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RESEARCH ARTICLE
The HECT E3 ubiquitin ligase NEDD4 interacts with
andubiquitylates SQSTM1 for inclusion body autophagyQiong Lin1,*,
Qian Dai1, Hongxia Meng1, Aiqin Sun1, Jing Wei1, Ke Peng1, Chandra
Childress2, Miao Chen3,Genbao Shao1 and Wannian Yang1,*
ABSTRACTOur previous studies have shown that the HECT E3
ubiquitin ligaseNEDD4 interacts with LC3 and is required for
starvation and rapamycin-induced activation of autophagy. Here, we
report that NEDD4 directlybinds to SQSTM1 via its HECT domain and
polyubiquitylates SQSTM1.This ubiquitylation is through K63
conjugation and is not involved inproteasomal degradation.
Mutational analysis indicates that NEDD4interacts with and
ubiquitylates the PB1 domain of SQSTM1. Depletionof NEDD4 or
overexpression of the ligase-defective mutant of NEDD4induced
accumulation of aberrant enlarged SQSTM1-positive inclusionbodies
that are co-localizedwith the endoplasmic reticulum (ER)markerCANX,
suggesting that the ubiquitylation functions in the SQSTM1-mediated
biogenic process in inclusion body autophagosomes. Takentogether,
our studies show that NEDD4 is an autophagic E3 ubiquitinligase
that ubiquitylates SQSTM1, facilitating SQSTM1-mediatedinclusion
body autophagy.
KEY WORDS: Autophagy, E3 ubiquitin ligase, Inclusion
bodies,NEDD4, PB1 domain, SQSTM1, p62, Ubiquitylation
INTRODUCTIONSQSTM1 (p62) is an autophagic cargo receptor that
plays a key role inselective autophagy (Kirkin et al., 2009a; Rogov
et al., 2014). Earlystudies have shown that SQSTM1 is
associatedwith protein inclusionsand aggregates, such as
Mallory–Denk bodies and Lewy bodies(Stumptner et al., 1999, 2007;
Nakaso et al., 2004; Tanji et al., 2015),and is considered to be a
universal component of protein inclusions(Zatloukal et al., 2002).
Further studies found that SQSTM1 functionsas a receptor for
ubiquitylated protein inclusion bodies or aggregatesand recruits
them to autophagosomes for degradation via interactionswith LC3-II
(Komatsu et al., 2007; Pankiv et al., 2007). Now, weknow that
SQSTM1 is a universal autophagic cargo receptor involvedin multiple
types of selective autophagy, such as mitophagy,pexophagy,
xenophagy and aggrephagy (Zheng et al., 2009; Geisleret al., 2010;
Bartlett et al., 2011; Ishimura et al., 2014; Zhang et al.,2015).
As a key cargo receptor in selective autophagy of proteininclusions
and aggregates, malfunction of SQSTM1 is associated withmultiple
diseases, such as Parkinson’s disease, Huntington’s
disease,Alzheimer’s disease, alcoholic hepatitis and cirrhosis
(Kuusisto et al.,2002; Stumptner et al., 2002; Zatloukal et al.,
2002; Nakaso et al.,2004; Du et al., 2009; Geisler et al., 2010;
Cuyvers et al., 2015).
Ubiquitylation is an important biochemical process in
SQSTM1-mediated selective autophagy. Multiple studies have shown
that theubiquitylation of protein inclusion bodies, aggregates or
otherautophagic cargos is pivotal for recognition by SQSTM1 in
theautophagic degradation process (Kim et al., 2008; Johansen
andLamark, 2011; Rogov et al., 2014). In addition, SQSTM1 is
capableof recruiting E3 ubiquitin ligases, such as TRAF6 and KEAP1,
forubiquitylating autophagic cargos or autophagic proteins
duringinitiation, formation or transportation of selective
autophagosomes(Kirkin et al., 2009a; Fan et al., 2010; Fusco et
al., 2012; Isaksonet al., 2013; Stolz et al., 2014). As an
autophagic cargo receptor,SQSTM1 is transported along with the
autophagic cargos inautophagosomes to lysosomes for degradation
(Bjørkøy et al., 2005,2006; Ichimura et al., 2008). Therefore,
degradation of SQSTM1sometimes is used as a molecular marker for
activation of autophagy(Bjørkøy et al., 2009).
While the role of SQSTM1 in selective autophagy is
wellestablished, it remains poorly understood how the receptor
activity ofSQSTM1 is regulated during selective autophagy. A recent
studyfound that casein kinase 2 (CK2) phosphorylates S403 in the
Ubadomain of SQSTM1 and enhances the binding capacity of SQSTM1
tothe polyubiquitin chain (Matsumoto et al., 2011). This
phosphorylationpromotes SQSTM1 to target polyubiquitylated proteins
and recruitubiquitylated cargos to autophagosomes (Matsumoto et
al., 2011).Recent studies indicate that ubiquitylation also
regulates the autophagyreceptor function of SQSTM1 for recognition
of autophagic cargos. Ithas been found that SQSTM1 is ubiquitylated
by the ring family E3ubiquitin ligases TRIM21 (Pan et al., 2016),
KEAP1–CULLIN3 (Leeet al., 2017), PARKIN (Song et al., 2016), and
the E2 conjugatingenzymes UBE2D2/3 (Peng et al., 2017). The
ubiquitylation producesdiversified effects on SQSTM function,
including suppression andactivation of the autophagic receptor
activity (Pan et al., 2016; Leeet al., 2017; Peng et al., 2017) and
promotion of the proteasomaldegradation of SQSTM1 (Song et al.,
2016). However, howubiquitylation of SQSTM1 regulates cellular
inclusion bodyautophagy remains unknown. Furthermore, SQSTM1 also
participatesin other cellular signaling pathways, such as atypical
PKC andNF-κB signaling pathways (Puls et al., 1997; Sanchez et al.,
1998;Sanz et al., 1999). Whether these pathways are
regulatedindependently or are connected to autophagy has not been
clarified.
Our recent studies found that NEDD4 (also known as NEDD4-1),a
member of the HECT E3 ubiquitin ligase family, interacts with
theautophagic protein LC3 through an LIR domain and is essential
forstarvation or rapamycin-induced activation of autophagy (Sun et
al.,2017). Knockdown of NEDD4 by shRNA caused aggregation ofGFP–LC3
puncta in the ER and deformation of mitochondria. Itappears that
interaction of NEDD4with LC3 is not only necessary forassociation
with autophagosomes, but also for activation of the E3ubiquitin
ligase. Our preliminary data also demonstrate that
NEDD4ubiquitylates SQSTM1, but not LC3 (Sun et al., 2017). These
resultsReceived 5 June 2017; Accepted 27 September 2017
1School of Medicine, Jiangsu University, Zhenjiang 212013,
China. 2Department ofBiology, Susquehanna University, 514
University Ave, Selinsgrove, PA 17870, USA.3Department of
Pathology, Affiliated People’s Hospital, Jiangsu
University,Zhenjiang 212013, China.
