p62 and Suppresses Protein Sequestration to Regulate Redox Homeostasis

Article

TRIM21 Ubiquitylates SQSTM1/p62 and Suppresses Protein Sequestration to Regulate Redox Homeostasis Graphical Abstract

Authors Ji-An Pan, Yu Sun, Ya-Ping Jiang, ..., Keiko Ozato, Richard Z. Lin, Wei-Xing Zong

Correspondence [email protected] (J.-A.P.), [email protected] (W.-X.Z.)

In Brief Pan et al. show that TRIM21 interacts with and ubiquitylates p62 at K7 via K63linkage. This p62 K7 ubiquitylation prevents p62 dimerization and sequestration function and negatively regulates the Keap1-Nrf2 redox pathway. TRIM21-null mice are protected from oxidative liver and heart damage.

Highlights d

SQSTM1/p62 is ubiquitylated by TRIM21 at K7 via K63linkage

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p62 K7 ubiquitylation prevents its dimerization and sequestration

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TRIM21 negatively regulates Keap1 sequestration and antioxidative response

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TRIM21 null liver and heart are protected from oxidative tissue damage

Pan et al., 2016, Molecular Cell 61, 720–733 March 3, 2016 ª2016 Elsevier Inc. http://dx.doi.org/10.1016/j.molcel.2016.02.007

Molecular Cell

Article TRIM21 Ubiquitylates SQSTM1/p62 and Suppresses Protein Sequestration to Regulate Redox Homeostasis Ji-An Pan,1,2,3,* Yu Sun,1,2,3 Ya-Ping Jiang,4 Alex J. Bott,1,2,3 Nadia Jaber,3 Zhixun Dou,3 Bin Yang,5 Juei-Suei Chen,3 Joseph M. Catanzaro,3 Chunying Du,6 Wen-Xing Ding,7 Maria T. Diaz-Meco,8 Jorge Moscat,8 Keiko Ozato,9 Richard Z. Lin,4,10 and Wei-Xing Zong1,2,3,* 1Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers University, 164 Frelinghuysen Road, Piscataway, NJ 08854, USA 2Rutgers Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08903, USA 3Department of Molecular Genetics & Microbiology, Stony Brook University, Stony Brook, NY 11794, USA 4Department of Physiology and Biophysics, Stony Brook University, Stony Brook, NY 11794, USA 5Key Laboratory of Artificial Cells, Tianjin Third Central Hospital, Tianjin 300170, China 6Department of Cancer Biology, University of Cincinnati, Cincinnati, OH 45267, USA 7Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160, USA 8Cancer Metabolism and Signaling Networks Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA 9Division of Developmental Biology, NICHD, National Institutes of Health, Bethesda, MD 20892, USA 10Department of Veterans Affairs Medical Center, Northport, NY 11768, USA *Correspondence: [email protected] (J.-A.P.), [email protected] (W.-X.Z.) http://dx.doi.org/10.1016/j.molcel.2016.02.007

SUMMARY

TRIM21 is a RING finger domain-containing ubiquitin E3 ligase whose expression is elevated in autoimmune disease. While TRIM21 plays an important role in immune activation during pathogen infection, little is known about its inherent cellular function. Here we show that TRIM21 plays an essential role in redox regulation by directly interacting with SQSTM1/p62 and ubiquitylating p62 at lysine 7 (K7) via K63-linkage. As p62 oligomerizes and sequesters client proteins in inclusions, the TRIM21-mediated p62 ubiquitylation abrogates p62 oligomerization and sequestration of proteins including Keap1, a negative regulator of antioxidant response. TRIM21deficient cells display an enhanced antioxidant response and reduced cell death in response to oxidative stress. Genetic ablation of TRIM21 in mice confers protection from oxidative damages caused by arsenic-induced liver insult and pressure overload heart injury. Therefore, TRIM21 plays an essential role in p62-regulated redox homeostasis and may be a viable target for treating pathological conditions resulting from oxidative damage.

et al., 2015), which can be regulated by SQSTM1/p62 (Katsuragi et al., 2015). p62 is a ubiquitin-binding protein that is enriched in protein aggregates (Shin, 1998) whose expression was found to be induced by oxidative stress (Ishii et al., 1996). One of Keap1’s functions is to act as a scaffold for the ubiquitin ligase Cullin 3, Ring-box 1 (Rbx1), and Nrf2 complex. Nrf2 is the transcriptional factor that, when in the nucleus, activates the expression of antioxidant enzymes by binding to the antioxidant response element (ARE) in their promoter region. Under normal conditions, Keap1 interacts with Nrf2 to retain the latter in the cytoplasm where Nrf2 is degraded via the ubiquitin-proteasome pathway (Kobayashi et al., 2004; Zhang et al., 2004). In response to proteotoxic and oxidative stress, p62 forms a homodimer, which facilitates its oligomerization and interaction with ubiquitylated proteins that promotes their aggregation, thereby sequestering the damaged or toxic proteins. A known mechanism for p62 dimerization is via the K7-D69 hydrogen bond in its PB1 domain (Wilson et al., 2003). The p62-mediated protein aggregate cargo can be subsequently delivered to autophagosomes for autophago-lysosomal degradation. As one of the protein inclusion client proteins is Keap1, the p62-mediated Keap1 sequestration and degradation frees Nrf2 from the Keap1 inhibition, allowing for its stabilization, nuclear translocation, transcriptional activation, and antioxidant response (Komatsu et al., 2010; Lau et al., 2010). In this study, we found that TRIM21 (Tripartite motif-containing protein 21), a ubiquitin E3 ligase, directly interacts with and ubiquitylates p62 to regulate its sequestration and cellular redox homeostasis.

