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Research Article

Ubiquitin-specific protease 19 regulates the stability of the E3 ubiquitin ligase MARCH6 Nobuhiro Nakamuran, Kumi Harada, Masako Kato, Shigehisa Hirose Department of Biological Sciences, Tokyo Institute of Technology, 4259-B13 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan

article information

abstract

Article Chronology:

Ubiquitin-specific protease (USP)19 is a recently identified deubiquitinating enzyme (DUB) having

Received 3 May 2014

multiple splice variants and cellular functions. One variant encodes an endoplasmic reticulum (ER)-

Received in revised form

anchored DUB that rescues misfolded transmembrane proteins from ER-associated degradation

20 July 2014

(ERAD), but the underlying mechanism remains to be elucidated. Here, we show that USP19 interacts

Accepted 22 July 2014

with the ERAD-associated E3 ubiquitin ligase MARCH6. Overexpression of USP19 delayed the

Available online 1 August 2014

degradation of MARCH6, leading to an increase in its protein level. In contrast, USP19 depletion

Keywords: ER-associated degradation E3 ubiquitin ligase Endoproteolytic processing Deubiquitinating enzyme Proteasomal degradation

resulted in decreased expression of MARCH6. We also show that USP19 overexpression reduced ubiquitination of MARCH6, while its knockdown had the opposite effect. In particular, USP19 was found to protect MARCH6 by deubiquitination from the p97-dependent proteasomal degradation. In addition, USP19 knockdown leads to increased expression of mutant ABCB11, an ERAD substrate of MARCH6. Moreover, USP19 is itself subjected to endoproteolytic processing by DUB activity, and the processing cleaves off an N-terminal cytoplasmic region of unknown function. However, elimination of this processing had no evident effect on MARCH6 stabilization. These results suggest that USP19 is involved in the regulation of ERAD by controlling the stability of MARCH6 via deubiquitination. & 2014 Elsevier Inc. All rights reserved.

Introduction In eukaryotic cells, secretory and membrane proteins are synthesized in the endoplasmic reticulum (ER) and nascent polypeptides fold into their mature conformation with the assistance of molecular chaperones and folding enzymes. Both unfolded and misfolded proteins are recognized and translocated back to the cytosol for proteasomal degradation by ER quality control processes and ER-associated

degradation (ERAD) [1]. In addition to aberrant ER proteins, degradation via ERAD is also carried out on normal, short-lived ER proteins, such as hydroxymethylglutaryl-CoA reductase (HMGR), in order to regulate their enzymatic activities [2]. Polyubiquitination is required for dislocation (or retrotranslocation) of most of the ERAD substrates from the ER into the cytosol. The cytosolic ATPases associated with diverse cellular activities (AAA), cdc48 in yeast and p97 (also termed VCP) in mammals, recognize polyubiquitinated ERAD substrates and

Abbreviations: USP, ubiquitin-specific protease; DUB, deubiquitinating enzyme; ER, endoplasmic reticulum; ERAD, ER-associated degradation; E3, E3 ubiquitin ligase; NPT II, neomycin phosphotransferase II; FBS, fetal bovine serum; siRNA, small interference RNA; NEM, N-ethylmaleimide; K48-Ub, K48-linked ubiquitin; UBL, ubiquitin-like n

Corresponding author. Fax: þ81 45 924 5824. E-mail address: [email protected] (N. Nakamura).

http://dx.doi.org/10.1016/j.yexcr.2014.07.025 0014-4827/& 2014 Elsevier Inc. All rights reserved.

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drive their extraction from the ER membrane for delivery to the 26S proteasome [3–7]. Ubiquitination is catalyzed by the sequential activities of three enzymes, an E1 ubiquitin activating enzyme, an E2 ubiquitin activating enzyme and an E3 ubiquitin ligase that confers substrate specificity [8]. The E3 ubiquitin ligases (hereafter, “E3s”) involved in ERAD have been identified and are currently being studied [9]. Yeast contains the two E3 ubiquitin ligases Hrd1 and Doa10, which have been shown to degrade all of the identified ERAD substrates [10–12]. Mammals contain many more types of E3s that participate in ERAD, including HRD1 (synoviolin), GP78 (AMFR), MARCH6 (TEB4), RMA1, RFP2 and TRC8, which diversity apparently reflects an expansion of the ERAD substrates [13–19]. Ubiquitination can be reversed by deubiquitination via deubiquitinating enzymes (DUBs) in a variety of cellular processes. Recent studies have reported that DUBs have a role in ERAD. For example, Rpn11, UCH37 and USP14 are proteasome-associated DUBs involved in removal of polyubiquitin from proteasomal substrates prior to proteolysis for the recycling of free ubiquitin [20]. Ataxin-3 and YOD1 promote the deubiquitination of p97-associated ERAD substrates, and facilitate delivery to the proteasome [21–24]. Recently, USP19 and USP25 were shown to rescue transmembrane ERAD substrates from proteasomal degradation [25,26]. USP19 was first characterized as being upregulated in the atrophying skeletal muscle of rats, a process in which USP19 controls the expression levels of myofibrillar proteins [27,28]. USP19 has multiple functions, in part due to the presence of its variant forms. For example, a soluble isoform of USP19 has been shown to control fibroblast cell proliferation [29,30]. Its DUB activity stabilizes the KPC1 E3 ubiquitin ligase, resulting in the downregulation of the cyclin inhibitor p27Kip1 and hence cell cycle progression [29,30]. On the other hand, Hassink et al. [26] have identified another USP19 isoform which is a transmembrane protein anchored to the ER membrane. The transmembrane USP19 has been shown to stabilize transmembrane ERAD substrates, such as the mutant CFTR channel and T-cell receptor α-chain, possibly by preventing their proteasomal degradation [26]. Thus, ER-anchored USP19 is thought to contribute to ER quality control and/or ERAD, but the precise mechanism involved remains to be elucidated. However, little is known about how the protein expression and activity of ERAD E3s are regulated. A number of studies have reported that various DUBs interact with certain E3s so as control their stability and functions [31]. Indeed, USP19 has been shown to associate with several of the soluble E3s, such as KPC1, c-IAPs and SIAHs, in order to regulate the cell cycle [29,30], apoptosis [32] and stability of USP19 [33]. Thus, the ER pattern of localization led us to hypothesize that USP19 affects the stability of the ERAD E3(s). In this study, we show that USP19 deubiquitinates and stabilizes MARCH6. In addition, USP19 knockdown resulted in an increased expression of mutant ABCB11, an ERAD substrate of MARCH6. These findings provide insight into the detailed regulatory mechanisms underlying the stability and activity of the ERAD E3s.