*Authors for correspondence ([email protected];
[email protected])
Q.L., 0000-0002-4393-2495; W.Y., 0000-0002-1246-7260
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clearly indicate that NEDD4 is an important E3 ubiquitin
ligaseinvolved in autophagic activation. In this report, we
continueinvestigating the role of NEDD4 in autophagy by
characterizinginteraction and ubiquitylation of SQSTM1 and defining
the functionof SQSTM1 ubiquitylation. We found that NEDD4 interacts
withSQSTM1 through the HECT (homologous to E6-AP carboxylterminus)
domain. The PB1 domain in SQSTM1 appears to be theNEDD4 interactive
and ubiquitylating region. The polyubiquitylationof SQSTM1 byNEDD4
ismainly throughK63 conjugation, which isimportant for the
SQSTM1-mediated inclusion body autophagy,rather than proteasomal
degradation. Our studies demonstrateNEDD4 as a key E3 ubiquitin
ligase in selective autophagy thatinteracts with and ubiquitylates
the autophagy receptor SQSTM1.
RESULTSNEDD4 interacts with SQSTM1Our previous studies have
shown that NEDD4 ubiquitylates SQSTM1(Sun et al., 2017). To
determine whether this ubiquitylation resultsfrom interaction
between NEDD4 and SQSTM1, we characterized thebinding of NEDD4 to
SQSTM1 using a co-immunoprecipitationassay. As shown in Fig. 1A,
NEDD4 is co-immunoprecipitated withSQSTM1 when both were
co-expressed in cells, indicating thatSQSTM1 binds to NEDD4.
Interestingly, the ligase-dead (LD) mutantNEDD4-C867A bound to
SQSTM1 with much higher affinity thanwild-type NEDD4 (Fig. 1B).
This suggests that the ligase-dead mutantof NEDD4 has a ‘trap’
effect on SQSTM1, which is a typical bindingmode for a
catalysis-defective enzyme with a substrate, as observed inbinding
of tyrosine phosphatase-defectivemutants with their
substrates(Flint et al., 1997).To determine the SQSTM1-binding
region in NEDD4 more
specifically, we made a series of truncation mutants of
NEDD4(Fig. 1C), and tested the binding of these mutants to SQSTM1.
HA-tagged NEDD4 or its truncation mutants were co-expressed with
GFP-tagged SQSTM1 in HEK293 cells and binding was detected by
co-immunoprecipitation assay. As shown in Fig. 1D,E, all the
truncationmutants are capable of binding to SQSTM1. As the mutant
NEDD4-N4Δ contains only the HECT domain (see Fig. 1C), this
indicates thatNEDD4 interacts with SQSTM1 through the HECT domain.
Toconfirm this, we co-expressed the HECT domain-deletion
mutant,NEDD4-HECTΔ, with SQSTM1 in HEK293 cells, and
examinedinteraction of the mutant with SQSTM1. As shown in Fig. 1F,
whilethe NEDD4 ligase-dead mutant NEDD4-C867A (NEDD4-LD)
wasco-immunoprecipitated with SQSTM1, NEDD4-HECTΔ showedlittle
co-precipitation with SQSTM1, confirming that the HECTdomain
interacts with SQSTM1. We also examined the bindingof NEDD4 to
another autophagy receptor NBR1 using a co-immunoprecipitation
assay, and found no detectable binding (Fig. 1G).
NEDD4 polyubiquitylates SQSTM1 through K63 chainconjugation and
the ubiquitylation does not causeproteasomal degradationSQSTM1 is a
key autophagic protein that interacts with LC3 and itplays an
important role in selective autophagy, including mitophagy(Kirkin
et al., 2009a; Johansen and Lamark, 2011; Stolz et al., 2014).Here,
we characterized the ubiquitylation of SQSTM1 by NEDD4using both
immunoprecipitation and GST–Uba pulldown assays.GST–Uba pulldown
assay has been successfully used for detection ofubiquitylated
proteins in our previous studies (Lin et al., 2010). Asshown in
Fig. 2A, GFP-tagged SQSTM1 was ectopically expressedwith or without
NEDD4 in HEK293 cells. Without NEDD4,SQSTM1 had a low level of
ubiquitination (lane 4). When co-expressed with NEDD4, SQSTM1 was
heavily polyubiquitylated
(lane 2). This result confirms that SQSTM1 is a
ubiquitylationsubstrate of NEDD4. We further examined whether
endogenousSQSTM1 is the substrate of NEDD4 upon activation of
autophagy inthe lung cancer cell line A549. To elevate the
ubiquitylation ofSQSTM1, we treated cells with rapamycin to
activate autophagy, andwith chloroquine to block the autophagic
degradation of SQSTM1. Asshown in Fig. 2B, upon treatment with
rapamycin and chloroquine,endogenous SQSTM1 was significantly
polyubiquitylated (lane 2),whereas in the NEDD4 shRNA cell line,
SQSTM1 was no longerpolyubiquitylated upon treatment with rapamycin
and chloroquine,suggesting that endogenous SQSTM1 is ubiquitylated
by NEDD4 inresponse to activation of autophagy.
The carboxyl terminus of SQSTM1 contains anUba domain that
canbind to other ubiquitylated proteins. To exclude the possibility
thatNEDD4-dependent ubiquitylation detected in SQSTM1 is from
theSQSTM1Uba domain-associated proteins, wemade theUba
truncationmutant of SQSTM1, SQSTM1-UbaΔ. Co-expression of
SQSTM1-UbaΔ with NEDD4 in HEK293 cells showed polyubiquitylationof
SQSTM1-ΔUba (lane 2, Fig. 2C), confirming that
thepolyubiquitylation of SQSTM1 by NEDD4 is not from the
SQSTM1Uba-associated ubiquitylated proteins. In fact,
immunoprecipitation ofSQSTM1 or the Uba truncation mutant without
NEDD4 co-expressionshowed no detectable polyubiquitylation (lane 4
in Fig. 2A and lane 3 inFig. 2C), indicating that the
SQSTM1Uba-associated ubiquitin proteins(if any) produced little
interference with NEDD4-mediatedpolyubiquitylation of SQSTM1.
