Epidithiodiketopiperazines Inhibit Protein Degradation by Targeting Proteasome Deubiquitinase Rpn11
Abstract
The 26S proteasome is the major proteolytic machine for breaking down cytosolic and nuclear proteins in eukaryotes. Due to the lack of a suitable assay, it is difficult to measure routinely and quantitatively the breakdown of proteins by the 26S proteasome in vitro. In the present study, we developed an assay to monitor proteasome-mediated protein degradation. Using this assay, we discovered that epidithiodiketopiperazine (ETPs) blocked the degradation of our model substrate in vitro. Further characterization revealed that ETPs inhibited proteasome function by targeting the essential proteasomal deubiquitinase Rpn11 (POH1/PSMD14). ETPs also inhibited other JAMM (JAB1/MPN/Mov34 metalloenzyme) proteases such as Csn5 and AMSH. An improved ETP with fewer non-specific effects, SOP11, stabilized a subset of proteasome substrates in cells, induced the unfolded protein response, and led to cell death. SOP11 represents a class of Rpn11 inhibitor and provides an alternative route to develop proteasome inhibitors.
Additional Information
© 2018 Elsevier Ltd. Under an Elsevier user license. Received 15 February 2018, Revised 23 May 2018, Accepted 25 July 2018, Available online 23 August 2018. We thank J.M. Huibregtse (The University of Texas at Austin) for the vector expressing GST-Wbp2-C-K222, Rsp5, and Rsp5-E6AP, A. Martin (University of California, Berkeley) for the pETDuet-1 vector expressing the Rpn11-Rpn8 dimer, T.M. Kapoor (The Rockefeller University) for providing the RPE1 WT and BTZ-resistant cell lines, H. Park for testing the cell lines for mycoplasma contamination, A. Bowers (University of North Carolina) for the stimulating discussion on gliotoxin, and D. Sherman (Amgen Inc.) for critical reading of our manuscript. This work was supported by grants from the Caltech Gates Grubstake Fund and Amgen to R.J.D. and from the NIH (CA164803) to R.J.D. and S.M.C. S.T.H. and B.D.S.S.D.S. were supported by the funding from FCT (grant no. SFRH/BD/65630/2009). Author Contributions: J.L. designed, executed, and interpreted the experiments using Wbp2 as proteasome activity substrate, in vitro Rpn11 assay and AMSH assay, and western blot. Y.Z. performed and analyzed the in vitro Csn5 assay, real-time qPCR assay, and western blot. B.D.S.S.D.S. and S.T.H. synthesized ETP compounds SOP1-11. Y.Y. and J.P. quantified the linkage type of ubiquitin chain on Wbp2. F.W. and T.-F.C performed and analyzed the UbG76V-GFP degradation assay. Y.M., C.P. and S.M.C. performed and analyzed the hCAII and MMP-2 assay. R.J.D. designed, interpreted, and oversaw the experiments for the entire study. The manuscript was drafted by J.L. and R.J.D with input from all authors. Declaration of Interests: R.J.D. is a founder, shareholder, and member of the Scientific Advisory Board of Cleave Biosciences, which is engaged in discovery and development of drugs that target enzymes involved in ubiquitin-dependent protein degradation. R.J.D. is currently Senior Vice President of discovery research at Amgen Inc.. S.M.C. is a co-founder and has an equity interest, and receives income as a member of the Scientific Advisory Board for Cleave Biosciences and is a co-founder, has an equity interest, and a member of the Scientific Advisory Board for Forge Therapeutics. Both companies may potentially benefit from the research results of certain projects in the laboratory of S.M.C. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies.Attached Files
Accepted Version - nihms-1510970.pdf
Supplemental Material - 1-s2.0-S2451945618302642-mmc1.pdf
Files
| Name | Size | Download all |
|---|---|---|
|
md5:4d8af72f5306763e4fada903711e7250
|
1.4 MB | Preview Download |
|
md5:659cc0198d025753bf40b5508d0c3948
|
2.9 MB | Preview Download |
Additional details
- PMCID
- PMC6309308
- Eprint ID
- 89092
- Resolver ID
- CaltechAUTHORS:20180823-134532623
- Caltech Gates Grubstake Fund
- Amgen
- CA164803
- NIH
- SFRH/BD/65630/2009
- Fundação para a Ciência e a Tecnologia (FCT)
- Created
-
2018-08-23Created from EPrint's datestamp field
- Updated
-
2022-02-16Created from EPrint's last_modified field