The highly abundant ~2 MDa 26S proteasome is the proteolytic arm of the UPS. It is made of two sub-complexes, the 19S regulatory particle (RP) and the 20S catalytic particle (CP), and in many cases two RPs cap either end of a CP. The CP is made of two β rings that contain the catalytic sites, each is made of seven subunits (β1-7
) flanked on both sides by two α-rings, also made of seven subunits each. Thus, the structure of the 20S CP is α1-7
. The RP includes a ‘base’ and a ‘lid’. The base is composed of a hexameric ring of ATPases that function to unfold the substrate and open the gate of the interlacing N-terminal segments of the α subunits, thus allowing entry of the unfolded substrate into the catalytic chamber. The lid is involved mostly in specific recognition of the ubiquitin signal (reviewed in REF. 88
) (for structure of the 26S complex see ). Because of its complex structure, numerous targets, and the need for rapid adaptation to various pathophysiological conditions, this multi-catalytic enzyme complex is stable and not regulated by degradation. Rather, it is primarily regulated by compositional variation.
The 26S proteasome and its regulation by degradation
Some of the integral 20S proteolytic subunits can be replaced in an inducible and tissue-specific manner that alters proteolytic specificities and adapts it to changing needs, most notably immune challenges. In addition to the 19S cap, other proteins and complexes, such as PA28, bind to the end of the 20S cylinder and activate it by opening the ‘gate’. Furthermore, proteasome-associated DUBs and E3s can remodel substrate-anchored polyubiquitin chains, which may modulate their susceptibility for degradation. Other proteins, such as the chaperone Ecm29, stabilize the association between different sub-complexes of the 26S proteasome (reviewed in REF. 88
). Consistent with its longevity, the proteasome appears to be degraded by the lysosome, probably through microautophagy 89
Recent studies reported the specific ubiquitylation of distinct subunits of the proteasome; however, these modifications appear to serve non-proteolytic functions. Monoubiquitylation of RPN10 regulates the ability of this subunit to bind substrates by sterically inhibiting its UIM (ubiquitin-interacting motif) 90
. Ubiquitylation by the RING finger E3 Not4 is essential to the integrity and function of the 26S proteasome, probably by affecting the function of Ecm29, such that, in the absence of the ligase, Ecm29 is ubiquitylated and degraded. A ubiquitylation target(s) of Not4 has not been identified; thus, the underlying mechanism of its action is unknown91
. It is possible that Not4 targets the Ecm29 ligase, and in the absence of Not4, the ligase targets Ecm29. Cyclin- dependent kinase-associated protein 1 (Cks1) plays a role in transcriptional activation that is independent from its role in regulating the cell cycle 92
. This requires the Cks1 ubiquitin-binding domain, which allows it to bind to the proteasome via its ubiquitylated subunits. Cks1 can probably bind to other ubiquitylated complexes, thus displaying a broad array of transcription-regulating activities.
Several studies suggest that selective degradation of critical components of the 26S proteasome, or induced dissociation of sub-complexes, are involved in the regulation of its activity. Treatment of hippocampal neurons with the neurotransmitter NMDA (N-Methyl-D-Aspartate) leads to dissociation of the 26S to the 19S cap and the 20S core, and to proteasomal degradation of the 19S (REF. 93
). The mechanism that underlies the NMDA effect is not known. It is possible that since there is also a decrease in ubiquitin conjugates following NMDA treatment (see below), the effect on the proteasome is indirect, and proteasome levels decrease when there are fewer substrates to degrade. Interestingly, it was reported that binding of polyubiquitylated substrates to the 19S RP activates proteasomal activity. This probably occurs by inducing conformational changes in the 20S CP that stabilize the gate opening of the α subunits and thereby facilitate channeling of substrates into the 20S and their access to its active sites. Although it has not been shown experimentally, this crosstalk between the 19S RP and 20S CP, and the stabilization of protrusion of the N-termini of the α subunits into the 19S RP, may contribute to the strengthening and stabilization of the association between the two sub-complexes 94,95
. Also, since the proteasome is involved in the endocytosis of glutamate receptor (for which NMDA is a ligand), the effect of NMDA on the proteasome may serve to potentiate the excitatory influence of the transmitter by inhibiting receptor endocytosis and subsequent degradation. It is assumed that the 19S dissociates into individual subunits prior to its degradation, although evidence for their ubiquitylation is lacking. Along with the 19S, two of its associated E3s, E6-AP and HUWE1 (HECT, UBA, and WWE domain-containing protein 1), are also degraded in response to NMDA93
. It is possible that the destruction of the proteasome-associated ligases suppresses conjugation and degradation, and stabilizes a subset of proteins required for synaptic activity during NMDA excitation.
In another study it was reported that activation of apoptosis results in caspase-mediated cleavage of the proteasomal subunits S6’ (Rpt5), S5a (Rpn10), and S2 (Rpn1), resulting in proteasome inactivation96
. As a result, pro-apoptotic proteins, such as Smac that are targeted by the UPS are stabilized, which is assumed to facilitate the execution of the apoptotic program. Interestingly, in myotubes, caspase-3-mediated cleavage of Rpt2 and Rpt6 increases proteasomal activity. This appears to be a specific feed-forward mechanism that accelerates proteolysis in muscle during catabolic states97
. Oxidative stress has been shown to induce disassembly of the proteasome to its sub-complexes98
. It was suggested that this dissociation protects cells by enabling the released 20S CP to degrade the oxidized proteins that are generated under these conditions, which bypasses the need for ubiquitylation, as the 20S can degrade proteins in a ubiquitin-independent manner in vitro
. It is unclear, however, whether the 20S can degrade cellular substrates in vivo
, as several strong lines of evidence suggest that even unfolded, oxidized and otherwise damaged proteins are degraded in the cell via a ubiquitin-dependent mechanism. Thus, in one study it was demonstrated that degradation of damaged cellular proteins exposed to heat, cadmium or paraquat, required the E2s Ubc4 and Ubc5, the proteasomal subunit RPN10, and the CDC48-UfD1-NPL4 complex99
. Also, the absolute requirement for ATP for all types of protein degradation suggests a need for ubiquitylation, which is ATP-dependent, and/or the 26S complex, which requires ATP for its assembly and function. By contrast, degradation by the 20S CP is energy-independent. An independent study that supports the notion that the 20S proteasome is inactive in cells was described in yeast, when during the stationary phase, the 26S proteasome similarly dissociates100
. In this case, the released 20S is inactive, as the N-termini of the α chains at either end of the CP remain interlaced, thus the entry gate to the CP is closed. This study suggests that dissociation is essential to slow proteolysis to maintain viability during nutrient shortage in the stationary phase. Regulation of the 26S proteasome by association-dissociation of its sub-complexes and degradation of its different subunits is described in .