We further determined the type of ubiquitin chain linkage
ofSQSTM1 catalyzed by NEDD4. As shown in Fig. 2D, NEDD4catalyzed
the K63-linked polyubiquitylation of SQSTM1 (toppanel), not the K48
polyubiquitylation (second panel), suggestingthat NEDD4 catalyzed
polyubiquitylation of SQSTM1 may not beinvolved in proteasomal
degradation. However, we detected a minorK63 and K48
polyubiquitylation of SQSTM1with expression of theligase-dead
mutant NEDD4-LD or without exogenous NEDD4(lanes 3 and 4, the top
two panels) that might be produced byendogenous ubiquitylation.
To confirm that NEDD4-dependent polyubiquitylation ofSQSTM1 does
not lead to proteasomal degradation, we examinedlevels of
ubiquitylation and SQSTM1 protein upon treating the cellswith the
specific proteasomal inhibitor bortezomib. As shown inFig. 2E,
treatment with bortezomib did not induce accumulation ofeither
NEDD4-dependent ubiquitylation or protein of SQSTM1. Infact,
bortezomib eliminated the NEDD4-dependent ubiquitylationof SQSTM1
(compare lane 7 with lane 8 in the right top panel inparallel with
lane 2 and lane 3 in the left top panel). These datafurther suggest
that NEDD4-dependent polyubiquitylation ofSQSTM1 is involved in
autophagy, not proteasomal degradation.
NBR1 is not a ubiquitylation substrate of NEDD4NBR1 is another
autophagic cargo receptor that contains similarstructural domains
to SQSTM1 and functions in selective autophagy,particularly in
recruiting ubiquitylated protein inclusions toautophagosomes
(Kirkin et al., 2009b). We wondered whetherNBR1 was ubiquitylated
by NEDD4. As shown in Fig. 3A, whileSQSTM1 was significantly
ubiquitylated when co-expressed withNEDD4 (compare lane 2 with lane
4, second panel), NBR1 showedno increase in ubiquitylation when
co-expressed with NEDD4(compare lane 3 with lane 5, top panel),
indicating that NBR1was notubiquitylated by NEDD4. Interestingly,
endogenous NBR1 washeavily ubiquitylated independent of NEDD4 (top
panel). We furtherconfirmed that NBR1 showed NEDD4-independent
ubiquitylationwhen co-expressed with NEDD4 mutants (Fig. 3B).
NEDD4-
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independent ubiquitylation and NBR1 protein level
weredramatically enhanced by treatment with proteasomal
inhibitorMG-132, but not lysosomal inhibitor chloroquine (Fig.
3B–D),suggesting that NBR1 has an active turnover by
proteasomal
degradation through ubiquitylation by a non-NEDD4 E3
ubiquitinligase. NEDD4 seems to antagonize the E3 ubiquitin ligase
forNBR1, because co-expression with NEDD4 markedly
reducedubiquitylation of NBR1 (compare lane 9 with lane 8, top
panel,
Fig. 1. NEDD4 interacts with SQSTM1 but not NBR1. (A,B)
HA-tagged NEDD4 or the ligase-deadmutant NEDD4-C867A (NEDD4-LD) was
co-transfected withSQSTM1 or GFP-SQSTM1 in HEK293 cells. SQSTM1 or
GFP-SQSTM1 was immunoprecipitated with an anti-SQSTM1 antibody and
co-immunoprecipitatedHA-tagged NEDD4 or the mutant was detected by
immunoblotting with an anti-HA antibody. (C) Truncation constructs
of human NEDD4. C2, C2 domain; I,II, III and IV, 4 WW domains;
HECT, the HECT domain; the numbers labeled in NEDD4 structure
sketches indicate the amino acid residue positions.(D–F) HA-tagged
NEDD4, the ligase-dead mutant NEDD4-C867A (labeled as NEDD4-LD) or
the truncation mutants were co-transfected with GFP–SQSTM1
inHEK293T cells. GFP–SQSTM1 was immunoprecipitated with an
anti-SQSTM1 antibody and co-immunoprecipitated HA-tagged NEDD4 or
the mutant wasdetected by immunoblotting with an anti-HA antibody.
In E, white asterisks indicate the NEDD4 truncation mutant bands.
ACTB, β-actin. (G) HA-taggedNEDD4 or the truncation mutants were
co-transfected with NBR1 in HEK293T cells. NBR1 was
immunoprecipitated with an anti-NBR antibody
andco-immunoprecipitated HA-tagged NEDD4 or the mutant was detected
by immunoblotting with an anti-HA antibody.
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Fig. 3B). In addition, co-expression with the N-terminal
truncationmutant of NEDD4, NEDD4-N1Δ, which is defective in binding
toLC3 (Sun et al., 2017), dramatically enhanced the amount of
NBR1protein through an unknown mechanism (see lane 6, top
panel,Fig. 3C,D). These data indicate that NBR1 is not a
ubiquitylationsubstrate of NEDD4.
NEDD4 interacts with the PB1 domain of SQSTM1 and
thisinteraction is required for ubiquitylation of SQSTM1To identify
the region in SQSTM1 that interacts with and isubiquitylated by
NEDD4, we made a series of SQSTM1 mutants inits functional domains
and tagged with GFP (Fig. 4A). These
mutants include the PB1 deletion mutant N43Δ, the PB1
domainpoint mutants K7A, K13E, R21A/R22A (named as 2R2A),
K13E/R21A/R22A (named as K13E/2R2A), D69A, the LIR domain
pointmutant L341A, the LIR deletion mutant [334–342]Δ, and theUba
domain point mutant L417V (Seibenhener et al., 2004).Ubiquitylation
of the SQSTM1 domain mutants by NEDD4 wasfirst examined by the
GST–Uba pulldown assay (Fig. 4B). Deletionof PB1 (N43Δ) diminished
the ubiquitylation of SQSTM1 (lane 5).Mutations in the LIR and the
Uba domain produced an insignificanteffect on the ubiquitylation.
These results suggest that the PB1domain is essential for
ubiquitylation by NEDD4, while interactionwith LC3 or ubiquitin is
dispensable.
Fig. 2. NEDD4 ubiquitylates SQSTM1with K63 linked polyubiquitin
chains and the ubiquitylation is not involved in proteasomal
degradation.(A) NEDD4 was co-transfected with GFP-SQSTM1 in HEK293T
cells. SQSTM1 was immunoprecipitated with an anti-SQSTM1 antibody
and ubiquitylatedSQSTM1 was detected by immunoblotting with an
anti-ubiquitin antibody. (B) Ubiquitylation of endogenous SQSTM1 is
dependent on NEDD4. shNEDD4 or thevector (pLKO.1) cell line
established in lung cancer A549 cells was treated with or without
chloroquine plus rapamycin for 18 h, and endogenous SQSTM1was
immunoprecipitated. Ubiquitylation of SQSTM1 was detected by
immunoblotting with an anti-ubiquitin antibody. Amount of SQSTM1 in
theimmunoprecipitation (middle panel) and NEDD4 in the lysates
(bottom panel) was detected by immunoblotting. The band labeled IgG
is the anti-SQSTM1 IgGcontaining both heavy and light chains due to
incomplete cleavage of di-sulfide bonds by the sample buffer.
