© 2017. Published by The Company of Biologists Ltd | Journal of Cell Science (2017) 130, 3839-3850 doi:10.1242/jcs.207068

RESEARCH ARTICLE The HECT E3 ubiquitin ligase NEDD4 interacts with and ubiquitylates SQSTM1 for inclusion body autophagy Qiong Lin1,*, Qian Dai1, Hongxia Meng1, Aiqin Sun1, Jing Wei1, Ke Peng1, Chandra Childress2, Miao Chen3, Genbao Shao1 and Wannian Yang1,*

ABSTRACT Ubiquitylation is an important biochemical process in SQSTM1- Our previous studies have shown that the HECT E3 ubiquitin ligase mediated selective autophagy. Multiple studies have shown that the NEDD4 interacts with LC3 and is required for starvation and rapamycin- ubiquitylation of protein inclusion bodies, aggregates or other induced activation of autophagy. Here, we report that NEDD4 directly autophagic cargos is pivotal for recognition by SQSTM1 in the binds to SQSTM1 via its HECT domain and polyubiquitylates SQSTM1. autophagic degradation process (Kim et al., 2008; Johansen and This ubiquitylation is through K63 conjugation and is not involved in Lamark, 2011; Rogov et al., 2014). In addition, SQSTM1 is capable proteasomal degradation. Mutational analysis indicates that NEDD4 of recruiting E3 ubiquitin ligases, such as TRAF6 and KEAP1, for interacts with and ubiquitylates the PB1 domain of SQSTM1. Depletion ubiquitylating autophagic cargos or autophagic proteins during of NEDD4 or overexpression of the ligase-defective mutant of NEDD4 initiation, formation or transportation of selective autophagosomes induced accumulation of aberrant enlarged SQSTM1-positive inclusion (Kirkin et al., 2009a; Fan et al., 2010; Fusco et al., 2012; Isakson bodies that are co-localized with the endoplasmic reticulum (ER) marker et al., 2013; Stolz et al., 2014). As an autophagic cargo receptor, CANX, suggesting that the ubiquitylation functions in the SQSTM1- SQSTM1 is transported along with the autophagic cargos in mediated biogenic process in inclusion body autophagosomes. Taken autophagosomes to lysosomes for degradation (Bjørkøy et al., 2005, together, our studies show that NEDD4 is an autophagic E3 ubiquitin 2006; Ichimura et al., 2008). Therefore, degradation of SQSTM1 ligase that ubiquitylates SQSTM1, facilitating SQSTM1-mediated sometimes is used as a molecular marker for activation of autophagy inclusion body autophagy. (Bjørkøy et al., 2009). While the role of SQSTM1 in selective autophagy is well KEY WORDS: Autophagy, E3 ubiquitin ligase, Inclusion bodies, established, it remains poorly understood how the receptor activity of NEDD4, PB1 domain, SQSTM1, p62, Ubiquitylation SQSTM1 is regulated during selective autophagy. A recent study found that casein kinase 2 (CK2) phosphorylates S403 in the Uba INTRODUCTION domain of SQSTM1 and enhances the binding capacity of SQSTM1 to SQSTM1 (p62) is an autophagic cargo receptor that plays a key role in the polyubiquitin chain (Matsumoto et al., 2011). This phosphorylation selective autophagy (Kirkin et al., 2009a; Rogov et al., 2014). Early promotes SQSTM1 to target polyubiquitylated proteins and recruit studies have shown that SQSTM1 is associated with protein inclusions ubiquitylated cargos to autophagosomes (Matsumoto et al., 2011). and aggregates, such as Mallory–Denk bodies and Lewy bodies Recent studies indicate that ubiquitylation also regulates the autophagy (Stumptner et al., 1999, 2007; Nakaso et al., 2004; Tanji et al., 2015), receptor function of SQSTM1 for recognition of autophagic cargos. It and is considered to be a universal component of protein inclusions has been found that SQSTM1 is ubiquitylated by the ring family E3 (Zatloukal et al., 2002). Further studies found that SQSTM1 functions ubiquitin ligases TRIM21 (Pan et al., 2016), KEAP1–CULLIN3 (Lee as a receptor for ubiquitylated protein inclusion bodies or aggregates et al., 2017), PARKIN (Song et al., 2016), and the E2 conjugating and recruits them to autophagosomes for degradation via interactions enzymes UBE2D2/3 (Peng et al., 2017). The ubiquitylation produces with LC3-II (Komatsu et al., 2007; Pankiv et al., 2007). Now, we diversified effects on SQSTM function, including suppression and know that SQSTM1 is a universal autophagic cargo receptor involved activation of the autophagic receptor activity (Pan et al., 2016; Lee in multiple types of selective autophagy, such as mitophagy, et al., 2017; Peng et al., 2017) and promotion of the proteasomal pexophagy, xenophagy and aggrephagy (Zheng et al., 2009; Geisler degradation of SQSTM1 (Song et al., 2016). However, how et al., 2010; Bartlett et al., 2011; Ishimura et al., 2014; Zhang et al., ubiquitylation of SQSTM1 regulates cellular inclusion body 2015). As a key cargo receptor in selective autophagy of protein autophagy remains unknown. Furthermore, SQSTM1 also participates inclusions and aggregates, malfunction of SQSTM1 is associated with in other cellular signaling pathways, such as atypical PKC and multiple diseases, such as Parkinson’s disease, Huntington’s disease, NF-κB signaling pathways (Puls et al., 1997; Sanchez et al., 1998; Alzheimer’s disease, alcoholic hepatitis and cirrhosis (Kuusisto et al., Sanz et al., 1999). Whether these pathways are regulated 2002; Stumptner et al., 2002; Zatloukal et al., 2002; Nakaso et al., independently or are connected to autophagy has not been clarified. 2004; Du et al., 2009; Geisler et al., 2010; Cuyvers et al., 2015). Our recent studies found that NEDD4 (also known as NEDD4-1), a member of the HECT E3 ubiquitin ligase family, interacts with the 1School of Medicine, Jiangsu University, Zhenjiang 212013, China. 2Department of autophagic protein LC3 through an LIR domain and is essential for Biology, Susquehanna University, 514 University Ave, Selinsgrove, PA 17870, USA. starvation or rapamycin-induced activation of autophagy (Sun et al., 3Department of Pathology, Affiliated People’s Hospital, Jiangsu University, Zhenjiang 212013, China. 2017). Knockdown of NEDD4 by shRNA caused aggregation of GFP–LC3 puncta in the ER and deformation of mitochondria. It *Authors for correspondence ([email protected]; [email protected]) appears that interaction of NEDD4 with LC3 is not only necessary for Q.L., 0000-0002-4393-2495; W.Y., 0000-0002-1246-7260 association with autophagosomes, but also for activation of the E3 ubiquitin ligase. Our preliminary data also demonstrate that NEDD4

