Abstract
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.
Highlights
The proteasome, the primary protease for ubiquitin-dependent proteolysis in eukaryotes, is usually found as a mixture of 30S, 26S, and 20S complexes
The 26S proteasome holoenzyme consists of a 19S regulatory particle (RP; a.k.a. proteasome activator 700), which is responsible for recognizing the ubiquitin signal and unfolding the target protein, and a 20S core particle (CP), which hydrolyzes the unfolded polypeptide into short peptides of varying lengths[6,7,8,9]
We addressed the possibility of a branched peptide entering into the proteolytic chamber, by adopting the MSFragger tool to search for variable ubiquitin remnants on proteasome-digested cyclin B1 (Supplementary Data 6)
Summary
The proteasome, the primary protease for ubiquitin-dependent proteolysis in eukaryotes, is usually found as a mixture of 30S, 26S, and 20S complexes. It has been suggested that the 20S complex plays a role under stress conditions that correlates with a greater need for removing damaged/misfolded proteins. It is unclear, how this role is carried out, as the 20S complex has no associated ATPase domain or dedicated ubiquitin receptors. Some archaea and prokaryotes, which lack ubiquitin, have free 20S complexes alongside other ATP-dependent proteases This suggests that the 20S is a primordial proteindegrading machine, which is possibly aided by loosely associated ATPase activators[24,25]. We find, using proteasome-trapped peptides (PTP), that some of these substrates may be proteolyzed along with the conjugated ubiquitin tag
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