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 (humandock11@gmail.com; sh3px1@yahoo.com)

Q.L., 0000-0002-4393-2495; W.Y., 0000-0002-1246-7260

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mailto:humandock11@gmail.commailto:sh3px1@yahoo.comhttp://orcid.org/0000-0002-4393-2495http://orcid.org/0000-0002-1246-7260

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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