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3 C. -L. Kuo and A. L. Goldberg. (2017) Ubiquitinated proteins promote the association of proteasomes with the deubiquitinating enzyme Usp14 and the ubiquitin ligase Ube3c. Proc. Natl. Acad. Sci. U.S.A.114, E3404–E3413. 2 The abbreviations used are:
UbubiquitinMEFmouse embryonic fibroblastUBLubiquitin-likeDUBdeubiquitinating enzymeUIMubiquitin-interacting motifUb-alubiquitin aldehydeATPγSadenosine 5′-O-(thiotriphosphate)USPubiquitin-specific proteaseNi-NTAnickel-nitrilotriacetic acidBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolamc7-amino-4-methylcoumarinBoct-butoxycarbonylSucsuccinylNle/nLnorleucineUb-VSubiquitin vinyl sulfonePANproteasome-activating nucleotidase.
The proteasome-associated deubiquitinating enzyme Usp14/Ubp6 inhibits protein degradation by catalyzing substrate deubiquitination and by poorly understood allosteric actions. However, upon binding a ubiquitin chain, Usp14 enhances proteasomal degradation by stimulating ATP and peptide degradation. These studies were undertaken to clarify these seemingly opposite regulatory roles of Usp14 and their importance. To learn how the presence of Usp14 on 26S proteasomes influences its different activities, we compared enzymatic and regulatory properties of 26S proteasomes purified from wild-type mouse embryonic fibroblast cells and those lacking Usp14. The proteasomes lacking Usp14 had higher basal peptidase activity than WT 26S, and this activity was stimulated to a greater extent by adenosine 5′-O-(thiotriphosphate) (ATPγS) than with WT particles. These differences were clear even though Usp14 is present on only a minor fraction (30–40%) of the 26S in WT mouse embryonic fibroblast cells. Addition of purified Usp14 to the WT and Usp14-defficient proteasomes reduced both their basal peptidase activity and the stimulation by ATPγS. Usp14 inhibits these processes allosterically because a catalytically inactive Usp14 mutant also inhibited them. Proteasomes lacking Usp14 also exhibited greater deubiquitinating activity by Rpn11 and greater basal ATPase activity than WT particles. ATP hydrolysis by WT proteasomes is activated if they bind a ubiquitinated protein, which is loosely folded. Surprisingly, proteasomes lacking Usp14 could be activated by such proteins even without a ubiquitin chain present. Furthermore, proteasomes lacking Usp14 are much more active in degrading non-ubiquitinated proteins (e.g. Sic1) than WT particles. Thus, without a ubiquitinated substrate present, Usp14 suppresses multiple proteasomal activities, especially basal ATP consumption and degradation of non-ubiquitinated proteins. These allosteric effects thus reduce ATP hydrolysis by inactive proteasomes and nonspecific proteolysis and enhance proteasomal specificity for ubiquitinated proteins.
). The cylindrical 20S proteasome is a hollow four-ring particle that contains in each of its central β-rings three types of active sites, which are chymotrypsin-like, trypsin-like, and caspase-like in specificity (
). The 19S regulatory particle performs several enzymatic and non-enzymatic functions that are required for the degradation of ubiquitinated proteins, including binding and disassembly of the Ub chain, ATP hydrolysis, and unfolding and translocating the substrate protein (
). The ATPases bind, unfold, and translocate the polypeptide through their central channel and then through the gated entry channel into the inner chamber of the 20S particle where proteolysis occurs (
During degradation of a Ub conjugate, the Ub chain is disassembled so that the Ub molecules can be reutilized. Three different deubiquitinating enzymes (DUBs) are associated with the 19S regulatory particle in higher eukaryotes. Two are cysteine proteases, Usp14/Ubp6 (
). Rpn11 is an integral 19S subunit, but Usp14 and Uch37 associate reversibly with the proteasome and are usually present at substoichiometric amounts. Like its homolog, Ubp6, in budding yeast, Usp14 contains an N-terminal UBL domain, which binds to Rpn1 on the proteasome (
). Rpn11 appears to remove the Ub chain en bloc by cleaving the isopeptide bond adjacent (or near) to the substrate and facilitates the translocation of the polypeptide through the ATPase ring into the 20S particle (
). This stimulatory role of Usp14 requires occupancy of its active site by either a Ub chain or the inhibitor ubiquitin aldehyde (Ub-al). This step, and not the initial binding of the ubiquitinated substrate to the particle, somehow enhances substrate entry into the 20S (“gate opening”) (
). Unlike gate opening, this stimulation of ATP hydrolysis, which drives conjugate degradation, thus requires two signaling events, a Ub chain (or Ub-al) binding to Usp14 and a polypeptide interacting probably with the ATPase (
). The DUB activity of Ubp6/Usp14 thus helps prevent the prolonged residence of a ubiquitinated protein on the 26S, and it also serves as a timing device that limits the time for hydrolysis of a potential substrate and that can promote the release of the substrate without its degradation (
) that revealed that the association of Ub-al with Usp14/Ubp6 induces marked changes in the structure of the 19S complex. After binding of this substrate analog, the central channel in the ATPase ring is enlarged, and the catalytic domain of Ubp6 becomes located closer to the ATPase ring and Rpn11.