Received 5 June 2017; Accepted 27 September 2017 ubiquitylates SQSTM1, but not LC3 (Sun et al., 2017). These results Journal of Cell Science

3839 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 3839-3850 doi:10.1242/jcs.207068 clearly indicate that NEDD4 is an important E3 ubiquitin ligase (lane 2). This result confirms that SQSTM1 is a ubiquitylation involved in autophagic activation. In this report, we continue substrate of NEDD4. We further examined whether endogenous investigating the role of NEDD4 in autophagy by characterizing SQSTM1 is the substrate of NEDD4 upon activation of autophagy in interaction and ubiquitylation of SQSTM1 and defining the function the lung cancer cell line A549. To elevate the ubiquitylation of of SQSTM1 ubiquitylation. We found that NEDD4 interacts with SQSTM1, we treated cells with rapamycin to activate autophagy, and SQSTM1 through the HECT (homologous to E6-AP carboxyl with chloroquine to block the autophagic degradation of SQSTM1. As terminus) domain. The PB1 domain in SQSTM1 appears to be the shown in Fig. 2B, upon treatment with rapamycin and chloroquine, NEDD4 interactive and ubiquitylating region. The polyubiquitylation endogenous SQSTM1 was significantly polyubiquitylated (lane 2), of SQSTM1 by NEDD4 is mainly through K63 conjugation, which is whereas in the NEDD4 shRNA cell line, SQSTM1 was no longer important for the SQSTM1-mediated inclusion body autophagy, polyubiquitylated upon treatment with rapamycin and chloroquine, rather than proteasomal degradation. Our studies demonstrate suggesting that endogenous SQSTM1 is ubiquitylated by NEDD4 in NEDD4 as a key E3 ubiquitin ligase in selective autophagy that response to activation of autophagy. interacts with and ubiquitylates the autophagy receptor SQSTM1. The carboxyl terminus of SQSTM1 contains an Uba domain that can bind to other ubiquitylated proteins. To exclude the possibility that RESULTS NEDD4-dependent ubiquitylation detected in SQSTM1 is from the NEDD4 interacts with SQSTM1 SQSTM1 Uba domain-associated proteins, we made the Uba truncation Our previous studies have shown that NEDD4 ubiquitylates SQSTM1 mutant of SQSTM1, SQSTM1-UbaΔ. Co-expression of SQSTM1- (Sun et al., 2017). To determine whether this ubiquitylation results UbaΔ with NEDD4 in HEK293 cells showed polyubiquitylation from interaction between NEDD4 and SQSTM1, we characterized the of SQSTM1-ΔUba (lane 2, Fig. 2C), confirming that the binding of NEDD4 to SQSTM1 using a co-immunoprecipitation polyubiquitylation of SQSTM1 by NEDD4 is not from the SQSTM1 assay. As shown in Fig. 1A, NEDD4 is co-immunoprecipitated with Uba-associated ubiquitylated proteins. In fact, immunoprecipitation of SQSTM1 when both were co-expressed in cells, indicating that SQSTM1 or the Uba truncation mutant without NEDD4 co-expression SQSTM1 binds to NEDD4. Interestingly, the ligase-dead (LD) mutant showed no detectable polyubiquitylation (lane 4 in Fig. 2A and lane 3 in NEDD4-C867A bound to SQSTM1 with much higher affinity than Fig. 2C), indicating that the SQSTM1 Uba-associated ubiquitin proteins wild-type NEDD4 (Fig. 1B). This suggests that the ligase-dead mutant (if any) produced little interference with NEDD4-mediated of NEDD4 has a ‘trap’ effect on SQSTM1, which is a typical binding polyubiquitylation of SQSTM1. mode for a catalysis-defective enzyme with a substrate, as observed in We further determined the type of ubiquitin chain linkage of binding of tyrosine phosphatase-defective mutants with their substrates SQSTM1 catalyzed by NEDD4. As shown in Fig. 2D, NEDD4 (Flint et al., 1997). catalyzed the K63-linked polyubiquitylation of SQSTM1 (top To determine the SQSTM1-binding region in NEDD4 more panel), not the K48 polyubiquitylation (second panel), suggesting specifically, we made a series of truncation mutants of NEDD4 that NEDD4 catalyzed polyubiquitylation of SQSTM1 may not be (Fig. 1C), and tested the binding of these mutants to SQSTM1. HA- involved in proteasomal degradation. However, we detected a minor tagged NEDD4 or its truncation mutants were co-expressed with GFP- K63 and K48 polyubiquitylation of SQSTM1 with expression of the tagged SQSTM1 in HEK293 cells and binding was detected by co- ligase-dead mutant NEDD4-LD or without exogenous NEDD4 immunoprecipitation assay. As shown in Fig. 1D,E, all the truncation (lanes 3 and 4, the top two panels) that might be produced by mutants are capable of binding to SQSTM1. As the mutant NEDD4- endogenous ubiquitylation. N4Δ contains only the HECT domain (see Fig. 1C), this indicates that To confirm that NEDD4-dependent polyubiquitylation of NEDD4 interacts with SQSTM1 through the HECT domain. To SQSTM1 does not lead to proteasomal degradation, we examined confirm this, we co-expressed the HECT domain-deletion mutant, levels of ubiquitylation and SQSTM1 protein upon treating the cells NEDD4-HECTΔ, with SQSTM1 in HEK293 cells, and examined with the specific proteasomal inhibitor bortezomib. As shown in interaction of the mutant with SQSTM1. As shown in Fig. 1F, while Fig. 2E, treatment with bortezomib did not induce accumulation of the NEDD4 ligase-dead mutant NEDD4-C867A (NEDD4-LD) was either NEDD4-dependent ubiquitylation or protein of SQSTM1. In co-immunoprecipitated with SQSTM1, NEDD4-HECTΔ showed fact, bortezomib eliminated the NEDD4-dependent ubiquitylation little co-precipitation with SQSTM1, confirming that the HECT of SQSTM1 (compare lane 7 with lane 8 in the right top panel in domain interacts with SQSTM1. We also examined the binding parallel with lane 2 and lane 3 in the left top panel). These data of NEDD4 to another autophagy receptor NBR1 using a co- further suggest that NEDD4-dependent polyubiquitylation of immunoprecipitation assay, and found no detectable binding (Fig. 1G). SQSTM1 is involved in autophagy, not proteasomal degradation.

