Facilitated Tau Degradation by USP14 Aptamers via Enhanced Proteasome Activity

Abstract

The ubiquitin-proteasome system (UPS) is the primary mechanism by which intracellular proteins, transcription factors, and many proteotoxic proteins with aggregation-prone structures are degraded. The UPS is reportedly downregulated in various neurodegenerative disorders, with increased proteasome activity shown to be beneficial in many related disease models. Proteasomes function under tonic inhibitory conditions, possibly via the ubiquitin chain-trimming function of USP14, a proteasome-associated deubiquitinating enzyme (DUB). We identified three specific RNA aptamers of USP14 (USP14-1, USP14-2, and USP14-3) that inhibited its deubiquitinating activity. The nucleotide sequences of these non-cytotoxic USP14 aptamers contained conserved GGAGG motifs, with G-rich regions upstream, and similar secondary structures. They efficiently elevated proteasomal activity, as determined by the increased degradation of small fluorogenic peptide substrates and physiological polyubiquitinated Sic1 proteins. Additionally, proteasomal degradation of tau proteins was facilitated in the presence of the UPS14 aptamers in vitro. Our results indicate that these novel inhibitory UPS14 aptamers can be used to enhance proteasome activity, and to facilitate the degradation of proteotoxic proteins, thereby protecting cells from various neurodegenerative stressors.

Full text

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SCIENTIFIC RepoRts | 5:10757 | DOI: 10.1038/srep10757
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Facilitated Tau Degradation by
USP14 Aptamers via Enhanced
Proteasome Activity
Jung Hoon Lee1, Seung Kyun Shin2, Yanxialei Jiang2, Won Hoon Choi1, Chaesun Hong3,
Dong-Eun Kim3 & Min Jae Lee1
The ubiquitin-proteasome system (UPS) is the primary mechanism by which intracellular proteins,
transcription factors, and many proteotoxic proteins with aggregation-prone structures are degraded.
The UPS is reportedly downregulated in various neurodegenerative disorders, with increased
proteasome activity shown to be benecial in many related disease models. Proteasomes function
under tonic inhibitory conditions, possibly via the ubiquitin chain-trimming function of USP14, a
proteasome-associated deubiquitinating enzyme (DUB). We identied three specic RNA aptamers of
USP14 (USP14-1, USP14-2, and USP14-3) that inhibited its deubiquitinating activity. The nucleotide
sequences of these non-cytotoxic USP14 aptamers contained conserved GGAGG motifs, with G-rich
regions upstream, and similar secondary structures. They eciently elevated proteasomal activity, as
determined by the increased degradation of small uorogenic peptide substrates and physiological
polyubiquitinated Sic1 proteins. Additionally, proteasomal degradation of tau proteins was facilitated
in the presence of the UPS14 aptamers in vitro. Our results indicate that these novel inhibitory
UPS14 aptamers can be used to enhance proteasome activity, and to facilitate the degradation of
proteotoxic proteins, thereby protecting cells from various neurodegenerative stressors.
Proteasomes are the primary proteolytic machinery used by cells for homeostasis of various regulatory
proteins. is catabolic process is mainly mediated by ubiquitin (Ub)-dependent proteolysis, but when the
proteins are intrinsically disordered proteins, they can be degraded by proteasomes in a Ub-independent
manner1. e 26S proteasome is a multimeric complex, composed of a 28-subunit core particle (CP or
20S complex) and a 19-subunit regulatory particle (RP, PA700, or 19S complex)2. e RP recognizes the
polyubiquitin (polyUb) chains of its substrates, deubiquitinates them before proteasomal proteolysis is
initiated, and translocates the substrates to the catalytic cavity of the CP.
e ubiquitin-proteasome system (UPS) appears to have its own quality control mechanisms, one
of which is accomplished by the proteasome-associated deubiquitinating enzymes (DUBs) USP14 and
UCH373. UPS14 interacts with RPN1, while UCH37 binds to ADRM1/RPN134,5. Both are located on
the RP, relatively far from the substrate entry pore and the Ub receptors RPN1 and RPN10. UPS14
and UCH37 are thought to mediate stepwise disassembly of the Ub chain from the distal end6. is
“chain-trimming” eect can delay proteasomal degradation by weakening the interaction between the
Ub receptors of the proteasomes, and the polyUb chains of the substrates. Deletion of the USP14 gene,
or chemical treatment with USP14 inhibitors, results in accelerated proteasomal degradation of various
target substrates7. ese ndings suggest that USP14 is a potential therapeutic target for treating diseases
where toxic proteins accumulate. However, it has also been reported that the trimming of Ub chains
might promote proteasomal degradation8. e mechanism regulating this remains to be elucidated, but
1Department of Biochemistry and Molecular Biology, Neuroscience Research Institute, Seoul National University
College of Medicine, Seoul 110-799, Republic of Korea. 2Department of Applied Chemistry, College of Applied
Sciences, Kyung Hee University, Yongin 446-701, Republic of Korea. 3Department of Bioscience and Biotechnology,
Konkuk University, Seoul 143-701, Republic of Korea. Correspondence and requests for materials should be
addressed to D.-E.K. (email: kimde@konkuk.ac.kr) or M.J.L. (email: minjlee@snu.ac.kr)
Received: 15 September 2014
Accepted: 28 April 2015
Published: 04 June 2015
OPEN

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SCIENTIFIC RepoRts | 5:10757 | DOI: 10.1038/srep10757
probably involves the rate of Ub chain-trimming on the proteasome in coordination with substrate trans-
location.
Aptamers are molecules composed of single-stranded nucleic acids (15–50 bases) that have been
generated by an in vitro selection process from a large pool of random sequences. is technique is
known as systemic evolution of ligands by exponential enrichment (SELEX)9,10. Since the introduction
of SELEX technology, a wide range of biological targets, including small molecules, peptides, proteins,
nucleic acids, cells, tissues and organisms, have been reported to bind to aptamers with high specicity.