INTRODUCTION RESULTS Cellular redox regulation plays an essential role in organismal homeostasis. A major mechanism for cell redox homeostasis regulation is the Keap1 (Kelch-like ECH-associated protein 1)-Nrf2 (Nuclear factor erythroid 2-related factor 2) pathway (Harder 720 Molecular Cell 61, 720–733, March 3, 2016 ª2016 Elsevier Inc.

p62 Is Ubiquitylated at Residue K7 via the K63-Linkage In our laboratory practice, we have constantly noticed that certain stressors, such as the proteasome inhibitor MG132,

Figure 1. p62 Is Ubiquitylated at K7 via the K63-Linkage (A) Baby mouse kidney (BMK) cells were transfected with His-Ub for 24 hr, then treated with MG132 (0.25 mM) and subjected to His pull down (Ni-NTA) and immunoblotting (IB). (B) Flag and His double-tagged p62 WT, K7R, and K13R were transfected into HEK293T cells for 36 hr. Cells were subjected to His pull down and IB. (C) Flag and His double-tagged p62 WT and K7R were co-transfected with HA-Ub WT, K48-only, and K63-only mutants in HEK293T cells. Cells were subjected to His pull down and IB. (D) Indicated p62 mutants were co-expressed in HEK293T cells. Cells were subjected to Flag IP and IB. (E) Flag-tagged p62 WT or indicated mutants were expressed in HEK293T cells. HA-Ub was expressed in another set of HEK293T cells. 24 hr post transfection, p62 or mutant-expressing cell lysates were incubated with the same amount of the HA-Ub expressing cell lysates for 3 hr. The mixtures were subjected to IP with FLAG antibody then IB with HA antibody. (F) p62 / MEFs stably expressing GFP-LC3 were reconstituted with p62 WT and p62K7R. Cells were treated with MG132 (0.25 mM) for 12 hr, then observed under deconvolution microscope. The percentage of cells with GFP-LC3 aggregates was quantified by counting over 50 cells in each sample. Data shown are the mean plus SD of three countings. ***p < 0.01.

enhance an upper shift of p62 in SDS-PAGE, suggesting a post-translational modification. Indeed, by transfecting cells with His-tagged ubiquitin and pull down with nickel beads, p62 ubiquitylation was detected, which was further enhanced upon MG132 treatment (Figure 1A). Serial deletion mutants of p62 revealed that deletion of the N-terminal 3–117 residues led to significant decrease of p62 ubiquitylation (Figure S1A). Lysine (K) to arginine (R) point mutations in the 3–117 region identified

residue K7 as the critical residue for p62 ubiquitylation (Figure S1B and Figure 1B). To determine whether p62(K7) ubiquitylation is via K48- or K63-linkage, K48-only or K63-only ubiquitin was expressed together with Flag and His double-tagged p62 (Figure 1C). K48-only ubiquitin led to reduced levels of p62 ubiquitylation, possibly owing to the increased proteasomal degradation promoted by K48-linked ubiquitylation, whereas K63-only ubiquitin led to enhanced p62 ubiquitylation (Figure 1C, Molecular Cell 61, 720–733, March 3, 2016 ª2016 Elsevier Inc. 721

p62 WT lanes of Ni-NTA). This result suggests that p62 is ubiquitylated by both K48- and K63-linkages. Markedly, the K63-linked ubiquitylation was largely decreased in p62K7R mutant (Figure 1C), indicating that K7 is ubiquitylated via K63-linkage. Next, we determined whether the K7 ubiquitylation affects p62 functions, one of which is to bind polyubiquitylated proteins and sequester them in aggregates (Moscat and Diaz-Meco, 2009). This is dependent on the ubiquitin-binding and dimerization properties of p62 (Bjørkøy et al., 2005). Using the p62 mutants tagged by either Flag or HA, we found that the K7R mutant lost its ability to form a homo-dimer (Figure 1D), similar to the D69A dimerization-deficient mutant (Bjørkøy et al., 2005). Importantly, the failure of dimerization led to the failure of p62 binding to polyubiquitylated proteins as seen for both the K7R and D69A mutants, similar to the ubiquitin-binding-deficient mutant I431A (Seibenhener et al., 2004) (Figure 1E). As ubiquitin-binding is essential for p62’s function to pack polyubiquitylated proteins into aggregates, we then tested whether K7R is able to mediate aggregate formation. Using MEFs that stably express GFP-LC3, we observed that while starvation-induced formation of LC3+ autophagosomes was not markedly affected, MG132-induced aggregate formation was abolished in p62 / MEFs (Figure S1C). Reconstitution of p62 / MEFs with wildtype p62 restored MG132-induced aggregate formation indicated by large-size LC3 puncta using either GFP-LC3 or endogenous LC3, whereas the K7R mutant failed to do so (Figures 1F and S1D). Taken together, these data indicate that p62 is ubiquitylated at K7 via K63-linkage and that K7 is critical for p62 dimerization, ubiquitin binding, as well as aggregate formation. TRIM21 Directly Ubiquitylates p62 at Residue K7 to Inhibit Its Oligomerization and Sequestration Function The failure of the K7R mutant to dimerize suggests two opposite possible scenarios: (1) modification of K7 by ubiquitylation promotes p62 dimerization via the ubiquitin chain; or (2) unmodified K7 is essential for p62 dimerization, and its modification by ubiquitylation leads to the loss of function of p62. Importantly, an X-ray crystallography analysis has demonstrated that K7 is a basic cluster residue that would form a hydrogen bond with its corresponding acidic cluster residue D69 (Wilson et al., 2003). This supports the latter option that the non-ubiquitinated form of K7 is critical for p62 dimerization. To further understand the nature of p62 K7 ubiquitylation, and to unequivocally demonstrate that p62 K7 ubiquitylation negatively regulates p62 dimerization and aggregation, we sought to identify the ubiquitin E3 ligase that catalyzes the p62 K7 ubiquitylation. In an in vitro ubiquitylation assay, we noticed that the immunoprecipitated Flag-p62 samples from HEK293T cells led to p62 ubiquitylation in the presence of purified E1, E2, and ubiquitin without the addition of an exogenous E3 (Figure 2A). This suggests that an E3 enzyme was co-immunoprecipitated in the Flag-p62 IP samples. Indeed, mass spectrometry of Flag-p62 IP samples identified two putative E3 ligases: TRIM21 and RNF138. Expression of TRIM21, but not RNF138, led to p62 ubiquitylation (Figures 2B and 2C), suggesting that TRIM21 is the E3 ligase for p62. A ligase-dead (LD) mutant TRIM21 (C16A, C31A, and H33W) (Wada and Kamitani, 2006) revealed that the E3 ligase ac722 Molecular Cell 61, 720–733, March 3, 2016 ª2016 Elsevier Inc.