NEDD4 polyubiquitylates SQSTM1 through K63 chain NBR1 is not a ubiquitylation substrate of NEDD4 conjugation and the ubiquitylation does not cause NBR1 is another autophagic cargo receptor that contains similar proteasomal degradation structural domains to SQSTM1 and functions in selective autophagy, SQSTM1 is a key autophagic protein that interacts with LC3 and it particularly in recruiting ubiquitylated protein inclusions to plays an important role in selective autophagy, including mitophagy autophagosomes (Kirkin et al., 2009b). We wondered whether (Kirkin et al., 2009a; Johansen and Lamark, 2011; Stolz et al., 2014). NBR1 was ubiquitylated by NEDD4. As shown in Fig. 3A, while Here, we characterized the ubiquitylation of SQSTM1 by NEDD4 SQSTM1 was significantly ubiquitylated when co-expressed with using both immunoprecipitation and GST–Uba pulldown assays. NEDD4 (compare lane 2 with lane 4, second panel), NBR1 showed GST–Uba pulldown assay has been successfullyusedfordetectionof no increase in ubiquitylation when co-expressed with NEDD4 ubiquitylated proteins in our previous studies (Lin et al., 2010). As (compare lane 3 with lane 5, top panel), indicating that NBR1 was not showninFig.2A,GFP-taggedSQSTM1 was ectopically expressed ubiquitylated by NEDD4. Interestingly, endogenous NBR1 was with or without NEDD4 in HEK293 cells. Without NEDD4, heavily ubiquitylated independent of NEDD4 (top panel). We further SQSTM1 had a low level of ubiquitination (lane 4). When co- confirmed that NBR1 showed NEDD4-independent ubiquitylation expressed with NEDD4, SQSTM1 was heavily polyubiquitylated when co-expressed with NEDD4 mutants (Fig. 3B). NEDD4- Journal of Cell Science

3840 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 3839-3850 doi:10.1242/jcs.207068

Fig. 1. NEDD4 interacts with SQSTM1 but not NBR1. (A,B) HA-tagged NEDD4 or the ligase-dead mutant NEDD4-C867A (NEDD4-LD) was co-transfected with SQSTM1 or GFP-SQSTM1 in HEK293 cells. SQSTM1 or GFP-SQSTM1 was immunoprecipitated with an anti-SQSTM1 antibody and co-immunoprecipitated HA-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 in HEK293T cells. GFP–SQSTM1 was immunoprecipitated with an anti-SQSTM1 antibody and co-immunoprecipitated HA-tagged NEDD4 or the mutant was detected by immunoblotting with an anti-HA antibody. In E, white asterisks indicate the NEDD4 truncation mutant bands. ACTB, β-actin. (G) HA-tagged NEDD4 or the truncation mutants were co-transfected with NBR1 in HEK293T cells. NBR1 was immunoprecipitated with an anti-NBR antibody and co-immunoprecipitated HA-tagged NEDD4 or the mutant was detected by immunoblotting with an anti-HA antibody. independent ubiquitylation and NBR1 protein level were degradation through ubiquitylation by a non-NEDD4 E3 ubiquitin dramatically enhanced by treatment with proteasomal inhibitor ligase. NEDD4 seems to antagonize the E3 ubiquitin ligase for MG-132, but not lysosomal inhibitor chloroquine (Fig. 3B–D), NBR1, because co-expression with NEDD4 markedly reduced suggesting that NBR1 has an active turnover by proteasomal ubiquitylation of NBR1 (compare lane 9 with lane 8, top panel, Journal of Cell Science

3841 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 3839-3850 doi:10.1242/jcs.207068

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 ubiquitylated SQSTM1 was detected by immunoblotting with an anti-ubiquitin antibody. (B) Ubiquitylation of endogenous SQSTM1 is dependent on NEDD4. shNEDD4 or the vector (pLKO.1) cell line established in lung cancer A549 cells was treated with or without chloroquine plus rapamycin for 18 h, and endogenous SQSTM1 was immunoprecipitated. Ubiquitylation of SQSTM1 was detected by immunoblotting with an anti-ubiquitin antibody. Amount of SQSTM1 in the immunoprecipitation (middle panel) and NEDD4 in the lysates (bottom panel) was detected by immunoblotting. The band labeled IgG is the anti-SQSTM1 IgG containing 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 Uba deletion mutant SQSTM1-UbaΔ was co-expressed with NEDD4 in HEK293 cells. The ubiquitylated SQSTM1 was precipitated with GST–ACK1Uba and detected by immunoblotting with anti-SQSTM1. (D) HA-tagged NEDD4 or its ligase-dead mutant NEDD4-C867A (NEDD4-LD) was co-expressed with SQSTM1 in HEK293T cells. SQSTM1 was immunoprecipitated with anti-SQSTM1. Polyubiquitylation of SQSTM1 was detected by immunoblotting with an antibody against either K63-linked or K48-linked polyubiquitin. The expression of SQSTM1, NEDD4 or the ligase-dead mutant was determined by immunoblotting of the cell lysates 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) for 12 h prior to harvesting the cells. Ubiquitylation of SQSTM1 was detected by immunoblotting with an anti-ubiquitin antibody.

Fig. 3B). In addition, co-expression with the N-terminal truncation mutants include the PB1 deletion mutant N43Δ, the PB1 domain mutant of NEDD4, NEDD4-N1Δ, which is defective in binding to point mutants K7A, K13E, R21A/R22A (named as 2R2A), K13E/ LC3 (Sun et al., 2017), dramatically enhanced the amount of NBR1 R21A/R22A (named as K13E/2R2A), D69A, the LIR domain point protein through an unknown mechanism (see lane 6, top panel, mutant L341A, the LIR deletion mutant [334–342]Δ, and the Fig. 3C,D). These data indicate that NBR1 is not a ubiquitylation Uba domain point mutant L417V (Seibenhener et al., 2004). substrate of NEDD4. Ubiquitylation of the SQSTM1 domain mutants by NEDD4 was first examined by the GST–Uba pulldown assay (Fig. 4B). Deletion NEDD4 interacts with the PB1 domain of SQSTM1 and this of PB1 (N43Δ) diminished the ubiquitylation of SQSTM1 (lane 5). interaction is required for ubiquitylation of SQSTM1 Mutations in the LIR and the Uba domain produced an insignificant To identify the region in SQSTM1 that interacts with and is effect on the ubiquitylation. These results suggest that the PB1 ubiquitylated by NEDD4, we made a series of SQSTM1 mutants in domain is essential for ubiquitylation by NEDD4, while interaction its functional domains and tagged with GFP (Fig. 4A). These with LC3 or ubiquitin is dispensable. Journal of Cell Science

3842 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 3839-3850 doi:10.1242/jcs.207068

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 in HEK293T 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 cell lysates 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.