Aptamers are oen referred to as “chemical antibodies,” and are one of only a few classes of biomolecules
that can be manufactured to bind to multiple dierent targets. RNA aptamers have been isolated and
shown to have stable conformations in vivo, following some modication. ey can specically bind
to proteins such as human immunodeciency virus Tat11, reverse transcriptase12, hepatitis C virus NS3
protease/helicase13,14, NS5B RNA-dependent RNA polymerase15, and severe acute respiratory syndrome
NTPase/helicase16. In addition, RNA aptamers can bind to prostate cancer cells through the extracellular
portion of the prostate-specic membrane antigen17, and to brain tissue18.
Aptamers that bind to specic proteins can repress the enzymatic activity of those proteins or
protein-protein interactions. e active sites or interacting motifs usually oer more exposed heteroa-
toms, which mediates hydrogen bonds or other strong interactions with the aptamers19. For therapeutic
applications, inhibitory aptamers are oen chemically modied to be resistant to degradation mediated
by serum. e age-related macular degeneration drug pegaptanib is a 27-nt aptamer that targets vascular
endothelial growth factor. It is conjugated with 40 kDa polyethylene glycol and contains inverted nucle-
otides at the 3′ terminus20. Considering the rapid progress in aptamer biology and related technologies,
aptamers are now considered essential for understanding and modulating various pathophysiological
processes.
To overcome the limitation of small-molecule USP14 inhibitors, we identied three novel USP14-binding
RNA aptamers that suppressed the deubiquitinating activity of USP14 in vitro. Consistent with the eects
of USP14 upon the proteasome, these USP14 aptamers enhanced proteasome activity, and facilitated the
degradation of Alzheimer’s disease (AD)-implicated tau proteins. e inhibitory USP14 aptamers were
non-cytotoxic and eectively relieved proteopathic stress in cultured cells. erefore, UPS14 aptamers
could oer an interesting alternative to delay the aggregation process of toxic, aggregation-prone pro-
teins.
Methods
Purication of recombinant USP14 proteins. We transformed pGEX-2T, pGEX-2T-USP14, and
pGEX-2T-USP14(C114A) into Escherichia coli strain BL21 (DE3). We puried USP14 and USP14(C114A)
and their glutathione S-transferase (GST)-conjugated counterparts [GST-USP14 and GST-USP14(C114A),
respectively] using previously described methods7, with some modications. Cultures were incubated at
37 °C, and when the optical density at 600 nm (OD600) was 0.6–0.8, we added IPTG to each culture
(nal concentration of 1 mM), and allowed cultures to incubate overnight at 25 °C. Cells were harvested
in phosphate-buered saline (PBS) containing protease inhibitor cocktails and lysed by sonication.
Following centrifugation, lysates were ltered and supernatants incubated with GST sepharose 4B resin
(GE Healthcare, Little Chalfont, UK) at 4 °C for 1 h. Aer washing with PBS, GST-USP14 was eluted using
10 mM reduced glutathione (50 mM Tris-HCl pH 8.0). e GST tag was removed by incubating protein
fractions with thrombin in a cleavage buer [50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2.5 mM CaCl2, and
0.1% (v/v) 2-mercaptoethanol] for 3 h at room temperature (RT). Aer centrifugation, supernatants were
incubated with benzamidine sepharose resin (GE Healthcare) to remove any residual thrombin. Puried
GST (data not shown), GST-USP14, GST-USP14(C114A), USP14, and USP14(C114A) were separated
by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and gels were stained with
Coomassie Brilliant Blue R-250 (CBB) to determine the size and purity of proteins (Fig.1A).
In vitro selection of RNA aptamers. A random RNA library was generated as previously described21
using polymerase chain reaction (PCR), T7 in vitro transcription, and a DNA template (109 bp) con-
taining 40 random nucleotides. Briey, the aptamer library template DNA (5′ -GGG TTC ACT GCA
GAC TTG ACG AAG CTT -40 N-A ATG GAT CCA CAT CAT CTA CGA ATT C-3′ ) was generated
by PCR using a forward primer (5′ -GAT AAT ACG ACT CAC TAT AGG GTT CAC TGC AGA CTT
GAC GAA-3′ ) containing the T7 promoter sequence (underlined) and a reverse primer (5′ -TTA CCT
AGG TGT AGA TGC TTA AG-3′ ) (Fig. 1B). e RNA aptamer library was prepared using T7 RNA
polymerase in in vitro transcription buer (50 mM Tris-HCl pH 7.5, 15 mM MgCl2, 2 mM spermidine,
5 mM DTT, and 2 mM NTPs) at 37 °C for 4 h. Products were treated with DNase I (ermo Scientic)
at 37 °C for 30 min, subjected to 12% denaturing urea PAGE using, and puried from gels. A 6-μ g
aliquot of each generated RNA pool was incubated with 100 μ L of GST sepharose 4B resin in binding
buer [30 mM Tris-HCl pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 2 mM DTT, and 1% (w/v) bovine serum
albumin (BSA)] for 30 min at RT with occasional shaking. e RNA-bead complexes were transferred
to porous centrifuge columns (Pierce), and RNA pools that were unbound to sepharose resin were col-
lected by centrifugation. e same procedure was performed using 2 μ g of GST proteins to remove
RNAs that were nonspecically bound to GST or glutathione resin. During each round of selection,
GST-tagged proteins were incubated with 6 μ g of pre-cleared RNA pools in 100 μ L of binding buer

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SCIENTIFIC RepoRts | 5:10757 | DOI: 10.1038/srep10757
for 30 min at RT, and then with 100 μ L of GST sepharose 4B resin for an additional 30 min at RT. e
quantity of GST-tagged proteins was gradually decreased (2 μ g, rounds 2–8; 1 μ g, round 9; 0.5 μ g, rounds
10–11; 0.25 μ g, rounds 12–13; and 0.125 μ g, rounds 14–15) to achieve more stringent conditions. e
RNA-GST-USP14 complexes were then washed three times with PBS, and eluted with 20 mM glutathione
in binding buer. Eluted RNAs were puried by phenol:chloroform extraction and ethanol precipitation,
reverse-transcribed with AccuPower RT PreMix (Bioneer), and amplied by PCR. e amplied cDNA
was puried using the AccuPrep Gel purication kit (Bioneer) and transcribed with T7 RNA polymerase
for the next round of selection (Fig.1C).
