UBL domain of Usp14 and other proteins stimulates proteasome activities and protein degradation in cells

Contributed by Alfred L. Goldberg, October 30, 2018 (sent for review May 30, 2018; reviewed by George DeMartino and Andreas Martin)
November 28, 2018
115 (50) E11642-E11650


26S proteasomes catalyze most of the protein degradation in eukaryotic cells, and their activity is precisely regulated. Upon binding ubiquitinated proteins, proteasomes become activated. This activation is triggered by binding of the ubiquitin chain to the proteasome-associated deubiquitinating enzyme Usp14/Ubp6. In studying this activation mechanism, we discovered that Usp14’s ubiquitin-like (UBL) domain by itself stimulates multiple proteasome activities and thus appears to mediate the activation by ubiquitinated proteins. Many other proteins contain UBL domains, and we show that the UBL domains of other proteins also stimulate the proteasomes’ degradative capacity. Thus, activation of proteasomes may represent a general new role for UBL-containing proteins. Furthermore, overexpressing the UBL domain in cells increases overall protein breakdown, which may have therapeutic applications.


The best-known function of ubiquitin-like (UBL) domains in proteins is to enable their binding to 26S proteasomes. The proteasome-associated deubiquitinating enzyme Usp14/UBP6 contains an N-terminal UBL domain and is an important regulator of proteasomal activity. Usp14 by itself represses multiple proteasomal activities but, upon binding a ubiquitin chain, Usp14 stimulates these activities and promotes ubiquitin-conjugate degradation. Here, we demonstrate that Usp14’s UBL domain alone mimics this activation of proteasomes by ubiquitin chains. Addition of this UBL domain to purified 26S proteasomes stimulated the same activities inhibited by Usp14: peptide entry and hydrolysis, protein-dependent ATP hydrolysis, deubiquitination by Rpn11, and the degradation of ubiquitinated and nonubiquitinated proteins. Thus, the binding of Usp14’s UBL (apparently to Rpn1’s T2 site) seems to mediate the activation of proteasomes by ubiquitinated substrates. However, the stimulation of these various activities was greater in proteasomes lacking Usp14 than in wild-type particles and thus is a general response that does not involve some displacement of Usp14. Furthermore, the UBL domains from hHR23 and hPLIC1/UBQLN1 also stimulated peptide hydrolysis, and the expression of hHR23A’s UBL domain in HeLa cells stimulated overall protein degradation. Therefore, many UBL-containing proteins that bind to proteasomes may also enhance allosterically its proteolytic activity.
In mammalian cells, 26S proteasomes are the major site for protein degradation (1). Most proteins digested by proteasomes are first tagged with ubiquitin (Ub) chains. The 26S proteasome is composed of the 20S proteolytic core particle and one or two 19S regulatory particles (PA700) (2). The cylindrical 20S proteasome is a hollow four-ring particle that contains in its two central β-rings six proteolytic sites, two of which are chymotrypsin-like, two trypsin-like, and two caspase-like in specificity (3). Its outer α-rings contain a central gated channel for substrate entry (4). The 19S regulatory particle performs several enzymatic and nonenzymatic functions required for the degradation of ubiquitinated proteins, including binding and disassembly of the Ub chain, ATP hydrolysis, and unfolding of the substrate protein (2). The base of the 19S regulatory particle contains a ring of six homologous AAA ATPases (5, 6). The ATPases bind, unfold, and translocate the polypeptide through their central channel and then through a gated entry channel into the 20S particle (7, 8). Many steps in this degradation process are linked to ordered cycles of ATP binding and hydrolysis (2).
Three different deubiquitinating enzymes (DUBs) are associated with the 19S regulatory particle of higher eukaryotes: Two are cysteine proteases, Usp14/Ubp6 (9) and Uch37/UchL5 (10, 11), and the third, Rpn11/PSMD14, is a metalloprotease (12, 13). Usp14 contains an N-terminal UBL domain and a C-terminal catalytic USP (ubiquitin-specific protease) domain (14). The deubiquitinating activity of Usp14 is dramatically stimulated upon its association with the 19S particle, which requires the UBL domain (15, 16). Rpn11 removes the Ub chain en bloc in an ATP-dependent manner and is essential for the degradation of ubiquitinated proteins (17). Thus, Rpn11 facilitates the translocation of the polypeptide through the ATPase ring into the 20S particle (12, 18), whereas the other DUBs are thought to restrict the time substrates bind to the proteasome before processing (2, 8).
Efficient degradation of a Ub conjugate requires a tightly integrated series of steps (2, 19) whose order and coordination are still poorly understood. After a Ub chain reversibly binds to the receptor subunits, the polypeptide, if it exposes a loosely folded domain, becomes tightly bound to the particle through an ATP-dependent reaction that commits the substrate to degradation (20). Surprisingly, the binding of a Ub conjugate to Usp14 or Uch37 activates the entry of small peptides through the ATPase ring and the gated channel into the 20S (21). If the ubiquitinated protein also contains a loosely folded domain, ATP hydrolysis is also activated (19).
This role of Usp14 in proteasome activation by Ub conjugates was surprising, because Usp14/Ubp6 was previously shown to inhibit the degradation of ubiquitinated proteins both through its DUB activity and by an allosteric mechanism (22, 23). Because the rapid trimming of Ub chains by Usp14 can lead to substrate release without its degradation, Finley, King, and coworkers (16, 24) showed that inhibitors of Usp14’s catalytic activity (IU1) can enhance the proteasomal degradation of certain proteins by slowing substrate deubiquitination. We recently demonstrated that in the absence of ubiquitinated substrates, Usp14 allosterically inhibits multiple proteasomal activities, including peptide entry and hydrolysis, ATP hydrolysis, and, surprisingly, deubiquitination by Rpn11 (25). These inhibitory actions of Usp14 in the absence of a substrate may help reduce wasteful ATP consumption and the degradation of nonubiquitinated proteins (25).
Recent cryoelectron microscopic studies have revealed that the binding of Usp14 or its yeast homolog Ubp6 to the 19S complex induces marked changes in 19S structure (2628). Upon binding the transition-state inhibitor Ub-aldehyde to the active site of Usp14/Ubp6, its C-terminal USP domain relocates near the ATPase ring and Rpn11. However, without the binding of Ub-aldehyde or a Ub chain, this domain is more dynamic, and fails to interact with the ATPases (26, 27). Presumably, these conformational changes provide a mechanistic explanation of Usp14’s opposing allosteric actions, both its basal inhibition of the proteasome and its activation upon substrate binding (19, 21, 25).
Although several roles have been suggested for the UBL domains (2931) present in many cell proteins, the best-documented function for the UBL domain is to provide affinity for the 26S proteasome, where the UBL domain can bind to the Ub receptor subunits Rpn10, Rpn13, and Rpn1 (3234). Among the many functions served by UBL-containing proteins is acting as shuttling factors (i.e., the UBL-UBA proteins), which bring Ub-conjugated proteins to the proteasome, as Ub ligases (e.g., Parkin) or as proteasomal DUBs (3537). Because of the importance of Usp14’s UBL domain in its association with the proteasome and in the resulting stimulation of Usp14’s deubiquitinating activity, we hypothesized that its UBL domain might also be important in mediating the activation of the proteasome upon substrate binding to Usp14. Therefore, we have investigated whether upon binding to the 26S proteasome, Usp14’s UBL domain by itself activates these various proteasomal activities. We demonstrate here that Usp14’s UBL domain allosterically activates the same proteasomal functions that are activated upon Ub-chain binding to the proteasome and thus enhances protein degradation by the UPS. These findings imply that Usp14’s UBL domain mediates the activation by Ub conjugates. However, because an even stronger activation was found in particles lacking Usp14, and because the UBL domains of other proteins were also shown to stimulate 26S activity, these findings also strongly suggest a new general role for UBL domains: that they not only promote the binding of proteins to the proteasome, but also activate the proteasome for protein degradation.