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full-length USP19 was amplified by PCR and was then subcloned into pcDNA3 (Invitrogen, Carlsbad, CA), yielding a mammalian expression vector for USP19. FLAG–USP19 and Myc-USP19 were constructed by cloning the open reading frame of USP19 into p3  FLAGCMV-10 (Sigma-Aldrich, St. Louis, MO) and pcDNA3 encoding a 6  Myc epitope tag, respectively. USP19–FLAG was generated by cloning cDNA fragments encoding residues 1–1360 of USP19 into p3  FLAGCMV-14 (Sigma-Aldrich). Point mutations were introduced by oligonucleotide-directed mutagenesis. FLAG– MARCH6 was constructed by cloning a cDNA fragment encoding human MARCH6 into the EcoRV site of p3  FLAGCMV-10. Hisp97 was constructed by a cDNA fragment encoding human p97 into the EcoRI and XbaI sites of pcDNA3 encoding a 6  His epitope tag. His-p97 QQ was generated by introducing E305Q and E578Q mutations by oligonucleotide-directed mutagenesis into the pcDNA3-His-p97 plasmid. FLAG-ABCB11G238V was constructed by cloning cDNA fragments encoding human ABCB11 containing a G238V mutation into the KpnI/XbaI sites of p3  FLAGCMV-10. The sequences of all of the plasmids were verified by DNA sequencing.

Antibodies The rabbit anti-USP19 polyclonal antibody (A301-587A) was purchased from Bethyl Laboratories (Montgomery, TX), which had been raised against residues 900–950 of human USP19, and was used for detecting endogenous human USP19 by Western blotting. The anti-USP19 polyclonal antibody (#799) was raised in a rabbit against GST-fusion proteins of residues 409–1222 of mouse USP19 (GST-DUB) and was antigen-affinity purified with HiTrap NHS-activated HP (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). The anti-MARCH6 polyclonal antibody (#917) was raised in a rabbit against GST-His6-fusion proteins of residues 2–78, 164–276 and 866–910 of human MARCH6 and was antigen-affinity purified. The animal protocols and procedures were approved by the Institutional Animal Care and Use Committee of Tokyo Institute of Technology. The following mouse monoclonal antibodies were purchased: anti-α-tubulin and anti-FLAG M2 antibodies (Sigma-Aldrich); anti-c-Myc antibody (Roche, Indianapolis, IN); anti-neomycin phosphotransferase II (NPT II; clone AC113) and anti-K48-linked ubiquitin antibodies (clone Apu2; Merck Millipore, Billerica, MA).

Cell culture and plasmid transfection 293T cells were cultured in Dulbecco modified Eagle medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. Plasmid transfection was performed with TransFectin reagent (Bio-Rad, Hercules, CA) according to the manufacturer's instruction.

RNA interference

Materials and methods Construction of mammalian expression plasmids Mouse Usp19 cDNA (IMAGE clone 6400986) was obtained from OpenBiosystems (Huntsville, AL). A cDNA fragment encoding

The human USP19-specific Stealth small interference RNA (siRNA) duplex oligonucleotides (50 -ggaggcaugauugguggccacuaca-30 ) were purchased from Invitrogen. Stealth RNAi Negative Control Medium GC Duplex #3 (Invitrogen) was used as a negative control. One day before siRNA transfection, 293T cells (0.5  105 cells) were seeded onto a 24-well plate. Cells were incubated in 500 μl

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of DMEM supplemented with 10% FBS, and were then transfected with 60 pmol of the siRNA duplex oligonucleotides, which were mixed with 2 μl of Lipofectamine 2000 (Invitrogen) in 100 μl of Opti-MEM I (Invitrogen). 4 h after transfection, growth medium was changed with DMEM supplemented with 10% FBS and antibiotics. At 2 or 3 days after transfection, cells were further transfected with plasmids as needed.

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Tris–HCl, pH 6.8, 2% SDS, 8% glycerol, and 0.005% bromophenol blue) in the presence of 1% β-mercaptoethanol, and then separated by SDS-PAGE. The gels were exposed to an imaging plate (GE Healthcare), and the signals were analyzed with a FLA7000 phosphorimager (Fujifilm, Tokyo, Japan) and quantified using MultiGene software (Fujifilm Medical Systems, Stanford, CT).