Figure 1. Purication of USP14 and human proteasomes, preparation of vme-proteasomes, and SELEX
for USP14 aptamers. (A) Approximately 1 μ g of puried recombinant USP14, USP14(C114A), GST-USP14,
and GST-USP14(C114A) was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) and Coomassie Brilliant Blue (CBB) staining. Bovine serum albumin (BSA) was used as a standard.
(B) RNA sequences used for in vitro selection. e random RNA library contained random 40-nt sequences,
anked by a 3’ region (23 bp) and a sequence containing the T7 promoter (46 bp, underlined). (C) Scheme
for the SELEX strategy. (1) Puried RNAs were incubated with GST-USP14. (2) USP14-RNA complexes
were captured by glutathione agarose beads. (3) Unbound RNA molecules were removed by centrifugation.
(4) USP14-RNA complexes were dissociated with elution buer containing excess imidazole. (5) RNAs
bound to USP14 were prepared by phenol:chloroform extraction and ethanol precipitation. Recovered
RNAs were reverse transcribed, amplied by polymerase chain reaction (PCR), and in vitro transcribed.
(6) Following 15 rounds of selection, the resultant cDNA was amplied and cloned into pcDNA 3.1. (D)
Human proteasomes (PTSM) were puried from β 4-tagged HEK293 cell lines. Some of the proteasomes
were treated with 1 μ M ubiquitin-vinylmethylester (Ub-vme) to yield vme-PTSMs. PTSMs (1 μ g) and vme-
PTSMs (1 μ g) were separated on 12% gradient gels and stained with CBB. (E) Western blotting analysis of
USP14 and proteasome subunit α 3. Approximately 250 ng of PTSMs or vme-PTSMs were subjected to SDS-
PAGE. Cropped gels/blots are used in (E).

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Cloning and sequencing. Aer 15 rounds of selection, the resulting cDNA was PCR-amplied using
forward (5′ -GAT AAT ACG ACT CAC TAT AGG GTT CAC TGC AGA CTT GAC GAA-3′ ) and reverse
(5′ -TTA CCT AGG TGT AGA TGC TTA AG-3′ ) primers. Amplicons were digested with HindIII and
EcoRI, and cloned into the pcDNA 3.1 vector (Invitrogen) for sequencing. e secondary structure of
selected RNA aptamers was predicted by the Mfold program based on the Zuker algorithm (http:\\mfold.
rna.albany.edu). For subsequent activity testing and inhibition assays, individual RNA aptamers were
amplied by PCR using forward (5′ -ATT AAT ACG ACT CAC TAT AGG G-3′ ) and reverse (5′ -TTA
CCT AGG TGT AGA TGC TTA AG-3′ ) primers, and in vitro transcribed using T7 RNA polymerase. We
used 40-nt sequences that were irrelevant to USP14 aptamers as controls (UUU GUC UAG CGC GUA
GUG GGG AGA UGU UGU GAU ACU GGG G).
Measurement of RNA aptamer binding to USP14. We loaded and immobilized 2 pmol of
GST-USP14 or GST onto a GST sepharose 4B resin, and then added 20 pmol of RNA aptamers. e
RNA eluted from the resin was reverse-transcribed and the resulting cDNA subjected to PCR. Agarose
gel electrophoresis, followed by ethidium bromide staining, was carried out to visualize protein-bound
RNA in 2% (w/v) agarose gels. For cloning, we used primers USP14-1 mutant (5′ -GCA GTG ATG TGC
TTC TAA AAA ACA ACC TAA AAA AAT TGC C-3′ ), USP14-2 mutant, (5′ -CTG AAA AAA AGT
TAG TTT CGC TGG TTT AAA ATC GGT GCG G-3′ ), and USP14-3 mutant (5′ -AAA AAA AAG GCT
CGT TTG GCC TGC CGA AAA AAG GCC GGG A-3′ ). e USP14 aptamer mutants were generated by
insertion of corresponding oligonucleotides into the pcDNA 3.1 vector, amplied by PCR using the same
forward and reverse primers, and in vitro transcribed using T7 RNA polymerase as described above.
Purication of human proteasomes and preparation of vinylmethylester-proteasomes.
Human proteasomes were puried by anity chromatography from a stable HEK293 cell line harbor-
ing biotin-tagged human β 4 as previously described7, with some modications. Cells were cultured in
15-cm dishes, harvested in lysis buer [50 mM NaH2PO4 pH 7.5, 100 mM NaCl, 10% glycerol, 5 mM
MgCl2, 0.5% NP-40, 5 mM ATP, and 1 mM DTT] containing protease inhibitors, and homogenized using
a Dounce homogenizer. Aer centrifugation, supernatants were incubated with streptavidin agarose resin
(Millipore) for 5 h at 4 °C. Beads were washed with lysis buer and TEV buer (50 mM Tris-HCl pH
7.5, 1 mM ATP, and 10% glycerol). e 26S proteasome was eluted from the resin by incubating with
TEV protease (Invitrogen) in TEV buer for 1 h at 30 °C, and concentrated using an Amicon ultra-spin
column (Millipore). To inhibit the deubiquitinating activity of proteasomes, ubiquitin-vinylmethylester
(Ub-vme; LifeSensors) was added as previously described22. Residual Ub-vme was removed by wash-
ing the beads three times with 50 bed volumes of washing buer. Proteasomes (240 ng) were sepa-
rated by SDS-PAGE using a 4–20% gradient gel. Gels were stained using the EzWay silver staining kit
(Koma Biotech) or CBB. e obtained proteasomes, which we designated vme-proteasomes, were tested
to conrm the elimination of deubiquitinating activity using the Ub-rhodamine 110 hydrolysis assay
(Ub-rho110, LifeSensors) (data not shown).