The UBL Domain of Usp14 Enhances Peptide Hydrolysis by 26S Proteasomes.

To dissect the roles of Usp14’s UBL and USP domains in the regulation of proteasome function, we tested whether binding of the UBL domain alone can influence its several enzymatic activities. When the purified GST-tagged UBL domain from Usp14 (residues 2 to 79) was incubated in the presence of ATP with 26S proteasomes purified from mouse embryonic fibroblasts (MEFs) lacking Usp14 (Usp14KO), it stimulated the proteasomes’ chymotrypsin-like activity in a similar fashion to a ubiquitin chain (hexaubiquitin) in the presence of Usp14. The hexa-Ub caused a larger stimulation with the enzymatically inactive C114A mutant Usp14 than with WT enzyme. By contrast, adding either WT Usp14 or the inactive C114A mutant without a substrate inhibited 26S peptidase activity, as reported previously (25). Thus, these regulatory effects are not related to Usp14’s DUB activity. Unlike the UBL domain, hexa-Ub then had only little effect in the absence of Usp14, and this effect was probably through its binding to Uch37 (19) (Fig. 1A).
Fig. 1.
By binding to 26S proteasomes, Usp14’s UBL domain stimulates peptide cleavage by all three peptidase sites, especially in proteasomes lacking Usp14. Activities of WT and Usp14KO 26S were assayed by measuring cleavage of the fluorogenic peptide-amc substrates (10 μM) in the presence of ATP or ATPγS (100 μM). Control (100%) indicates the peptidase activity of each type of proteasome (1 nM) without added proteins. (A) Addition of GST-UBL from Usp14 (200 nM) by itself stimulates the chymotrypsin-like activity of 26S similar to addition of Usp14 (1 μM) plus a linear hexa-Ub chain (200 nM). Addition of WT Usp14 or the enzymatically inactive C114A mutant by themselves inhibited, but together with hexa-Ub stimulated, peptidase activity, especially with the inactive Usp14 mutant. LLVY-amc: Suc-Leu-Leu-Val-Tyr-amc as the substrate for the chymotrypsin-like activity. *P < 0.05 and **P < 0.01 by Student’s t test. Data are the means ± SD; n = 4. (B) The UBL of Usp14 stimulates the chymotrypsin-like activity of both WT and Usp14KO 26S. The UBL seems to bind with similar affinities (∼100 nM) to both, but causes a much larger stimulation of the Usp14KO 26S. Data are the means ± SD; n = 5. (C) All three peptidase activities are stimulated more by the UBL (200 nM) in the Usp14KO 26S than in the WT with ATP present. LRR-amc: Boc-Leu-Arg-Arg-amc as the substrate for the trypsin-like activity; nLPnLD-amc: Ac-Nle-Pro-Nle-Asp-amc as the substrate for the caspase-like activity. *P < 0.05 and **P < 0.01 by Student’s t test. Data are the means ± SD; n = 6. (D) In the presence of ATPγS, the UBL domain further enhanced (five- to eightfold) the three peptidase activities of Usp14KO 26S. Peptide hydrolysis by WT 26S was also stimulated, but to a smaller extent. *P < 0.05 and **P < 0.01 by Student’s t test. Data are the means ± SD; n = 6. (E) Full-length Usp14 inhibits the activation of peptide hydrolysis by UBL. Addition of increasing concentrations of WT Usp14 or the inactive C114A mutant decreased the chymotrypsin-like activity of 26S proteasomes in the presence of UBL and ATPγS. Usp14KO proteasomes (1 nM) were preincubated with the UBL (100 nM), and different concentrations of Usp14 were then added to the mixture. Data are the means ± SD; n = 4. (F) Full-length Usp14 competes with the GST-UBL for binding to the 26S proteasome. Usp14KO 26S bound to UBL on resin was eluted by adding increasing concentrations of Usp14 (55). Proteasomes eluted were quantitated using LLVY-amc hydrolysis (20). Data are represented as means ± SD; n = 6. (G) The UBL domain from Usp14 stimulates the chymotrypsin-like activity of yeast WT and T1 mutant (ARR) 26S but not of the T2 mutant (AKAA) (32). Activities of the resin-bound yeast 26S were measured after adding the UBL derived from mammalian Usp14 (200 nM). Data are the means ± SD; n = 3.
This stimulation by the UBL domain increased with higher concentration and was consistently about twofold larger in Usp14KO proteasomes than in WT 26S (Fig. 1B). The greater enhancement of 26S activity in the absence of Usp14 contrasts with the activation by Ub conjugates, which in yeast 26S requires Ubp6 (21) and in mammalian proteasomes requires Usp14 or Uch37 (Fig. 1A) (19). By itself, the purified UBL domain had no peptidase activity (SI Appendix, Fig. S1A) and did not stimulate peptide hydrolysis by 20S proteasomes without the 19S regulatory particle present (SI Appendix, Fig. S1B). These observations are not due to the linkage to GST, a dimeric protein, because a similar stimulation of the proteasomes’ chymotrypsin-like activity was observed upon addition of the monomeric His6-tagged UBL domain (SI Appendix, Fig. S1C). In addition, the UBL increased the trypsin-like and especially the caspase-like activities of the 26S, and these effects were also consistently larger in the Usp14KO 26S (Fig. 1 C and D). This enhanced hydrolysis by all three types of active sites is most likely due to greater substrate entry through the ATPase ring and gated entry channel into the 20S particle (i.e., “gate opening”), rather than by allosteric activation of the individual catalytic sites (4, 38).
This simultaneous large enhancement of all three peptidase activities resembles the stimulatory effects of PA28αβ and nucleotide binding, especially ATPγS, which enhances peptide entry into the 20S particle (39). However, the UBL domain stimulated peptide hydrolysis even further in the presence of ATPγS (Fig. 1D), which had been assumed to cause maximal gate opening and rates of peptide hydrolysis (SI Appendix, Fig. S1D). By contrast, the addition of full-length Usp14 to 26S proteasomes reduced the stimulation of peptide hydrolysis by ATPγS (25). The UBL domain in the presence of ATPγS surprisingly caused a much greater stimulation of the Usp14-deficient 26S than of the WT 26S (Fig. 1D). Thus, ATPγS and Usp14’s UBL domain appear to stimulate synergistically peptide hydrolysis by proteasomes, which implies that the stimulation by the UBL domain requires the conformation of the 19S particle induced by ATPγS (40).