These findings indicate that Usp14 has seemingly opposite actions and that it functions both as an inhibitor and an activator of proteolysis. The simplest hypothesis to resolve these apparently conflicting roles would be that Usp14/Ubp6 normally inhibits proteolysis, but when a Ub chain on a potential substrate is bound, Usp14 allosterically activates the proteasome's degradative functions (
). This activated state is then maintained until the Ub chain is no longer bound to the DUB as occurs when proteolysis is complete or the substrate is released. The present studies were undertaken to examine further this hypothesis and to determine how Usp14 functions as an allosteric inhibitor of proteolysis and how it influences key steps in conjugate degradation. By comparing the enzymatic properties of WT and Usp14-deficient proteasomes, we show here that 26S particles lacking Usp14 exhibit a greater capacity to degrade non-ubiquitinated proteins, greater ATPase activity, and even greater DUB activity than WT proteasomes. Together, these findings imply that, in the absence of a substrate, Usp14 normally maintains the 26S proteasome in a quiescent state, but upon binding a Ub conjugate, it allosterically activates several enzymatic processes essential for efficient destruction of Ub conjugates.
Usp14 deficiency increases 26S content of Uch37 and Rpn13 and overall proteolysis
To understand the regulatory role of Usp14, we compared subunit composition and various enzymatic activities of 26S proteasomes purified from WT mouse embryonic fibroblast (MEF) cells and MEFs lacking Usp14 (Usp14KO). In yeast, Ubp6 is present on a small fraction of the proteasomes (
). In the WT MEF cells, we determined that Usp14 was present on ∼40% of the 26S proteasomes based on Western blotting using increasing amounts of purified Usp14 as a standard and a proteasome with molecular mass of 2.5 MDa (Fig. 1A). The proteasomes purified from the WT and Usp14KO MEFs appeared very similar in subunit composition (Fig. 1, B and C) with two clear exceptions. The other cysteine protease DUB, Uch37, and the Ub receptor, Rpn13, to which Uch37 binds were both increased. Interestingly, the total quantity of Uch37 in the two types of cell lysates did not differ, although Rpn13 expression did increase in the Usp14KO cells (Fig. 1D). This increased association of Uch37 and Rpn13 with the proteasome presumably occurred to compensate for the loss of Usp14. In the Δubp6 yeast, degradation of a number of model proteins was increased (
) above control levels. We therefore tested whether overall rates of protein degradation differed in WT and KO MEF cells by pulse-chase methods using [3H]phenylalanine. To follow the breakdown of long-lived proteins, which comprise the bulk of cell proteins (
), cell proteins were radiolabeled for 24 h and regrown in chase medium (DMEM containing non-radioactive phenylalanine and cycloheximide to repress protein synthesis) for 3 h to allow breakdown of short-lived cell components. Then the cells were regrown in the fresh chase medium, and degradation rates were assayed. The total protein degradation rate was about 45% greater in Usp14KO cells (p < 0.05) than in controls (Fig. 1F).