USP14 binding to proteasomes. e eect of USP14 aptamers on the interaction of USP14 with the
proteasome was investigated using proteasome anity pulldown assays. Aer HEK293 cells were har-
vested in lysis buer, cell lysates were homogenized using a 1 mL syringe with a 26G × 1/2′ ′ needle. Aer
centrifugation, supernatants were incubated with RNA aptamers for 10 min, and then with streptavidin
agarose resin (Millipore) for 5 h at 4 °C. Beads were washed with lysis buer and boiled in SDS sample
buer. e binding of USP14 to the 26S proteasome was monitored by western blotting using an anti-
body against USP14 (Bethyl Laboratories, Inc).
Activity of proteasomes and DUBs. Based on the hydrolysis of uorogenic succinyl-Leu-Leu-Val-Tyr-
7-amido-4-methylcoumarin (suc-LLVY-AMC) peptides, the chymotrypsin-like activity of proteasomes
was measured to determine their proteolytic activity. A suc-LLVY-AMC hydrolysis assay was carried out
using puried proteasome and 12.5 μ M suc-LLVY-AMC (Enzo Life Sciences) in assay buer (50 mM
Tris-HCl pH 7.5, 1 mM EDTA, 1 mg/mL BSA, 1 mM ATP, and 1 mM DTT). e Ub-rho110 hydrolysis
reaction was carried out using proteasomes or USP14 activated by vme-proteasomes, along with 20 nM
or 100 nM Ub-rho110 in the presence or absence of 1 μ g/mL (equivalent to 33 nM) RNA aptamers. RNA
aptamers were incubated with proteasomes for 5 min before they were added to substrates. To examine
the eects of RNA aptamers on USP14, RNA aptamers were incubated with USP14 for 5 min, then with
vme-proteasomes for 5 min, before they were added to substrates. UCHL3 and USP47 were kindly pro-
vided by Eunice Eun-Kyeong Kim; USP5 was provided by Kyeong Kyu Kim. Proteasomal activity and
deubiquitinating activity were monitored by measuring free AMC or rho110 uorescence, respectively,
in black 96-well plates using a TECAN innite m200 uorometer.
Kinetic analysis of USP14-mediated deubiquitinating activity. For kinetic analysis, the
Ub-rho110 hydrolysis activity of USP14 on USP14 aptamers was monitored over a concentration course.
Normalization of uorescence intensity to the concentration of rhodamine 110 was achieved using a free
rhodamine 110 standard curve (Santa Cruz Biotechnology). e KM and kcat parameters were determined

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by nonlinear regression t, with a model derived from the Michaelis-Menten equation, using GraphPad
Prism 5 (GraphPad Inc., USA)
Ubiquitination of recombinant Sic1 and tau. Polyubiquitinated Sic1 with PY motifs (Ub-Sic1) was
prepared as previously described23, with some modications. e Ub conjugation reaction was conducted
by incubating 10 pmol of Sic1PY, 2 pmol of Uba1, 5 pmol of Ubc4, 5 pmol of Rps5, and 1.2 nmol of Ub
in a buer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM DTT, 5 mM ATP, and 10 mM MgCl2) for 4 h
at 25 °C. Conjugates were absorbed to a Ni-NTA resin (Qiagen, Germany), washed with buer (50 mM
Tris-HCl pH 8.0, 50 mM NaCl, and 40% glycerol), eluted with 200 mM imidazole in wash buer, and dia-
lyzed into wash buer containing 10% glycerol. Recombinant tau and CHIP proteins were expressed and
puried from pET29b vectors using conventional purication methods. Unphosphorylated tau (0.5 μ g)
was incubated with 200 ng of Uba1, 4 μ g of UbcH5b, 2 μ g of CHIP, 1 μ g of HSP70, and 10 μ g of Ub for
2 h at RT in a 100-μ L reaction.
In vitro degradation assays. Puried human proteasomes (5 nM) were incubated with 20 nM
Ub-Sic1 or Ub-tau in proteasome assay buer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10% glycerol,
2 mM ATP, 10 mM MgCl2, and 1 mM DTT). In certain instances, USP14 was incubated with proteasomes
for 5 min before reactions were initiated. To examine the eects of RNA aptamers on in vitro degrada-
tion assays, aptamers were incubated with USP14 for 5 min before USP14 was added to proteasomes.
Reactions were terminated by adding 2 × SDS-PAGE sample buer and subjected to SDS-PAGE. Ub-Sic1
and Ub-tau degradation was monitored by immunoblotting, using antibodies against T7 (Millipore) and
tau (clone Tau-5, Invitrogen), respectively.
Cell culture and RNA aptamer transfection. All cells in this study were cultured in Dulbecco’s
modied Eagle’s medium supplemented with 10% fetal bovine serum. USP14 aptamer transfection was
performed in 6-well plates using Lipofectamine 2000 (Invitrogen). Cells were transfected for 6 h and
whole cell extracts were collected 24 h aer the media was changed.
Assessment of cell viability. Cell viability was assessed using a modied 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) assay24. HeLa cell cultures that were approximately 90% conu-
ent in 96-well plates were treated with control or USP14 aptamers complexed with Lipofectamine 2000
at various concentrations (up to 50 μ g/mL) for 4 h. We then added 10 μ L of MTT solution to each well
and incubated plates at 37 °C/5% CO2 for 2.5 h. Media was discarded and 200 μ L of dimethyl sulfoxide
was added to each well and plates were incubated for 30 min at RT. e absorbance of the solution at
570 and 630 nm was determined. Triplicate wells were assayed for each condition. Cell survival was also
assessed when tau was induced by Dox, and when oxidative stress was induced by paraquat. Inducible
tau cell lines were incubated with Dox (250 pg/mL) and paraquat (1 mM) for 3 h and then transfected
with USP14 aptamers.