Usp14’s UBL Domain Binds to the T2 Site of Rpn1 to Activate the 26S.

Because the Usp14 UBL had no effect on the peptidase activity of the 20S core particles (SI Appendix, Fig. S1B), its activating mechanism must differ from that of PA28 or that of the ATPase’s C-terminal HbYX motif, which binds to 20S’s α-ring (4, 41). More likely, the UBL stimulates by binding to the same site on Rpn1 as does full-length Usp14 (32, 42), and thus it does not directly affect the 20S gating mechanism. Accordingly, the stimulation of peptide hydrolysis was much smaller in the WT particles than in the 26S lacking Usp14, probably because in the WT particle the Usp14 and the free UBL bind to the same site, which reduces the stimulation by the UBL domain (Fig. 1 C and D). Accordingly, modification of Usp14 with the covalent inhibitor Ub-vinyl sulfone, which enhances its association with the 26S particle (27), partially inhibits the stimulation of the chymotrypsin-like activity by the added UBL (SI Appendix, Fig. S2). To determine if in activating peptide hydrolysis the UBL binds to the same site as is occupied by Usp14, we tested whether adding full-length Usp14 competitively inhibits the stimulation of gate opening by the UBL. As predicted, incubation of the Usp14KO 26S with increasing amounts of full-length WT Usp14 or its C114A active-site mutant together with the UBL domain reduced the stimulation of the chymotrypsin-like activity by the UBL (Fig. 1E). Furthermore, when Usp14KO proteasomes were first bound to a GST-UBL resin, they could be eluted by addition of increasing amounts of the full-length WT Usp14 or the inactive C114A mutant (Fig. 1F). This activation thus is not related to Usp14’s DUB activity, which is located in its USP domain. The findings that Usp14 competes with GST-UBL for binding to the proteasome and inhibits the stimulatory effect of UBL suggest that the binding of a Ub chain or Ub-aldehyde to Usp14 allosterically stimulates peptide entry (21) through its UBL domain, while its inhibitory USP domain prevents this stimulation by the UBL until a Ub chain binds to the USP domain.
Recently, it was shown that yeast Rpn1 has two separate Ub binding sites, T1 and T2 (32). T1 is the binding site for Ub and the UBL domain of Rad23, while T2 is the site for binding of Ubp6’s UBL domain (32) and therefore presumably mediates this activation. We therefore tested whether T1 or T2 may be important in the activation of peptide hydrolysis. Upon incubation with 26S proteasomes purified from yeast bearing mutations that inactivate the T1 or the T2 site (32), the UBL of Usp14 stimulated the chymotrypsin-like activity of purified 26S proteasomes of both WT (SY1214) and the T1-mutant strain (SY1210, rpn1-ARR), but not those from the T2-mutant strain (SY1724, rpn1-AKAA) (Fig. 1G). Although the absolute stimulation was much smaller in the yeast particles than mammalian 26S, presumably because of our use of a mammalian UBL domain, these findings indicate that binding to the T2 site, but not the T1 site, is required for this stimulation by Usp14’s UBL.

Usp14’s UBL Domain Together with a Loosely Folded Protein Enhances ATP Hydrolysis.

A general feature of hexameric AAA ATP-dependent proteases is that their ATPase activity is stimulated upon binding a loosely folded protein substrate (4345). However, the stimulation of the proteasomal ATPases requires the simultaneous binding of a Ub chain to Usp14 (or to Uch37) and an unstructured region of the polypeptide to the 19S ATPases, although the Ub chain and polypeptide need not be covalently linked (19). Thus, as we showed recently (25), Usp14 represses proteasomal ATP hydrolysis, but upon binding of a Ub chain to Usp14 the ATPases are activated, provided an unfolded polypeptide is also present (2).
Therefore, we tested whether the Usp14’s UBL domain can also stimulate the ATPase activity of the 26S as it stimulates peptidase activities. Although the UBL alone had no effect, the UBL with β-casein present stimulated ATP hydrolysis in MEF proteasomes (Fig. 2A) and did so to a similar extent as hexameric Ub (Fig. 2A). Thus, stimulation of the 19S ATPases by the UBL does not require Ub-chain binding but still requires the binding of a loosely folded protein, probably to ATPases (2). Thus, the UBL domain by itself mimics the ability of substrate-bound Usp14 to activate both peptide and ATP hydrolysis. These findings further suggest that the UBL domain mediates this activation by substrate, and therefore that Usp14’s catalytic (USP) domain is probably responsible for the basal inhibition of these activities until it binds a Ub chain and interacts with ATPases (2628). By contrast, in 26S lacking Usp14, the binding of casein alone fully stimulated ATP hydrolysis (25), and the addition of UBL had no further effect (Fig. 2A, Right). Interestingly, a similar stimulation of the ATPases by a nonubiquitinated polypeptide has been observed in reconstituted yeast proteasomes (27). Such behavior is clearly not seen in our preparations from WT mammalian cells (2, 19), where the presence of Usp14 makes the activation dependent on a ubiquitinated protein or a protein plus a ubiquitin chain.
Fig. 2.
UBL enhances ATP hydrolysis by WT proteasomes provided an unstructured protein (casein) is also present and stimulates the degradation of nonubiquitinated proteins. (A) Upon binding of casein (1 μM) and a hexa-Ub chain (1 μM), which together mimic the binding of Ub conjugates (20), ATP hydrolysis by WT and Usp14KO 26S (20 nM) increased about two- to threefold. The UBL (500 nM) plus casein also increased ATP hydrolysis in both to a similar extent as hexa-Ub plus casein. However, UBL or hexa-Ub alone did not increase ATP hydrolysis by either type of proteasome. However, in the Usp14KO 26S, in the absence of UBL or hexa-Ub chain, casein alone could increase ATP hydrolysis. ATP hydrolysis was measured using the malachite green assay (62). 6Ub: linear hexa-Ub. *P < 0.05 and **P < 0.01 by Student’s t test. Data are represented as means ± SD; n = 3. (B) The UBL domain, like a Ub chain, stimulates the degradation of an intrinsically unstructured protein (Sic1) by 26S proteasomes. Effects of UBL (500 nM) or linear hexa-Ub chain (1 μM) on the degradation of PY-Sic1 (100 nM) by WT 26S (2 nM) were assayed by Western blotting. Proteasomes were incubated with the UBL or Ub chain at room temperature for 15 min before the reaction started. (B, Left) At the indicated times, the remaining Sic1 was measured. IB, immunoblotting. (B, Right) Degradation of Sic1 was measured using ImageJ software and plotted. Similar data were obtained in three independent experiments.

