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Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples
Published on: May 10, 2015
COP9-associated CSN5 regulates exosomal protein deubiquitination and sorting
Yuelong Liu1, Spandan V Shah, Xiaoyu Xiang
1Division of Clinical Immunology and Rheumatology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
The American Journal of Pathology
|February 28, 2009
Summary
The COP9 signalosome (CSN)-associated protein CSN5 regulates the sorting of proteins into exosomes. CSN5 influences exosomal protein levels and HIV-1 release, challenging current ubiquitin-dependent sorting models.
Area of Science:
- Cell Biology
- Molecular Biology
- Protein Trafficking
Background:
- Proteins in multivesicular bodies are sorted for degradation or exosome secretion.
- Mechanisms governing cellular cargo sorting into exosomes remain largely unknown.
- Ubiquitination is a key factor in protein sorting pathways.
Purpose of the Study:
- To investigate the role of COP9 signalosome (CSN)-associated protein CSN5 in exosomal protein sorting.
- To determine if CSN5 influences the ubiquitination status of exosomal proteins.
- To explore the impact of CSN5 on HIV Gag sorting and HIV-1 release.
Main Methods:
- Western blot analysis of exosomal proteins.
- Small interfering (si)RNA knockdown of CSN5 in 293 cells.
- Transfection of 293 cells with JAB1/MPN/Mov34 metalloenzyme domain-deleted CSN5.
Main Results:
- CSN5 knockdown increased ubiquitinated and non-ubiquitinated exosomal proteins, including heat shock protein 70.
- CSN5 lacking the JAB1/MPN/Mov34 metalloenzyme domain increased ubiquitinated heat shock protein 70 in exosomes.
- Loss of CSN5's deubiquitinating activity enhanced HIV Gag sorting and HIV-1 release.
Conclusions:
- CSN5 quantitatively regulates proteins sorted into exosomes.
- CSN5 influences exosomal protein sorting via both deubiquitinating activity-dependent and -independent mechanisms.
- This finding contrasts with the prevailing model of ubiquitin-dependent exosomal sorting.
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