All eukaryotic cells recycle proteins (functional as well as damaged) continuously for survival. The ubiquitin proteasome system (UPS) is the major proteolytic pathway for the removal of cytosolic, nuclear, and many membrane-associated proteins. Target proteins are selected by tagging them covalently with ubiquitin, typically with lysine48-linked tetraubiquitin chains, followed by proteolysis within the 26S proteasome. 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. To do so, the 19S RP utilizes three ubiquitin receptors (PSMD2/Rpn1, PSMD4/Rpn10, and ADRM1/Rpn13), several deubiquitinases that remove the ubiquitin signal, and a hexameric ring of AAA ATPases (RPT1-6 subunits) that unfolds the substrate and translocates it to the barrel-shaped (α 7 β 7 β 7 α 7) 20S CP. After traversing the channel of the ATPase ring, unfolded substrates pass through the α-ring at the outer surface of the 20S CP, enter the antechamber, and cross yet another aperture (the β-annulus) to access the proteolytic chamber of the 20S CP. Three of the seven β subunits in each of the two β-rings that align this proteolytic chamber (β1, β2, and β5) are proteolytically active, and between them hydrolyze most substrate peptide bonds to generate a variety of short peptide products.
Physiological conditions that require broad changes to the proteome necessitate a greater proteolytic capacity to remove the unnecessary proteins. In addition, common stressors such as oxidation, temperature, ionization, or toxins cause protein damage as well as damage to the ubiquitin–proteasome machinery. Thus, such stressors cause a need for alternative pathways for removing damaged proteins. Interestingly, the 20S CP is relatively resistant to oxidation damage compared to the 26S holoenzyme and persists as a stable complex under many stress conditions. Hence, 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, however, how this role is carried out, as the 20S complex has no associated ATPase domain or dedicated ubiquitin receptors.
Although the 20S subcomplex is an integral part of the 26S holoenzyme, it is also quite abundant as a free complex in many cell types. Free 20S complexes have been suggested to be proteasome assembly intermediates, 26S breakdown products (due to disassembly), or stand-alone proteolytic enzymes. For instance, some archaea and prokaryotes, which lack ubiquitin, have free 20S complexes alongside other ATP-dependent proteases.This suggests that the 20S is a primordial protein-degrading machine, which is possibly aided by loosely associated ATPase activators. Some reports suggest that the 20S complex functions independently, even in eukaryotes, by acting directly on disordered or oxidized/damaged proteins. In eukaryotic cells, the 20S may be augmented by non-ATPase activators such as 11Sreg/PA28 or PA200 in addition to the more abundant ATPase-containing 19S. Attachment of proteasome activators influences substrate selection and may also affect product outcome due to allosteric effects on β-catalytic active sites. Thus, changes in the cellular ratio between the 20S and the 26S proteasomes may be part of an adaptive response to meet cellular needs. However, without an associated unfoldase activity, in vitro 20S CP proteolyzes only unstructured proteins in a ubiquitin-independent manner.
Since the two proteasome species (26S and 20S) have the same catalytic active sites, understanding their distinctive roles has been challenging. To this end, we chemically synthesized a panel of polyubiquitinated conjugates that are potential substrates for either enzyme. Since the location of the ubiquitin tag on the substrate could influence its binding efficiency to the 26S proteasomes and possibly even the proteolytic outcome, we attached lysine48-linked ubiquitin/ubiquitin chains to a natural (i.e., published) site on a known substrate for ubiquitin-dependent 26S proteasome degradation, cyclin B1. Here, we describe the signature activities of the 20S proteasome that could be used to study its function in vivo. For instance, we find that under hypoxia, the 20S proteasome aids clearance of damaged proteins and improves cell viability. We also find, using proteasome-trapped peptides (PTP), that some of these substrates may be proteolyzed along with the conjugated ubiquitin tag.
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