Tau degradation assays. An inducible tau cell line (HEK293-trex-htau40)25 was transfected with
USP14 aptamers and treated with various concentrations of Dox. Aer 24 h, whole-cell lysates were pre-
pared in RIPA buer and used for immunoblotting. Where necessary, cells were treated with 80 μ g/mL
cycloheximide (CHX) (Enzo Life Sciences) before harvesting. For SDS-PAGE, each lane was loaded with
the extract from an equal cell number, generally corresponding to 10-20 μ g/lane, or 1/10 of the sample
recovered from one well of a 6-well plate.
Results
Preparation of recombinant USP14 proteins and selection of RNA aptamers. We p r e v i -
ously showed that yeast and mammalian USP14, a proteasome-associated DUB enzyme, functions as
an endogenous inhibitor of proteasomes6,7. e Ub chain-trimming eect of USP14 might antagonize
the degradation of many proteasome substrates, with small molecule inhibitors facilitating proteolysis.
However, small molecule inhibitors exhibit relatively high cytotoxicity and low inhibitory ecacy7. To
identify USP14 aptamers, we used SELEX techniques with GST-USP14 (78 kDa), while USP14 (53 kDa)
was also expressed and puried for subsequent analysis with aptamers and proteasomes (Fig.1A).
To select for RNA aptamers with a high anity that were specic to USP14, we generated a pool
of RNAs (approximately 1014 molecules) containing a 40 nt random core sequence that was anked by
the T7 promoter site at the 5′ end and primer-binding sites at the 3′ end (Fig. 1B)16. Prior to initial
screening, we performed negative selection over 15 rounds to remove non-specic RNAs bound to the
glutathione-sepharose resin or to GST proteins from the RNA pool (Fig. 1C). e stringency of RNA
binding to the USP14 protein was increased as the rounds progressed (see Methods).
Human 26S proteasomes were puried from a HEK293-derived cell line that stably expressed
polyhistidine- and biotin-tagged β 4 subunits26. Puried proteasomes showed an approximate 1:1 molar
ratio between the RP and CP complexes (Fig.1D). Basal deubiquitinating activity of USP14 is strongly
activated by proteasomes, although the subunit responsible for activation remains to be elucidated4,21.
e majority of proteasomal deubiquitinating activity was irreversibly blocked using Ub-vme5, which
forms an adduct with Cys in the active site of the thiol protease DUB. e resulting vme-proteasomes

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SCIENTIFIC RepoRts | 5:10757 | DOI: 10.1038/srep10757
appeared to retain the components of normal 26S proteasomes (Fig.1D). Large amounts of USP14 were
co-puried with the RP of proteasomes (Fig.1E). When covalently modied by Ub-vme, the electropho-
retic mobility of USP14 was reduced by 7.5 kDa, which corresponded to the size of Ub (Fig.1E), which
indicates that the DUB enzymes on the proteasomes, including USP14, were fully inactivated.
Identication of USP14 aptamers and their inhibitory eects on deubiquitinating activ-
ity. We examined the RNA sequences of 20 randomly selected clones, and identied three dierent
aptamers. ese RNA aptamers were designated USP14-1, USP14-2, and USP14-3, and represented 30
(6/20), 45 (9/20), and 25% (5/20) respectively, of the identied aptamers (Fig.2A). All of the aptamers
contained conserved sequences and similar structural motifs, as predicted by Mfold27. In addition to two
stem-loops in their secondary structures (Fig. 2B), there was a sequence of ve conserved nucleotides
Figure 2. USP14-specic aptamers, predicted secondary structures, and inhibition of USP14
deubiquitinating activity. (A) ree dierent sequences of RNA aptamers were identied from the random
RNA pool by SELEX. All sequences contained a conserved GGAGG motif (red) and a G-rich region (grey).
(B) e secondary structures of the USP14 aptamers were calculated using Mfold. (C) Validation that the
USP14 RNA aptamers bound to USP14. Control, 40 nt RNA oligonucleotides containing a sequence that
was irrelevant to USP14 aptamers. (D) Ub-rho110 hydrolysis by recombinant USP14 or USP14(C114A).
Prior to activation by vme-PTSM (1 nM), various concentrations of USP14 proteins were pre-incubated
with RNA aptamers (1 μ g/mL). As a control, 1 μ g/mL random RNA pools were used. Note that USP14 alone
(without activation by proteasomes) showed only basal Ub-rho hydrolysis activity. (E) Ub-rho110 hydrolysis
by various deubiquitinating enzymes (DUBs) in the presence or absence of USP14 aptamers (1 μ g/mL). (F)
Ub-rho110 hydrolysis as in (D), except that USP14-3, at various concentrations, was incubated with USP14
(10 nM) or USP14(C114A) (10 nM) and vme-PTSM (1 nM). (G) e deubiquitinating activity of USP14 was
monitored by Ub-rho110 (20 nM) hydrolysis in the presence of PTSMs and 1 μ g/mL USP14 aptamers. e
original RNA aptamer library (random RNA pool) was used as a control. All data represent the mean ± SD
from three independent experiments. *p < 0.01 (one-way analysis of variance ANOVA with Bonferroni’s
multiple comparison test). RFU, relative uorescence unit. PTSM, 26S human proteasomes. vme-PTSM, Ub-
vme-treated 26S human proteasomes.

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SCIENTIFIC RepoRts | 5:10757 | DOI: 10.1038/srep10757
(GGAGG), followed by a G-rich sequence. Our results suggest that the exposed GGAGG and G-rich
sequences in RNA aptamers could be important for interactions with USP14.