We then determined the contribution of the ubiquitin-proteasome pathway using bortezomib to selectively inhibit the proteasome as described elsewhere (
). Total substrate flux through the proteasome was increased (Fig. 1F) by about 27% in the Usp14KO MEFs (p < 0.01). However, in addition to the enhanced proteasomal proteolysis, there was also greater protein degradation by lysosomes (calculated by subtracting the proteasomal degradation from the total proteolysis) in the Usp14KO MEFs, which presumably reflects the greater rate of autophagy recently reported in other Usp14-deficient cells (
), the great majority of the intracellular proteolysis in the MEF cells was by proteasomes.
To also evaluate the degradation rate of short-lived proteins, the MEF cells were initially labeled for 20 min with [3H]phenylalanine. During the subsequent chase period, the most short-lived cell proteins were hydrolyzed about 53% faster in Usp14KO cells (p < 0.05) than in controls (Fig. 1G). Despite the greater rates of proteolysis by the ubiquitin-proteasome and autophagic pathways and despite the loss of a major proteasomal DUB in the Usp14KO MEF cells, the content of ubiquitinated proteins did not differ significantly in the Usp14KO and WT cells (Fig. 1E).
Binding of Usp14 to the 26S proteasomes inhibits basal peptide hydrolysis
The chymotrypsin-like activity of the proteasomes from Usp14KO cells was slightly (35%) higher than that of the particles from WT MEFs (Fig. 2A). Because this difference in peptidase activities was seen even though Usp14 is present on less than half the WT particles (Fig. 1A), this difference must underestimate the actual increase in peptide hydrolysis in the Usp14KO cells. We therefore studied the effects of increasing Usp14 content by adding recombinant Usp14 to the purified 26S proteasomes. With WT Usp14 added, the absolute chymotrypsin-like activity of the 26S decreased to a similar extent in WT and KO particles (17–33%) (Fig. 2B). In general, the present inhibition appeared larger in Usp14KO 26S where the basal activity was greater. Addition of the enzymatically inactive Cys → Ala Usp14 mutant also decreased the chymotrypsin-like activity in both WT and Usp14KO proteasomes. Thus, this inhibition is due to allosteric action of Usp14 (Fig. 2B).
Unlike ATP, the non-hydrolyzable ATP analog ATPγS causes a marked stimulation of proteasomal peptide hydrolysis because it induces major conformational changes in the 19S particle, leading to an alignment of the ATPases' central channel with the 20S gate (
). Adding increasing amounts of ATPγS stimulated the chymotrypsin-like activities of both the WT and Usp14KO particles (Fig. 2C). However, the maximum stimulation of Usp14KO 26S proteasomes was consistently larger than that of WT particles (Fig. 2C). With ATPγS (100 μm), the chymotrypsin-like activity was stimulated maximally 5–6-fold, the trypsin-like activity was stimulated 11–15-fold, and the caspase-like activity was stimulated 15–20-fold over those activities with ATP (Fig. 2D), and in each case, the maximal activity and the degree of stimulation were greater in the Usp14KO proteasomes than in WT particles (Fig. 1D).
We also tested the effects of increasing Usp14 content on this stimulation of proteasomal peptide hydrolysis by ATPγS. Incubation with the recombinant Usp14 in the presence of ATPγS decreased by 20–26% the chymotrypsin-like activity of both types of proteasomes (Fig. 2E). Thus, although Usp14 increases peptide hydrolysis (
) when it binds a Ub chain, the presence of Usp14 by itself inhibits 20S gate opening and the activation by nucleotides.
Usp14 inhibits the basal activity of proteasomal ATPases
A general feature of bacterial ATP-dependent proteases and the archaeal PAN ATPase, which are all hexameric complexes homologous to the 26S proteasomal ATPases Rpt1–6, is that their ATPase activity is stimulated upon binding a loosely folded polypeptide (
). All these ATPases also bind and translocate polypeptides into an associated proteolytic compartment. Unlike these enzymes, the stimulation of the 26S ATPases requires the simultaneous binding of a Ub chain to Usp14 (or to Uch37) in addition to the unstructured region of the polypeptide, although the Ub chain and polypeptide need not be covalently linked (
). To evaluate the role of Usp14 in regulating the ATPases, we compared the activation of ATP hydrolysis by WT and Usp14KO particles (Fig. 3). Surprisingly, the Usp14KO 26S had a 60% higher basal ATPase activity than WT 26S (33.4 versus 20.7 nm ATP/nm 26S/min; p < 0.05) (Fig. 3). When either type of the proteasomes was incubated with linear hexa-Ub chains, there was no significant stimulation of ATPase (unlike some previous reports (
)). However, when the Ub chains were added with the structureless protein β-casein, there was a 2.7-fold stimulation of ATP hydrolysis with the WT and a 1.8-fold stimulation with Usp14KO 26S (Fig. 3). As a result, the stimulated rates of ATP hydrolysis by the 26S from the two cell types were both about 60 μm ATP/μm 26S/min.