Previous studies have shown that the PB1 domain functions in because K7A, which is also a dimerization defective mutant homo- or hetero-dimerization of SQSTM1 through the interaction (Lamark et al., 2003), retains NEDD4 binding capacity (lane 3). between the basic cluster and the OPCA (OPR–PC–AID) motif We further characterized NEDD4-dependent ubiquitylation of within the PB1 domain and is required for localization on the PB1 mutants of SQSTM1 by both immunoprecipitation autophagosomes (Lamark et al., 2003; Itakura and Mizushima, (Fig. 4D) and GST–Uba pulldown assays (Fig. 4E). The results 2011). The results in Fig. 4B indicate that the PB1 domain is from both assays were consistent, and showed that mutations on required for either binding to or ubiquitylation by NEDD4 or both. R21/R22 (lane 4 in Figs. 4D and 4E) and mutation on K7 (lane 3 in Thus, we used the PB1 truncation mutant N43Δ and the Fig. 4D, lane 5 in Fig. 4E) significantly reduced the ubiquitylation, dimerization defective mutants K7A and 2R2A for testing the whereas the mutation on K13 had no effect (lane 5 in Fig. 4D, lane 3 binding to NEDD4. As shown in Fig. 4C, the PB1 deletion mutant in Fig. 4E). The results indicate that the PB1 domain and the R21/ N43Δ or the point mutant 2R2A failed to co-immunoprecipitate R22 residues are essential for the binding to and ubiquitylation by NEDD4 (lanes 2 and 4), whereas wild-type SQSTM1 or the mutant NEDD4, and that K7 is one of the major NEDD4 ubiquitylation K7A co-immunoprecipitated NEDD4 (lanes 1 and 3). These data sites. It has been shown that the RING family E3 ubiquitin ligase demonstrate that PB1 is the NEDD4-interactive domain and that TRIM21 also ubiquitylates K7 of SQSTM1, and the ubiquitylation R21 and R22 in the PB1 domain are the residues essential for impairs oligomerization of SQSTM1 thus suppressing the binding to NEDD4. In addition, the defect in dimerization in the SQSTM1-mediated sequestration of KEAP1 (Pan et al., 2016). In

2R2A mutant is unlikely to be the cause of loss of NEDD4 binding, future studies, it would be interesting to examine whether the Journal of Cell Science

3843 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 3839-3850 doi:10.1242/jcs.207068

Fig. 4. NEDD4 interacts with and ubiquitylates SQSTM1 through the PB1 domain. (A) SQSTM1 mutants. PB1, PHOX and BEM1P domain; ZZ, ZZ-type zinc finger domain; TB, TRAF6 binding domain; LIR, LC3-interactive region; KIR, KEAP1 interactive region; UBA, ubiquitin-associated domain. (B–E) NEDD4 was 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, lightly exposed. In C and D, SQSTM1 or its mutants in the lysates were immunoprecipitated with an anti-SQSTM1 antibody. The co-immunoprecipitated NEDD4 was detected with an anti-NEDD4 antibody and ubiquitylation of SQSTM1 or its mutants was detected with an anti-ubiquitin antibody. (F) GFP–SQSTM1 or its mutant was expressed in HEK293T cells with or without NEDD4. GST–LC3 was used to precipitate GFP–SQSTM1 or its mutants and results detected by immunoblotting with anti-SQSTM1 and anti-NEDD4 antibodies. ubiquitylation of K7 by NEDD4 has the same effect on SQSTM1 as biogenesis, and causes aggregation of GFP–LC3 puncta (Sun et al., that of TRIM21 2017). Here, we further determined the effect of NEDD4 knockdown We also examined whether the ubiquitylation affects binding of on the cellular localization and morphology of SQSTM1-positive SQSTM1 to LC3 by co-expression of the SQSTM1 mutants with fluorescent puncta in response to treatment with rapamycin. Similar NEDD4. The GST–LC3 pulldown assay confirmed that SQSTM1- to GFP–LC3, SQSTM1 was observed as tiny fluorescent puncta L341A or [334–342]Δ is defective in LC3 binding (lanes 6–10, localized at para-nuclei in the vector control cells without rapamycin Fig. 4F) and other mutants are capable of binding to LC3 (lanes 5,6 treatment, while in the NEDD4 knockdown cells, significant and 11–14, Fig. 4F). Co-expression with NEDD4 did not affect accumulation of heterogeneous large SQSTM1 puncta was seen binding of SQSTM1 or the mutants to LC3, although the protein (Fig. 5A). Quantification analysis indicates that the average size of the level of wild-type SQSTM1 and the PB1 domain truncation mutant SQSTM1 puncta increased ∼4-fold up to ∼1 µm, upon knockdown of was slightly reduced by co-expression with NEDD4 (lanes 4 and 6, NEDD4, but the average number of SQSTM1 puncta per cell did not second panel, Fig. 4D). This result suggests that ubiquitylation of change significantly (Fig. 5B). Furthermore, SQSTM1 puncta in the SQSTM1 by NEDD4 is not involved in regulation of the LC3 NEDD4 knockdown cells were randomly distributed in the cells, no binding, which is consistent with our previous studies with para-nuclear localization was observed (Fig. 5A). These large immunofluorescence staining (Sun et al., 2017). SQSTM1-positive puncta in NEDD4 knockdown cells are likely to be protein inclusion bodies, which are the common autophagic cargos Knockdown of NEDD4 causes accumulation of the SQSTM1- associated with SQSTM1 (Zatloukal et al., 2002). Upon treatment positive inclusion bodies with rapamycin for 18 h, SQSTM1 puncta in the vector control cells Our recent studies have shown that knockdown of NEDD4 impairs was distributed at one side of the nucleus (bottom left panel, Fig. 5A) rapamycin- and starvation-induced autophagy and autophagosomal and average numbers of the SQSTM1 puncta per cell increased Journal of Cell Science

3844 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 3839-3850 doi:10.1242/jcs.207068

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 is shown at the bottom. NEDD4 (LM), low molecular weight NEDD4; NEDD4 (HM), high molecular weight NEDD4. NEDD4 (LM) is a degradation product of NEDD4 (HM) (Sun et al., 2017). Endogenous SQSTM1 was immunostained with an anti-SQSTM1 antibody followed by a fluorescent dye-conjugated secondary antibody, and the fluorescence was visualized under an inverted Nikon fluorescent microscope. Scale bars: 10 µm. (B) Quantification of numbers and sizes of the SQSTM1 fluorescent 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 and measured 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<0.01; ***P<0.001. (C,D) Lentiviral vector-loaded GFP–SQSTM1 was stably transfected and expressed in both the vector control and NEDD4 shRNA cell lines and treated with DMSO (solvent control) or 1 µM rapamycin for 18 h. The cells were fixed and stained with anti-GOLGA2 (Golgi marker) (C), anti-CANX (ER marker) (D) and DAPI. Scale bars: 20 µm. Journal of Cell Science