IgG-HC, IgG heavy chain. (C) SQSTM1 or its Ubadeletion mutant
SQSTM1-UbaΔwas co-expressed with NEDD4 in HEK293 cells. The
ubiquitylated SQSTM1 was precipitated with GST–ACK1Uba and
detectedby immunoblotting with anti-SQSTM1. (D) HA-tagged NEDD4 or
its ligase-dead mutant NEDD4-C867A (NEDD4-LD) was co-expressed with
SQSTM1 inHEK293T cells. SQSTM1 was immunoprecipitated with
anti-SQSTM1. Polyubiquitylation of SQSTM1 was detected by
immunoblotting with an antibody againsteither K63-linked or
K48-linked polyubiquitin. The expression of SQSTM1, NEDD4 or the
ligase-dead mutant was determined by immunoblotting of the
celllysates with anti-SQSTM1 or anti-HA antibody. (E) Inhibition of
proteasomes does not cause accumulation of SQSTM1 and
NEDD4-dependent ubiquitylation.The experimental procedures were the
same as in D except cells were treated with the proteasomal
inhibitor bortezomib (10 µM) or DMSO (solvent control) for12 h
prior to harvesting the cells. Ubiquitylation of SQSTM1 was
detected by immunoblotting with an anti-ubiquitin antibody.
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Previous studies have shown that the PB1 domain functions
inhomo- or hetero-dimerization of SQSTM1 through the
interactionbetween the basic cluster and the OPCA (OPR–PC–AID)
motifwithin the PB1 domain and is required for localization
onautophagosomes (Lamark et al., 2003; Itakura and Mizushima,2011).
The results in Fig. 4B indicate that the PB1 domain isrequired for
either binding to or ubiquitylation by NEDD4 or both.Thus, we used
the PB1 truncation mutant N43Δ and thedimerization defective
mutants K7A and 2R2A for testing thebinding to NEDD4. As shown in
Fig. 4C, the PB1 deletion mutantN43Δ or the point mutant 2R2A
failed to co-immunoprecipitateNEDD4 (lanes 2 and 4), whereas
wild-type SQSTM1 or the mutantK7A co-immunoprecipitated NEDD4
(lanes 1 and 3). These datademonstrate that PB1 is the
NEDD4-interactive domain and thatR21 and R22 in the PB1 domain are
the residues essential forbinding to NEDD4. In addition, the defect
in dimerization in the2R2A mutant is unlikely to be the cause of
loss of NEDD4 binding,
because K7A, which is also a dimerization defective
mutant(Lamark et al., 2003), retains NEDD4 binding capacity (lane
3).
We further characterized NEDD4-dependent ubiquitylation ofthe
PB1 mutants of SQSTM1 by both immunoprecipitation(Fig. 4D) and
GST–Uba pulldown assays (Fig. 4E). The resultsfrom both assays were
consistent, and showed that mutations onR21/R22 (lane 4 in Figs. 4D
and 4E) and mutation on K7 (lane 3 inFig. 4D, lane 5 in Fig. 4E)
significantly reduced the ubiquitylation,whereas the mutation on
K13 had no effect (lane 5 in Fig. 4D, lane 3in Fig. 4E). The
results indicate that the PB1 domain and the R21/R22 residues are
essential for the binding to and ubiquitylation byNEDD4, and that
K7 is one of the major NEDD4 ubiquitylationsites. It has been shown
that the RING family E3 ubiquitin ligaseTRIM21 also ubiquitylates
K7 of SQSTM1, and the ubiquitylationimpairs oligomerization of
SQSTM1 thus suppressing theSQSTM1-mediated sequestration of KEAP1
(Pan et al., 2016). Infuture studies, it would be interesting to
examine whether the
Fig. 3. NBR1 is not an ubiquitylation substrate of NEDD4-1.
HA-tagged NEDD4-1 or its mutant was co-transfected with SQSTM1 or
NBR1 for 48 h inHEK293T cells. Treatment with MG-132 (10 µM) or
chloroquine (50 µM) was carried out 18 h before harvesting the
cells. (A,B) Ubiquitylated proteins in the celllysates were
precipitated with GST–Uba-conjugated beads. Ubiquitylated SQSTM1 or
NBR1 was detected by immunoblotting with an anti-SQSTM1 or an
anti-NBR1 antibody. (C,D) NBR1 was detected directly from cell
lysates by immunoblotting with an anti-NBR1 antibody.
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ubiquitylation of K7 by NEDD4 has the same effect on SQSTM1
asthat of TRIM21We also examined whether the ubiquitylation affects
binding of
SQSTM1 to LC3 by co-expression of the SQSTM1 mutants withNEDD4.
The GST–LC3 pulldown assay confirmed that SQSTM1-L341A or
[334–342]Δ is defective in LC3 binding (lanes 6–10,Fig. 4F) and
other mutants are capable of binding to LC3 (lanes 5,6and 11–14,
Fig. 4F). Co-expression with NEDD4 did not affectbinding of SQSTM1
or the mutants to LC3, although the proteinlevel of wild-type
SQSTM1 and the PB1 domain truncation mutantwas slightly reduced by
co-expression with NEDD4 (lanes 4 and 6,second panel, Fig. 4D).
This result suggests that ubiquitylation ofSQSTM1 by NEDD4 is not
involved in regulation of the LC3binding, which is consistent with
our previous studies withimmunofluorescence staining (Sun et al.,
2017).