Usp14’s UBL Domain Stimulates the Degradation of a Nonubiquitinated Unfolded Protein.

Because the Usp14 UBL domain stimulates gate opening and ATP hydrolysis, both of which are essential for efficient proteolysis (2), the UBL might also stimulate the proteasomal degradation of some nonubiquitinated proteins. In fact, the addition of either hexa-Ub chains or the UBL alone to WT 26S stimulated dramatically (up to 10-fold) the breakdown of the intrinsically disordered protein Sic1 (46) (Fig. 2B). However, in proteasomes lacking Usp14 (SI Appendix, Fig. S3A), the addition of the UBL did not stimulate further the degradation of Sic1, probably because this process is already activated in these particles (25), as is the rate of ATP hydrolysis (25), which determines the rate of protein degradation (47). Another noteworthy feature of the Usp14-deficient proteasomes is that the activation of ATP hydrolysis requires only the presence of a loosely folded protein and does not also require a Ub chain (Fig. 2A, Right). These findings provide further evidence that Usp14 inhibits Ub-independent proteolysis until binding of a Ub chain reverses this inhibition, which seems to occur via Usp14’s UBL domain.

Usp14’s UBL Domain Stimulates Deubiquitination by Rpn11.

Upon binding a Ub chain, Usp14’s USP domain moves into proximity with Rpn11 and sterically hinders Rpn11 (27), whose activity and accessibility to substrates depend on ATP hydrolysis (18). Surprisingly, even though Usp14KO proteasomes lack an important DUB, they have a much greater capacity to disassemble Ub chains than WT due to a stimulation of Rpn11’s activity (25). To learn if the UBL domain may also influence Rpn11 activity, we studied the effects of GST-UBL on the hydrolysis of tetra-Ub chains. The UBL stimulated the breakdown of both K48 and K63 Ub chains by both the WT and Usp14KO 26S proteasomes (Fig. 3A). The UBL even increased the proteasomes’ ability to hydrolyze tetra-Ub (Fig. 3A) in the presence of ATPγS, which by itself enhances Rpn11 activity (25). Accordingly, this stimulation of tetra-Ub breakdown by the UBL was blocked by an inhibitor of Rpn11, o-phenanthroline, but not by the inhibitor of cysteine DUBS, Ub-VS (Fig. 3B). In addition, the Usp14KO proteasomes disassembled another DUB substrate, K63 FRET di-Ub, faster upon UBL addition (SI Appendix, Fig. S3B). Thus, Usp14’s UBL domain allosterically stimulates Rpn11’s catalytic activity, while its USP domain upon binding a Ub chain limits Rpn11 to handling only one Ub chain at a time by preventing Rpn11 from interacting with a new substrate (27). Because Rpn11-mediated release of the Ub chain is essential for substrate translocation into the 20S (18), this activation of Rpn11 is likely to have a major effect in enhancing the degradation of ubiquitinated proteins.
Fig. 3.
UBL domain stimulates both Ub-chain disassembly by Rpn11 and the degradation of Ub5-DHFR. (A) UBL increases the breakdown of K48 and K63 tetra-Ub chains (368 nM) by MEF 26S with ATP or ATPγS present. 26S proteasomes (5 nM) were incubated with UBL as indicated at room temperature for 15 min before the reaction, which was carried out at 37 °C for 20 min. The generation of tri, di-, and mono-Ubs was analyzed by Western blotting as previously reported (25). Similar data were obtained in three independent experiments. (B) The UBL stimulates breakdown of K48 tetra-Ub by WT 26S with ATP present, and this stimulation must involve Rpn11 because it was inhibited by o-phenanthroline (OPT; 1 mM) but not by Ub-vinyl sulfone (Ub-VS; 200 nM). Similar data were obtained in two independent experiments. (C) 26S proteasomes disassemble the Ub chain on ubiquitinated DHFR to generate monomeric Ubs. Upon inhibition of Usp14 and Uch37 by Ub-aldehyde (300 nM), tetra-Ub chains are disassembled by Rpn11, and the Rpn11 inhibitor OPT (1 mM) blocked completely deubiquitination of DHFR. Degradation of Ub5-DHFR (100 nM) by 26S proteasomes (2 nM) in 20 min was assayed by Western blotting. The asterisk indicates nonspecific signal from Ub-aldehyde. (D) Illustration of why degradation of Ub5-DHFR is dependent on Rpn11. Neither Usp14 nor Uch37 can catalyze disassembly of the tetra-Ub chain. Only after Rpn11 cleaves tetraubiquitin chains en bloc off the protein can these other deubiquitinating enzymes disassemble the chain. Therefore, degradation of Ub5-DHFR depends on Rpn11 activity. (E) Although WT 26S degrades Ub5-DHFR rapidly, the addition of UBL (500 nM) enhances this process further. However, inhibition of Rpn11 activity by OPT (1 mM) completely blocks degradation. Similar data were obtained in three independent experiments.

