Systematic identification of 20S proteasome substrates

For years, proteasomal degradation was predominantly attributed to the ubiquitin-26S proteasome pathway. However, it is now evident that the core 20S proteasome can independently target proteins for degradation. With approximately half of the cellular proteasomes comprising free 20S complexes, this degradation mechanism is not rare. Identifying 20S-specific substrates is challenging due to the dual-targeting of some proteins to either 20S or 26S proteasomes and the non-specificity of proteasome inhibitors. Consequently, knowledge of 20S proteasome substrates relies on limited hypothesis-driven studies. To comprehensively explore 20S proteasome substrates, we employed advanced mass spectrometry, along with biochemical and cellular analyses. This systematic approach revealed hundreds of 20S proteasome substrates, including proteins undergoing specific N- or C-terminal cleavage, possibly for regulation. Notably, these substrates were enriched in RNA- and DNA-binding proteins with intrinsically disordered regions, often found in the nucleus and stress granules. Under cellular stress, we observed reduced proteolytic activity in oxidized proteasomes, with oxidized protein substrates exhibiting higher structural disorder compared to unmodified proteins. Overall, our study illuminates the nature of 20S substrates, offering crucial insights into 20S proteasome biology. A systematic investigation of the repertoire of protein substrates degraded by the 20S proteasome under basal and oxidized conditions reveals hundreds of substrates and differential functionality of na & iuml;ve and oxidized 20S proteasome complexes.20S proteasome substrates are primarily RNA- and DNA-binding proteins with intrinsically disordered regions. The 20S proteasome not only fully degrades substrates but also cleaves proteins at their N- and C-termini, indicating regulatory actions. Oxidized proteasomes show lower proteolytic activity in comparison to na & iuml;ve proteasomes. Oxidized protein substrates display higher structural disorder than na & iuml;ve substrates. A systematic investigation of the repertoire of protein substrates degraded by the 20S proteasome under basal and oxidized conditions reveals hundreds of substrates and differential functionality of na & iuml;ve and oxidized 20S proteasome complexes.