Statistical analysis Preparation of whole cell lysates and Western blotting Cells were homogenized in TNE buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, and 1 mM EDTA) containing 1 M urea, 10 μM N-ethylmaleimide (Wako Pure Chemical Industries, Osaka, Japan), 2 μM epoxomicin (Peptide Institute, Osaka, Japan) and protease inhibitors [10 mM leupeptin (Peptide Institute), 1 mM pepstatin A (Peptide Institute), 5 mg/ ml aprotinin (Sigma-Aldrich), and 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich)] for 30 min on ice, which were then centrifuged at 13,000  g for 30 min at 4 1C. The samples were dissolved in Laemmli buffer (60 mM Tris–HCl, pH 6.8, 2% SDS, 8% glycerol, and 0.005% bromophenol blue) in the presence of 1% βmercaptoethanol, heated at 70 1C for 5 min and electrophoresed. Proteins were transferred to an Immobilon-P membrane (Merck Millipore) with semidry or tank blot apparatus as described previously [34]. After blocking in TBST (150 mM NaCl, 10 mM Tris–HCl, pH 7.6, and 0.05% Tween 20) for 30 min at room temperature, the membrane was incubated with primary antibodies in TBST overnight at 10 1C, followed by secondary antibodies conjugated with horseradish peroxidase in TBST. Signals were developed with Luminata Forte (Merck Millipore) or ECL Plus (GE Healthcare), and imaged using a Kodak ImageStation (Eastman Kodak, Rochester, NY) or Hyperfilm ECL (GE Healthcare). Quantification of the band intensity was performed with ImageJ software (National Institutes of Health, Bethesda, MD).

Statistical analysis was performed with Graphpad Prism software (GraphPad Software, La Jolla, CA). Data are represented as the mean7SEM of at least three independent experiments. Student's t test was used to determine statistical significance. A value of po0.05 was considered significant.

N-terminal sequence analysis Two days after transfection with USP19–FLAG, 293T cells were extracted with 1% Triton-X 100 in PBS and were then subjected to immunoprecipitation with anti-FLAG M2 agarose beads. The eluate was separated by SDS-PAGE on a 10% polyacrylamide gel and blotted on a polyvinylidene difluoride membrane [Immobilon-PSQ (0.2-μm pore size); Merck Millipore]. After washing in MilliQ water, the filter was stained with a Coomassie brilliant blue (CBB) solution (45% methanol, 10% acetic acid, and 0.1% CBB-R250) for 5 min, and was then destained in a solution of 45% methanol and 7% acetic acid for 15 min followed by a 90% methanol solution for 40 s. The 120-kDa protein band was excised and subjected to an N-terminal sequence analysis, which was performed by Aproscience (Tokushima, Japan) by Edman degradation on a Procise 494 HT protein sequencing system (Applied Biosystems, Foster City, CA).

Results Immunoprecipitation USP19 stabilizes MARCH6 Protein samples extracted in TNE buffer containing 1 M urea, 10 μM N-ethylmaleimide, 2 μM epoxomicin and the protein inhibitors were incubated with 20 μl of anti-FLAG M2 agarose beads (Sigma-Aldrich) or anti-c-Myc agarose beads (Sigma-Aldrich) overnight at 10 1C. After washing 3 times with the same buffer, immunoprecipitates were eluted with 25 μl of 0.1 M glycine–HCl, pH 2.7 and 1% Nonidet P-40, and were subsequently neutralized by adding 5 μl of 1 M Tris–HCl, pH 8.0.

Pulse-chase experiments 293T cells were seeded onto 6-well dishes and were transiently transfected with plasmids. One day after transfection, cells were starved in DMEM for 2 h at 37 1C. Cells were then labeled for 30 min at 37 1C in cysteine/methionine-free DMEM supplemented with 5% dialyzed FBS and 0.1 mCi/ml [35S]cysteine/[35S]methionine (PerkinElmer, Waltham, MA). After washing with ice-cold PBS, cells were chased in normal culture medium at 37 1C. Cells were washed with ice-cold PBS and lysed in 1 ml of TNE buffer. Cell lysates were cleared by centrifugation at 13,000 rpm and the resulting supernatants were incubated with 20 μl of anti-FLAG M2 beads overnight at 4 1C. Immunoprecipitates were washed 3 times with TNE buffer, heated at 70 1C in Laemmli buffer (60 mM

Since transmembrane USP19 and MARCH6 have been shown to be localized to the ER, we first assessed the possibility that USP19 interacts with MARCH6. We performed coimmunoprecipitation experiments designed to analyze their complex formation. Myctagged mouse USP19 was expressed along with FLAG-tagged MARCH6 in human embryonic kidney 293T cells. Cell lysates were immunoprecipitated with anti-Myc antibody, followed by Western blotting. FLAG–MARCH6 was coimmunoprecipitated with MycUSP19 (Fig. 1A). We next examined whether the DUB activity of USP19 has any impact on the protein expression level of MARCH6. 293T cells were transfected with FLAG–MARCH6 along with either a pcDNA3 vector (as a control), Myc-USP19 or Myc-USP19C548S (an enzymatically inactive mutant containing a C548S substitution). Western blotting of the cell lysates demonstrated that Myc-USP19 overexpression resulted in a marked increase in the expression levels of FLAG–MARCH6 compared to control cells (Fig. 1B, lane 2). No such effect was observed by Myc-USP19C548S overexpression (Fig. 1B, lane 3), suggesting that the DUB activity of USP19 appears to stabilize MARCH6. We confirmed the increased protein levels of endogenous MARCH6 in cells overexpressing Myc-USP19 (Fig. 1C). Interestingly, Myc-USP19 was detected as two major bands; one at 185-kDa, corresponding to the full-length protein and another at