The UBL Domain of Usp14 Stimulates Ub-Dependent Degradation.

Because the Usp14 UBL domain stimulates several steps critical for proteasomal degradation of ubiquitinated proteins (i.e., Rpn11, gate opening and protein-activated ATP hydrolysis), we tested whether the UBL domain also enhances the degradation of a Ub conjugate, Ub5-DHFR (Fig. 3C), in which an N-terminal Ub fusion of dihydrofolate reductase (DHFR) is conjugated to a K48-linked tetra-Ub chain (48). 26S proteasomes digest this ubiquitinated protein and disassemble its Ub chain to monomeric Ubs (Fig. 3C, lane 3). However, with Ub-aldehyde present to inhibit Usp14 and Uch37, unattached tetra-Ub chains were generated (Fig. 3C, lane 4). Because the Ub chain was not digested when o-phenanthroline was present (Fig. 3C, lanes 5 and 6), Usp14 and Uch37 surprisingly are unable to deubiquitinate this substrate until Rpn11 first cleaves off the tetra-Ub from the DHFR [perhaps because of Usp14’s preference for multiply ubiquitinated proteins (24)] (illustrated in Fig. 3D). Thus, the deubiquitination of Ub5-DHFR requires Rpn11 activity, as does the degradation of DHFR (Fig. 3E, lane 4). Even though WT 26S hydrolyzed Ub5-DHFR rapidly, as predicted, the addition of the UBL domain further enhanced its degradation (Fig. 3E).

The UBL Domains of Other UBL-UBA Proteins Can Also Stimulate 26S Peptidase Activity.

Like Usp14/Ubp6, a variety of other proteins also contain a UBL domain, such as the shuttling factors hHR23A and B, which bind to Ub conjugates through their UBA domains and also to proteasomes through their UBL domains (SI Appendix, Fig. S4). To determine whether the UBL domains of these other proteins can also stimulate proteasomal activities, we assayed peptide hydrolysis by Usp14KO 26S proteasomes with or without the GST-UBL of hHR23B and hPLIC1/UBQLN1 (Fig. 4A). Like the UBL of Usp14, hHR23B’s UBL enhanced all three peptidase activities of Usp14KO 26S three- to fivefold. The UBL of hPLIC1 also increased these activities, but to a much smaller extent (1.5- to 2-fold) than hHR23B’s UBL. A phylogenetic analysis of UBL domains indicated that the primary sequences of UBLs from hHR23A and B and their yeast homolog Rad23 are evolutionarily close to those of Usp14 and Ubp6 (SI Appendix, Fig. S4). By contrast, the UBL of hPLIC1 is more closely related to Ub, which has no stimulatory effect (21). These findings strongly suggest that proteasomal activation is a common property of the UBL domains found in many proteins that bind to the 26S.
Fig. 4.
UBL domains of other UBL-UBA proteins, hHR23 and hPLIC1, also stimulate peptide hydrolysis by 26S proteasomes, and expression of EGFP-UBL increases the degradation of long-lived proteins in HeLa cells. (A) With ATPγS (100 μM) and Usp14KO 26S (1 nM), the UBL domain (500 nM) of hHR23B stimulates proteasomes’ three peptidase activities three- to fivefold, while the UBL domain of hPLIC1 stimulates them twofold or less. Peptidase activities were measured as in Fig. 1. *P < 0.05 and **P < 0.01 by Student’s t test. Data are the means ± SD; n = 6. (B) EGFP and EGFP-UBL derived from hHR23A were expressed in HeLa cells (48 h after transfection). (C) HeLa cells expressing EGFP-UBL degrade long-lived proteins faster than control cells. Proteins were labeled with [3H]phenylalanine (5 μCi/mL) for 24 h. After 1 h in chase medium (DMEM containing 2 mg/mL nonradioactive phenylalanine) to allow short-lived proteins to be degraded, the breakdown of cellular proteins was measured in fresh chase medium for up to 3 h. Data are the means ± SD; n = 3. (D) The content of ubiquitin conjugates in HeLa cell lysates was lower after expression of EGFP-UBL than after EGFP. Forty-eight hours after transfection, equal amounts of proteins (2 μg) in lysates were electrophoresed and then Western blotted using anti-Ub antibody. Similar results were obtained from two independent experiments. (E) Summary of Usp14’s allosteric regulation of proteasomal degradation of ubiquitinated proteins. Without a Ub-conjugate bound, Usp14 (through its USP domain) inhibits ATP hydrolysis, substrate entry into the 20S, and deubiquitination by Rpn11 (25). However, upon binding Ub chains, Usp14 undergoes major structural transitions (2), and its UBL domain activates peptide entry into 20S, Rpn11, and ATP hydrolysis, if an unfolded protein also binds. These actions promote efficient degradation of ubiquitinated substrates. This activated state should be maintained as long as Usp14 binds the Ub chain. These processes return to the quiescent basal state when the substrate is degraded or is deubiquitinated and dissociates. These allosteric actions are not dependent on Usp14’s catalytic activity, which functions to degrade the Ub chain and thus limits the duration of the active state (25). Also shown is the proposed activation of proteasomes by UBL-containing proteins, which should be independent of the presence of Usp14.

Expression of the UBL Domain in HeLa Cells Stimulates Protein Degradation.

Because the UBL domain of Usp14 can stimulate several proteasome activities that are essential for Ub-conjugate degradation and can promote the hydrolysis of a model ubiquitinated substrate, we tested whether expression of a UBL domain in cultured cells can increase the degradation of cellular proteins generally. HeLa cells were transfected with pEGFP-UBLhHR23A (Fig. 4B), and the rates of degradation of long-lived proteins, which comprise the bulk of cell proteins, were measured (Fig. 4C). To follow overall rates of proteolysis, we initially radiolabeled most cell proteins by exposure to [3H]phenylalanine for 24 h and followed the subsequent degradation of radiolabeled proteins by measuring their conversion to TCA-soluble material in the presence of a large excess of nonradioactive phenylalanine to prevent reincorporation of radiolabeled amino acids (49). The HeLa cells expressing EGFP-UBLhHR23A degraded endogenous proteins 60% more rapidly than control cells expressing EGFP (Fig. 4C). Most likely, this enhanced proteolysis occurred as a consequence of the enhanced proteasome activity, because the cells expressing EGFP-UBL also had a much lower level of Ub conjugates (Fig. 4D). This enhancement of overall degradation is surprising, since overexpression of GST-UBL might be expected to inhibit many important UBL-dependent interactions in the cells, such as the binding of the shuttling factors to the proteasome. In fact, when HeLa cells were transfected with a greater amount of the pEGFP-UBLhHR23A plasmid to increase further UBL levels, the degradation of long-lived cellular proteins was inhibited (SI Appendix, Fig. S5). These findings further indicate that binding of UBL to proteasomes enhances their capacity to degrade ubiquitinated substrates in vivo, as was seen in vitro.


