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. 2014 May 16;33(10):1159-76.
doi: 10.1002/embj.201386906. Epub 2014 May 8.

Autoubiquitination of the 26S proteasome on Rpn13 regulates breakdown of ubiquitin conjugates

Affiliations

Autoubiquitination of the 26S proteasome on Rpn13 regulates breakdown of ubiquitin conjugates

Henrike C Besche et al. EMBO J. .

Abstract

Degradation rates of most proteins in eukaryotic cells are determined by their rates of ubiquitination. However, possible regulation of the proteasome's capacity to degrade ubiquitinated proteins has received little attention, although proteasome inhibitors are widely used in research and cancer treatment. We show here that mammalian 26S proteasomes have five associated ubiquitin ligases and that multiple proteasome subunits are ubiquitinated in cells, especially the ubiquitin receptor subunit, Rpn13. When proteolysis is even partially inhibited in cells or purified 26S proteasomes with various inhibitors, Rpn13 becomes extensively and selectively poly-ubiquitinated by the proteasome-associated ubiquitin ligase, Ube3c/Hul5. This modification also occurs in cells during heat-shock or arsenite treatment, when poly-ubiquitinated proteins accumulate. Rpn13 ubiquitination strongly decreases the proteasome's ability to bind and degrade ubiquitin-conjugated proteins, but not its activity against peptide substrates. This autoinhibitory mechanism presumably evolved to prevent binding of ubiquitin conjugates to defective or stalled proteasomes, but this modification may also be useful as a biomarker indicating the presence of proteotoxic stress and reduced proteasomal capacity in cells or patients.

Keywords: 26S proteasomes; Ube3c/Hul5; proteasome inhibitors; proteasome regulation; ubiquitination.

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Figures

Figure 1
Figure 1. Five ubiquitin ligases accumulate on the 26S proteasome upon inhibition with BTZ
  1. HEK293F parental cells and cells stably overexpressing Dss1-FLAG were treated with different concentrations of BTZ for various times. Levels of proteasomes and ligases in the crude extracts were analyzed by Western blot.

  2. Proteasomes were isolated from cells generated in (A) using anti-FLAG affinity resin. Equal amounts of proteasomes were analyzed by Western blot. Huwe1 was not detectable under these conditions, most likely due to the small amounts of cells used in this experiment compared to those used in our initial purifications (e.g. Supplementary Fig S2C).

Source data are available online for this figure.
Figure 2
Figure 2. Ubiquitination of proteasome subunits in cells
  1. 26S proteasomes were isolated from Dss1-FLAG-expressing HEK293F cells that were treated with or without 1 μM BTZ for 4 h, and ubiquitination of different subunits analyzed by Western blot (WB). To confirm that these subunits were modified by ubiquitination, these particles were incubated with the deubiquitinating enzyme Usp2 (1 μM) for 1 h at 4°C.

  2. Schematic view of Rpn13. K34 is located in, and K21 adjacent to the Pru domain responsible for binding of Ub conjugates.

  3. Proteasomes isolated from normal and BTZ-treated cells were treated with 1 μM Usp2 for 1 h at either 4°C or 37°C. Samples were analyzed by Western blot.