tivity of TRIM21 was required for p62 ubiquitylation (Figure 2D). An in vitro ubiquitylation assay further revealed that TRIM21 directly ubiquitylates p62 (Figure 2E) via the K63-linkage (Figure 2F), which was abolished in the p62K7R mutant (Figure 2G). Silencing of TRIM21 abolished p62 ubiquitylation both at the basal level and upon MG132 treatment (Figure 2H). Therefore, TRIM21 is a direct ubiquitin E3 ligase for the p62 residue K7. As the p62K7R mutant was unable to dimerize (Figure 1D) and to promote protein aggregation (Figure 1F), we tested whether TRIM21 plays a positive or negative role in p62 dimerization and aggregation using three approaches. First, we used the bimolecular fluorescence complementation (BiFC) assay (Huang et al., 2010). p62 WT or the K7R mutant was fused to the split Venus proteins, Venus N (1–173) and Venus C (155–239), and expressed in HEK293T cells (Figure 3A). BiFC was observed when p62 WT-VN and p62 WT-VC were co-expressed (Figures S2A and 3B), but not with the K7R mutants (Figure 3B). Induction of wild-type TRIM21 using the estrogen receptor inducing system led to reduced BiFC, whereas the ubiquitin ligase-dead (LD) mutant TRIM21-LD failed to do so (Figures S2B and 3C), indicating that TRIM21 decreases p62 oligomerization in an E3 ligase activity-dependent manner. Alternatively, we examined p62 oligomerization by coexpressing p62 tagged with either Flag or HA epitopes. Since a significant amount of p62 resides in protein aggregates, especially under the condition of proteotoxic stress such as treatment of MG132, we used the reversible crosslinking agent dithiobis succinimidyl propionate (DSP) to treat the cells and then lysed the cells in strong detergent (1% SDS) to solubilize all proteins. The lysates were subjected to coimmunoprecipitation (coIP) upon diluting the detergent to 0.1% SDS. Indeed, TRIM21 silencing led to increased p62 oligomerization both at basal condition and upon MG132 treatment (Figure 3D). Moreover, since p62 oligomerization is important for its sequestration function, we determined whether p62-mediated aggregation is regulated by TRIM21. To this end, we detected the presence of p62 in aggregates by treating cells with the crosslinking agent DSP and running the cell lysates under either reducing or nonreducing condition (Figure S2C). Under the reducing condition, total p62 would be detected with a migration of 62 kDa, whereas under the non-reducing condition, only p62 monomers would be detected at 62 kDa, and the oligomers or those conjugated with other proteins would display such a slow migration that it would not run through the gel (illustrated in Figure S2C). Therefore, a higher ratio of the 62 kDa band in the non-reducing versus reducing conditions would indicate reduced p62 oligomerization or sequestration. MG132 treatment led to less p62 monomer in the non-reducing condition (Figure 3E), indicating p62 aggregation and sequestration. TRIM21 silencing led to further decreased p62 monomer, hence increased aggregation and sequestration (Figure 3E). In TRIM21 knockdown cells, reconstitution of TRIM21 WT, but not the LD mutant, led to increased p62 monomer, indicating decreased p62 aggregation and sequestration (Figure 3E). The role of TRIM21 in negatively regulating p62 oligomerization was further supported using immunofluorescence analysis and fractionation assay, which shows that reconstitution of TRIM21

Figure 2. p62 Is Ubiquitylated by TRIM21 (A) Flag-p62 was expressed in HEK293T cells and subjected to IP using the Flag antibody and eluted using Flag peptides. The eluates were incubated with in vitro ubiquitination assay system containing E1, E2, and ubiquitin, then subjected to IB. (B and C) His-Ub was transfected with vector and HA-TRIM21 (B) or HA-RNF138 (C) into HEK293T cells. 24 hr later, cells were subjected to His pull down followed by IB. (D) His-Ub was co-transfected with HA-TRIM21 WT or HA-TRIM21-LD mutant in HEK293T cells. Cells were subjected to His pull down and IB. (E) Purified Flag-p62 was incubated with IgG control, purified HA-TRIM21 WT, or LD mutant in the ubiquityliation assay mix, and probed for indicated proteins. (F) His-Ub (WT, K48R, and K63R) mutants were expressed with or without HA-TRIM21 in HEK293T cells. Cells were subjected to His pull down and IB. (G) Flag and His double-tagged p62 WT or K7R mutant was co-expressed with vector or TRIM21 in HEK293T cells. Cells were subjected to His pull down and IB. (H) MD-MBA-231 with stable expression of Flag-p62-His was infected with lentiviral non-targeting control (NTC) shRNA or shTRIM21. Cells were treated with MG132 then subjected to His pull down and IB.