None of the USP14 RNA aptamers was found to bind to GST-tagged proteins from the binding assay
similar to the SELEX strategy (Fig.2C), however they all exhibited a high anity to USP14. e con-
trol RNA aptamers did not bind to USP14 (Fig.2C). Mutations in the consensus GGAGG and G-rich
sequences in the USP14 RNA aptamers abolished their ability to bind to USP14 (Fig.2C). ese ndings
suggest that the exposure of GGAGG and G-rich sequences in RNA aptamers is critical for their inter-
action with USP14.
To investigate the eects of USP14 aptamers on USP14-mediated deubiquitinating activity, the
RNA aptamers were transcribed and puried in vitro. We used recombinant USP14 proteins and
vme-proteasomes for hydrolysis of Ub-rho110, a highly sensitive uorogenic substrate of DUBs. In the
absence of vme-proteasomes, USP14 exhibited little deubiquitinating activity. In contrast, USP14 was
activated in the presence of vme-proteasomes, exhibiting higher Ub-rho110 hydrolysis activity than
for vme-proteasomes alone (Fig. 2D). e addition of USP14-1, USP14-2, or USP14-3 to the reaction
resulted in a decrease in USP14 deubiquitinating activity (Fig.2D and Supplementary Fig. 1). Inhibition
of deubiquitinating activity by USP14 aptamers was specic to USP14, with the USP14-3 aptamer failing
to inhibit the DUB activity of UCHL3, USP47, USP5, and UCHL5/UCH37 (Fig.2E). USP14-3 inhibited
USP14 deubiquitinating activity in a dose-dependent manner (Fig.2F and Supplementary Fig. 2). e
catalytically inactive mutant of USP14, USP14(C114A), exhibited little deubiquitinating activity and was
unaected by the USP14 aptamers (Fig. 2D,F), indicating that the USP14 aptamers specically inhibit
USP14 DUB activity.
Using Ub-rho110, we also measured the enzyme kinetics of recombinant USP14 with vme-proteasomes
in the presence and absence of the aptamers (Supplementary Fig. 3). e KM of USP14 bound to the
aptamers (1716 ± 300 nM) was signicantly higher than that for free USP14 (1124 ± 248 nM). However,
kcat values were comparable regardless of whether USP14 aptamers were present (0.948 ± 0.079 s−1 for
USP14-3 vs. 0.938 ± 0.073 s−1 for control RNA aptamers), suggesting that the USP14 aptamers might
aect substrate binding to USP14 to a greater extent than deubiquitinating activity. Further work will be
required to determine the mechanism of inhibition of the USP14 aptamers. Our results strongly indi-
cate that the newly identied USP14 aptamers eectively inhibit the deubiquitinating activity of USP14
in vitro.
We tested whether the total deubiquitinating activity of the proteasome was aected by the three
USP14 aptamers. Proteasomes that were not treated with Ub-vme showed signicant deubiquitinating
activity, possibly from USP14 and UCH37, another proteasomal DUB that interacts with RPN133 (Fig.2G
and Supplementary Fig. 4). e enzymatic redundancy between these two enzymes on the proteasome
is unclear, but is considered insignicant because UCH37 exhibits much weaker Ub hydrolysis activ-
ity than USP147. Addition of the USP14 aptamers strongly inhibited proteasome-mediated Ub-rho110
hydrolysis (Fig.2G and Supplementary Fig. 4). USP14-3 showed the strongest inhibitory activity for all
tested proteasome concentrations. e strong inhibition exhibited by USP14-3 could be a result of its
ability to simultaneously bind to UCH37 (data not shown). is would indicate that UCH37 plays a
more signicant role in deubiquitinating polyubiquitinated substrates than previously thought3. Further
study is required to determine whether chain-trimming by UCH37 can suppress proteasome activity
as eectively as USP14. Taken together, our results provide strong evidence that USP14 RNA aptamers
block the activity of USP14 and proteasomal DUBs.
RNA aptamers accelerate proteasomal degradation of substrates in vitro. Our results suggest
that the RNA aptamers bind to USP14 and inhibit its deubiquitinating activity. We previously reported
that inhibition of USP14’s Ub-chain trimming eects might enhance the proteolytic activities of protea-
somes in vitro and in vivo7. To explore the possibility that inhibitory USP14 aptamers might enhance
proteasome activity, we used suc-LLVY-AMC, a uorogenic reporter substrate of 26S proteasomes. e
uorescent intensity as a result of suc-LLVY-AMC hydrolysis gradually increased over time (Fig.3A and
Supplementary Fig. 5). Proteasomal activity was higher when the USP14 RNA aptamers were present
than when control aptamers were used, indicating that inhibiting USP14 with an RNA aptamer results in
facilitated proteasomal degradation. Potentially USP14 aptamers inhibit USP14 by preventing its docking
on the proteasome. However, direct pulldown assays indicated that the scenario is negative (Fig. 3B),
indicating that the inhibitory eect by the RNA aptamer is mainly on USP14’s DUB activity.
We also used physiologically relevant polyubiquitinated proteins to simultaneously monitor prote-
olysis and Ub chain-trimming. Sic1, a cyclin-dependent kinase inhibitor in Saccharomyces cerevisiae,
was polyubiquitinated using UBA1, UBC4, and RSP5 as cognate E1, E2, and E3 enzymes, respectively,
along with other reconstitution components such as Ub and ATP23. e resulting Ub-Sic1 was gradually
degraded when puried proteasomes were added. Addition of the proteasome inhibitor MG132 mutant
protein in the reaction delayed Ub-Sic1 degradation (Fig. 3C). Mixing puried proteasomes with cat-
alytically inactive recombinant USP14(C114A) facilitated the degradation of Ub-Sic1. In the control
reaction, approximately 70% of Ub-Sic1 proteins were degraded by proteasomes aer 10 min. However,
in the presence of USP14 aptamers, Ub-Sic1 was almost completely degraded under the same conditions,
showing similar kinetics for proteasomal degradation as USP14(C114A) was added in the reaction. ese

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SCIENTIFIC RepoRts | 5:10757 | DOI: 10.1038/srep10757
results suggest that USP14 aptamers function by inhibiting the catalytic activity of USP14, representing
a unique biochemical and therapeutic potential for enhancing proteasome function.