3845 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 3839-3850 doi:10.1242/jcs.207068 significantly (Fig. 5B), indicating that biogenesis of the SQSTM1- with the Golgi marker GOLGA2/GM-130 (Fig. 5C), and had little positive autophagosomes was induced by rapamycin. In NEDD4 change upon treatment with rapamycin (Fig. 5D), suggesting that knockdown cells, large SQSTM1 puncta remained randomly SQSTM1-positive inclusion bodies are retained in ER membrane distributed, and numbers of the SQSTM1 puncta did not change vesicles upon NEDD4 knockdown, which is consistent with our significantly upon rapamycin treatment (Fig. 5A,B). These results previous observation on aggregation of the LC3-positive puncta in indicate that knockdown of NEDD4 blocks the rapamycin-induced ER membrane vesicles upon depletion of NEDD4 (Sun et al., 2017). biogenesis of autophagosomes and suggest that knockdown of These results suggest that the defect in inclusion body autophagy NEDD4 might impair the SQSTM1-mediated inclusion body caused by NEDD4 depletion might occur in the early stage of the autophagy, thus causing accumulation of the large SQSTM1- autophagosomal biogenic process in the ER. positive inclusion bodies in cells. Our previous studies showed that knockdown of NEDD4 caused Ubiquitylation of SQSTM1 by NEDD4 is required for inclusion aggregation of GFP–LC3 puncta that was co-localized with ER body autophagy membrane markers, but not with Golgi marker, and suggested that As knockdown of NEDD4 caused aberrant aggregation of the NEDD4 is required for biogenesis of autophagosomes (Sun et al., SQSTM1-positive inclusion bodies retained in the ER (Fig. 5), we 2017). Here, we examined the effect of NEDD4 knockdown on wondered whether the large aggregates of the SQSTM1-positive localization of the SQSTM1-positive puncta in cells. As shown in protein inclusion bodies in NEDD4 knockdown cells were formed Figs. 5A, 5C and 5D, a portion of the SQSTM1 puncta was upon defective ubiquitylation of SQSTM1 by NEDD4. To investigate aberrantly aggregated into large inclusion bodies in the shNEDD4 this hypothesis, we used HEK293T or HEK293A cells to transiently cell line, but not in the vector control cell line. Furthermore, the overexpress GFP–LC3, SQSTM1 and the PB1 defective mutant enlarged SQSTM1-positive inclusion bodies in the shNEDD4 cell SQSTM1-2R2A with wild-type NEDD4 or its ligase-dead mutant line were co-localized with the ER marker CANX (Fig. 5D), but not NEDD4-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). Journal of Cell Science

3846 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 3839-3850 doi:10.1242/jcs.207068 with wild-type SQSTM1 in HEK293T cells led to typical localization autophagosomes, and caused deformation of mitochondria, as we of GFP-LC3 and SQSTM1 on autophagosomes. Co-expression of have shown previously (Sun et al., 2017). In this report, we have GFP–LC3 with the PB1-defective mutant SQSTM1-2R2A resulted demonstrated a new role of ubiquitylation of SQSTM1 by the HECT in diffused fluorescence of both GFP–LC3 and SQSTM1-2R2A, E3 ubiquitin ligase NEDD4 in regulation of inclusion body confirming that homo-oligomerization, which is defective in autophagy. Depletion of endogenous NEDD4 or ectopic SQSTM1-2R2A, is required for SQSTM1 to localize on overexpression of the ligase-dead mutant of NEDD4 caused autophagosomes. However, co-expression of GFP–LC3 and formation of aberrant gigantic aggregates of both LC3- and SQSTM1 with NEDD4 ligase-dead mutant C867A resulted in SQSTM1-positive inclusion bodies, pointing to an important role formation of multiple gigantic inclusion bodies (1–10 µm diameter) of NEDD4 in the SQSTM1-mediated inclusion body autophagy. containing the GFP–LC3 and SQSTM1 fluorescence in cells (the SQSTM1 is known to be involved in inclusion body autophagy bottom panels, Fig. 6A). These data, together with the data in Fig. 5, (Zatloukal et al., 2002; Komatsu et al., 2007; Pankiv et al., 2007). suggest that, first, defect in ubiquitylation of SQSTM1 by NEDD4 Early studies found that SQSTM1 associated with ubiquitylated may impair inclusion body autophagy; and second, ubiquitylation of Mallory–Denk bodies in alcoholic liver and Lewy bodies in SQSTM1 is not required for homo-oligomerization, as either Parkinson’s disease tissue (Stumptner et al., 1999, 2007; Zatloukal knockdown of NEDD4 or overexpression of the ligase-dead mutant et al., 2002). Thus, SQSTM1 was defined as an inclusion body of NEDD4 did not result in diffused localization of SQSTM1 and marker protein (Zatloukal et al., 2002). Subsequently, after interaction GFP–LC3 in cells (Fig. 5 and Fig. 6A). In addition, overexpression of of SQSTM1 with the autophagosomal protein LC3 was discovered, the ligase-dead mutant induced much larger inclusion bodies than this inclusion body association was linked to the function of that induced by knockdown of NEDD4 (Fig. 5A-D, Fig. 6A), SQSTM1 in mediating inclusion body autophagy (Komatsu et al., indicating that the trapping of SQSTM1 by the ligase-dead mutant of 2007; Pankiv et al., 2007). As both the LC3 (the LIR domain) and the NEDD4, as