Source data are available online for this figure.
Figure 3
Figure 3. Ubiquitination of Rpn13 induced by proteasome inhibition is slowly reversible in vivo
A HEK293F cells overexpressing Dss1-FLAG were treated with DMSO or proteasome inhibitors (10 μM MG132, 1 μM BTZ, 400 nM epoxomycin) for 4 h. The inhibitor was then washed out, and the cells were grown for additional 1, 4, and 20 h. Crude cell extracts were prepared and analyzed for proteasomal peptidase activity (suc-LLVY-amc). B–E Cells as in (A) were analyzed by Western blot for accumulation of ubiquitin conjugates, modification of proteasome subunits, and content of proteasome-associated ubiquitin ligases (B, C). The amount of poly-ubiquitin conjugates and ubiquitinated Rpn13 was quantified by densitometry analysis (D, E). For MG132-treated cells, Rpn13 ubiquitination was reduced to less than 25% control level (0 h after washout) 20 h after washout, when proteasome activity and poly-ubiquitin conjugate level were restored to almost normal level. For BTZ-treated cells, 20-h incubation after washout was much less efficient in restoring proteasome activity and poly-ubiquitin conjugate level. Source data are available online for this figure.
Figure 4
Figure 4. BTZ treatment causes poly-ubiquitination of Rpn13 by Ube3c and mono-ubiquitination of Rpt1 by RNF181
A–E HEK293F cells were transfected with siRNA against proteasome-associated ubiquitin ligases Ube3c/Hul5 (A), Ube3a/E6AP (B), Rnf181 (C), Huwe1 (D), and Ubr4 (E). After BTZ treatment (1 μM, 4 h), cells were lysed, and ubiquitination of Rpn13 (A–E) was measured by WB. Knockdown of Ube3c blocked Rpn13 ubiquitination. F From the same knockdown experiments as in (A–E), mono-ubiquitination of Rpt1 was also measured by WB. Knockdown of RNF181 blocked Rpt1 mono-ubiquitination. G When WT Ube3c, but not the catalytically inactive C1051A mutant Ube3c, was re-expressed in Ube3c knockdown cells, basal and BTZ-induced Rpn13 ubiquitination were both restored. Source data are available online for this figure.
Figure 5
Figure 5. Reconstitution of Rpn13 ubiquitination using affinity-purified 26S and activation of this process by proteasome inhibition
  1. Rpn13 ubiquitination was stimulated by inhibiting 20S function with BTZ, 19S ATPases with ATPγS, or Rpn11 with a Zn2+-chelating agent. Isolated proteasomes were incubated at 37°C for the times indicated in the presence of ATP, Ub, E1, and Ubch5a as the E2. Rpn13 ubiquitination was then analyzed by WB. Left panel, ubiquitination carried out with or without BTZ. Central panel, ATP was substituted with ATPγS where indicated. Right panel, ubiquitination carried out with or without a zinc-chelating agent.

  2. Only Rpn13 is extensively modified by poly-ubiquitination. Isolated 26S proteasomes were incubated in the presence of ATP, Ub, and E1 with or without the E2 for 2 h at 37°C and analyzed by Western blot. BTZ and Ub aldehyde were included in all reactions.

  3. Use of single-lysine Ub mutants or Ub mutants lacking specific lysines indicates that Rpn13 was poly-ubiquitinated by predominantly K29 and K48-linked Ub chains. Proteasomes were ubiquitinated as in (A) and (B) with or without wild-type or mutant Ub as indicated, and Rpn13 ubiquitination was analyzed by Western blot. Top panel, Ub point mutants. Bottom panel, single-lysine Ub mutants.

  4. No deubiquitination of immobilized 26S proteasomes (FLAG) is observed after removing the ubiquitination mixture (S/N) and replacing it with buffer, followed by incubation at 37°C. Usp2 treatment readily deubiquitinates Rpn13 under these conditions.