Although β-casein by itself had no effect on ATP hydrolysis by the WT proteasomes, surprisingly, ATP hydrolysis by the Usp14KO 26S was consistently stimulated by β-casein alone (1.7-fold), which is similar to the stimulation seen with a Ub conjugate or when a Ub chain and casein were present together. In this respect, the Usp14KO particles resemble the AAA ATPases that regulate proteolysis in Escherichia coli and the archaeal PAN proteasome-regulatory ATPases, which hydrolyze ATP faster in the presence of casein (
). This inhibition by Usp14 of both the basal ATP hydrolysis and its activation by proteins in the absence of a Ub chain must help prevent ATP consumption that is not linked to degradation of ubiquitinated proteins.
Usp14 reduces the degradation of non-ubiquitinated unfolded proteins
These findings predict that proteasomes lacking Usp14 should degrade some non-ubiquitinated proteins faster than the WT 26S. In fact, in the absence of Ub, the intrinsically disordered protein PY-Sic1 (
) is degraded very slowly by WT proteasomes; however, as predicted, Usp14KO 26S degraded Sic1 at least 6-fold faster than WT 26S did (Fig. 4, A and C). By contrast, the addition of linear hexa-Ub chains to mimic substrate ubiquitination stimulated the breakdown of Sic1 by WT 26S about 10-fold and by Usp14KO 26S at most 2-fold (Fig. 4, B and C). Consequently, the maximal rates of degradation achieved by the two types of particles with the Ub chains present were similar. These findings provide further evidence that Usp14 inhibits proteasomal proteolysis until a Ub chain binds and reverses this inhibition. These inhibitory effects are allosteric because a specific inhibitor of Usp14's catalytic activity, IU1, did not enhance the degradation of non-ubiquitinated Sic1 (Fig. 4D). Furthermore, the addition of the inactive Cys → Ala mutant Usp14 to Usp14KO 26S decreased the degradation of Sic1 (Fig. 4E).
Usp14 inhibits deubiquitination by Rpn11
Rapid degradation of Ub conjugates by the proteasome requires the removal of the Ub chain from the substrate (
). Because Usp14 upon binding a Ub chain stimulates proteasomal peptide and ATP hydrolysis, we tested whether it may also enhance the proteasome's other DUB activities by comparing the rates of breakdown of free tetra-Ub chains (chains not conjugated to a protein) by WT and Usp14KO 26S (Fig. 5A). Surprisingly, even though Usp14KO proteasomes lack the important DUB Usp14, they showed a much greater capacity to disassemble Lys-63 Ub chains (Fig. 5B) than WT particles as shown by their more rapid generation of tri-, di-, and mono-Ubs. The loss of Usp14 also mildly increased the rates of disassembly of Lys-48 tetra-Ub (Fig. 5C). Because the Zn2+ chelator o-phenanthroline markedly suppressed this accelerated breakdown of tetra-Ub to the level seen with WT 26S, the faster chain hydrolysis in the Usp14KO must be due to activation of the Zn2+-containing metalloprotease Rpn11. In fact, Rpn11 is the most active proteasomal DUB against Lys-63 chains (
). Furthermore, although Uch37 and Rpn13 levels were increased in the Usp14KO proteasomes (Fig. 1C), Ub-VS, which inhibits Uch37, did not decrease the rapid breakdown of tetra-Ub chains by Usp14KO 26S (Fig. 5B). Thus, the activity of Rpn11 (but not Uch37) is stimulated by the absence of Usp14 in the 26S. These findings are also consistent with the recent structure of the 26S by cryo-EM, which shows that Ubp6 can normally block substrate access to Rpn11 (
). However, in addition to this catalytic role, as emphasized by the present results, Usp14 is a major allosteric regulator of proteasome function that has the unusual capacity to both activate and inhibit multiple steps in Ub conjugate degradation. The first evidence of this regulatory role was the finding in yeast that Ubp6 inhibits the degradation of several proteins (
), presumably by slowing their deubiquitination and providing more time for proteasomal proteolysis. However, unlike a Usp14 substrate or substrate analog (e.g. Ub-al), IU1 does not cause allosteric activation of ATP hydrolysis (
), peptide entry, or degradation of non-ubiquitinated PY-Sic1 (Fig. 4D). Nevertheless, by slowing substrate deubiquitination by Usp14, IU1 may prolong substrate association with Usp14 and thus prolong proteasomal activation.