Usp14’s UBL Domain Is an Allosteric Stimulator of Multiple Proteasomal Activities.

Despite its catalytic and regulatory importance, Usp14 is found on only a small fraction of 26S proteasomes in vivo, apparently on those particles actively involved in degradation of ubiquitinated proteins (50). When proteasomes bind ubiquitin conjugates, their association with Usp14 increases, and hydrolysis of the conjugates promotes dissociation of the Usp14. The binding of Usp14 to the proteasome has been reported to dramatically stimulate its catalytic activity and to require its UBL domain (16). However, addition of the UBL domain does not cause dissociation of Usp14 copurified with the proteasome. By helping remove Ub chains from the protein bound to the proteasome, Usp14/Ubp6 decreases the wasteful degradation of Ub by the proteasome and promotes Ub recycling (51). However, Usp14 can both allosterically inhibit and activate multiple steps in Ub-conjugate degradation (19, 21, 25). We showed here that Usp14’s UBL domain by itself allosterically stimulates the same proteasome activities as are inhibited by Usp14 in the absence of a substrate and are activated when a Ub chain binds to Usp14: peptide hydrolysis, ATPase activity, degradation of nonubiquitinated and ubiquitinated proteins, and Rpn11 activity. The coordinated increase in the three peptidase activities strongly suggests that the association of the UBL domain enhances the entry of substrate peptides into the 20S chamber (Fig. 1), rather than causing an allosteric stimulation of the six peptidase sites. It has long been assumed that ATPγS opens this gate maximally (39). However, the UBL domain stimulates peptide hydrolysis most effectively in the presence of ATPγS (Fig. 1D). This nonhydrolyzed nucleotide causes not only 20S’s gate opening by promoting the docking of the ATPases’ C-terminal HbYX motifs into the intersubunit pockets in the 20S’s outer ring (4), but also by triggering the reorganization of the central pore in the ATPase ring, leading to its enlargement and coaxial alignment with the 20S gate (40). A similar structural reorganization occurs during degradation of a ubiquitinated substrate (7). The present findings may suggest that this entry channel is much more accessible to substrate translocation when a UBL domain and the nonhydrolyzable nucleotide are present together than with either alone. Such a large increase (five- to eightfold) in the substrate entry channel should be clearly evident by cryoEM.
Alternatively, this large increase in peptidase activity when a UBL is present with ATPγS may occur through the activation of a distinct population of the proteasomes that are otherwise not engaged in degradation. Even purified proteasome preparations are heterogeneous [e.g., only a small fraction contain Usp14 or Ube3C (50)]. Cryoelectron microscopy of isolated neurons indicated that most cellular 26S proteasomes are not in a substrate-engaged conformation (52). Possibly the UBL domain in vitro or when expressed in vivo induces a more active conformation in this population.
During the degradation of a ubiquitinated protein, Rpn11 is located above the ATPase channel (7) and cleaves primarily between the proximal Ub and the substrate to release the Ub chain (12). It thus facilitates translocation of the protein substrate through the ATPases into the 20S particle (18). These actions of Rpn11 are all stimulated by ATPγS (25) but, even in the presence of ATPγS, Usp14’s UBL domain stimulates further Rpn11’s capacity to disassemble Ub chains. This stimulation was observed in the absence of Usp14’s catalytic USP domain, which normally inhibits Rpn11 sterically (27) upon binding a Ub chain. These observations thus reveal an unexpected coordination between the actions of these two 26S DUBs (Fig. 3A). Because the stimulation of peptide hydrolysis and of Ub-chain disassembly by the UBL are additive with the effects of ATPγS, the UBL domain probably promotes these activities by a mechanism distinct from that of nucleotide binding. However, the cryoEM analyses thus far have focused on defining the large changes that occur similarly upon binding of ATPγS, Ub-aldehyde, or a Ub-conjugated substrate and involve steric inhibition of Rpn11 by Usp14 (7, 26, 27, 40), but they have not as yet exposed large additive effects, as demonstrated here by biochemical assays (Figs. 1D and 3A).
Proteasome activation upon binding a Ub conjugate is a multistep process (2) involving sequential recognition of the ubiquitin chain and then the protein substrate (2, 19). After the initial binding of the Ub chain to Usp14, the 19S ATPases become activated and commit the protein for degradation (2, 19). With casein present, the UBL alone was found to stimulate ATP hydrolysis by the proteasomes (Fig. 2A). Thus, the UBL provides a very similar positive allosteric signal to the ATPases as does Usp14 upon binding a Ub chain or Ub-aldehyde (21).
Like a Ub chain, the UBL does not by itself cause ATPase activation, which still requires the presence of a polypeptide with a loosely folded domain. Most likely this domain interacts directly with the proteasomal AAA ATPases to activate them, as occurs with the homologous bacterial and archaeal ATP-dependent proteases (2). Like these AAA ATPases, in proteasomes lacking Usp14 (unlike the wild-type particles), casein by itself can stimulate ATP hydrolysis without a Ub chain, Ub-aldehyde, or UBL domain present. Interestingly, in the yeast 26S reconstituted in vitro, a nonubiquitinated protein substrate could activate ATP hydrolysis by itself (27). Such behavior does not correlate with and cannot account for the requirement for both a ubiquitin chain and a loosely folded domain for protein degradation by proteasomes in vivo (53).
Together, the present observations make it very likely that the proteasome activation upon substrate binding to Usp14 is mediated by its UBL domain (Fig. 4E). Accordingly, adding WT or catalytically inactive Usp14 reduces the ability of the exogenous UBL to stimulate substrate entry and to bind to the 26S (Fig. 1E), apparently by competing for the same binding site on Rpn1 (Fig. 1F) (42, 54). Despite this competition for the same site, UBL does not act by displacing Usp14 from the particles, since incubation of purified 26S with a high concentration of GST-UBL from Usp14 did not dissociate the bound Usp14 from the proteasome (SI Appendix, Fig. S6). Also, our method for purification of the proteasomes uses GST-UBL from Usp14 or hHR23B as an affinity ligand (55) but fails to dislodge Usp14 from the proteasomes, and before and after this step about 20 to 30% of the particles contain Usp14 (25, 50). This tight binding of inhibitory Usp14 to the 26S can explain why the stimulation of different activities by the added UBL is consistently greater in proteasomes lacking Usp14 than in the WT. Consequently, the stimulation of overall proteolysis in cells by expression of UBL is likely to involve these Usp14-deficient particles.
We cannot exclude an alternative possibility that binding of a Ub chain to Usp14’s inhibitory USP domain might also contribute to the increased ATP and peptide hydrolysis by temporarily reversing the basal inhibition of these processes (27). However, based on the present evidence, we favor a single common mechanism for UBL’s actions on proteasomes lacking Usp14 and those where Usp14’s UBL domain mediates the activation by ubiquitinated substrates.
Most likely, the UBL domain activates substrate entry into the 20S, ATP hydrolysis, and Rpn11 by inducing conformational changes in Rpn1, since Usp14 interacts with Rpn1 through its UBL domain (26, 32, 42). Indeed, Usp14’s UBL failed to enhance peptidase activity of yeast 26S having an AKAA mutation in Rpn1’s T2 site where the UBL of Ubp6 binds (32) but activated normally in the 26S mutated in the T1 binding site (Fig. 1G). Because these three proteasomal functions activated by the UBL are catalyzed by subunits located at some distance from Usp14, Rpn1 must mediate these allosteric actions since it interacts with both the ATPases and the UBL domain (54). Furthermore, the same activities are all dependent on the six ATPase subunits, which function coordinately in proteolysis and in 20S gating (56). Therefore, the Rpn1-mediated enhancement of ATPase activity may be responsible for both the activation of substrate entry and Rpn11.
In addition to UBL’s activating role, the full-length Usp14 has inhibitory functions through its USP domain’s interactions with the particle that are not shared by the UBL domain (25). This allosteric inhibition observed with proteasomes in vitro accounts for the greater degradation of cell proteins in Usp14KO MEF cells than in WT (25). Interestingly, the UBL increased the degradation by proteasomes of both Ub5-DHFR (Fig. 3E) and the nonubiquitinated protein Sic1 (Fig. 2B). The enhancement of overall proteolysis by expression of EGFP-UBL in HeLa cells (Fig. 4C) most likely results from proteasome activation, since it was accompanied by a fall in Ub-conjugate levels. This stimulation of proteolysis is particularly striking, because overexpression of UBL would be expected to compete with the binding of shuttling factors to the 26S and to inhibit proteolysis, as we actually observed with higher expression levels.