Source data are available online for this figure.
Figure 6
Figure 6. Rpn13 ubiquitination is a sensitive cellular response to impairment of protein degradation
A HEK293F cells were treated with different concentrations of BTZ for 4 h. The concentration of BTZ that causes a 50% inhibition of the chymotrypsin-like 26S peptidase activity is 20 nM, while the concentration that causes a 50% inhibition of the caspase-like activity is 40 nM. B Degradation of cellular proteins (prelabeled with 3H-phenylalanine) by the proteasome (see Materials and Methods) is markedly inhibited by BTZ when used at above 20 nM and at 40 nM 60% inhibition is observed. C–E The effects of different concentrations of BTZ on levels of poly-ubiquitin conjugates and ubiquitinated Rpn13 were assayed by Western blotting and quantified by densitometry. BTZ causes maximal accumulation of poly-ubiquitin conjugates between 20 and 40 nM (D), and 20 nM is also the concentration at which Rpn13 ubiquitination begins to elevate (2-fold of control) (E). At 40 nM, BTZ causes near-maximal level of Rpn13 ubiquitination (5-fold of control). Quantification of ubiquitin conjugate level and Rpn13 ubiquitination was done by densitometric analysis of Western blotting (see Materials and Methods). Source data are available online for this figure.
Figure 7
Figure 7. Heat-shock and arsenite treatment cause Rpn13 ubiquitination and accumulation of Ub conjugates
  1. HEK293F cells were treated with 0.5 μM Na3AsO3 together with indicated concentrations of BTZ for 3 h. Na3AsO3 dramatically caused the accumulation of poly-ubiquitinated proteins and induced Rpn13 ubiquitination as well.

  2. HEK293F cells were treated with increasing concentrations of BTZ for 3 h at 37°C or under 43°C (heat shock) for 1 or 3 h. Heat shock and arsenite also caused the accumulation of poly-ubiquitinated proteins as well as Rpn13 ubiquitination.

Source data are available online for this figure.
Figure 8
Figure 8. Rpn13 ubiquitination markedly inhibits the ability of 26S proteasomes to bind and degrade ubiquitinated proteins, but not peptides or non-ubiquitinated proteins
  1. Isolated proteasomes were preincubated for 0–2 h at 37°C in the presence of ATP and Ub, with or without E1 and Ubch5a (E2) as indicated and analyzed by Western blot. (Ub aldehyde was included in all reactions to block deubiquitination, but its presence had no significant effect.)

  2. Proteasomes preincubated for 2 h as in (A) were diluted 20-fold, and the degradation of 32P-labeled Ubn-Sic1 was assayed by measuring the production of TCA-soluble radioactive peptides.

  3. After preincubation, degradation of 32P-labeled Ub5-DHFR was assayed as in (B).

  4. To evaluate the effects of Rpn13 modification on hydrolysis of short peptides, proteasomes were preincubated for 2 h as in (A) and diluted 20-fold, and the rate of suc-LLVY-amc hydrolysis was measured in the presence of ATP or ATPγS, which stimulates gate opening and peptide substrate entry (Smith et al, 2007).

  5. To follow the degradation of a non-ubiquitinated substrate, 26S proteasomes were preincubated as in (A), and the breakdown of 32P-labeled casein to TCA-soluble peptides was assayed as in (B).

  6. To measure the binding of ubiquitinated proteins, proteasomes were preincubated for 2 h as in (A) and bound to immobilized Ub5-DHFR at 4°C. The resin was washed in a low-salt buffer (0 mM NaCl), high-salt buffer (300 mM NaCl) or in the presence of UIM, and bound proteasome activity was assayed by measuring suc-LLVY-amc hydrolysis (Peth et al, 2010).

Source data are available online for this figure.
Figure 9
Figure 9. Proposed mechanism for Rpn13 based on the present findings
Normally, Ub chains on the substrate bind initially to Rpn13 and S5a/Rpn10, and once the polypeptide becomes committed to degradation, it is translocated through the ATPase ring into the 20S (Finley, ; Peth et al, 2013). The Ub ligase, Ube3c, presumably functions to extend Ub chains to facilitate substrate degradation and ensure its processivity (Crosas et al, 2006). However, when proteasomes are stalled due to difficult-to-degrade substrates, or during proteotoxic stresses (heat-shock or arsenite exposure), or in vitro when 20S function is inhibited with bortezomib, 19S function slowed with ATPγS, or substrate deubiquitination by Rpn11 is blocked with a Zn2+ chelator, Ube3c ubiquitinates Rpn13 and prevents further substrate binding in vivo.

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