Activity-Guided Proteomic Profiling of Proteasomes Uncovers a Variety of Active (and Inactive) Proteasome Species.

Proteasomes are multisubunit, multicatalytic protein complexes present in eukaryotic cells that degrade misfolded, damaged, or unstructured proteins. In this study, we used an activity-guided proteomic methodology based on a fluorogenic peptide substrate to characterize the composition of proteasome complexes in WT yeast and the changes these complexes undergo upon the deletion of Pre9 (Δα3) or of Sem1 (ΔSem1). A comparison of whole-cell proteomic analysis to activity-guided proteasome profiling indicates that the amounts of proteasomal proteins and proteasome interacting proteins in the assembled active proteasomes differ significantly from their total amounts in the cell as a whole. Using this activity-guided profiling approach, we characterized the changes in the abundance of subunits of various active proteasome species in different strains, quantified the relative abundance of active proteasomes across these strains, and charted the overall distribution of different proteasome species within each strain. The distributions obtained by our mass spectrometry-based quantification were markedly higher for some proteasome species than those obtained by activity-based quantification alone, suggesting that the activity of some of these species is impaired. The impaired activity appeared mostly among 20SBlm10 proteasome species which account for 20% of the active proteasomes in WT. To identify the factors behind this impaired activity, we mapped and quantified known proteasome-interacting proteins. Our results suggested that some of the reduced activity might be due to the association of the proteasome inhibitor Fub1. Additionally, we provide novel evidence for the presence of nonmature and therefore inactive proteasomal protease subunits ß2 and ß5 in the fully assembled proteasomes.

Isolation of Proteasome-Trapped Peptides (PTPs) for Degradome Analysis.

Analyzing intracellular peptides generated by proteasomes is highly informative to understand the spatiotemporal regulation of protein homeostasis. A large portion of eukaryotic proteins is proteolyzed within the 20S core particle of the 26S holoenzyme, where proteins are cleaved into peptides of varying lengths. A small percentage of these peptides are presented to the immune system as a representation of the proteome content of the cell. Therefore, understanding the rules that govern proteolytic specificity and product diversity is of relevance not only to biochemistry and proteostasis but also to physiology and immunology. One of the greatest challenges is to separate such proteasome-generated peptides from the total intracellular peptidome due to the susceptibility of short unstructured peptides to myriad proteases and peptidases that are activated upon cell lysis. Here, we describe a simple and rapid method to isolate peptides that are closely associated with proteasomes or trapped inside the core particle of proteasomes in eukaryotic cells. This approach termed PTPs, for proteasome-trapped peptides, requires a limited number of cells as starting materials compared to other published methods yet still provides sufficient yields for mass spectrometry-based proteomic analysis. A single sample obtained from cultured mammalian cells allowed the identification of 1000-2000 different PTPs following LC-MS analysis with high-resolution mass spectrometer.

The 20S as a stand-alone proteasome in cells can degrade the ubiquitin tag.

The proteasome, the primary protease for ubiquitin-dependent proteolysis in eukaryotes, is usually found as a mixture of 30S, 26S, and 20S complexes. These complexes have common catalytic sites, which makes it challenging to determine their distinctive roles in intracellular proteolysis. Here, we chemically synthesize a panel of homogenous ubiquitinated proteins, and use them to compare 20S and 26S proteasomes with respect to substrate selection and peptide-product generation. We show that 20S proteasomes can degrade the ubiquitin tag along with the conjugated substrate. Ubiquitin remnants on branched peptide products identified by LC-MS/MS, and flexibility in the 20S gate observed by cryo-EM, reflect the ability of the 20S proteasome to proteolyze an isopeptide-linked ubiquitin-conjugate. Peptidomics identifies proteasome-trapped ubiquitin-derived peptides and peptides of potential 20S substrates in Hi20S cells, hypoxic cells, and human failing-heart. Moreover, elevated levels of 20S proteasomes appear to contribute to cell survival under stress associated with damaged proteins.