Journal of Proteomics & Bioinformatics
Open Access
ISSN: 0974-276X
ISSN: 0974-276X
Jeroen A A Demmers
Erasmus University Medical Center, The Netherlands
Posters-Accepted Abstracts: J Proteomics Bioinform
The 26S proteasome is a 2.5 MDa protein complex, which degrades unneeded and damaged proteins in the cell. As such, it is critical in regulating proteostasis and controls key regulator abundance levels. Malfunctioning of the ubiquitin��?proteasome system has been implicated in diseases such as cancer and neurodegenerative disorders. On the other hand, in cancer therapeutics the induction of apoptosis by proteasome inhibition using drugs is widely used. Current strategies are directed towards the development of more selective inhibitors that target the proteasome regulatory subcomplex and have less side-effects. We take a proteomics approach to dissect the molecular mechanisms of the proteasome regulatory subcomplex, which is essential for the development of better proteasome inhibitors. Targeted proteasome inactivation by selective RNAi knockdown or drugs is monitored at the proteome and ubiquitinome levels using a SILAC approach in Drosophila. Over 5,000 proteins and 10,000 diGly peptides were identified and quantified. After brief inactivation by drugs, proteins involved in stress response, cell cycle regulation, apoptosis and the UPS were upregulated (e.g., Hsp proteins) and accumulated. After prolonged inactivation, the abundances of several 100s of proteins were altered. Similar effects were observed after inactivation of the proteasome with RNAi knockdown of different subunits. Protein ubiquitination dramatically increased upon proteasome inactivation. Interestingly, many proteins showed dynamic ubiquitination changes in opposite directions on different target lysine residues within the same protein. Proteomic analysis of individual RNAi knockdown of three proteasome bound deubiquitinating enzymes indicated that each of them has a different and specific function. Finally, proteasome interactome profiling under different experimental conditions using LFQ based quantitation suggested that the proteasome itself is a dynamic complex that recruits different partners and/or (sub)complexes under specific conditions. Global analysis of the dynamic proteome and ubiquitinome after proteasome inactivation gives detailed insight into regulatory mechanisms of the proteasome.
Email: j.demmers@erasmusmc.nl