shown in Fig. 1B, which reduces the level of free ubiquitin (the Uba domain) binding ability are possessed by SQSTM1, produces a much more severe defect in autophagy- SQSTM1, it is possible that SQSTM1 functions as a ubiquitylated mediated removal of cellular inclusion bodies than that by depletion inclusion body autophagy receptor by recruiting the inclusion body to of NEDD4 only, suggesting that SQSTM1 in the NEDD4 autophagosomes through interaction with ubiquitin and LC3 (Kirkin knockdown cells may still retain partial function in facilitating the et al., 2009a). In later studies, this functional mode has been extended removal of inclusion bodies. to the other autophagic cargos, such as invaded bacteria and To confirm that ubiquitylation of SQSTM1 is required for peroxisomes, whose autophagy is also regulated by SQSTM1 (Zheng inclusion body autophagy, we co-expressed SQSTM1 or SQSTM1- et al., 2009; Zhang et al., 2015). Interestingly, NEDD4 was 2R2A plus GFP–LC3 with or without NEDD4 or its ligase-dead previously found to facilitate the endosome-mediated lysosomal mutant C867A in HEK293A cells (Fig. 6B). Upon co-expression of degradation of α-synuclein, a major component of Lewy bodies in SQSTM1 plus GFP–LC3 or NEDD4, the cells showed localization Parkinson’s disease, by directly interacting with and ubiquitylating of SQSTM1 and GFP–LC3 on normal autophagosomes (Fig. 6Bi– α-synuclein (Tofaris et al., 2011; Chung et al., 2013; Sugeno et al., iii). However, upon co-expression of SQSTM1 with the NEDD4 2014). NAB2, a small chemical that is an activator of NEDD4, ligase-dead mutant C867A, the cells formed huge inclusion bodies reversed a mutated α-synuclein-induced cytotoxicity in neurons containing SQSTM1 and GFP–LC3 (Fig. 6B iv–vi, Fig. 6Ci–iii), derived from Parkinson’s disease patients (Chung et al., 2013). confirming that lack of SQSTM1 ubiquitylation by NEDD4 impairs Although it has been proposed that NEDD4-facilitated degradation of inclusion body autophagy. Consistent with Fig. 6A, the PB1- α-synuclein is through an endosomal/lysosomal route, not autophagy defective mutant SQSTM1-2R2A and the co-expressed GFP–LC3 (Sugeno et al., 2014), our work suggest that the role of autophagy in displayed diffused distribution in cells in either the presence or the NEDD4-facilitated α-synuclein degradation might need to be re- absence of NEDD4 or the ligase-dead mutant (Fig. 6Bvii–xii, examined. Furthermore, our studies also suggest that NEDD4 might Fig. 6Ci–iii). These data suggest that ubiquitylation of SQSTM1 is be a universal selective autophagic E3 ubiquitin ligase that is required for its function in inclusion body autophagy, but not for its involved in other types of SQSTM-mediated selective autophagy, oligomerization or localization on autophagosomes. such as xenophagy, pexophagy and mitophagy. In fact, we have observed that knockdown of NEDD4 in lung cancer A549 cells DISCUSSION induced aberrant enlargement and deformation of mitochondria (Sun Ubiquitylation is an important biochemical process in selective et al., 2017). Thus, NEDD4 might be an effective therapeutic target autophagy. Most of the studies on ubiquitylation in selective for SQSTM1-mediated selective autophagy-related diseases, autophagy have been focused on the role of ubiquitylation in particularly neuronal degenerative diseases. recognition of autophagic cargos (Kirkin et al., 2009a; Johansen and Our studies presented in this report added a new mechanism Lamark, 2011; Stolz et al., 2014). Recently, ubiquitylation of underlying the SQSTM1-mediated selective autophagy, i.e. the autophagy receptors has been studied and found to be involved in a ubiquitylation by NEDD4 via the PB1 domain regulates the cargo diversified regulatory function in autophagy. Ubiquitylation by receptor activity of SQSTM1. The PB1 domain of SQSTM1 RING family E3 ubiqiuitin ligases and E2 conjugating enzymes functions in homodimerization and heterodimerization with either regulates the autophagic receptor activity (Pan et al., 2016; atypical PKCs (aPKCs), NBR1 and MAP2K5 (MEK5) (Lamark Lee et al., 2017; Peng et al., 2017) or promotes the proteasomal et al., 2003; Moscat et al., 2006, 2009). The PB1 domain of SQSTM1 degradation of SQSTM1 (Song et al., 2016). Our previous studies has two regions that are involved in dimerization: one region at the have shown that NEDD4, a member of the HECT E3 ubiquitin N-terminus of the PB1 contains several positively charged residues, ligase family, not only directly binds to autophagosomal protein such as K7, R21 and R22 in SQSTM1, the other region is at the LC3 (Sun et al., 2017), but also interacts with SQSTM1 through the C-terminus of the PB1 the so-called OPCA motif that is conserved in HECT domain and polyubiquitylates SQSTM1. Knockdown of a number of the PB1-containing proteins (Lamark et al., 2003). It has NEDD4 in lung cancer A549 cells impaired both rapamycin- and been demonstrated that K7, R21 or R22 in the PB1 of SQSTM1 is starvation-induced activation of autophagy and formation of essential for either homo-dimerization or heterodimerization through Journal of Cell Science