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Figure 3. TRIM21 Inhibits p62 Dimerization and Its Sequestration Function (A) Schematic for the strategy constructing p62 fused with Venus N terminus (p62-VN) or C terminus (p62-VC). p62-VN and p62-VC were expressed individually or simultaneously in HEK293T cells. The expression was determined by IB. (B) Wild-type or K7R p62-VN/p62-VC was expressed in HEK293T cells. Representative fluorescence microscopic images are shown. (C) p62-VN or p62-VC was expressed with estrogen receptor (ER)-fused TRIM21 WT or LD mutant in HEK293T cells. 16 hr later, cells were treated with 4-OHT for indicated times to induce TRIM21 expression (top panel). Venus fluorescence was determined by flow cytometry and quantified (bottom panel). (D) HeLa cells stably expressing both Flag- and HA-p62 were infected with lentivirus shNTC and shTRIM21. Cells were used for Flag IP, and IB for HA. The amount of HA-p62 was quantified by LI-COR Odyssey and is shown in the right panel. *p < 0.05. (E) HeLa cells with stable shTRIM21 were reconstituted with shRNA-resistant TRIM21 WT or TRIM21 LD. Cells were treated with MG132 then crosslinked with DSP and lysed in IP lysis buffer with 1% SDS. The cells lysates mixed with loading buffer containing or not containing b-mercaptoethanol were subjected to IB. (F) TRIM21 / MEFs reconstituted with TRIM21 WT or LD were treated with MG132 (0.25 mM) for 12 hr. Cells were used for IF using anti-TRIM21 (green) and antiHA (red) antibodies and observed under deconvolution microscope. Cells with aggregates were quantified by blind counting. Shown on the right is the mean plus SD of three countings. *p < 0.05, **p < 0.01. Cells with high expression of TRIM21 WT or LD are marked with an asterisk.

WT, but not the LD mutant, led to diminishment of MG132induced p62 aggregates (Figures 3F and S2D). Consistent with these findings, TRIM21 silencing led to increased p62 binding 724 Molecular Cell 61, 720–733, March 3, 2016 ª2016 Elsevier Inc.

to ubiquitylated proteins (Figure S2E), and reconstitution of TRIM21 WT, but not the LD mutant, led to diminished ubiquitin aggregates in TRIM21 / cells (Figure S2F).

In addition to the proteasome inhibitor MG132, we also examined other conditions that are known to induce p62 aggregation including genetic ablation of autophagy-essential proteins Atg7 and Vps34. In Atg7flox/flox (Komatsu et al., 2005) and Vps34flox/flox (Jaber et al., 2012) MEFs, ablation of respective genes by Cre expression led to decreased p62 monomer in the nonreducing condition as a result of accumulated p62 aggregation due to the failure of autophagic clearance of p62 (Figures S3A and S3B). Overexpression of wild-type TRIM21, but not the LD mutant, led to increased amount of p62 monomer (Figures S3A and S3B), indicating that TRIM21 prevents p62 aggregation under the condition of genetic inhibition of autophagy. Consistently, the increased amount of insoluble polyubiquitylated proteins in Atg7 / cells was reduced by the expression of TRIM21 WT, but not the LD mutant (Figure S3C). Taken together, these data indicate that residue K7 is critical for p62 dimerization, aggregation, and sequestration function and that TRIM21 negatively regulates these functions by ubiquitylating p62 at residue K7. TRIM21 Interaction with p62 Is via the PRYSPRY Domain and Is Enhanced by Proteotoxic Stress We next sought to characterize the molecular nature of the interaction between p62 and TRIM21. Two p62 deletion mutants D3–117 and D117–200 showed significantly decreased interaction with TRIM21 (Figure 4A). Further deletion mutation revealed that the two domains aa3–61 and 163–200 had lost TRIM21 interaction (Figure 4B). In TRIM21, deletion of aa252–476, which spans the PRYSPRY domain that is conserved among numerous TRIM family members and is important for their anti-viral function (Nisole et al., 2005; Song et al., 2005), led to a near-complete loss of p62 interaction (Figure 4C). It has been shown that residues D355, W381, W383, and F450 within the TRIM21 PRYSPRY domain are clustered at the center of the interface that interacts with IgG Fc (James et al., 2007). We reasoned that these residues may also be critical for TRIM21 interaction with other proteins. Indeed, the TRIM21W381/383A mutant abolished p62 interaction (Figure 4D), p62 ubiquitylation (Figure 4E), and the suppression of p62 aggregation (Figure 4F). The importance of the TRIM21 ligase activity was also determined by the autophagic degradation of p62. Wild-type p62 was degraded by starvation-induced autophagy, but p62K7R, which is deficient in oligomerization (Figure 1C), was not (Figure S4A). This is consistent with the notion that p62 autophagic degradation is dependent on p62 dimerization and aggregation (Itakura and Mizushima, 2011). Importantly, wild-type TRIM21, but not the W381/383A mutant, suppressed starvation-induced p62 autophagic degradation (Figure S4B). These data indicate that W381 and W383 are required for TRIM21 interaction with p62 and for its function to ubiquitylate p62 and suppress p62 oligomerization. We then studied how TRIM21-mediated p62 ubiquitylation might be regulated. In a co-overexpression system, treatment of MG132, but not serum starvation, rapamycin, or tunicamycin, led to increased interaction between TRIM21 and p62 (Figure 4G). Since the available TRIM21 and p62 antibodies were not suitable for immunoprecipitation, we introduced a Flag tag to the endogenous TRIM21 gene via CRISPR gene editing in