Because of Usp14's inhibitory actions, it was surprising to find that binding of a Ub chain to Usp14/Ubp6 allosterically stimulates peptide entry into the 20S (
). The present observations bridge these seemingly contradictory roles and show that Usp14 by itself inhibits at least three key functions in substrate degradation, but upon binding a Ub chain, Usp14 releases this inhibition and activates these processes further.
Interestingly, in Usp14-deficient cells, the proteasomes contain significantly greater amounts of Rpn13 and Uch37 (Fig. 1B) even though the cells contained only slightly increased cellular levels of Rpn13 (Fig. 1C). Although this Ub receptor is often classified as a stoichiometric subunit, several investigators have reported that the Ub receptor Rpn13 is present in proteasomes in substoichiometric levels (
). Presumably, the increased Uch37 and Rpn13 levels on the proteasome help compensate for the loss of Usp14's catalytically activity, but how this loss of Usp14 is recognized and leads to increased Uch37 and Rpn13 binding are interesting questions for future study.
In the absence of a substrate and thus independently of its role as a DUB, Usp14 was found to inhibit multiple proteasomal processes. For example, the 26S lacking Usp14 has a 20–30% greater peptidase activity than WT 26S (Fig. 2A). Because at most 30–40% of the WT proteasomes contain Usp14 (Fig. 1E), this greater activity must underestimate the actual effect of Usp14 on peptide entry into the 20S particle and ATP hydrolysis. In addition, all three peptidase activities of the Usp14KO 26S were consistently stimulated by ATPγS more than the WT (Fig. 2D). Accordingly, addition of recombinant WT or Cys → Ala Usp14 further represses allosterically both the basal and ATPγS-stimulated peptidase activities (Fig. 2, B and E). Nucleotides, like ATPγS, open the gated entry of the 20S particle by stimulating interaction of ATPases' C-terminal HbYX motifs with intersubunit pockets in the 20S α-ring (
). Because Usp14 suppresses all three peptidase activities in the presence of ATPγS (Fig. 2D), Usp14 must inhibit substrate entry rather than altering the catalytic activity of the three sites. Although ATPγS was previously assumed to cause maximal increases in substrate entry, Usp14KO can be stimulated further than WT 26S in the presence of ATPγS. Thus, Usp14 must influence substrate entry by additional mechanisms distinct from those activated by ATPγS.
One important surprising finding was that after loss of Usp14 the 26S exhibited a greater ability to disassemble tetra-Ub chains due to increased activity of Rpn11 (Fig. 5). Thus, under basal conditions, Usp14 allosterically inhibits Rpn11 and reduces its capacity to digest Ub conjugates. Recently, the catalytic domain of Usp14 (the “USP domain”) upon association with Ub-VS was shown by cryo-EM to block sterically substrate access to Rpn11 (
). Therefore, the loss of Usp14 probably exposes the active site of Rpn11 and enables it to digest Ub chains more readily. However, it seems likely that in addition to this steric effect Usp14 may allosterically inhibit Rpn11's catalytic activity just as it inhibits the particle's other proteasomal activities.