Knockdown of NEDD4 causes accumulation of the SQSTM1-positive
inclusion bodiesOur recent studies have shown that knockdown of
NEDD4 impairsrapamycin- and starvation-induced autophagy and
autophagosomal
biogenesis, and causes aggregation of GFP–LC3 puncta (Sun et
al.,2017). Here, we further determined the effect of NEDD4
knockdownon the cellular localization and morphology of
SQSTM1-positivefluorescent puncta in response to treatment with
rapamycin. Similarto GFP–LC3, SQSTM1 was observed as tiny
fluorescent punctalocalized at para-nuclei in the vector control
cells without rapamycintreatment, while in the NEDD4 knockdown
cells, significantaccumulation of heterogeneous large SQSTM1 puncta
was seen(Fig. 5A). Quantification analysis indicates that the
average size of theSQSTM1puncta increased∼4-fold up to∼1 µm, upon
knockdown ofNEDD4, but the average number of SQSTM1 puncta per cell
did notchange significantly (Fig. 5B). Furthermore, SQSTM1 puncta
in theNEDD4 knockdown cells were randomly distributed in the cells,
nopara-nuclear localization was observed (Fig. 5A). These
largeSQSTM1-positive puncta in NEDD4 knockdown cells are likely
tobe protein inclusion bodies, which are the common autophagic
cargosassociated with SQSTM1 (Zatloukal et al., 2002). Upon
treatmentwith rapamycin for 18 h, SQSTM1 puncta in the vector
control cellswas distributed at one side of the nucleus (bottom
left panel, Fig. 5A)and average numbers of the SQSTM1 puncta per
cell increased
Fig. 4. NEDD4 interacts with and ubiquitylates SQSTM1 through
the PB1 domain. (A) SQSTM1mutants. PB1, PHOX and BEM1P domain; ZZ,
ZZ-type zincfinger domain; TB, TRAF6 binding domain; LIR,
LC3-interactive region; KIR, KEAP1 interactive region; UBA,
ubiquitin-associated domain. (B–E) NEDD4was co-expressed with
GFP–SQSTM1 or the mutant into HEK293T cells. In B and E,
ubiquitylated proteins in the lysates were precipitated by
GST–Uba-conjugated beads. The ubiquitylated SQSTM1 or its mutant
was detected by immunoblotting with an anti-SQSTM1 antibody. HE,
heavily exposed; LE, lightlyexposed. In C and D, SQSTM1 or its
mutants in the lysates were immunoprecipitated with an anti-SQSTM1
antibody. The co-immunoprecipitated NEDD4was detected with an
anti-NEDD4 antibody and ubiquitylation of SQSTM1 or its mutants was
detected with an anti-ubiquitin antibody. (F) GFP–SQSTM1 or
itsmutant was expressed in HEK293T cells with or without NEDD4.
GST–LC3 was used to precipitate GFP–SQSTM1 or its mutants and
results detected byimmunoblotting with anti-SQSTM1 and anti-NEDD4
antibodies.
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Fig. 5. Knockdown of NEDD4 induces aggregates of the SQSTM1
puncta that co-localize with the ER membrane marker CANX. (A) The
vector control(pLKO.1) or the NEDD4 shRNA A549 cell line was
treated with 1 µM rapamycin for 18 h to activate autophagy.
Knockdown effect on NEDD4 by shNEDD4 isshown at the bottom. NEDD4
(LM), lowmolecular weight NEDD4; NEDD4 (HM), high molecular weight
NEDD4. NEDD4 (LM) is a degradation product of NEDD4(HM) (Sun et
al., 2017). Endogenous SQSTM1was immunostained with an anti-SQSTM1
antibody followed by a fluorescent dye-conjugated secondary
antibody,and the fluorescencewas visualized under an inverted Nikon
fluorescent microscope. Scale bars: 10 µm. (B) Quantification of
numbers and sizes of the SQSTM1fluorescent puncta from fluorescence
microscopy images. A total of 3159 SQSTM1 puncta in 47 vector
control cells, 4163 SQSTM1 puncta in 29 rapamycin-treated vector
control cells, 2830 SQSTM1 puncta in 41 shNEDD4 cells, and 880
SQSTM1 puncta in 25 rapamycin-treated shNEDD4 cells were counted
andmeasured with ImageJ for statistical analysis. The statistical
analysis was performed based on the numbers and average sizes of
the puncta in each of the cells.**P
-
significantly (Fig. 5B), indicating that biogenesis of the
SQSTM1-positive autophagosomes was induced by rapamycin. In
NEDD4knockdown cells, large SQSTM1 puncta remained
randomlydistributed, and numbers of the SQSTM1 puncta did not
changesignificantly upon rapamycin treatment (Fig. 5A,B). These
resultsindicate that knockdown of NEDD4 blocks the
rapamycin-inducedbiogenesis of autophagosomes and suggest that
knockdown ofNEDD4 might impair the SQSTM1-mediated inclusion
bodyautophagy, thus causing accumulation of the large
SQSTM1-positive inclusion bodies in cells.Our previous studies
showed that knockdown of NEDD4 caused
aggregation of GFP–LC3 puncta that was co-localized with
ERmembrane markers, but not with Golgi marker, and suggested
thatNEDD4 is required for biogenesis of autophagosomes (Sun et
al.,2017). Here, we examined the effect of NEDD4 knockdown
onlocalization of the SQSTM1-positive puncta in cells. As shown
inFigs. 5A, 5C and 5D, a portion of the SQSTM1 puncta wasaberrantly
aggregated into large inclusion bodies in the shNEDD4cell line, but
not in the vector control cell line. Furthermore, theenlarged
SQSTM1-positive inclusion bodies in the shNEDD4 cellline were
co-localized with the ER marker CANX (Fig. 5D), but not
with the Golgi marker GOLGA2/GM-130 (Fig. 5C), and had
littlechange upon treatment with rapamycin (Fig. 5D), suggesting
thatSQSTM1-positive inclusion bodies are retained in ER
membranevesicles upon NEDD4 knockdown, which is consistent with
ourprevious observation on aggregation of the LC3-positive puncta
inERmembrane vesicles upon depletion of NEDD4 (Sun et al.,
2017).These results suggest that the defect in inclusion body
autophagycaused by NEDD4 depletion might occur in the early stage
of theautophagosomal biogenic process in the ER.
Ubiquitylation of SQSTM1 by NEDD4 is required for inclusionbody
autophagyAs knockdown of NEDD4 caused aberrant aggregation of
theSQSTM1-positive inclusion bodies retained in the ER (Fig. 5),
wewondered whether the large aggregates of the
SQSTM1-positiveprotein inclusion bodies in NEDD4 knockdown cells
were formedupon defective ubiquitylation of SQSTM1byNEDD4. To
investigatethis hypothesis, we used HEK293T or HEK293A cells to
transientlyoverexpress GFP–LC3, SQSTM1 and the PB1 defective
mutantSQSTM1-2R2A with wild-type NEDD4 or its ligase-dead
mutantNEDD4-C867A. As shown in Fig. 6A, co-expression of
GFP–LC3
Fig. 6. Overexpression of the ligase defective mutant of NEDD4
causes formation of gigantic SQSTM1-positive inclusion bodies.
(A–C) GFP–LC3,SQSTM1 or its PB1-defective mutant 2R2A expressed or
co-expressed with NEDD4 or its ligase-dead mutant NEDD4-C867A
(NEDD4-LD) in HEK293A cells.The cells were immunostained with
anti-SQSTM1 (A–C) or NEDD4 (C). Scale bars: 10 µm (A); 7.5 µm (B
and C).