MEFs (Figure S4C) so that Flag-tagged TRIM21 is expressed at the endogenous level. CoIP analysis using a Flag antibody showed that MG132 induced the interaction between TRIM21 and p62 (Figure 4H). These results are consistent with our observation that p62 ubiquitylation is enhanced upon MG132 treatment and suggest that the TRIM21-mediated p62 ubiquitylation is a cell stress adaptation mechanism in response to proteasome inhibition in order to maintain a hemostatic level of functional p62. TRIM21 Negatively Regulates p62-Mediated Sequestration of Keap1 and Antioxidant Response An important function of p62 is to retain proteins in aggregates (sequestosomes) for subsequent autophagic degradation. One of the client proteins is Keap1, which negatively regulates the antioxidant response by interacting and preventing the nuclear accumulation of the transcriptional factor Nrf2, the master regulator to activate antioxidant gene transcription (Komatsu et al., 2010). As our results thus far indicate that TRIM21 negatively regulates p62-mediated protein sequestration by ubiquitylating p62 at K7, we sought to study whether this mechanism has an impact on a cell’s antioxidant response. To this end, we used sodium arsenate (As(III)), which induces a detrimental level of intracellular reactive oxygen species (Barchowsky et al., 1996; Lynn et al., 1998). We first used p62 / MEFs reconstituted with either p62 WT or p62K7R. While As(III) treatment did not induce Keap1 aggregation in p62 / MEFs, reconstitution of p62 WT, but not p62K7R, led to aggregation of both p62 and Keap1 and their accumulation in the insoluble fraction (Figures 5A and 5B). The ability of p62 WT to sequester Keap1 was also indicated by increased Nrf2 and NQO1 expression (Figures 5C and 5D). This As(III)-induced p62 sequestration is likely to be negatively regulated by TRIM21 since As(III) induced p62-TRIM21 interaction at the endogenous level in the TRIM21-Flag knockin cells (Figure 5E). This As(III)-induced p62 aggregation is suppressed by TRIM21 WT, but not the W381/383A mutant (Figures 5F and S5A). TRIM21 reconstitution led to decreased p62 and Keap1 in the insoluble fraction (Figure 5G), decreased Nrf2 in the nucleus (Figure 5H), and decreased Nrf2 stability (Figures 5I and S5B). Consistently, wild-type TRIM21 showed a low level of NQO1 promoter activation (Figure 5J) and increased oxidative stress (Figure 5K). Together, these data indicate that p62 plays an antioxidant role by sequestrating Keap1 in response to As(III) and that TRIM21 negatively regulates this function by ubiquitylating p62. Ablation of TRIM21 Leads to Protection against Arsenic-Induced Cell Death and Liver Injury We next examined the effect of TRIM21 on As(III)-induced cell death. Acute As(III) treatment induces ROS, DNA damage, and activation of poly(ADP-ribose) polymerase (PARP) as well as depletion of NAD and ATP, leading to bioenergetic catastrophe and cell death (Bernstam and Nriagu, 2000; Chou et al., 2004; Kang et al., 2004). Indeed, in wild-type cells, As(III) treatment led to DNA damage indicated by increased H2A.X phosphorylation (Figure 6A), PARP activation indicated by increased poly(ADP) ribosylation (PAR) (Figure 6B), and cell death in a manner that was inhibitable by ROS scavengers Molecular Cell 61, 720–733, March 3, 2016 ª2016 Elsevier Inc. 725

Figure 4. TRIM21 Interacts with p62 via the PRYSPRY Domain (A–C) Flag-tagged TRIM21 and HA-tagged p62 WT or their truncation mutants were expressed in indicated combinations in HEK293T cells for 24 hr. Cells were subjected to Flag IP and IB. p62 lost interaction with TRIM21 D213–117 and D117–200 (A). TRIM21 lost interaction with p62 D3–61, D117–162, and D163–200 (B). p62 lost interaction with TRIM21 D252–476 (C). (D) Flag-tagged TRIM21 WT or W381/383A mutant and HA-tagged p62 were expressed in HEK293T cells for 24 hr. Cells were subjected to Flag IP and IB. (E) HA-tagged TRIM21 WT or W381/383A mutant and His-Ub were expressed in HEK293T cells. Cells were subjected to His pull down and IB. (F) TRIM21 / MEFs reconstituted with vector, TRIM21 WT, and TRIM21 W381/383A mutant were treated with DSP and lysed in IP lysis buffer with 1% SDS. Lysates were mixed with loading buffer with/without b-mercaptoethanol and subjected to IB. (G) HA-tagged p62 and Flag-tagged TRIM21 were expressed in HEK293T cells for 24 hr. Cells were treated with MG132 (0.5 mM) for 16 hr, rapamycin (50 nM) for 16 hr, tunicamycin (0.5 mg/ml) for 16 hr, or cultured in serum-free medium for 6 hr. Cells were subjected to Flag IP and IB. (H) A vector control or Flag tag was introduced into MEFs to fuse with the endogenous TRIM21 by CRISPR. Cells were treated with MG132 (0.25 mM) for 12 hr, then subjected to Flag IP and IB.

N-acetylcysteine (NAC) and 2,2,6,6-tetramethylpiperidine-Noxyl (TEMPO) (Figure S6A) and by PARP inhibitors 3,4-Dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone (DPQ) and 1,5-dihydroxyisoquinilone (DHIQ) (Figure S6B). Importantly, 726 Molecular Cell 61, 720–733, March 3, 2016 ª2016 Elsevier Inc.

As(III)-induced DNA damage, PARP activation, and cell death were markedly suppressed in TRIM21 / cells (Figures 6A– 6D). Reconstitution of TRIM21 WT, but not the LD mutant, resensitized cells to As(III) (Figure 6E). Similarly, silencing of p62

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led to increased sensitivity to As(III) (Figure S6C). Similar to TRIM21 / MEFs, silencing of TRIM21 in MDA-MB-231 and MCF-7 cells also led to protection against As(III) toxicity (Figures S6D and S6E). The As(III)-induced cell death was not inhibited by necrostatin-1 (Nec-1) (Figure S6F). Interestingly, while TRIM21deficient cells were refractory to As(III)-induced cell death, they were more susceptible to cell death induced by MNNG (Figure S6G), which was blocked by PARP inhibitor, but not Nec-1 (Figure S6H), and to cell death induced by TNFa/cycloheximide/z-VAD (TCZ) (Figure S6I), which was blocked by Nec-1 (Figure S6J), suggesting that TRIM21 may play a protective role in response to harmful conditions other than oxidative stress. Nonetheless, the above data indicate that TRIM21 inhibition leads to protection against As(III)-induced oxidative stress. TRIM21 is ubiquitously expressed in all tissues (Figure S7A). To test the physiological relevance of TRIM21, we tested arsenic trioxide (As2O3)-induced liver injury using the wholebody TRIM21 knockout mouse (Yoshimi et al., 2009). Sublethal dosage of As2O3 (30 mg/kg) was given to wild-type or TRIM21 / littermates for 24 hr. A significant body weight loss was observed in wild-type mice, which was significantly alleviated in TRIM21 / mice (Figure 6F). H&E staining revealed a moderate level of necrosis near the central vein in the wild-type mice, which was blocked in TRIM21 / mice (Figure 6G). Immunohistochemistry (IHC) analysis revealed that TRIM21 / mice displayed more profound aggregation of p62 and ubiquitylated proteins, as well as reduced oxidative DNA damage indicated by 8-oxodG staining (Figure 6G). Cell fractionation revealed that As2O3 treatment led to increased insoluble p62 and Keap1, which was markedly further enhanced and correlated with the higher NQO1 expression in TRIM21 / livers (Figures 6H and 6I). This was consistent with the increased p62-Keap1 colocalization (Figure S7B) as well as increased expression of a number of antioxidant genes upon As(III) treatment (Figure S7C). Together, these data indicate that ablation of TRIM21 confers