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65-kDa (Fig. 1B and C, arrows). The characterization of the smaller band is described in a later section. We investigated the correlation between the USP19 DUB activity and the degradation rate of MARCH6 by pulse-chase experiments. 293T cells were transfected with the FLAG–MARCH6

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along with either an empty vector, Myc-USP19, or MycUSP19C548S. Cells were metabolically labeled for 30 min with [35S]methionine and [35S]cysteine, and were then chased for various time points. The labeled FLAG–MARCH6 was immunoprecipitated from cell lysates and was then analyzed by SDS-PAGE

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followed by autoradiography. The half-life of FLAG–MARCH6 was estimated to be approximately 2 h in control cells (Fig. 1D). Coexpression of Myc-USP19 significantly delayed the degradation (a half-life of approximately 25 h), while Myc-USP19C548S overexpression had no effect (a half-life of approximately 2 h) (Fig. 1D). Furthermore, we addressed whether knockdown of the endogenous USP19 protein with small interfering RNA (siRNA) would reduce the levels of endogenous MARCH6. 293T cells were transfected with control non-silencing siRNA or human USP19 siRNA duplexes and were then cultivated for three days. The reduced USP19 levels resulted in a decrease in the steady-state levels of endogenous MARCH6 (Fig. 1E). These results suggest that USP19 stabilizes MARCH6 in a manner dependent on its DUB activity.

USP19 deubiquitinates MARCH6 It is plausible that the stabilization of MARCH6 by USP19 may result from reduced ubiquitination. To evaluate this, we determined the effect of Myc-USP19 overexpression on the extent of the ubiquitination of FLAG–MARCH6. FLAG–MARCH6 was transfected into 293T cells along with either an empty vector, MycUSP19 or Myc-USP19C548S. The cells were treated with 10 μM epoxomicin, a potent inhibitor of the proteasome, for 2 h, to accumulate the ubiquitinated forms of proteins. The cell lysates were then subjected to immunoprecipitation with an anti-FLAG antibody in the presence of urea, EDTA (to inactivate RING-type E3s), N-ethylmaleimide (NEM; to block DUB activities) and epoxomicin. The immunoprecipitates were analyzed by Western blotting with antibodies specific to the K48-linked ubiquitin (K48-Ub) chains, which facilitate dislocation and proteasomal degradation of ERAD substrates [35–37]. The K48-Ub signals on FLAG–MARCH6 were sharply decreased by Myc-USP19 overexpression, whereas they were not affected by Myc-USP19C548S (Fig. 2A). Conversely, to test the effect of USP19 knockdown on MARCH6 ubiquitination, USP19 siRNA-treated cells were transfected with FLAG–MARCH6 and were then subjected to immunoprecipitation with an anti-FLAG antibody followed by Western blotting with an anti-Ub-K48 antibody. Reduced USP19 expression

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resulted in an increase in ubiquitination of FLAG–MARCH6 (Fig. 2B). Taken together, these results suggest that USP19 deubiquitinates MARCH6.

USP19 protects MARCH6 from p97-dependent ERAD MARCH6 has been shown to be an unstable protein that undergoes K48-linked self-ubiquitination and subsequent proteasomal degradation [13]. However it is unclear whether, as with other ERAD substrates, ubiquitinated MARCH6 is dislocated from the ER membrane into the cytosol for proteasomal degradation in a manner dependent on the AAA ATPase p97, which is known to promote substrate extraction and delivery to the 26S proteasome during ERAD. We thus investigated whether p97 is involved in MARCH6 degradation. FLAG–MARCH6 was transfected into 293T cells along with either a pcDNA3 vector, hexa-histidine-tagged p97 (His-p97), its dominant negative ATPase mutant (His-p97QQ), or Myc-USP19. p97QQ has been shown to inhibit proteasomal degradation of ERAD substrates due to impaired dislocation [3,4]. Western blotting of cell lysates demonstrated that overexpression of His-p97QQ resulted in an increase in the FLAG–MARCH6 levels to a similar extent when Myc-USP19 was transfected (Fig. 3A, lanes 3 and 4), suggesting that p97 is involved in ubiquitinmediated proteasomal degradation of MARCH6. On the other hand, His-p97 overexpression did not affect FLAG–MARCH6 expression compared with control cells (Fig. 3A, lanes 1 and 2), suggesting that the activity of endogenous p97 is sufficient for MARCH6 degradation. The same phenomenon was observed for endogenous MARCH6 (Supplementary Fig. S1). Expression of p97QQ is also known to cause an accumulation of ubiquitinated dislocation intermediates. Indeed, when FLAG–MARCH6 was transfected into 293T cells along with either a pcDNA3 vector or His-p97QQ, the amount of K48-linked ubiquitination on FLAG– MARCH6 was increased by His-p97QQ expression, compared with control cells (Fig. 3B, lanes 1 and 2). Moreover, the accumulation of ubiquitinated FLAG–MARCH6 was potently inhibited by MycUSP19 overexpression, whereas no such effect was observed by Myc-USP19C548S overexpression (Fig. 3B, lanes 3 and 4). Taken together, these results suggest that USP19 protects MARCH6 from p97-mediated proteasomal degradation.

