradation play a key role in cellular stress responses, but the impact of Hsp90 inhibition on the ubiquitinome has not been characterized on a global scale. We used stable isotope labeling and antibody-based peptide enrichment to quantify more than 1500 protein sites modified with a Gly-Gly motif, the remnant of ubiquitination, in human T-cells treated with an Hsp90 inhibitor. We observed rapid changes in GlyGly-modification sites, with strong increases for some Hsp90 clients but also decreases for a majority of cellular proteins. A comparison with changes in total protein levels and protein synthesis and decay rates from a previous study revealed a complex picture with different regulatory patterns observed for different protein families. Overall the data support the notion that for Hsp90 clients GlyGly-modification correlates with targeting by the ubiquitin-proteasome system and decay, while for other proteins levels of GlyGly-modification appear to be mainly influenced by their synthesis rates. Therefore a correct interpretation of changes in ubiquitination requires knowledge of multiple parameters. Data are available via ProteomeXchange with identifier PXD001549.

Biological significance: Proteostasis, i.e. the capacity of the cell to maintain proper synthesis and maturation of proteins, is a fundamental biological process and its perturbations have far-reaching medical implications e.g. in cancer or neurodegenerative diseases. Hsp90 is an essential chaperone responsible for the correct maturation and stability of a number of key proteins. Inhibition of Hsp90 triggers a global stress response caused by accumulation of misfolded chains, which have to be either refolded or eliminated by protein degradation pathways such as the Ubiquitin-Proteasome System (UPS). We present the first global assessment of the changes in the ubiquitinome, the subset of ubiquitin-modified proteins, following Hsp90 inhibition in human T-cells. The results provide clues on how cells respond to a specific proteostasis challenge. Furthermore, our data also suggest that basal ubiquitination levels for most proteins are influenced by synthesis rates. This has broad significance as it implies that a proper interpretation of data on ubiquitination levels necessitates simultaneous knowledge of other parameters.

Keywords: Chaperone; Hsp90; Mass spectrometry; Quantitation; Stress response; Ubiquitin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Gene Expression Regulation, Enzymologic / physiology
  • HSP90 Heat-Shock Proteins / antagonists & inhibitors*
  • HSP90 Heat-Shock Proteins / metabolism*
  • Humans
  • Jurkat Cells
  • Proteome / metabolism*
  • Ubiquitin / metabolism*
  • Ubiquitinated Proteins / metabolism*
  • Ubiquitination / physiology*


  • HSP90 Heat-Shock Proteins
  • Proteome
  • Ubiquitin
  • Ubiquitinated Proteins