Dysfunction in the UPS is closely related with tau degradation and neurodegeneration in AD28,29. Tau
proteins are thought to be degraded by the UPS, especially during the early phases of tauopathy or AD
progression30. In the human AD brain, proteasome activity is impaired28, and might be related to the
age-dependent decrease of proteasome activity. However, it could also be the consequence of an accumu-
lation of tau protein. To verify that proteasome activation by USP14 aptamers facilitates tau degradation,
we prepared Ub-tau. e ability of proteasomes to degrade Ub-tau was enhanced in the presence of the
USP14 aptamers (Fig.3D,E). ese results strongly suggest that inhibition of USP14 by RNA aptamers
might antagonize Ub chain-trimming on proteasomes, consequently facilitating the degradation of many
UPS substrates.
USP14 aptamers enhanced tau degradation in cultured cells and protected against oxida-
tive stress. We investigated the eects of USP14 aptamers on HeLa cell viability. e small molecule
USP14 inhibitor IU1 exhibited signicant cytotoxicity at 50 μ g/mL (~167 μ M), while the USP14 aptam-
ers exhibited no noticeable cytotoxicity at concentrations up to 100 μ g/mL (Fig. 4A). e eect of the
USP14 aptamers on the proteasomal degradation of tau was examined using a HEK293-derived cell
line expressing the longest isoform of human tau (htau40) upon induction with doxycycline25. ese
cells expressed htau40 in a dose-dependent manner and produced SDS-resistant tau aggregates aer
about 2 days in culture31. When USP14-3 aptamers were transfected into cells, the levels of induced tau
proteins were signicantly decreased in a dose-dependent manner (Fig.4B). e turnover of tau protein
was facilitated by USP14-3 aptamers, as shown in our CHX chase analysis (Fig.4C,D). Under those
Figure 3. USP14 aptamers facilitate proteasomal degradation of polyubiquitinated substrates in vitro.
(A) Proteasome activity was monitored by hydrolysis of the uorogenic substrate suc-LLVY-AMC (12.5 μ M)
in the presence of aptamers (1 μ g/mL). e random RNA pool was used as a control. *p < 0.05, **p < 0.01
(one-way analysis of variance with Bonferroni’s multiple comparison test). (B) e eect of USP14 aptamers
on USP14 binding to the 26S proteasome was determined by immunoblotting aer pulldown assays. (C) In
vitro degradation assays using 20 nM polyubiquitinated T7-Sic1PY (Ub-Sic1), PTSMs (5 nM), recombinant
USP14 or USP14(C114A) (75 nM), and/or MG132 (1 μ M) in the presence or absence of USP14-3 (20 μ g/
mL). Reaction mixtures at the indicated times were analyzed by SDS-PAGE and western blotting using
antibodies against T7. Relative quantitation results are shown below. (D) In vitro Ub-tau degradation assay
using PTSMs without USP14 reconstitution in the presence of USP14-3 (20 or 40 μ g/mL). Ub-tau proteins
were analyzed by SDS-PAGE and western blotting using antibodies against tau. Long exp, long exposure.
Short exp, short exposure. Cropped gels/blots are used in the loading control panels of (B) and (C). (E)
Quantitation of in vitro tau degradation in the presence or absence of USP14 aptamers. Data represent the
mean ± SD from three independent experiments. *p < 0.05 (paired Student’s t-test).

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conditions, tau mRNA levels were comparable (Fig. 4E), indicating that accelerated tau degradation
occurs post-translationally. e USP14 aptamers reduced cytotoxicity, which was induced by oxidative
stress, in the presence of induced tau (Fig.4F). Treatment with the proteasome inhibitor MG132 reversed
the protective eects of USP14 aptamers. ese results indicate that USP14 aptamer-induced proteasome
activation might protect cells under various stressful conditions, including neurodegeneration. Future
work is necessary to determine whether the RNA aptamers we identied can delay the formation of tau
aggregates in the mouse brain.
Discussion
We identied three RNA aptamers that specically bind to USP14, a proteasome-associated DUB. ese
USP14-specic aptamers eectively inhibited deubiquitinating activity and enhanced proteasome activity
in vitro. Cells treated with the aptamers showed facilitated degradation of soluble tau, delayed accumu-
lation of tau aggregates, and enhanced cellular resistance to proteotoxic stress (Fig.4). Treatment of cells
with IU1 and USP14 aptamers yielded similar results, suggesting that activation of cellular proteasomes
is possible through USP14 inhibition. RNA aptamers have some advantages over small molecules as ther-
apeutic agents, and our ndings suggest that USP14 aptamers could be used in the treatment of diseases
associated with abnormal proteasome function.
Mammalian proteasomes contain three distinct DUBs: RPN11, USP14, and UCH37. RPN11 is a met-
alloprotease, and a constituent component of the RP. USP14 and UCH37 are cysteine proteases that are
reversibly associated with RPN1 and RPN13 respectively, their cognates on the RP. It is thought that
the 26S proteasome regulatory subunit RPN11 promotes substrate degradation, while other DUBs delay
degradation. RPN11 cleaves the base of a polyUb chain and enables the substrate to enter the CP for
Figure 4. USP14 aptamers facilitated tau degradation and protected cells from tau-mediated
cytotoxicity. (A) MTT assays were used to assess the cytotoxicity of USP14-3 aptamers on HeLa cells. (B)
Inducible tau cell lines were treated with doxycycline (Dox, 250 pg/mL) for 24 h and then transfected with
USP14-3 aptamers (0, 25, 50, or 100 nM). Levels of soluble tau proteins were analyzed by SDS-PAGE and
western blotting. (C) Cycloheximide (CHX) chase analysis was used to examine the eects of USP14-3 on
tau degradation. Inducible tau cell lines were transfected with the indicated aptamer (100 nM), treated with
250 pg/mL Dox for 24 h, and then 80 μ g/mL CHX. Random 40 nt RNA oligonucleotides (100 nM) were
used as controls. Cropped gels/blots are used. (D) Quantitation of tau levels in (C), which were normalized
to actin. Data represent the mean ± SD from three independent experiments. (E) Post-translation tau
regulation by USP14 aptamers. Quantitative reverse transcription PCR assays were performed using primers
specic for tau and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Values indicate the means ± SD
from three independent experiments. (F) Tau was induced with 250 pg/mL Dox. Oxidative stress was
induced with 1 mM paraquat for 3 h prior to transfection with USP14-3 or random 40 nt RNA controls
(100 nM each). Values are presented as the mean ± SD from ve independent experiments. *p < 0.01 (one-
way analysis of variance with Bonferroni’s multiple comparison test).

