Autoubiquitination of the 26S proteasome on Rpn13 regulates breakdown of ubiquitin conjugates

Area of Science:

• Cellular biology
• Biochemistry
• Molecular mechanisms of protein degradation

Background:

• Protein degradation rates in eukaryotic cells are primarily governed by ubiquitination.
• The regulatory mechanisms controlling the proteasome's degradation capacity remain underexplored, despite the clinical use of proteasome inhibitors.

Purpose of the Study:

• To investigate the regulation of mammalian 26S proteasome capacity.
• To identify factors influencing proteasome function under conditions of inhibited proteolysis or proteotoxic stress.

Main Methods:

• Analysis of ubiquitination patterns in mammalian 26S proteasomes and their subunits.
• In vitro and in vivo experiments using proteasome inhibitors, heat shock, and arsenite treatment.
• Biochemical assays to measure proteasome binding and degradation activity.

Main Results:

• Mammalian 26S proteasomes are associated with five ubiquitin ligases.
• The proteasome subunit Rpn13 is selectively poly-ubiquitinated by Ube3c/Hul5 upon proteolysis inhibition or proteotoxic stress.
• Rpn13 ubiquitination impairs the proteasome's ability to bind and degrade ubiquitinated proteins, without affecting its activity on peptide substrates.

Conclusions:

• A novel autoinhibitory mechanism for the 26S proteasome is identified, involving Rpn13 ubiquitination.
• This mechanism likely prevents substrate binding to compromised proteasomes and serves as a biomarker for proteotoxic stress and diminished proteasomal capacity.