Fig. 1 – USP19 interacts with and stabilizes MARCH6. (A) FLAG–MARCH6 was transfected into 293T cells along with either a pcDNA3 vector (lanes 1 and 3) or Myc-USP19 (lanes 2 and 4). Whole cell lysates were subjected to immunoprecipitation (IP) with an antiMyc antibody. The immunoprecipitates (50% of the eluates; lanes 1 and 2) and the lysates (10% of the input; lanes 3 and 4) were analyzed by Western blotting with antibodies against FLAG (top panel), Myc (middle panel) and α-tubulin (bottom panel). (B) FLAG–MARCH6 was transfected into 293T cells along with either a pcDNA3 vector (lane 1), Myc-USP19 (lane 2) or Myc-USP19C548S (lane 3). Whole cell lysates (20 μg of proteins) were analyzed by Western blotting with antibodies against FLAG (top panel), Myc (second top panel), α-tubulin (an internal loading control; third top panel) and neomycin phosphotransferase II (NPT II, the Neor gene product as a transfection control; bottom panel). The arrow indicates the position of the short fragment of USP19 that was detected in only the Myc-USP19-transfected cells. (C) 293T cells were transfected with either pcDNA3 vector (lane 1), Myc-USP19 (lane 2) or Myc-USP19C548S (lane 3). Whole cell lysates (20 μg of proteins) were analyzed by Western blotting with antibodies against MARCH6 (top panel), Myc (second top panel), α-tubulin (third top panel) and NPT II (bottom panel). The arrow indicates the position of the short fragment of Myc-USP19. (D) FLAG–MARCH6 was transfected into 293T cells along with either a pcDNA3 vector, Myc-USP19 (WT) or Myc-USP19C548S (C548S). The cells were pulse labeled with 35S for 30 min, chased for indicated periods of time and lysed. FLAG–MARCH6 was immumoprecipitated with anti-FLAG beads and then analyzed by SDS-PAGE followed by autoradiography. The data were plotted as a percentage of the remaining FLAG–MARCH6 proteins relative to time zero from at least three independent experiments (mean7SEM). (E) 293T cells were transfected with control siRNA (lane 1) or USP19-specific siRNA duplexes (lane 2). Three days after transfection, whole cell lysates (40 μg of proteins) were analyzed by Western blotting with antibodies against MARCH6 (top panel), USP19 (middle panel) and α-tubulin (bottom panel).

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type II [38]. Since enzyme activity is generally proportional to the protein expression levels, we expected that knockdown of USP19 would reduce MARCH6 expression, which in turn would stabilize the ABCB11G238V mutant due to a decrease in MARCH6 E3 activity. To confirm this, 293T cells treated with USP19–siRNA duplexes were transfected with FLAG-tagged ABCB11G238V and then subjected to Western blotting with an anti-FLAG antibody. As expected, the expression levels of FLAG-ABCB11G238V were increased in the USP19 knockdown cells compared to control siRNA-treated cells (Fig. 4A). This effect was counteracted by overexpression of FLAG–MARCH6 (Fig. 4B). These results suggest that USP19 is likely to positively regulate the MARCH6-mediated ERAD of ABCB11G238V.

The N-terminal region of USP19 is cleaved in a manner dependent on DUB activity

Fig. 2 – USP19 promotes the deubiquitination of MARCH6. (A) FLAG–MARCH6 was transfected into 293T cells along with either pcDNA3 (lanes 1, 4, and 7), Myc-USP19 (lanes 2, 5, and 8) or MycUSP19C548S (lanes 3, 6, and 9). The cells were incubated with 10 μM epoxomicin for 2 h to inhibit proteasomal degradation. Whole cell lysates were subjected to immunoprecipitation (IP) with an antiFLAG antibody. The immunoprecipitates (50% of the eluates) were analyzed by Western blotting with an anti-K48-Ub antibody (lanes 1–3) and anti-FLAG antibody (lanes 4–6). The lysates (50 μg of proteins; lanes 7–9) were analyzed by Western blotting with antibodies against Myc (top panel), α-tubulin (middle panel) and NPT II (bottom panel). (B) 293T cells were transfected with control siRNA (lanes 1, 3, and 5) or USP19-specific siRNA duplexes (lanes 2, 4, and 6). Two days after transfection, the cells were further transfected with FLAG–MARCH6 and then incubated for 1 day. After treatment with 10 μM epoxomicin for 6 h, the cells were lysed and then subjected to immunoprecipitation (IP) with an anti-FLAG antibody. The immunoprecipitates (50% of the eluates) were analyzed by Western blotting with antiK48-Ub antibody (lanes 1 and 2) and anti-FLAG antibody (lanes 3 and 4). The lysates (50 μg of proteins; lanes 5 and 6) were analyzed by Western blotting with antibodies against USP19 (top panel), α-tubulin (middle panel) and NPT II (bottom panel).