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proteolysis. USP14 and UCH37 trim Ub chains from the distal end of polyUb, thus decreasing the an-
ity of the chain for Ub receptors. USP14 has been reported to be capable of non-catalytically inhibiting
proteasomes32, although the molecular mechanisms responsible for this are unclear.
In general, USP14 and UCH37 ensure that short or non-degradable Ub chains from substrates
are released from the proteasome. e small molecule inhibitor of USP14, IU1, was identied by
high-throughput screening, and found to enhance proteasomal degradation of target substrates in vitro
and in vivo7. In vitro assays using Ub-Sic1 and Ub-tau with USP14 aptamers (Fig.3C,D) indicated that
elevated proteasomal activity might be due to catalytically inhibiting Ub chain-trimming of the protea-
some substrate. e eects of USP14 aptamers on in vitro Ub degradation were more prominent when
proteasomes were saturated with recombinant USP14 than when they were not. e dierent responses
of Ub-Sic1 and Ub-tau to USP14 aptamers suggests that chain-trimming may not be a universal mecha-
nism for regulating the rate of protein turnover. e weak response of tau proteins to USP14 inhibition
suggests that their Ub chains have a higher binding anity to the 26S proteasome compared with those
of Sic123. e USP14 aptamers enhance proteasomal degradation; however, it remains to be determined
what features of Ub substrates, or whether the geometric morphology of polyUb chains aects degra-
dation.
e in vivo stability of RNA aptamers can be improved by chemical modications, while their biodistri-
bution and clearance can be enhanced by conjugation with chemical moieties, such as polyethylene glycol
or cholesterol. Aptamers were rst reported in the early 1990s9 and have received widespread attention as
potential therapeutic agents. e aptamer GB1-10 has been shown to recognize a glioblastoma-associated
tenascin-C isoform, which is a useful marker for disease activity33. e expression of RNA aptamers
against Ku protein in MCF-7 breast carcinoma cells potentially sensitized them to the anticancer drug
etoposide34. e aptamer pegaptanib slows vision loss in people with neovascular age-related macular
degeneration35. It has been approved by the United States Food and Drug Administration, and has been
incorporated into European guidelines. Several other RNA and DNA aptamers have been developed for
use in preclinical and clinical trials19. e USP14 aptamer could be used for modulating proteotoxic
proteins in cells. e essential sequence and structure of USP14 aptamers that mediates the strong inter-
actions with USP14 requires elucidation. is would assist in determining how these aptamers can be
further truncated and modied without adversely aecting their binding activity in vivo.
Enhancing proteasome activity could be a therapeutic strategy to treat diseases caused by the accu-
mulation of damaged and misfolded proteins, such as neurodegenerative diseases and cardiomyopathies.
Li et al. showed that cellular proteasome levels were increased when the 11S proteasome was upregulated
in cardiomyocytes. is enhanced proteasome-mediated removal of oxidized proteins, and protected
cells against cardiac proteinopathy and myocardial ischemia36,37. In the current study, we observed that
inhibiting USP14 with specic aptamers did not appear to alter proteasome levels, but alleviated tau- and
paraquat-induced cytotoxicity. Our results indicate that enhanced proteasome activity can have a bene-
cial eect on cell viability under conditions of general proteotoxic stress. It remains to be determined
whether USP14-specic RNA aptamers, or their modied forms, can accelerate the degradation of other
proteopathic proteins. It was previously shown that IU1 was able to accelerate the degradation of tau,
TDP43, ataxin 3, and glial brillary acidic protein7. e application of USP14 aptamers is not necessarily
limited to cytoprotective roles against toxic protein accumulation. USP14 also plays an essential role in
recycling Ub monomers, and in the dynamic regulation of Ub pools in cells6,38. erefore, USP14 aptam-
ers could be used to understand molecular mechanisms of USP14 activity and Ub homeostasis in cells.
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Acknowledgements
We are grateful to Dain Bang, Ji Hyeon Kim and Dong Hoon Han for their assistance in the screening
experiments. We would also like to thank Kyeong Kyu Kim and Eunice Eun-Kyeong Kim for supplying
us with DUBs. is work was supported by grants from the Disease Oriented Translational Research
(HI14C0202 to M.J.L.) and Korea-UK R&D Collaboration grant (HI14C2036 to M.J.L.) of the Korea Health
Industry Development Institute, the Basic Science Research Program (2013R1A1A2059793 to J.H.L.),
and National Research Foundation grants funded by the Korean Government (2012M3A9B2028336 to
D.-E.K.).
Author Contributions
J.H.L. carried out the majority of the in vitro screening and activity assays. S.K.S., Y.J. and W.H.C.
performed cell-based experiments. S.K.S. and C.H. provided key reagents. D.-E.K. and M.J.L. were
responsible for the overall design and oversight of the project.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Lee, J. H. et al. Facilitated Tau Degradation by USP14 Aptamers via Enhanced
Proteasome Activity. Sci. Rep. 5, 10757; doi: 10.1038/srep10757 (2015).
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