protection against As2O3-induced oxidative burst, cell death, and liver injury. TRIM21 Knockout Mice Are Protected from Heart Damage Induced by Transverse Aortic Constriction Another in vivo condition where ROS plays an important role is cardiac dysfunction (Belch et al., 1991; von Harsdorf et al., 1999). A commonly used in vivo model in studying heart failure is pressure overload upon severe transverse thoracic aorta constriction (sTAC), which has been shown to induce ROS and can be alleviated by ROS scavengers (Date et al., 2002). We therefore examined sTAC-induced heart damage in TRIM21 / mice. In the control mice, sTAC caused cardiac dilatation (Figure 7A) associated with an acute drop in contractility and increased left ventricular diastolic and systolic volumes (Figures 7B and 7C). In sharp contrast, the heart size and cardiac contractility was completely preserved in the banded TRIM21 / mice with fractional shortening and ejection fraction levels similar to the sham-operated animals (Figures 7A–7C). Close examination of the heart tissue revealed that sTAC induced disarrangement of cardiomyocytes with sarcoplasmic vacuoles in the wild-type mice. These abnormalities were absent in the hearts of banded TRIM21 / mice (Figures 7D, 7H, and 7E). IHC analysis revealed that hearts from TRIM21 / mice displayed more profound aggregation of p62 and ubiquitylated proteins compared with hearts from wild-type mice (Figure 7D, IHC for p62 and ubiquitin). Consistently, while sTAC induced elevated H2A.X phosphorylation in wild-type hearts indicative of DNA damage, H2A.X phosphorylation was markedly suppressed and accompanied by increased antioxidant response indicated by increased NQO1 expression in TRIM21 / hearts (Figure 7E). These results suggest that loss of TRIM21 attenuated the ROS release from sTAC that leads to myocyte damage resulting in decreased cardiac contractility. Taken together with the protection from As2O3-induced liver damage in TRIM21 / mice (Figure 6), our

Figure 5. Trim21 Negatively Regulates p62-Mediated Keap1 Sequestration and Antioxidant Response (A) p62 / MEFs reconstituted with vector, Flag-p62 WT, or Flag-p62K7R mutant were untreated or treated with sodium arsenate (As(III), 10 mM) for 6 hr. Cells were subjected to IF with Flag and Keap1 antibodies and observed under deconvolution microscope. Cells with Keap1 aggregates were counted blindly. Data shown are the averages plus SD of at least three countings with over 200 cells. **p < 0.01, ***p < 0.001. (B) p62 / MEFs reconstituted with vector, Flag-p62 WT, and Flag-p62K7R were treated with As(III) (10 mM) for 2.5 hr. Cells were lysed in IP lysis buffer with 1% Triton X-100. The insoluble fraction was lysed in IP lysis buffer with 1% SDS. 30 mg soluble proteins and corresponding volume of insoluble proteins were used for IB. (C) p62 / MEFs reconstituted with vector, Flag-p62 WT, and Flag-p62K7R were left untreated or treated with As(III) (10 mM) for indicated times. Cells were lysed in RIPA (1% SDS) and probed for Nrf2 and tubulin. (D) p62 / MEFs reconstituted with vector, Flag-p62 WT, and Flag-p62K7R were transfected with NQO1-ARE-luc and pRL-TK (Renilla Luciferase Control Reporter Vector, Promega) plasmids. 16 hr post transfection, cells were treated with As(III) and assayed for luciferase activity. (E) MEFs without/with CRISPR Flag fused into TRIM21 gene were treated with As(III) (10 mM) for 4 hr and subjected to Flag IP and IB. (F) TRIM21 / MEFs reconstituted with vector, TRIM21 WT, and TRIM21W381/383A mutant were treated with the crosslinking agent DSP and lysed in IP lysis buffer with 1% SDS. The lysates were mixed with loading buffer with/without b-mercaptoethanol and subjected to SDS-PAGE and IB. (G) TRIM21 / MEFs reconstituted with vector and HA-TRIM21 WT were treated with As(III). Cells were lysed in IP lysis buffer containing 1% Triton X-100. The insoluble fraction was dissolved in RIPA buffer containing 1% SDS. Both the Triton X-100 soluble and insoluble fractions were subjected to IB. (H) TRIM21 / MEFs reconstituted with vector and HA-TRIM21 WT were treated with As(III) (10 mM). Cells were fractionated to obtain the cytosol and nuclear fractions and probed for indicated proteins. (I) TRIM21 / MEFs reconstituted with vector, HA-TRIM21 WT, or HA-TRIM21W381/383A mutants were treated with As(III) (10 mM) for 4 hr. Cells were lysed in RIPA buffer containing 1% SDS and probed for indicated proteins. (J) TRIM21 / MEFs reconstituted with vector, HA-TRIM21 WT, HA-TRIM21-LD, or HA-TRIM21W381/383A were transfected with NQO1-ARE-luc and Renilla luciferase pRL-TK constructs. 16 hr later, cells were treated with As(III) (10 mM) and assayed for luciferase activity. (K) TRIM21 / MEFs reconstituted with vector, HA-TRIM21 WT, HA-TRIM21-LD, or HA-TRIM21W381/383A mutants were treated with As(III) (10 mM) for 4 hr. Cells were stained with H2DCFDA and analyzed by flow cytometry. Quantification of relative H2DCFDA intensity (Geo mean plus SD of four repeats) is shown on the right. ***p < 0.001, ****p < 0.0001.