In the Usp14KO proteasomes, ATPase activities are also regulated differently than in WT (Fig. 3). Unlike the WT, which requires the binding of both a Ub chain and an unfolded protein to stimulate ATP hydrolysis (
), in the Usp14-deficient 26S, such a protein can stimulate this process even without a Ub chain present (Fig. 3). In this respect, the ATPases in the Usp14KO proteasomes resemble the homologous, ATP-dependent proteases from bacteria (Lon, ClpAP, ClpXP, and HslUV) and the direct 26S ancestor, PAN, which regulates the activity of archaeal 20S proteasomes. These enzymes are all protein substrate-activated AAA ATPases (
). During the evolution of eukaryotes, ubiquitin conjugation became linked to proteolysis, and through Usp14, the 26S proteasome evolved the requirement for Ub chain binding to activate peptide entry and ATP hydrolysis. This enhancement of ATPase activity is a critical event because it drives substrate translocation, and the amount of ATP consumed is directly proportional to the amount of Ub conjugates degraded (
). Although the structural basis for these different allosteric effects are not yet clear, it is noteworthy that Ubp6 binds to Rpn1, but when Ub aldehyde occupies the active site on Usp14, these proteins also interact with the ATPases Rpt1 and -2 (
The inhibition of multiple proteasomal activities by Usp14 can account for the finding that MEF cells lacking Usp14 degrade cellular proteins, both short-lived and long-lived components (Fig. 1, F and G), faster than WT MEFs. This observation of enhanced degradation of cell proteins generally by proteasomes extends the findings on breakdown of several model proteins in Δubp6 yeast (
). These observations are also intriguing because they are further evidence that under physiological conditions the ubiquitin-proteasome pathway and autophagy are coordinately regulated even though the proteasomes remain the primary site of protein hydrolysis (
It is also noteworthy that, unlike the WT, proteasomes lacking Usp14 can efficiently degrade certain unstructured proteins without ubiquitination (Fig. 4A). This degradation of non-ubiquitinated proteins must result from enhanced translocation of such unstructured proteins into the 20S particle and presumably from the ability of such non-ubiquitinated proteins to activate ATP hydrolysis. The inhibition of Ub-independent proteolysis by Usp14 must also be an allosteric effect because there is no Ub chain on these substrates to hydrolyze and because the inactive Cys → Ala mutant Usp14 also inhibited degradation (Fig. 4E).
Although these several inhibitory actions of Usp14 probably contribute to its ability to suppress overall protein degradation in vivo (
), its inhibition of ATPase activation by non-ubiquitinated proteins appears to be critical in preventing Ub-independent proteolysis in vivo. These findings together also suggest a clear rationale for these bidirectional effects of Usp14 on proteasome function (Fig. 6). In the absence of a substrate, Usp14 helps prevent wasteful ATP consumption and non-selective proteolysis by the proteasome, especially degradation of non-ubiquitinated proteins. The activation of the proteasome upon binding a Ub conjugate thus enhances the selectivity for ubiquitinated substrates, especially those with unfolded domains.
As noted above, the greater activity of the 26S from cells lacking Usp14 is striking because Usp14 is present on only a fraction of the WT particles. It seems likely, therefore, that additional factors can suppress the basal activities of the particles lacking Usp14. One possible inhibitory factor might be Uch37 because its content on the 26S is increased in the Usp14KO MEFs and because the interaction of Ub chains with Uch37 can activate gate opening and ATP hydrolysis (
C. -L. Kuo and A. L. Goldberg. (2017) Ubiquitinated proteins promote the association of proteasomes with the deubiquitinating enzyme Usp14 and the ubiquitin ligase Ube3c. Proc. Natl. Acad. Sci. U.S.A.114, E3404–E3413.
the subsequent interaction between the Ub chain and Usp14 can lead to more efficient and selective breakdown of the ubiquitinated substrates.
Purification of proteins
GST-UBL derived from hHR23B was expressed in E. coli and purified with GSH-Sepharose as described previously (
). Briefly, cell lysates prepared with sonication (15 s six times at 18 watts) were spun for 1 h at 100,000 × g. The soluble supernatants 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, washed, and incubated 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, and the remaining supernatant contained purified 26S proteasomes. Protein concentration was determined using BCA reagent. The molarity of 26S proteasome particles was calculated based on an average molecular mass of 2.5 MDa.