Knockdown of USP19 stabilizes mutant ABCB11 It has been shown that MARCH6 promotes degradation of the bile salt export pump (Bsep or ABCB11) containing the G238V mutation, which causes progressive familial intrahepatic cholestasis

As mentioned above, an N-terminal Myc-tagged USP19 was detected as two bands of 185 and 65 kDa on Western blotting with anti-Myc antibody (Fig. 5A, lane 1, arrowhead). When the blot was probed with anti-USP19 #799 polyclonal antibody, which had been raised against the C-terminal cytoplasmic portion of mouse USP19, a  120-kDa band was detected in addition to the  185-kDa band (Fig. 5A, lane 3, arrow). Since the sum of molecular weights of the two small bands (65 kDaþ120 kDa) approximates the size of the full-length protein ( 185 kDa), USP19 is likely to be endoproteolytically cleaved. Interestingly, the small bands were not observed when the DUB activity was inactivated by a C548S mutation (Fig. 5A, lanes 2 and 4), suggesting that the cleavage of USP19 depends on its DUB activity. To determine the cleavage site, we analyzed the N-terminal sequence of the cleaved C-terminal fragment. 293T cells were transfected with a C-terminal FLAG-tagged USP19 (USP19–FLAG) and were then subjected to immunoprecipitation with anti-FLAG antibodies (Fig. 5B). When the 120-kDa protein band in the immunoprecipitates (Fig. 5B, arrow) was sequenced by Edman degradation, its N-terminal sequence was found to be Ala-Lys-ValAla-Val, which is identical to the residues 435–439 that are positioned between the second CHORD-Sgt1 domain and the catalytic Cys-box domain (Fig. 5C). This result indicates that the cleavage site is located between Gly434 and Ala435. The amino acid sequence around the cleavage site is highly conserved across species (Fig. 5C). To determine if these conserved residues are important for the cleavage, we introduced single glutamine substitutions at positions 430–436 of Myc-USP19. Cell lysates of 293T cells transfected with these Myc-USP19 mutants were analyzed by Western blotting with anti-Myc and anti-USP19 #799 antibodies. The results show that the processing was abolished by G433Q and G434Q mutations but not by the other mutations examined (Fig. 5D), suggesting that the Gly433–Gly434 sequence, but not its adjacent sequences, is necessary for the cleavage of USP19. In addition to Gly433–Gly434, three Gly–Gly motifs are present at residues 421–422 and 452–454, among which two (Gly421–Gly422 and Gly433–Gly434) are conserved from fish to mammals (Fig. 5C). We thus introduced single alanine substitutions at the residues 421, 433, and 453, and then analyzed their effects on the USP19 cleavage. 293T cells were transfected with mock, wild-type USP19 or a mutant containing G421A, G433A, or G453A. Western blotting of the cell lysates with an anti-USP19 #799 antibody showed that the cleavage was abolished only by a G433A mutation (Fig. 5E).

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Fig. 3 – USP19 inhibits p97-dependent proteasomal degradation of MARCH6. (A) 293T cells were transfected with FLAG–MARCH6 along with either pcDNA3 (lane 1), His-p97 (lane 2), His-p97QQ (lane 3) or Myc-USP19 (lane 4). Whole cell lysates of the cells (25 μg of proteins) were analyzed by Western blotting with antibodies against FLAG (top panel), Myc (second top panel), His (third top panel), α-tubulin (second bottom panel) and NPT II (bottom panel). (B) FLAG–MARCH6 was transfected into 293T cells along with only pcDNA3 (lanes 1, 5, and 9) or with Hisp97QQ and either pcDNA3 (lanes 2, 6, and 10), Myc-USP19 (lanes 3, 7, and 11) or Myc-USP19C548S (lanes 4, 8, and 12). Whole cell lysates were subjected to immunoprecipitation (IP) with an anti-FLAG antibody followed by Western blotting with an antiK48-Ub antibody (lanes 1–4) and anti-FLAG antibody (lanes 5–8). The lysates (40 μg of proteins) were analyzed by Western blotting (lanes 9–12) with antibodies against Myc (top panel), His (second top panel), α-tubulin (third top panel) and NPT II (bottom panel).

This was also confirmed by pulse-chase experiments, which showed that the short fragment was not produced from the G433A mutant until at least 8 h after a pulse (Fig. 5F). In addition, we found that the cleaved N-terminal fragment exhibited a similar migration pattern to a deletion mutant of USP19 consisting 434 N-terminal amino acid residues (Fig. 5G), suggesting that USP19 is cleaved into two pieces at the location between Gly434 and Ala435. Collectively, these results suggest that USP19 effects cleavage at the conserved di-glycine sequence in a manner dependent on its DUB activity. Finally, we used pulse-chase experiment to examine whether an inhibition of the processing of USP19 would influence the degradation rate of FLAG–MARCH6. 293T cells transfected with Myc-USP19 or its G434Q mutant (Myc-USP19G434Q) were metabolically labeled for 30 min, and then chased for 0, 20, and 40 h. The result showed that the turnover of FLAG–MARCH6 was not significantly altered between Myc-USP19 and Myc-USP19G434Q (Fig. 5H). Thus, the processing of USP19 does not influence the stabilization of FLAG–MARCH6.