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Figure 6. Ablation of TRIM21 Leads to Protection from Arsenic Toxicity (A and B) TRIM21+/+ and TRIM21 / MEFs were treated with As(III) (10 mM) for 12 hr (A) or for the indicated times (B). Cells were collected, and the lysates were subjected to IB. (C and D) MEFs were treated with As(III) (10 mM). (C) Cells were observed under phase-contrast microscope 16 hr after the treatment. Representative micrographs are shown. (D) Cell viability was determined by trypan blue. Data shown are the mean plus SD of three countings. **p < 0.01. (E) TRIM21 / MEFs reconstituted with vector, HA-TRIM21 WT, or HA-TRIM21W381/383A mutant were treated with As(III) (10 mM). Cell viability was determined by trypan blue. Shown are the mean plus SD of three countings. **p < 0.01. (legend continued on next page)

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results strongly indicate that ablation of TRIM21 can increase the antioxidant response and protect against pathological conditions where dysregulated ROS plays a major etiological role. DISCUSSION In this study, we find that the ubiquitin E3 ligase TRIM21 directly interacts with and ubiquitylates p62 at residue K7, which inhibits p62 dimerization and sequestration function. K7 has been reported to form a hydrogen bond with D69 (Wilson et al., 2003), which is essential for p62 dimerization (Bjørkøy et al., 2005). Our findings demonstrate that there exists a naturally occurring post-translational modification of p62 K7. Our study also shows a molecular consequence is the failure of sequestering Keap1, leading to a reduced capacity of antioxidant response. TRIM21-deficient cells show enhanced p62 oligomerization and aggregation in response to proteotoxic and oxidative stresses (proteasome inhibition, arsenic), as well as upon ablation of autophagy-essential proteins Vps34 or Atg7. This is accompanied by enhanced antioxidant response and protection from superoxide-induced cell death and tissue damage. On the other hand, although ROS is often considered harmful, it is not entirely surprising that a cell needs a negative regulator of the reductive response such as TRIM21 since certain levels of ROS may be beneficial by serving a critical role in cell signaling and anti-pathogenic response. Moreover, hyperactivating the reductive response may facilitate cancer cell survival and proliferation, hence promoting oncogenesis and therapy resistance (Sayin et al., 2014; Schafer et al., 2009). Along this line, hyperactivation of p62 and Nrf2 was shown to promote tumorigenesis (DeNicola et al., 2011; Duran et al., 2008; Mathew et al., 2009), and genetic deletion of TRIM21 or reduced TRIM21 expression was associated with the development and poor prognosis in numerous cancers (Brauner et al., 2015; Ding et al., 2015; Zhao and Bepler, 2001). Therefore, by ubiquitylating p62 and suppressing cellular reductive response, TRIM21 may function as a pleiotropic antagonistic factor that is beneficial in maintaining developmental redox homeostasis yet detrimental under excessive oxidative insult. TRIM21 is a RING finger domain-containing E3 ligase that belongs to the family of the tripartite motif (TRIM) family (Wada and Kamitani, 2006). It was initially discovered as a 52 kDa antigen of autoantibody to the ribonucleoprotein Ro/SSA in systemic lupus erythematosus (SLE) (Ben-Chetrit et al., 1988), whose increased expression was also found in Sjo¨gren’s syndrome (Ben-Chetrit et al., 1990). TRIM21 can interact with numerous proteins involved in both innate and adaptive immunity. While no obvious gross developmental defects were observed, TRIM21 / mice showed increased dysregulation of NF-kB-mediated proinflammatory cytokine production (Espinosa et al., 2009; Yoshimi et al., 2009). An important molecular feature of TRIM21 is its

binding to immunoglobulin G (IgG) via its PRYSPRY domain (James et al., 2007), which plays an important role in innate immune activation during viral infection (Mallery et al., 2010; McEwan et al., 2013). While it was previously reported that p62 can modulate TRIM21 function and NF-kB activity (Kim and Ozato, 2009), we identified W381/383 in the TRIM21 PRYSPRY domain that are required for p62 interaction, implying a role of p62 in modulating TRIM21 IgG binding and immune response. p62 is a major stress response regulator. Its expression and activity are critically regulated to maintain proper cell function and organismal homeostasis. p62 is regulated at multiple levels. At the transcription level, p62 expression is activated in a feedforward loop by Nrf2 (Jain et al., 2010), by SP-1, AP-1, NF-kB, and Ets-1(Vadlamudi and Shin, 1998), by the Ras/MEK/ERK1/2 pathway (Duran et al., 2008), and by the JNK/c-Jun pathway (Puissant et al., 2010). At the protein stability level, p62 is degraded along with the protein aggregates by autophagy (Pankiv et al., 2007) and is often served as an indicator for autophagy induction. At the post-translational level, p62 is phosphorylated at numerous sites, which enhances its activity (Ichimura et al., 2013; Lim et al., 2015; Linares et al., 2011; Matsumoto et al., 2011; Tanji et al., 2014). Here, we show that p62 activity can be negatively regulated by TRIM21-mediated K7 ubiquitylation and that TRIM21-p62 interaction is induced by proteotoxic stress and oxidative stress. Our finding adds to the delicate system where p62 level needs to be critically regulated in order to maintain cellular homeostasis and suggests a therapeutic potential by targeting TRIM21 in diseases involving proteotoxic and redox stress. EXPERIMENTAL PROCEDURES Reagents The following reagents were used: MG132 (Sigma-Aldrich, C2211), sodium arsenate (Sigma-Aldrich, 35000-1L-R), arsenic trioxide (Sigma-Aldrich, 311383), UBE2N/UbcH13/Uev1a (BostonBiochem, E2-664), UBE1 (BostonBiochem, E-306), ubiquitin (BostonBiochem, U-100), UBE2D2 (BostonBiochem, E2622), creatine phosphate (Amresco, 0271), creatine kinase (Affymetrix, 13915-100mg), Ni-NTA agarose (QIAGEN, 133203974), Protein A (Roche, 11719408001), NAC (Sigma-Aldrich, A9165), Tempo (Sigma-Aldrich, 176141), DPQ (Sigma-Aldrich, D5314), and DHIQ (Sigma-Aldrich, I138). Lentiviral and Retroviral Infection Lentiviruses and retroviruses were generated in HEK293T cells. The infection was performed as previously described (Pan et al., 2011). Mouse Experiments All mouse experiments were done in compliance with the Institutional Animal Care and Use Committee guidelines at Stony Brook University and Rutgers University. Statistics Student’s t test was used to compare the differences between two groups. One-way ANOVA with Tukey’s post hoc test was used for comparisons