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withwild-type SQSTM1 inHEK293T cells led to typical
localizationof GFP-LC3 and SQSTM1 on autophagosomes. Co-expression
ofGFP–LC3 with the PB1-defective mutant SQSTM1-2R2A resultedin
diffused fluorescence of both GFP–LC3 and SQSTM1-2R2A,confirming
that homo-oligomerization, which is defective inSQSTM1-2R2A, is
required for SQSTM1 to localize onautophagosomes. However,
co-expression of GFP–LC3 andSQSTM1 with NEDD4 ligase-dead mutant
C867A resulted information of multiple gigantic inclusion bodies
(1–10 µm diameter)containing the GFP–LC3 and SQSTM1 fluorescence in
cells (thebottom panels, Fig. 6A). These data, together with the
data in Fig. 5,suggest that, first, defect in ubiquitylation of
SQSTM1 by NEDD4may impair inclusion body autophagy; and second,
ubiquitylation ofSQSTM1 is not required for homo-oligomerization,
as eitherknockdown of NEDD4 or overexpression of the ligase-dead
mutantof NEDD4 did not result in diffused localization of SQSTM1
andGFP–LC3 in cells (Fig. 5 and Fig. 6A). In addition,
overexpression ofthe ligase-dead mutant induced much larger
inclusion bodies thanthat induced by knockdown of NEDD4 (Fig. 5A-D,
Fig. 6A),indicating that the trapping of SQSTM1 by the ligase-dead
mutant ofNEDD4, as shown in Fig. 1B, which reduces the level of
freeSQSTM1, produces a much more severe defect in
autophagy-mediated removal of cellular inclusion bodies than that
by depletionof NEDD4 only, suggesting that SQSTM1 in the
NEDD4knockdown cells may still retain partial function in
facilitating theremoval of inclusion bodies.To confirm that
ubiquitylation of SQSTM1 is required for
inclusion body autophagy, we co-expressed SQSTM1 or SQSTM1-2R2A
plus GFP–LC3 with or without NEDD4 or its ligase-deadmutant C867A
in HEK293A cells (Fig. 6B). Upon co-expression ofSQSTM1 plus
GFP–LC3 or NEDD4, the cells showed localizationof SQSTM1 and
GFP–LC3 on normal autophagosomes (Fig. 6Bi–iii). However, upon
co-expression of SQSTM1 with the NEDD4ligase-dead mutant C867A, the
cells formed huge inclusion bodiescontaining SQSTM1 and GFP–LC3
(Fig. 6B iv–vi, Fig. 6Ci–iii),confirming that lack of SQSTM1
ubiquitylation by NEDD4 impairsinclusion body autophagy. Consistent
with Fig. 6A, the PB1-defective mutant SQSTM1-2R2A and the
co-expressed GFP–LC3displayed diffused distribution in cells in
either the presence orabsence of NEDD4 or the ligase-dead mutant
(Fig. 6Bvii–xii,Fig. 6Ci–iii). These data suggest that
ubiquitylation of SQSTM1 isrequired for its function in inclusion
body autophagy, but not for itsoligomerization or localization on
autophagosomes.
DISCUSSIONUbiquitylation is an important biochemical process in
selectiveautophagy. Most of the studies on ubiquitylation in
selectiveautophagy have been focused on the role of ubiquitylation
inrecognition of autophagic cargos (Kirkin et al., 2009a; Johansen
andLamark, 2011; Stolz et al., 2014). Recently, ubiquitylation
ofautophagy receptors has been studied and found to be involved in
adiversified regulatory function in autophagy. Ubiquitylation
byRING family E3 ubiqiuitin ligases and E2 conjugating
enzymeseither regulates the autophagic receptor activity (Pan et
al., 2016;Lee et al., 2017; Peng et al., 2017) or promotes the
proteasomaldegradation of SQSTM1 (Song et al., 2016). Our previous
studieshave shown that NEDD4, a member of the HECT E3
ubiquitinligase family, not only directly binds to autophagosomal
proteinLC3 (Sun et al., 2017), but also interacts with SQSTM1
through theHECT domain and polyubiquitylates SQSTM1. Knockdown
ofNEDD4 in lung cancer A549 cells impaired both rapamycin-
andstarvation-induced activation of autophagy and formation of
autophagosomes, and caused deformation of mitochondria, as
wehave shown previously (Sun et al., 2017). In this report, we
havedemonstrated a new role of ubiquitylation of SQSTM1 by the
HECTE3 ubiquitin ligase NEDD4 in regulation of inclusion
bodyautophagy. Depletion of endogenous NEDD4 or
ectopicoverexpression of the ligase-dead mutant of NEDD4
causedformation of aberrant gigantic aggregates of both LC3-
andSQSTM1-positive inclusion bodies, pointing to an important
roleof NEDD4 in the SQSTM1-mediated inclusion body autophagy.
SQSTM1 is known to be involved in inclusion body
autophagy(Zatloukal et al., 2002; Komatsu et al., 2007; Pankiv et
al., 2007).Early studies found that SQSTM1 associated with
ubiquitylatedMallory–Denk bodies in alcoholic liver and Lewy bodies
inParkinson’s disease tissue (Stumptner et al., 1999, 2007;
Zatloukalet al., 2002). Thus, SQSTM1 was defined as an inclusion
bodymarker protein (Zatloukal et al., 2002). Subsequently, after
interactionof SQSTM1 with the autophagosomal protein LC3 was
discovered,this inclusion body association was linked to the
function ofSQSTM1 in mediating inclusion body autophagy (Komatsu et
al.,2007; Pankiv et al., 2007). As both the LC3 (the LIR domain)
and theubiquitin (the Uba domain) binding ability are possessed
bySQSTM1, it is possible that SQSTM1 functions as a
ubiquitylatedinclusion body autophagy receptor by recruiting the
inclusion body toautophagosomes through interaction with ubiquitin
and LC3 (Kirkinet al., 2009a). In later studies, this functional
mode has been extendedto the other autophagic cargos, such as
invaded bacteria andperoxisomes, whose autophagy is also regulated
by SQSTM1 (Zhenget al., 2009; Zhang et al., 2015). Interestingly,
NEDD4 waspreviously found to facilitate the endosome-mediated
lysosomaldegradation of α-synuclein, a major component of Lewy
bodies inParkinson’s disease, by directly interacting with and
ubiquitylatingα-synuclein (Tofaris et al., 2011; Chung et al.,
2013; Sugeno et al.,2014). NAB2, a small chemical that is an
activator of NEDD4,reversed a mutated α-synuclein-induced
cytotoxicity in neuronsderived from Parkinson’s disease patients
(Chung et al., 2013).Although it has been proposed that
NEDD4-facilitated degradation ofα-synuclein is through an
endosomal/lysosomal route, not autophagy(Sugeno et al., 2014), our
work suggest that the role of autophagy inthe NEDD4-facilitated
α-synuclein degradation might need to be re-examined. Furthermore,
our studies also suggest that NEDD4 mightbe a universal selective
autophagic E3 ubiquitin ligase that isinvolved in other types of
SQSTM-mediated selective autophagy,such as xenophagy, pexophagy and
mitophagy. In fact, we haveobserved that knockdown of NEDD4 in lung
cancer A549 cellsinduced aberrant enlargement and deformation of
mitochondria (Sunet al., 2017). Thus, NEDD4 might be an effective
therapeutic targetfor SQSTM1-mediated selective autophagy-related
diseases,particularly neuronal degenerative diseases.
