Exploring the Dynamics of the Proteasome Interaction Network by Quantitative Proteomics

Abstract

Most cellular processes in a cell are mediated by large protein assemblies or multi‐protein complexes, not by individual proteins. The protein complexes resemble molecular machines with highly ordered dynamic structures that assemble, store and transduce biological information. Systematic identification and characterization of these macromolecular machines will provide the essential knowledge base and set the stage for linking proteome dynamics and architecture to cellular functions. The 26S proteasome is a macromolecular machine existing in all eukaryotes and is responsible for ubiquitin/ATP dependent protein degradation in both cytosol and nucleus. It consists of two subcomplexes, the 20S core particle (CP) and the 19S regulatory complex (RP). The 20S CP is responsible for various catalytic activities, while the 19S RP is involved in several biochemical functions including recognition and unfolding of polyubiquitinated substrates, assisting in opening the gate of the 20S chamber and subsequently translocating unfolded substrates into the 20S for degradation. Selective degradation in cells is tightly controlled and plays an important role for regulating cell cycle progression, signal transduction and maintaining genome stability, etc. Disruption of normal proteolytic destruction pathways can lead to a wide range of human disease, including cancer and neurodegenerative disorders. Despite intensive research, the unknown still exceeds what are currently known on ubiquitin/proteasome dependent degradation. In order to understand the regulation of the 26S proteasome function, we have developed an integrated proteomic approaches to comprehensively characterize the 26S proteasome complexes. In addition, the QTAX strategy, quantitative identification of tandem‐affinity purified cross‐linked (x) protein complexes was developed to capture protein interaction of all natures in living cells, which has been successfully applied to map the dynamic interaction network of the 26S proteasome under various physiological conditions. Coupled with protein interaction network analysis, validity of the identified proteasome interactions have been confirmed and the biological significance of the identified interactions has been suggested. The methodologies presented here can be applied for the study of other protein complexes and their interaction networks.