At this point, the only missing link was the identification of the downstream protease that would specifically recognize ubiquitinated substrates. Tanaka and colleagues identified a second ATP-requiring step in the reticulocyte proteolytic system, which occurred after ubiquitin conjugation,65 and Hershko and colleagues demonstrated that the energy was required for conjugate degradation.66 An important advance in the field was a discovery by Hough and colleagues, who partially purified and characterized a high-molecular-mass alkaline protease that Inhibitors,research,lifescience,medical degraded ubiquitin adducts of lysozyme, but not untagged lysozyme, in an ATP-dependent
mode.67 This protease, which was later called the 26S proteasome (see below), provided all the necessary criteria for being the specific proteolytic arm of the ubiquitin system. This finding was confirmed, and the protease was further characterized by Waxman and colleagues who found that it Inhibitors,research,lifescience,medical was an unusually large, ~1.5 MDa, enzyme, unlike any other known protease.68 A further advance in the field was the discovery69 that a smaller neutral multi-subunit 20S protease complex that was discovered together with the larger 26S complex was similar to a “multicatalytic proteinase complex” Inhibitors,research,lifescience,medical (MCP) that had been described earlier in bovine pituitary gland by Wilk and Orlowski.70 This 20S protease was ATP-independent
and has different catalytic activities, cleaving on the carboxy-terminal side of hydrophobic, basic, and acidic residues. Hough and colleagues raised the possibility—although they did not show it experimentally—that this 20S protease could be a part of the larger 26S protease that degrades
the ubiquitin adducts.69 Later studies showed that, indeed, the 20S complex is the core catalytic Inhibitors,research,lifescience,medical particle of the larger 26S complex.71,72 However, strong evidence that the active “AZD6244 mw mushroom”-shaped 26S protease was generated through the assembly of two distinct Inhibitors,research,lifescience,medical subcomplexes—the catalytic 20S cylinder-like MCP and an additional 19S ball-shaped subcomplex (that was predicted to have a regulatory role)—was provided only in the early 1990s by Hoffman and colleagues73 who mixed the two purified particles and generated the active 26S enzyme. The proteasome is a large, 26S, multicatalytic protease that degrades polyubiquitinated proteins to small peptides. It is composed of two subcomplexes: a 20S core particle (CP), that carries the catalytic activity, and a 19S regulatory particle (RP). The 20S CP is a barrel-shaped structure composed of heptaminol four stacked rings, two identical outer β rings and two identical inner β rings. The eukaryotic α and β rings are composed each of seven distinct subunits, giving the 20S complex the general structure of α1–7β1–7β1–7α1-7. The catalytic sites are localized to some of the β subunits. Each extremity of the 20S barrel can be capped by a 19S RP each composed of 17 distinct subunits, 9 in a “base” subcomplex and 8 in a “lid” subcomplex.