Discussion The molecular mechanisms for the ER quality control and ERAD processing systems have been extensively studied over the past

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Fig. 4 – Increased expression of mutant ABCB11 by USP19 knockdown. (A) 293T cells were transfected with control siRNA (lane 1) or USP19-specific siRNA duplexes (lane 2). Two days after transfection, the cells were further transfected with FLAGABCB11G238V and incubated for 1 day. Whole cell lysates (30 μg of proteins) were analyzed by Western blotting with antibodies against FLAG (top panel), USP19 (second top panel), α-tubulin (third top panel) and NPT II (bottom panel). The upper and lower bands of FLAG-ABCB11G238V represent the mature and immature glycosylated proteins, respectively. (B) 293T cells were transfected with USP19-specific siRNA duplexes. Two days after transfection, FLAG-ABCB11G238V was transfected into the cells along with either pcDNA3 (lane 1) or FLAG–MARCH6 (lane 2). After one-day incubation, whole cell lysates (30 μg of proteins) were analyzed by Western blotting with antibodies against FLAG (two top panels), USP19 (third top panel), α-tubulin (fourth top panel) and NPT II (bottom panel). two decades, and a number of factors involved have been identified and characterized. However, the role of DUBs in these systems has not been elucidated yet. In this study, we provide evidence that, in mammalian cells, ER-anchored USP19 controls the stability of MARCH6 by regulating deubiquitination. It has been reported that USP19 interacts with and stabilizes transmembrane ERAD substrates, such as mutant CFTR and T-cell receptor α [26]. We found that USP19 also interacts with and stabilizes MARCH6 (Fig. 1). It has been reported that USP19 stabilizes its transmembrane substrates through both a DUBactivity-dependent and -independent mechanism [26,32,39]. Since the enzymatically inactive mutant USP19 has no effect on protein turnover or the level of MARCH6, the stabilization is mediated by a DUB-activity-dependent mechanism. USP19 overexpression reduces the K48-linked ubiquitination of MARCH6 (Fig. 2). Moreover, we have confirmed the DUB activity toward K48-linked ubiquitin in vitro (Supplementary Fig. S2). K48-linked ubiquitin chains are known to target substrate proteins for proteasomal degradation [40]. Thus it is concluded that MARCH6 is a novel specific substrate for USP19 DUB activity, and that USP19-mediated deubiquitination protects it from proteasomal degradation. This effect was not an artifact of overexpression of USP19, since knockdown of endogenous USP19 using siRNA exhibited the opposite effect than the one that caused increased ubiquitination and reduced expression of MARCH6 (Figs. 1 and 2).

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Fig. 5 – Proteolytic processing of the N-terminal region of USP19. (A) 293T cells were transfected with Myc-USP19 (lanes 1 and 3) or Myc-USP19C548S (lanes 2 and 4). Whole cell lysates (20 μg of proteins) were analyzed by Western blotting (WB) with an anti-Myc antibody (lanes 1 and 2) and anti-USP19 #799 antibody (lanes 3 and 4; see (C) for the antigenic region). The arrow and arrowhead indicate the bands corresponding to the 120-kDa and 65-kDa proteins, respectively. (B) 293T cells were transfected with USP19– FLAG. Whole cell lysates were subjected to immunoprecipitation with anti-FLAG antibody followed by SDS-PAGE. Subsequently, the immunoprecipitated proteins were transferred to a polyvinylidene difluoride membrane and then visualized by CBB staining. The arrowhead and arrow indicate the positions of the full-length and smaller fragment of USP19–FLAG, respectively. The small fragment was subjected to N-terminal sequence analysis. (C) At the top, a schematic is shown of the domain structure of mouse USP19. The CS domains (residues 43–140 and 326–424), catalytic Cys box (residues 539–555), ubiquitin-like (Ubl) domain (residues 720–808), MYND-finger domain (residues 833–875), catalytic His box (residues 1190–1210) and transmembrane domain (residues 1334–1354) are indicated by the colored boxes. The cleavage site is also indicated. The broken line indicates the site of processing. At the bottom there is an alignment of the amino acid sequences of mouse USP19 (residues 420–455) and its homologs in other species. The conserved amino acid residues and the Gly–Gly sequences are indicated by asterisks and bold letters, respectively. The five most N-terminal residues of the small fragment of USP19–FLAG (arrow in B), as determined by Edman degradation, are indicated by the dark box. The GenBank accession numbers are as follows: mouse USP19, BC060613; human USP19, NM_006677; dog (Canis familiaris) USP19, XP_862305; Western clawed frog (Xenopus tropicalis) Usp19, NP_001072879; and zebrafish (Danio rerio) Usp19, XP_689922. (D) 293T cells were transfected with mutant Myc-USP19 containing glutamine substitutions at the indicated amino acids. Whole cell lysates (40 μg of proteins) were analyzed by Western blotting with anti-Myc (top panel) and antiUSP19 #799 antibodies (bottom panel). The arrowhead and arrow indicate the cleaved N- and C-terminal fragments of the mutant Myc-USP19, respectively. (E) 293T cells were transfected with pcDNA3 (lane 1), USP19 (lane 2), or its mutant containing G421A (lane 3), G433A (lane 4) or G453A (lane 5). Whole cell lysates (30 μg of proteins) were analyzed by Western blotting with an anti-USP19 #799 antibody. The arrowhead and arrow indicate the cleaved N- and C-terminal fragments, respectively. (F) 293T cells were transfected with USP19 (left three lanes) or USP19G433A (right three lanes). The cells were pulse labeled with 35S for 30 min, then chased for 0, 5 and 8 h. USP19 and USP19G433A were immumoprecipitated from whole cell lysates with an anti-USP19 #799 antibody and were then analyzed by SDS-PAGE followed by autoradiography. Arrow indicates the cleaved C-terminal fragments of USP19. (G) 293T cells were transfected with FLAG–USP19 (lane 1) and its deletion mutants containing N-terminal 422 (lane 2), 434 (lane 3) and 453 (lane 4) residues, respectively. Whole cell lysates were analyzed by Western blotting with anti-FLAG antibody. (H) 293T cells transfected with FLAG–MARCH6 along with Myc-USP19 (WT; left three lanes) or Myc-USP19G433Q (G434Q; right three lanes). The cells were pulse labeled with 35S for 30 min, then chased for 0, 20, and 40 h. FLAG–MARCH6 was immumoprecipitated from whole cell lysates with anti-FLAG antibody and were then analyzed by SDS-PAGE followed by autoradiography. The data was plotted as a percentage of the remaining FLAG–MARCH6 relative to that at time zero from six independent experiments (mean7SEM).