UBL Domains in Other Proteins Can Also Stimulate Proteasomal Activity.

Like the UBL domain of Usp14, the UBL domains of hHR23 and hPLIC1 also stimulated proteasomal peptidase activities, although to different extents. The UBL of hHR23B had as high a stimulatory effect as that of Usp14, while that of hPLIC1 stimulated to a lesser extent (Fig. 4A). Interestingly, phylogenetic analysis indicated that UBLs of hHR23A/B or Rad23 are evolutionarily closer to that of Usp14 (SI Appendix, Fig. S4). But the UBL of hPLIC1 resembles more closely the structure of Ub, which has no stimulatory effect as a free monomer (21). So, in addition to serving as a “shuttling factor” that delivers Ub conjugates to the proteasome, hHR23 may also activate proteasomes through its UBL domain, functioning in a similar fashion as Usp14’s upon substrate binding. Such an activation of the proteasome upon substrate delivery by hHR23 might be particularly important in activating the majority of cellular 26S particles, which lack Usp14 (26, 50).
These findings further suggest that stimulation of proteasomal activities might be a common property shared by the broad range of proteins containing UBL domains. For example, shuttling factors such as hHR23 and hPLIC1 may not only help 26S proteasomes bind Ub conjugates but also may help activate the proteasome for efficient degradation of substrates. Besides these shuttling factors, other proteins containing a UBL domain have been known to serve various catalytic functions, such as Ub ligation by Parkin (57), dephosphorylation by UBLCP1 (58), and proteolysis by Ddi2 (59). In fact, in our ongoing studies, we have obtained clear evidence that several such UBL-containing proteins also have the capacity to stimulate proteasome activities.

Materials and Methods

Purification of Proteins.

GST-UBL with the UBL domain derived from Usp14 (amino acids 2 to 79), hHR23A (amino acids 2 to 82), hHR23B (amino acids 2 to 82), or hPLIC1 (amino acids 38 to 111) was expressed in Escherichia coli and purified with GSH-Sepharose as described previously (55). GST-fused Usp14 (WT and active-site C114A mutant) was also expressed in E. coli and purified using GSH-Sepharose as described (16). Resin-bound Usp14 was treated with thrombin to cut off GST from Usp14. Residual thrombin was cleared from Usp14 using benzamidine-Sepharose. His-UIM derived from the UIM2 domain of S5a (55), His-tagged PY-Sic1 (60), and His-tagged Usp14 UBL was expressed in E. coli and purified with Ni-NTA resin. Ub5-DHFR was a kind gift from Takeda Pharmaceuticals.

Purification of 26S Proteasomes.

WT and Usp14KO mouse embryonic fibroblasts (16) grown in DMEM (supplemented with 10% FBS and 1% penicillin/streptomycin) were pooled to affinity purify 26S proteasomes using the method previously described (61). Briefly, cell lysates prepared with sonication (15 s six times at 18 W) were spun for 1 h at 100,000 × g. The soluble extracts were incubated at 4 °C with GST-UBL derived from hHR23B and a corresponding amount of GSH-Sepharose. The slurry containing 26S proteasomes bound to GST-UBL was poured into an empty column and washed, followed by incubation with His-UIM. The eluate was collected and incubated with Ni-NTA agarose for 20 min at 4 °C. The Ni-NTA–bound His-UIM was spun out by centrifugation at 3,000 × g, and the remaining supernatant contained purified 26S proteasomes. Protein concentration was determined using Bradford reagent. Molarity of 26S proteasome particles was calculated based on a molecular mass of a doubly capped 26S particle of 2.5 MDa.
Yeast 26S proteasomes were purified as previously described (32) with modification from SY1214 (Mata ProA-TEV-Rpt1::HIS3 rpn13-pru::natMX rpn10-uim::kanMX), SY1210 (MATa ProA-TEV-Rpt1::HIS3 rpn1-ARR::TRP1 rpn13-pru::natMX rpn10-uim::kanMX), and SY1724 (MATa ProA-TEV-Rpt1::HIS3 rpn1-AKAA::TRP1 rpn13-pru::natMX rpn10-uim::kanMX). 26S proteasomes were purified using IgG resin (Life Technologies) and were used for peptidase assays without elution.