3847 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 3839-3850 doi:10.1242/jcs.207068 binding to the OPCA motif of the other PB1 (Lamark et al., 2003). mutagenesis kit (QuikChange® Site-Directed Mutagenesis Kit) was Our studies have shown that deletion of the first 43 amino acid purchased from Strategene (200518). The NEDD4 shRNA (5′-AUUUGA- residues in the PB1 domain, which does not include the OPCA ACCGUAUAGUUCAGC-3′) in the lentiviral expression vector pLKO.1 was region, eliminated binding to NEDD4 and the ubiquitylation by purchased from Open Biosystems (RHS4533-EG4734). All the cell lines NEDD4 (Fig. 4), indicating that the OPCA motif of SQSTM1 is not were purchased from ATCC. required for interaction with NEDD4. Mutation of R21/R22 of the Cell culture and transfection PB1 domain, which are critical residues for interaction with the HEK293T, HEK293A and A549 cells were maintained in Dulbecco’s OPCA region in dimerization, significantly reduced interaction with modified Eagle’s medium (Gibco, 11965092) with 10% heat-inactived fetal and ubiquitylation by NEDD4 (Fig. 4), suggesting that an OPCA-like bovine serum (FBS), 100 units/ml penicillin and streptomycin at 37°C with region may be present in NEDD4. As the HECT domain of NEDD4 5% CO2. For transfection, the cells were seeded 1 day before transfection. is the region binding to SQSTM1 (Fig. 1), it is possible that the HECT The transfection procedures were the same as described previously (Lin domain contains an OPCA-like region that is capable of binding to et al., 2010; Sun et al., 2017). the positive residues of the PB1 of SQSTM1. In addition, NEDD4 does not interact and ubiquitylate another selective autophagy Construction of plasmids and mutagenesis receptor NBR1 that also contains a PB1 domain at the N-terminus Human SQSTM1 or MAP1LC3B cDNA was subcloned into the mammalian (Fig. 3) (Kirkin et al., 2009a). Interestingly, the PB1 domain of NBR1 expression vectors pcDNA3-HA, pcDNA3-MYC, lentiviral GFP vector has the OPCA motif but does not have the arginine residues pLVTHM-GFP (a gift from Dr Jihe Zhao at University of Central Florida) or corresponding to R21 and R22 in the PB1 of SQSTM1 (Lamark et al., GST fusion vector pGEX4T3 (GE Health Care Life Sciences, 28-9545-52). Human NEDD4 or the mutant cDNA was subcloned into the lentiviral 2003). This further supports the hypothesis that the HECT domain of expression vector pFUW (a gift from Dr Jihe Zhao at University of Central NEDD4 contains an OPCA-like region that interacts with the Florida) for establishing stable cell lines in A549 cells, and into the positively charged residues in the N-terminal region of the PB1 of mammalian expression vector pcDNA3-HA for transient transfection in SQSTM1. HEK293 cells. Point mutations and truncations of NEDD4 or SQSTM1 were The PB1 domain is essential for SQSTM1 to homo- and hetero- created using a mutagenesis kit from Stratagene. dimerize/oligomerize and to localize on autophagosomes (Lamark et al., 2003; Itakura and Mizushima, 2001). However, ubiquitylation Preparation of cell lysates, immunoprecipitation, immunoblot by NEDD4 is dispensable for autophagosomal localization of and GST-fusion protein affinity precipitation assay SQSTM1, because knockdown of NEDD4 or expression of the Cells were rinsed once with ice-cold PBS and lysed in ice-cold Mammalian ligase-dead mutant of NEDD4 did not produce a diffused distribution lysis buffer (40 mM Hepes, pH 7.4, 100 mM NaCl, 1% Triton X-100, like the dimerization/oligomerization-defective mutants of SQSTM1 25 mM glycerol phosphate, 1 mM sodium orthovanadate, 1 mM EDTA, (Fig. 6). We currently do not know how the NEDD4-mediated 10 µg/ml aprotinin, and 10 µg/ml leupeptin) or RIPA buffer (40 mM Hepes, pH 7.4, 1% Triton X-100, 0.5% sodium deoxylcholate, 0.1% SDS, 100 mM ubiquitylation regulates the exact molecular function of SQSTM1. NaCl, 1 mM EDTA, 25 mM β-glycerolphosphate, 1 mM sodium There are two possible molecular effects for the ubiquitylation. The orthovanadate, 10 µg/ml leupeptin and aprotinin) as indicated. The cell first effect facilitates the hetero-dimerization of SQSTM1with NBR1. lysates were cleared by centrifugation at 13,000 rpm for 15 min. For It has been observed that cooperation between SQSTM1 with NBR1 immunoprecipitation, primary antibodies were added to the lysates and plays an important role in inclusion body autophagy (Kirkin et al., incubated with rotation at 4°C for 30 min, followed by adding 20 µl of 2009b; Tanji et al., 2015). Ubiquitylation on the PB1 domain by protein-A–Sephorose bead slurry (1:1) to the lysates and incubating with NEDD4 may enhance the binding affinity of SQSTM1 to NBR1, thus rotation for an additional 3 h. The immunoprecipitates were washed three enabling SQSTM1 to recruit NBR1 for inclusion body autophagy, times with lysis buffer. The cell lysates or immunoprecipitated proteins were while knockdown of endogenous NEDD4 or overexpression of the denatured by addition of SDS-PAGE sample buffer and boiled for 5 min, – ligase-dead mutant of NEDD4 may interfere with interaction between resolved by 8 14% SDS-PAGE. The proteins in the gel were transferred to PVDF membranes (Millipore). Immunoblotting with chemiluminescence SQSTM1 and NBR1 and cause accumulation of inclusion bodies was performed as described previously (Lin et al., 2010; Sun et al., 2017). (Fig. 6). The second possible effect is changing conformational GST fusion protein expression, purification and affinity precipitation structure of SQSTM1 by the ubiquitylation for interaction with assay were performed as previously described (Lin et al., 2010; Sun et al., downstream effectors, such as TRAF6 and KEAP1, to activate 2017). inclusion body autophagy. The ubiquitylated PB1 domain could intramolecularly interact with the Uba domain at the C-terminus to Immunofluorescence staining expose the effector interactive regions that sit between, thus Cells were cultured in glass coverslip-bottomed culture dishes (MatTek, enhancing interaction with downstream effectors and activating Ashland, MA) to 50–80% confluence. After the culture medium was selective autophagic signaling. Our future studies will follow these aspirated, the cells were rinsed with PBS twice, fixed with 3.7% questions and determine how the ubiquitylation affects hetero- paraformaldehyde at 25°C for 10 min, and permeabilized with 0.2% Triton dimerization of SQSTM1 with NBR1 and interaction with X-100 in PBS at 25°C for 10 min. After washing with PBS, the cells were incubated with primary antibody at 8°C overnight. The cells were washed with downstream interactive effectors in inclusion body autophagy. PBS three times and incubated with secondary antibody conjugated with a fluorescent dye at 37°C for 1–2h.AfterwashingwithPBSthreetimes, MATERIALS AND METHODS fluorescent staining of the cells was visualized under a Zeiss LSM710 Materials confocal microscope or Nikon inverted fluorescent microscope. Anti-SQSTM1 (D3; SC-28359) antibody was purchased from Santa Cruz Biotech; anti-NBR1 from Proteintech (16004-1-AP); anti-NEDD4 from Millipore (07-049); anti-LC3 from Abgent (AP1802a); anti-GFP (MMS- Quantification of fluorescent puncta number and size 118R), anti-ubiquitin (P4G7; MMS-258R) and anti-HA (MMS-101R) from The analysis and quantification of fluorescent images were performed using BioLegend; anti-CANX and anti-GOLGA2/GM130 from ECM Biosciences ImageJ. The threshold in detection of the fluorescence was set to cover all (OK7670); anti-ACTB from Sigma-Aldrich (A5441). The dilution for the visible fluorescent puncta. Numbers of fluorescent puncta were counted antibodies was 1:1000 for western blotting; 2 µg antibody/ml lysate for from two randomly selected fluorescence microscopy fields (25 to 47 cells). immunoprecipitation; 1:50 for immunofluorescence staining. The DNA The size of the fluorescent puncta in each cell was measured and averaged. Journal of Cell Science