The precise mechanism for the substrate recognition, however, remains to be determined. It has been shown that the ER localization of USP19 is essential for the rescue effect

on transmembrane ERAD substrates [26]. Similarly, we have observed that the cytosolic isoform of USP19 does not affect the expression levels of MARCH6 (data not shown). The membrane

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anchoring may ensure close proximity between USP19 and its substrates, which then allows for physical interaction and deubiquitination. Polyubiquitinated luminal and membranous ERAD substrates are extracted from the ER membrane by p97 through the proteinconducting channel, including Sec61α and Derlins [41]. The ERAD machinery-associated DUBs, such as Ataxin-3 and YOD1, are hypothesized to trim the ubiquitin chains on the p97-associated dislocation intermediates, which facilitates dissociation of the ERAD substrates from p97 and eventually, targeting to the proteasome [21–23]. However, USP19 seems to have the opposite effect on MARCH6, the degradation of which depends on p97 (Fig. 3). Since the expression levels of enzymes often reflect their enzymatic activity, we speculate that USP19 may participate in the control of the ERAD pathway by regulating the stability, and possibly also activity, of MARCH6. In support of this hypothesis, we showed that ABCB11G238V was stabilized by the USP19 knockdown that reduced MARCH6 expression (Fig. 4). We showed that the USP19 DUB activity catalyzes the endoproteolytic processing that effects cleavage just after the internal, highly conserved Gly433–Gly434 sequence. Mutational analysis revealed that the Gly433 and Gly434 residues are critical for this processing (Fig. 5D), which resembles the phenomenon observed in the processing of the ubiquitin precursor proteins by DUBs in order to generate the free ubiquitin. Given that mutations in other Gly–Gly sequences did not compromise the processing of USP19 (Fig. 5E), the Gly433–Gly434 sequence may be the most accessible to the DUB catalytic center at the level of the tertiary structure. USP1 has also been reported to undergo self-processing by cleavage after a Gly–Gly sequence. In the case of USP1, a potential ubiquitin-like fold exists immediately upstream of the Gly–Gly sequence, and is thought to facilitate recognition of the cleavage site of USP1 [42]. Although, no such structure exists upstream of the USP19 cleavage site [43,44], the ubiquitin-like (UBL) domain is present in a region downstream, between the DUB catalytic core motifs (Fig. 5C). The UBL domain is structurally homologous to ubiquitin and is present in several USP family members [43]. The UBL domain might be involved in the recognition and cleavage of the Gly433–Gly434 sequence. The cleaved N-terminal region of USP19 contains two CHORD-Sgt1 (CS) domains (Fig. 5C). The CS domain is related to the HSP90 co-chaperone p23 and is proposed to mediate protein–protein interaction [45]. Although the role of the CS domains of USP19 is still unclear, the N-terminal region has been shown to bind to several different substrates, including c-IAPs and HIF-1α [32,39]. Such self-processing might be a mechanism for controlling substrate binding. However, MARCH6 stabilization was not affected by the abolishment of selfprocessing (Fig. 5H). At this point, we do not know how the processing of USP19 is regulated. The pulse-chase experiments showed that the amount of the cleaved fragment was increased over time, suggesting that this processing occurs continuously (Fig. 5F). The proteolytic processing of other DUBs has been shown to occur in response to various stimuli: 1) ultraviolet irradiation induces autocleavage of USP1 [42] and 2) USP7 is targeted for caspase-dependent proteolysis during apoptosis in thymocytes [46]. It might be that ER stress and muscle atrophy act on the processing of USP19 in a manner similar to that of the activation of the expression of the USP19 gene [26,27]. In conclusion, we have identified MARCH6 as a novel substrate for USP19, and have investigated the unresolved mechanism for

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the regulation of the protein expression and activity of MARCH6. The proposed mechanism suggests the rescue of MARCH6 from p97-mediated proteasomal degradation. USP19 may have an important role in the regulation of ERAD and ER protein quality control. Further investigation is needed to elucidate the physiological significance of USP19-mediated stabilization of MARCH6, and also to identify novel substrates and regulatory factors.

Acknowledgments We thank Yoko Yamamoto and Ayako Takada for DNA sequencing, and Yuriko Ishii and Tomoko Okada for secretarial assistance. This work was supported by Grants-in-Aid for Scientific Research 21026010, 22370029, 22770123, and 24570209 and the 21st Century and Global Center of Excellence Program of the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Pacific Edit reviewed the manuscript prior to submission.

Appendix A.

Supporting information

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.yexcr.2014.07.025.

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