Our studies presented in this report added a new
mechanismunderlying the SQSTM1-mediated selective autophagy, i.e.
theubiquitylation by NEDD4 via the PB1 domain regulates the
cargoreceptor activity of SQSTM1. The PB1 domain of SQSTM1functions
in homodimerization and heterodimerization withatypical PKCs
(aPKCs), NBR1 and MAP2K5 (MEK5) (Lamarket al., 2003;Moscat et al.,
2006, 2009). The PB1 domain of SQSTM1has two regions that are
involved in dimerization: one region at theN-terminus of the PB1
contains several positively charged residues,such as K7, R21 and
R22 in SQSTM1, the other region is at theC-terminus of the PB1 the
so-called OPCAmotif that is conserved ina number of the
PB1-containing proteins (Lamark et al., 2003). It hasbeen
demonstrated that K7, R21 or R22 in the PB1 of SQSTM1 isessential
for either homo-dimerization or heterodimerization through
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binding to the OPCA motif of the other PB1 (Lamark et al.,
2003).Our studies have shown that deletion of the first 43 amino
acidresidues in the PB1 domain, which does not include the
OPCAregion, eliminated binding to NEDD4 and the ubiquitylation
byNEDD4 (Fig. 4), indicating that the OPCA motif of SQSTM1 is
notrequired for interaction with NEDD4. Mutation of R21/R22 of
thePB1 domain, which are critical residues for interaction with
theOPCA region in dimerization, significantly reduced interaction
withand ubiquitylation by NEDD4 (Fig. 4), suggesting that an
OPCA-likeregion may be present in NEDD4. As the HECT domain of
NEDD4is the region binding to SQSTM1 (Fig. 1), it is possible that
the HECTdomain contains an OPCA-like region that is capable of
binding tothe positive residues of the PB1 of SQSTM1. In addition,
NEDD4does not interact and ubiquitylate another selective
autophagyreceptor NBR1 that also contains a PB1 domain at the
N-terminus(Fig. 3) (Kirkin et al., 2009a). Interestingly, the PB1
domain of NBR1has the OPCA motif but does not have the arginine
residuescorresponding to R21 and R22 in the PB1 of SQSTM1 (Lamark
et al.,2003). This further supports the hypothesis that the HECT
domain ofNEDD4 contains an OPCA-like region that interacts with
thepositively charged residues in the N-terminal region of the PB1
ofSQSTM1.The PB1 domain is essential for SQSTM1 to homo- and
hetero-
dimerize/oligomerize and to localize on autophagosomes (Lamarket
al., 2003; Itakura and Mizushima, 2001). However, ubiquitylationby
NEDD4 is dispensable for autophagosomal localization ofSQSTM1,
because knockdown of NEDD4 or expression of theligase-deadmutant of
NEDD4 did not produce a diffused distributionlike the
dimerization/oligomerization-defective mutants of SQSTM1(Fig. 6).
We currently do not know how the NEDD4-mediatedubiquitylation
regulates the exact molecular function of SQSTM1.There are two
possible molecular effects for the ubiquitylation. Thefirst effect
facilitates the hetero-dimerization of SQSTM1with NBR1.It has been
observed that cooperation between SQSTM1 with NBR1plays an
important role in inclusion body autophagy (Kirkin et al.,2009b;
Tanji et al., 2015). Ubiquitylation on the PB1 domain byNEDD4may
enhance the binding affinity of SQSTM1 toNBR1, thusenabling SQSTM1
to recruit NBR1 for inclusion body autophagy,while knockdown of
endogenous NEDD4 or overexpression of theligase-deadmutant of
NEDD4may interferewith interaction betweenSQSTM1 and NBR1 and cause
accumulation of inclusion bodies(Fig. 6). The second possible
effect is changing conformationalstructure of SQSTM1 by the
ubiquitylation for interaction withdownstream effectors, such as
TRAF6 and KEAP1, to activateinclusion body autophagy. The
ubiquitylated PB1 domain couldintramolecularly interact with the
Uba domain at the C-terminus toexpose the effector interactive
regions that sit between, thusenhancing interaction with downstream
effectors and activatingselective autophagic signaling. Our future
studies will follow thesequestions and determine how the
ubiquitylation affects hetero-dimerization of SQSTM1 with NBR1 and
interaction withdownstream interactive effectors in inclusion body
autophagy.
MATERIALS AND METHODSMaterialsAnti-SQSTM1 (D3; SC-28359)
antibody was purchased from Santa CruzBiotech; anti-NBR1 from
Proteintech (16004-1-AP); anti-NEDD4 fromMillipore (07-049);
anti-LC3 from Abgent (AP1802a); anti-GFP (MMS-118R), anti-ubiquitin
(P4G7; MMS-258R) and anti-HA (MMS-101R) fromBioLegend; anti-CANX
and anti-GOLGA2/GM130 from ECM Biosciences(OK7670); anti-ACTB from
Sigma-Aldrich (A5441). The dilution forantibodies was 1:1000 for
western blotting; 2 µg antibody/ml lysate forimmunoprecipitation;
1:50 for immunofluorescence staining. The DNA
mutagenesis kit (QuikChange® Site-Directed Mutagenesis Kit)
waspurchased from Strategene (200518). The NEDD4 shRNA
(5′-AUUUGA-ACCGUAUAGUUCAGC-3′) in the lentiviral expression vector
pLKO.1 waspurchased from Open Biosystems (RHS4533-EG4734). All the
cell lineswere purchased from ATCC.
Cell culture and transfectionHEK293T, HEK293A and A549 cells
were maintained in Dulbecco’smodified Eagle’s medium (Gibco,
11965092) with 10% heat-inactived fetalbovine serum (FBS), 100
units/ml penicillin and streptomycin at 37°C with5% CO2. For
transfection, the cells were seeded 1 day before transfection.The
transfection procedures were the same as described previously
(Linet al., 2010; Sun et al., 2017).