3848 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 3839-3850 doi:10.1242/jcs.207068

Statistical analysis was performed based on the numbers and average sizes Chung, C. Y., Khurana, V., Auluck, P. K., Tardiff, D. F., Mazzulli, J. R., Soldner, of puncta from each of the cells. F., Baru, V., Lou, Y., Freyzon, Y., Cho, S. et al. (2013). Identification and rescue of α-synuclein toxicity in Parkinson patient-derived neurons. Science 342, 983-987. Virus packaging and transduction Cuyvers, E., van der Zee, J., Bettens, K., Engelborghs, S., Vandenbulcke, M., The viral packaging was performed as described previously (Mi et al., 2015; Robberecht, C., Dillen, L., Merlin, C., Geerts, N., Graff, C. et al. (2015). Genetic Sun et al., 2017). Briefly, the lentiviral plasmids were co-transfected with variability in SQSTM1 and risk of early-onset Alzheimer dementia: a European psPAX2 (Addgene) and pMD2.G (Addgene) packaging plasmids into early-onset dementia consortium study. Neurobiol. Aging. 36, 2005. actively growing HEK293KT cells using Lipofectamine 2000 transfection e15-22005.22. Du, Y., Wooten, M. C. and Wooten, M. W. (2009). Oxidative damage to the reagent. Viral particle-containing culture medium was collected every 24 h promoter region of SQSTM1/p62 is common to neurodegenerative disease. three times. The medium was cleared by centrifugation at 1000 g for 5 min, Neurobiol. Dis. 35, 302-310. and used for infecting target cells in the presence of 6 µg/ml polybrene. The Fan, W., Tang, Z., Chen, D., Moughon, D., Ding, X., Chen, S., Zhu, M. and Zhong, infected cells were selected with puromycin. Q. (2010). Keap1 facilitates p62-mediated ubiquitin aggregate clearance via autophagy. Autophagy 6, 614-621. Flint, A. J., Tiganis, T., Barford, D. and Tonks, N. K. (1997). Development of Analysis of autophagy “substrate-trapping” mutants to identify physiological substrates of protein tyrosine Autophagy was activated by treatment of cells with the mTOR inhibitor phosphatases. Proc. Natl. Acad. Sci. USA. 94, 1680-1685. rapamycin (LC Laboratory, R5000) for the indicated time. LC3- or SQSTM1- Fusco, C., Micale, L., Egorov, M., Monti, M., D’Addetta, E. V., Augello, B., positive autophagosomes were visualized by either immunofluorescence Cozzolino, F., Calcagnì, A., Fontana, A., Polishchuk, R. S. et al. (2012). The staining or GFP-tag under Zeiss LSM710 confocal fluorescent microscope or E3-ubiquitin ligase TRIM50 interacts with HDAC6 and p62, and promotes the sequestration and clearance of ubiquitinated proteins into the aggresome. PLoS a Nikon inverted fluorescent microscope. ONE 7, e40440. Geisler, S., Holmström, K. M., Skujat, D., Fiesel, F. C., Rothfuss, O. C., Kahle, Detection of ubiquitylated proteins and in vitro E3 ubiquitin P. J. and Springer, W. (2010). PINK1/Parkin-mediated mitophagy is dependent ligase activity assay on VDAC1 and p62/SQSTM1. Nat. Cell Biol. 12, 119-131. Detection of ubiquitylated proteins was performed using both GST-Uba Ichimura, Y., Kominami, E., Tanaka, K. and Komatsu, M. (2008). Selective turnover of p62/A170/SQSTM1 by autophagy. Autophagy 4, 1063-1066. pulldown and immunoprecipitation assays as described previously (Lin et al., Isakson, P., Lystad, A. H., Breen, K., Koster, G., Stenmark, H. and Simonsen, A. 2010; Wang et al., 2010). Briefly, cells were lysed with RIPA buffer (40 mM (2013). TRAF6 mediates ubiquitination of KIF23/MKLP1 and is required for Hepes, pH 7.4, 1% Triton X-100, 0.5% sodium deoxylcholate, 0.1% SDS, midbody ring degradation by selective autophagy. Autophagy 9, 1955-1964. 100 mM NaCl, 1 mM EDTA, 25 mM β-glycerolphosphate, 1 mM sodium Ishimura, R., Tanaka, K. and Komatsu, M. (2014). Dissection of the role of p62/ orthovanadate, 10 µg/ml leupeptin and aprotinin) and the ubiquitylated proteins Sqstm1 in activation of Nrf2 during xenophagy. FEBS Lett. 588, 822-828. Itakura, E. and Mizushima, N. (2011). p62 Targeting to the autophagosome were detected either by immunoprecipitation with the primary antibody formation site requires self-oligomerization but not LC3 binding. J. Cell Biol. followed by immunoblotting with an anti-ubiquitin antibody (BioLegend, 192, 17-27. 646302), or by affinity precipitation with GST–UBA-conjugated glutathione Johansen, T. and Lamark, T. (2011). Selective autophagy mediated by autophagic beads followed by immunoblotting with anti-SQSTM1 antibody. adapter proteins. Autophagy 7, 279-296. Kim, P. K., Hailey, D. W., Mullen, R. T. and Lippincott-Schwartz, J. (2008). Statistical analysis Ubiquitin signals autophagic degradation of cytosolic proteins and peroxisomes. Proc. Natl. Acad. Sci. USA. 105, 20567-20574. The Student t-test was used in statistical analysis of experimental data. A P- Kirkin, V., McEwan, D. G., Novak, I. and Dikic, I. (2009a). A role for ubiquitin in value less than 0.05 was considered as statistically significant. selective autophagy. Mol. Cell. 34, 259-269. Kirkin, V., Lamark, T., Sou, Y.-S., Bjørkøy, G., Nunn, J. L., Bruun, J.-A., Shvets, Acknowledgements E., McEwan, D. G., Clausen, T. H., Wild, P. et al. (2009b). A role for NBR1 in We want to thank Dr Jihe Zhao of University of Central Florida for the lentiviral autophagosomal degradation of ubiquitinated substrates. Mol. Cell. 33, 505-516. expression vectors pLVTHM-GFP and pFUW. Komatsu, M., Waguri, S., Koike, M., Sou, Y.-S., Ueno, T., Hara, T., Mizushima, N., Iwata, J., Ezaki, J., Murata, S. et al. (2007). Homeostatic levels of p62 control Competing interests cytoplasmic inclusion body formation in autophagy-deficient mice. Cell 131, 1149-1163. The authors declare no competing or financial interests. Kuusisto, E., Salminen, A. and Alafuzoff, I. (2002). Early accumulation of p62 in neurofibrillary tangles in Alzheimer’s disease: possible role in tangle formation. Author contributions Neuropathol. Appl. Neurobiol. 28, 228-237. Conceptualization: Q.L., W.Y.; Methodology: Q.L., W.Y.; Validation: A.S.; Formal Lamark, T., Perander, M., Outzen, H., Kristiansen, K., Øvervatn, A., Michaelsen, analysis: Q.L., Q.D., G.S., M.C., W.Y.; Investigation: Q.L., Q.D., H.M., A.S., J.W., E., Bjørkøy, G. and Johansen, T. (2003). Interaction codes within the family of K.P., C.C., W.Y.; Resources: W.Y.; Data curation: Q.L., Q.D., H.M., W.Y.; Writing - mammalian Phox and Bem1p domain-containing proteins. J. Biol. Chem. 278, original draft: Q.L., W.Y.; Writing - review & editing: W.Y.; Visualization: W.Y.; 34568-34581. Supervision: Q.L., G.S., W.Y.; Project administration: Q.L., W.Y.; Funding Lee, Y. J., Chou, T.-F., Pittman, S. K., Keith, A. L., Razani, B. and Weihl, C. C. acquisition: Q.L., W.Y. (2017). 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