Polyclonal rabbit anti-Ub (A-100) was from Boston Biochem, and anti-Sic1 (sc-50441) was from Santa Cruz Biotechnology. Monoclonal mouse anti-DHFR (sc-74594) was from Santa Cruz Biotechnology. HRP-conjugated secondary antibodies were from Promega.

Immunoblot Analysis.

Samples for the immunoblots were run on 4 to 12% Bis-Tris gels (WG1403BOX10; Life Technologies) with Mes buffer (NP0002). Proteins were analyzed following SDS/PAGE and transferred onto 0.45-µm PVDF membranes (Whatman). Immunoblots were blocked and incubated with appropriate primary and secondary antibodies. Membranes were developed with enhanced chemiluminescence reagent (Immobilon Western HRP substrate and Luminol reagent WBKLS0500; Millipore) onto X-ray film. The ImageJ program (NIH; https://imagej.nih.gov/ij) was utilized to quantify the signal from the film.

Proteasome Activity Assays.

As indicated, proteasomes were generally incubated with the UBL domain, casein, linear hexa-Ub chains, or DUB inhibitors at room temperature for 15 min before the start of reactions. Peptide hydrolysis by MEF 26S proteasomes was measured with 10 μM LRR-amc (Boc-Leu-Arg-Arg-amc), LLVY-amc (Suc-Leu-Leu-Val-Tyr-amc), or nLPnLD-amc (Ac-Nle-Pro-Nle-Asp-amc) (Bachem) (λex 380 nm; λem 460 nm) and 1 nM 26S proteasomes at 37 °C. Proteasomal activities were calculated from 30 to 60 min after the start of the reaction. The reaction mixture was composed of 50 mM Tris (pH 7.6), 100 mM KCl, 0.1 mM ATP or ATPγS, 0.5 mM MgCl2, 1 mM DTT, and 25 ng/μL BSA (Sigma). Degradation of PY-Sic1 (100 nM) by 26S proteasomes (2 nM) was carried out in the presence of 50 mM Tris⋅HCl (pH 7.6), 5 mM MgCl2, 1 mM ATP, 1 mM DTT, and 10 μg/mL BSA (Sigma) for the indicated time (0 to 4 h) at 37 °C and measured by Western blotting with anti-Sic1 antibody. Degradation of Ub5-DHFR by 26S proteasomes was carried out under the same conditions for 20 min at 37 °C and measured by Western blotting with anti-DHFR antibody. ATP hydrolysis by proteasomes was measured using the malachite green assay (62). Deubiquitination by 26S particles (5 nM) was assayed with tetra-Ub chains (368 nM) in 25 mM Hepes buffer (pH 7.6) containing 100 mM KCl, 5 mM MgCl2, 1 mM DTT, and 1 mM ATP or ATPγS at 37 °C for 20 min. After the reaction, the products were analyzed by Western blotting with anti-Ub antibodies.

Usp14 Competition Assay.

26S proteasomes from MEF cells were incubated with GST-UBL derived from Usp14 and GSH-Sepharose. Unbound proteins were removed using washing buffer (50 mM Tris, pH 7.6, 100 mM KCl, 0.1 mM ATP or ATPγS, 0.5 mM MgCl2, 1 mM DTT, and 25 ng/μL BSA). Proteasomes were eluted with this same buffer containing increasing concentrations of Usp14 (WT or C114A mutant). Eluted fractions were used to measure peptidase activity.

Degradation of Long-Lived Cellular Proteins.

The overall rates of degradation of long-lived protein in HeLa cells were determined, as described previously, after transfection with pEGFP-C1 (control) or pEGFP-UBLhHR23A (25). hHR23A’s UBL (amino acids 2 to 82) was subcloned into pEGFP-C1 vector to construct pEGFP-UBLhHR23A.

Phylogenetic Tree Analysis.

Amino acid sequences of UBL domains of Usp14, Ubp6, hHR23A/B, Rad23, Dsk2, hPLIC1, Parkin, as well that of Ub were aligned, and their phylogenetic tree was generated with ClustalW2 (https://www.ebi.ac.uk/tools/msa/clustalw2/). The resulting Newick-format tree data were visualized with Phylodendron (iubio.bio.indiana.edu/treeapp/treeprint-form.html).


We are grateful to Megan LaChance for her valuable assistance in preparing the manuscript, Dan Finley and Byung Hoon Lee for providing cells and reagents, and Takeda Pharmaceuticals for providing Ub5-DHFR. This work was supported by grants from the National Institute of General Medical Science (R01 GM51923) and Target ALS Foundation.

Supporting Information

Appendix (PDF)


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Information & Authors


Published in

Go to Proceedings of the National Academy of Sciences
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Proceedings of the National Academy of Sciences
Vol. 115 | No. 50
December 11, 2018
PubMed: 30487212


Submission history

Published online: November 28, 2018
Published in issue: December 11, 2018


  1. proteasome activation
  2. UBL domain
  3. Usp14/Ubp6
  4. hHR23/Rad23
  5. hPLIC/ubiquilin


We are grateful to Megan LaChance for her valuable assistance in preparing the manuscript, Dan Finley and Byung Hoon Lee for providing cells and reagents, and Takeda Pharmaceuticals for providing Ub5-DHFR. This work was supported by grants from the National Institute of General Medical Science (R01 GM51923) and Target ALS Foundation.



Hyoung Tae Kim
Department of Cell Biology, Harvard Medical School, Boston, MA 02115
Alfred L. Goldberg1 [email protected]
Department of Cell Biology, Harvard Medical School, Boston, MA 02115


To whom correspondence should be addressed. Email: [email protected].
Author contributions: H.T.K. and A.L.G. designed research; H.T.K. performed research; H.T.K. contributed new reagents/analytic tools; H.T.K. and A.L.G. analyzed data; and H.T.K. and A.L.G. wrote the paper.
Reviewers: G.D., The University of Texas; and A.M., University of California, Berkeley.

Competing Interests

The authors declare no conflict of interest.

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    UBL domain of Usp14 and other proteins stimulates proteasome activities and protein degradation in cells
    Proceedings of the National Academy of Sciences
    • Vol. 115
    • No. 50
    • pp. 12537-E11885







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