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Updated: Jun 25, 2026
11:36
In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones
Published on: July 25, 2019
K63-specific deubiquitination by two JAMM/MPN+ complexes: BRISC-associated Brcc36 and proteasomal Poh1.
Eric M Cooper1, Colleen Cutcliffe, Troels Z Kristiansen
1Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA. emcooper@jhsph.edu
The EMBO Journal
|February 14, 2009
Summary
Researchers discovered a unique deubiquitinating enzyme activity specific for K63-linked polyubiquitin chains. This activity, found in a large protein complex, lacks a cysteine residue and is associated with proteasome and signalosome components.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Deubiquitinating enzymes (DUBs) play crucial roles in regulating cellular processes by removing ubiquitin chains.
- Polyubiquitin chain linkage types, such as K63 and K48, dictate distinct cellular fates and signaling pathways.
- Specific DUBs with defined linkage specificities are essential tools for dissecting ubiquitin signaling.
Purpose of the Study:
- To characterize an unusual deubiquitinating activity present in HeLa cell extracts.
- To determine the substrate specificity and biochemical properties of this novel DUB activity.
- To identify the protein complexes and components responsible for the observed K63-specific deubiquitination.
Main Methods:
- Biochemical assays using various polyubiquitin chain substrates (K63-linked, K48-linked, etc.).
- Enzyme inhibition assays using N-ethyl-maleimide and ubiquitin aldehyde.
- Gel filtration chromatography to determine the molecular weight of the active complex.
- Co-fractionation studies across multiple chromatographic steps to identify associated protein complexes.
Main Results:
- A DUB activity highly specific for K63-linked polyubiquitin chains was identified in HeLa cell extracts.
- The activity was insensitive to cysteine-reactive inhibitors, suggesting a non-cysteine active site.
- The DUB activity was found to reside in a high molecular weight complex (approx. 600 kDa) and co-fractionated with the 19S proteasome (PA700), COP9 signalosome (CSN), and a Brcc36-containing complex (BRISC).
- The K63-specific activity was intrinsic to PA700 and BRISC, and CSN-associated activity was mediated by Brcc36.
- Cleavage was specific to K63 linkages, even within mixed-linkage chains, while other linkages (K6, K11, K29, K48, alpha) remained intact.
Conclusions:
- The JAMM/MPN+ domain protein Brcc36 is a key component of K63-specific deubiquitinating activity.
- K63-specific deubiquitinating activity is associated with both the 19S proteasome and the COP9 signalosome, mediated by Brcc36.
- Specificity for K63-linked polyubiquitin chains appears to be a common characteristic of the JAMM/MPN+ family of DUBs.
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