Polyclonal rabbit anti-Ub (A-100) was purchased from Boston Biochem. Anti-Rpn1 (sc-68352), anti-Rpn2 (sc-166038), and anti-Sic1 (sc-50441) were from Santa Cruz Biotechnology. Anti-Rpn6 (14303), anti-Rpn11 (4197), and anti-Rpt5 (13923) were from Cell Signaling Technology. Anti-β5 (A303-847A), anti-Usp14 (A300-920A), and anti-Rpn13 (A302-554A) were purchased from Bethyl Laboratories. Anti-Uch37 (3904-1) was from Epitomics. HRP-conjugated secondary antibodies were from Promega.
Samples for immunoblotting were run on 4–12% Bis-Tris gels (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 (W. S. Rasband, National Institutes of Health) was utilized to quantify the signal from the film.
Proteasome activity assays
As indicated, proteasomes were generally incubated with or without casein, linear hexa-Ub chains, Usp14, or a DUB inhibitor at room temperature for 15 min before the start of reactions. Peptide hydrolysis by MEF 26S proteasomes was measured with 10 μm Boc-Leu-Arg-Arg-amc (Boc-LRR-amc), Suc-Leu-Leu-Val-Tyr-amc (Suc-LLVY-amc), or Ac-Nle-Pro-Nle-Asp-amc (Ac-nLPnLD-amc) (Bachem) (λex, 380 nm; λem, 460 nm) and the indicated concentration of 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 0.01 mg/ml BSA (Sigma) for the indicated time (0–4 h) at 37 °C and measured by Western blotting with an anti-Sic1 antibody. ATP hydrolysis by proteasomes was measured using the malachite green assay (
). Deubiquitination by 26S particles (5 nm) was assayed with tetra-Ub chains (368 nm) in HEPES buffer (25 mm) containing 100 mm KCl, 5 mm MgCl2, 1 mm DTT, and 1 mm ATP at 37 °C for 20 min. After the reaction, the products were analyzed by Western blotting with anti-Ub antibodies.
In vivo proteolysis
The overall rates of degradation of long-lived proteins in WT and Usp14KO MEF cells were determined as described previously (
). MEF cells were grown with [3H]phenylalanine (5 μCi/ml) for 24 h to label cell proteins and then washed twice with PBS and once with chase medium (i.e. fresh DMEM containing 2 mm nonradioactive phenylalanine and 100 μm cycloheximide). Cells were grown in chase medium for 2 h to let short-lived proteins be degraded. Then fresh chase medium containing DMSO or 10 μm bortezomib/Velcade was added. One hour after addition of the inhibitors, samples of the medium were collected for up to 4 h. Then each sample was mixed with 10% trichloroacetic acid (final concentration) to precipitate proteins. The acid-soluble radioactivity reflects the amount of prelabeled, long-lived cell proteins degraded at each time and was expressed relative to the total amount of radioactivity initially incorporated into protein. The absolute rate of proteasomal proteolysis was determined by subtracting the rates of proteolysis in cells treated with bortezomib from that in untreated cells. Lysosomal proteolysis was determined by subtracting the rates of proteasomal degradation from that of overall proteolysis.
The overall rates of degradation of short-lived proteins were determined by a pulse-chase protocol by incubating MEF cells with [3H]phenylalanine (10 μCi/ml) for 20 min to label cell proteins. The cells were then washed twice with PBS and once with the chase medium containing large amounts of nonradioactive phenylalanine to block incorporation of labeled amino acids. After incubation in chase medium for 20 min more, samples were collected every 10 min for up to 40 min. The degradation rate was calculated using the same method as used for evaluating the degradation rate of long-lived proteins.
H. T. K. and A. L. G. designed the studies and wrote the manuscript. H. T. K. performed the experiments.
We are grateful to Megan LaChance for valuable assistance in preparing the manuscript and to Dan Finley and Byung Hoon Lee for providing cells and reagents.
Recognition and processing of ubiquitin-protein conjugates by the proteasome.
This work was supported by NIGMS, National Institutes of Health Grant R01 GM51923, by the Cure Alzheimer's Fund, and by the Project ALS Foundation (2015-06). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.