Construction of plasmids and mutagenesisHuman SQSTM1 orMAP1LC3B
cDNAwas subcloned into the mammalianexpression vectors pcDNA3-HA,
pcDNA3-MYC, lentiviral GFP vectorpLVTHM-GFP (a gift fromDr Jihe
Zhao at University of Central Florida) orGST fusion vector pGEX4T3
(GE Health Care Life Sciences, 28-9545-52).Human NEDD4 or the
mutant cDNA was subcloned into the lentiviralexpression vector pFUW
(a gift from Dr Jihe Zhao at University of CentralFlorida) for
establishing stable cell lines in A549 cells, and into themammalian
expression vector pcDNA3-HA for transient transfection inHEK293
cells. Point mutations and truncations ofNEDD4 or SQSTM1werecreated
using a mutagenesis kit from Stratagene.
Preparation of cell lysates, immunoprecipitation, immunoblotand
GST-fusion protein affinity precipitation assayCells were rinsed
once with ice-cold PBS and lysed in ice-cold Mammalianlysis buffer
(40 mM Hepes, pH 7.4, 100 mM NaCl, 1% Triton X-100,25 mM glycerol
phosphate, 1 mM sodium orthovanadate, 1 mM EDTA,10 µg/ml aprotinin,
and 10 µg/ml leupeptin) or RIPA buffer (40 mMHepes,pH 7.4, 1%
Triton X-100, 0.5% sodium deoxylcholate, 0.1% SDS, 100 mMNaCl, 1 mM
EDTA, 25 mM β-glycerolphosphate, 1 mM sodiumorthovanadate, 10 µg/ml
leupeptin and aprotinin) as indicated. The celllysates were cleared
by centrifugation at 13,000 rpm for 15 min. Forimmunoprecipitation,
primary antibodies were added to the lysates andincubated with
rotation at 4°C for 30 min, followed by adding 20 µl
ofprotein-A–Sephorose bead slurry (1:1) to the lysates and
incubating withrotation for an additional 3 h. The
immunoprecipitates were washed threetimes with lysis buffer. The
cell lysates or immunoprecipitated proteins weredenatured by
addition of SDS-PAGE sample buffer and boiled for 5 min,resolved by
8–14% SDS-PAGE. The proteins in the gel were transferred toPVDF
membranes (Millipore). Immunoblotting with chemiluminescencewas
performed as described previously (Lin et al., 2010; Sun et al.,
2017).
GST fusion protein expression, purification and affinity
precipitationassay were performed as previously described (Lin et
al., 2010; Sun et al.,2017).
Immunofluorescence stainingCells were cultured in glass
coverslip-bottomed culture dishes (MatTek,Ashland, MA) to 50–80%
confluence. After the culture medium wasaspirated, the cells were
rinsed with PBS twice, fixed with 3.7%paraformaldehyde at 25°C for
10 min, and permeabilized with 0.2% TritonX-100 in PBS at 25°C for
10 min. After washing with PBS, the cells wereincubatedwith primary
antibody at 8°Covernight. The cells werewashedwithPBS three times
and incubated with secondary antibody conjugated with afluorescent
dye at 37°C for 1–2 h. After washing with PBS three
times,fluorescent staining of the cells was visualized under a
Zeiss LSM710confocal microscope or Nikon inverted fluorescent
microscope.
Quantification of fluorescent puncta number and sizeThe analysis
and quantification of fluorescent images were performed
usingImageJ. The threshold in detection of the fluorescence was set
to cover allthe visible fluorescent puncta. Numbers of fluorescent
puncta were countedfrom two randomly selected fluorescence
microscopy fields (25 to 47 cells).The size of the fluorescent
puncta in each cell was measured and averaged.
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Statistical analysis was performed based on the numbers and
average sizesof puncta from each of the cells.
Virus packaging and transductionThe viral packaging was
performed as described previously (Mi et al., 2015;Sun et al.,
2017). Briefly, the lentiviral plasmids were co-transfected
withpsPAX2 (Addgene) and pMD2.G (Addgene) packaging plasmids
intoactively growing HEK293KT cells using Lipofectamine 2000
transfectionreagent. Viral particle-containing culture medium was
collected every 24 hthree times. The medium was cleared by
centrifugation at 1000 g for 5 min,and used for infecting target
cells in the presence of 6 µg/ml polybrene. Theinfected cells were
selected with puromycin.
Analysis of autophagyAutophagy was activated by treatment of
cells with the mTOR inhibitorrapamycin (LC Laboratory, R5000) for
the indicated time. LC3- or SQSTM1-positive autophagosomes were
visualized by either immunofluorescencestaining or GFP-tag under
Zeiss LSM710 confocal fluorescent microscope ora Nikon inverted
fluorescent microscope.
Detection of ubiquitylated proteins and in vitro E3
ubiquitinligase activity assayDetection of ubiquitylated proteins
was performed using both GST-Ubapulldown and immunoprecipitation
assays as described previously (Lin et al.,2010; Wang et al.,
2010). Briefly, cells were lysed with RIPA buffer (40 mMHepes, pH
7.4, 1% Triton X-100, 0.5% sodium deoxylcholate, 0.1% SDS,100 mM
NaCl, 1 mM EDTA, 25 mM β-glycerolphosphate, 1 mM
sodiumorthovanadate, 10 µg/ml leupeptin and aprotinin) and the
ubiquitylated proteinswere detected either by immunoprecipitation
with the primary antibodyfollowed by immunoblotting with an
anti-ubiquitin antibody (BioLegend,646302), or by affinity
precipitation with GST–UBA-conjugated glutathionebeads followed by
immunoblotting with anti-SQSTM1 antibody.
Statistical analysisThe Student t-test was used in statistical
analysis of experimental data. AP-value less than 0.05 was
considered as statistically significant.
AcknowledgementsWe want to thank Dr Jihe Zhao of University of
Central Florida for the lentiviralexpression vectors pLVTHM-GFP and
pFUW.
Competing interestsThe authors declare no competing or financial
interests.
Author contributionsConceptualization: Q.L., W.Y.; Methodology:
Q.L., W.Y.; Validation: A.S.; Formalanalysis: Q.L., Q.D., G.S.,
M.C., W.Y.; Investigation: Q.L., Q.D., H.M., A.S., J.W.,K.P., C.C.,
W.Y.; Resources: W.Y.; Data curation: Q.L., Q.D., H.M., W.Y.;
Writing -original draft: Q.L., W.Y.; Writing - review &
editing: W.Y.; Visualization: W.Y.;Supervision: Q.L., G.S., W.Y.;
Project administration: Q.L., W.Y.; Fundingacquisition: Q.L.,
W.Y.
FundingThis work is supported by National Natural Science
Foundation of China (NSFC)(81372208 to Q.L. and 81472558 to
W.Y.).
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