(F–I) Nine pairs of litter mates of TRIM21+/+ and TRIM21 / mice were gavaged with 30 mg/kg As2O3. (F) Body weight was measured before and after 24 hr treatment, and loss of body weight was calculated and plotted. *p < 0.05. (G) Livers from water or As2O3-treated mice were collected after 24 hr. Tissues were processed for H&E staining and IHC. Representative images are shown. (H) TRIM21+/+ and TRIM21 / mice were treated with As2O3 for 24 hr. Liver tissues were homogenized and separated into detergent (1% Triton X-100)-soluble ‘‘S’’ and insoluble ‘‘I’’ fractions and subjected to IB. The ratio of insoluble/soluble band intensities was normalized to the corresponding tubulin band and is shown on the right. *p < 0.05, **p < 0.01. (I) Liver tissue was lysed in RIPA buffer containing 1% SDS and subjected to IB.

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Figure 7. Ablation of TRIM21 Leads to Protection against Heart Injury Induced by sTAC Sham or sTAC was performed on wild-type or TRIM21 further analyses. (A) Hearts from each group were photographed.

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mice. 4 days later, animals were subjected to echocardiography, then the hearts were isolated for

(legend continued on next page)

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between more than two groups. Results were considered significant when p < 0.05. Image Processing and Densitometry Measurements Images captured by deconvolution and confocal microscopes were viewed and processed by AxioVision LE and Zeiss LSM image browser, respectively. Images were processed in Adobe Photoshop to enhance the brightness and contrast. Densitometry of immunoblot bands was determined by ImageJ or by the Odyssey Infrared Imaging System. More detailed experimental procedures can be found in the Supplemental Information. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures and seven figures and can be found with this article online at http://dx.doi. org/10.1016/j.molcel.2016.02.007. AUTHOR CONTRIBUTIONS J.-A.P. and W.-X.Z. conceived the ideas and designed the experiments. J.-A.P., Y.S., and W.-X.Z. wrote the paper. C.D., J.M., and K.O. provided critical reagents. All authors performed experiments or data analysis. ACKNOWLEDGMENTS We thank Drs. Michael Frohman (Stony Brook University), Terje Johanson (University of Tromso, Norway), Huikuan Lin (M.D. Anderson Cancer Center), Noboru Mizushima (University of Tokyo), and Zhenyu Yue (Mt Sinai School of Medicine) for reagents. We thank Drs. Yanxiang Zhao (Hong Kong Polytechnic University) and David Goldfarb (University of Rochester) for insightful comments. This work was supported by grants from NIH (R01CA129536 and R01GM97355 to W.-X.Z., R01CA172025 and R01DK108743 to J.M., R01CA192642 to M.T.D.-M., 5P30CA030199 to M.T.D.-M., and J.M.), Department of Veterans Affairs Merit Review Program (to R.Z.L.), and a key project (12KG107) from the Municipal Healthy Bureau of Tianjin, China (to B.Y.). Received: September 15, 2015 Revised: January 13, 2016 Accepted: February 5, 2016 Published: March 3, 2016 REFERENCES Barchowsky, A., Dudek, E.J., Treadwell, M.D., and Wetterhahn, K.E. (1996). Arsenic induces oxidant stress and NF-kappa B activation in cultured aortic endothelial cells. Free Radic. Biol. Med. 21, 783–790. Belch, J.J., Bridges, A.B., Scott, N., and Chopra, M. (1991). Oxygen free radicals and congestive heart failure. Br. Heart J. 65, 245–248. Ben-Chetrit, E., Chan, E.K., Sullivan, K.F., and Tan, E.M. (1988). A 52-kD protein is a novel component of the SS-A/Ro antigenic particle. J. Exp. Med. 167, 1560–1571. Ben-Chetrit, E., Fox, R.I., and Tan, E.M. (1990). Dissociation of immune responses to the SS-A (Ro) 52-kd and 60-kd polypeptides in systemic lupus erythematosus and Sjo¨gren’s syndrome. Arthritis Rheum. 33, 349–355. Bernstam, L., and Nriagu, J. (2000). Molecular aspects of arsenic stress. J. Toxicol. Environ. Health B Crit. Rev. 3, 293–322.

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(B) Representative B-mode (left ventricle labeled) and M-mode echocardiographic images are shown. (C) M-mode images from nine cardiac cycles for each animal were used to calculate ventricular measurements: LVDV (left ventricular diastolic volume), LVSV (left ventricular systolic volume), FS (fractional shortening), and EF (ejection fraction). Individual data points and the mean are shown. n.s., non-significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (D) Hearts from sham or sTAC-treated mice were collected and examined with H&E and IHC staining. (E) Heart tissue was lysed in RIPA buffer containing 1% SDS and subjected to IB.

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