TRIM38 Inhibits Tnfα- and IL-1Β–Triggered NF-Κb Activation by Mediating Lysosome-Dependent Degradation of TAB2/3

TRIM38 inhibits TNFα- and IL-1β–triggered NF-κB activation by mediating lysosome-dependent degradation of TAB2/3

Ming-Ming Hua, Qing Yanga, Jing Zhanga, Shi-Meng Liua, Yu Zhanga, Heng Lina, Zhe-Fu Huangb, Yan-Yi Wangb, Xiao-Dong Zhanga, Bo Zhonga, and Hong-Bing Shua,1

aState Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China; and bWuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430072, China

Edited by George R. Stark, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, and approved December 23, 2013 (received for review September 26, 2013) TNFα and IL-1β are two proinflammatory cytokines that play crit- inflammatory responses (5, 7). Thus, the TAK1-TAB2/3 complex ical roles in many diseases, including rheumatoid arthritis and in- critically mediates TNFα- and IL-1β–triggered signaling. fectious diseases. How TNFα- and IL-1β–mediated signaling is TNFα- and IL-1β–triggered signaling is timely terminated or finely tuned is not fully elucidated. Here, we identify tripartite- down-regulated to avoid excessive inflammatory responses. motif protein 38 (TRIM38) as a critical negative regulator of TNFα- About 10 proteins have been reported to attenuate TNFα and and IL-1β–triggered signaling. Overexpression of TRIM38 inhibited IL-1β signaling by targeting various signaling molecules in the κ activation of NF- B and induction of downstream cytokines fol- pathways. MARCH8, cIAP1, cIAP2, and RBCK1 are E3 ubiq- α β lowing TNF and IL-1 stimulation, whereas knockdown or knock- uitin ligases that induce K48-linked polyubiquitination and pro- out of TRIM38 had the opposite effects. TRIM38 constitutively β– teasome-dependent degradation of ILRAcP, RIP1, and TAB2/3, interacted with critical components TGF- activated kinase 1 (TAK1)- – α α binding protein 2/3 (TAB2/3) and promoted lysosome-dependent respectively (8 10). Tripartite-motif protein 5 (TRIM 5 )in- degradation of TAB2/3 independent of its E3 ubiquitin ligase ac- teracts with the TAK1-TAB1-TAB2/3 complex and promotes IMMUNOLOGY tivity. Consistently, deficiency of TRIM38 resulted in abolished trans- degradation of TAB2 via the lysosomal pathway (11). Several location of TAB2 to the lysosome, increased level of TAB2 in cells, and deubiquitinating enzymes have also been reported to negatively enhanced activation of TAK1 after TNFα and IL-1β stimulation. We regulate TNFα and/or IL-1β signaling. A20, USP2a, USP4, and conclude that TRIM38 negatively regulates TNFα-andIL-1β– USP20 deubiquitinate TRAF6 (12–15), whereas CYLD medi- induced signaling by mediating lysosome-dependent degradation ates deubiquitination of TRAF6 and IKKγ (16, 17). In addition, of TAB2/3, two critical components in TNFα- and IL-1β–induced A20, CYLD, and USP4 also catalyze deubiquitination of RIP1, signaling pathways. Our findings reveal a previously undiscovered TRAF2, and TAK1, respectively (16, 18, 19). Recently, it has mechanism by which cells keep the inflammatory response in been shown that DUSP14 dephosphorylates TAK1, thereby check to avoid excessive harmful immune response triggered by inhibiting TNFα- and IL-1β–triggered signaling (20). Given the α β TNF and IL-1 . diverse mechanisms by which TNFα and IL-1β signaling is down- regulated, it is possible that different enzymes target distinct he proinflammatory cytokines TNFα and IL-1β play central signaling molecules in different types of cells after TNFα or IL-1β Troles in many diseases, including infectious diseases, auto- stimulation, and it is of great interest to identify additional immunity, and cancers (1–4). One hallmark following TNFα and β κ IL-1 stimulation is the activation of NF- B and mitogen-acti- Significance vated protein kinases (MAPKs) and the subsequent induction of cytokines and chemokines. TNFα binds to TNF receptor 1, which recruits tumor necrosis factor receptor-associated death domain Infection of pathogenic microbes induces the body to produce cytokines, which are mediators of inflammation. TNFα and IL- protein (TRADD) through its death domain. TRADD further re- β cruits TRAF2, TRAF5, cIAP1, cIAP2, and receptor-interacting 1 are two important proinflammatory cytokines that trigger protein 1 (RIP1) to form a large receptor complex, where RIP1 a series of cellular reactions, leading to induction of downstream genes and inflammation. Understanding how the cel- undergoes K63-linked ubiquitination. The TGF-β–associated ki- lular reactions are triggered by the proinflammatory cytokines nase 1 (TAK1)-associating chaperones, TAB2 and TAB3 (TAB2/ is important for deciphering the molecular mechanisms of in- 3), bind preferentially to the K63-linked polyubiquitin chains flammation. In this study, we identified a protein called TRIM38, of RIP1, which results in autophosphorylation and activation of which negatively regulates TNFα-andIL-1β–triggered cellular TAK1. TAK1 further phosphorylates the MAPK kinases such as reactions by causing degradation of TAB2/3, two cellular com- MEKK3 and MEKK6 and inhibitor kappa B kinase β (IKKβ). ponents essential for TNFα-andIL-1β–triggered cellular re- The MEKKs phosphorylate the MAPKs, including JNK and p38, sponse. This study reveals a mechanism by which cells keep to activate the transcription factor AP-1, whereas IKKβ phos- the inflammatory response in check to avoid excessive harmful phorylates IκBα, which results in its proteasome-dependent κ immune response and may provide clues on treatments of degradation, leading to the release of NF- B to the nucleus (5, 6). inflammation. IL-1β binds to IL-1 receptor (IL-1R) and results in recruitment of

the IL-1R accessory protein (IL-1RAcP) and the adaptor protein Author contributions: M.-M.H., Y.-Y.W., and H.-B.S. designed research; M.-M.H., Q.Y., J.Z., MyD88. MyD88 further recruits IRAK1, IRAK4, and TNF S.-M.L., Y.Z., H.L., and Z.-F.H. performed research; M.-M.H., Y.-Y.W., X.-D.Z., B.Z., and H.-B.S. receptor-associated factor 6 (TRAF6) to the receptor com- analyzed data; and M.-M.H., B.Z., and H.-B.S. wrote the paper. plex, where TRAF6 catalyzes K63-linked autoubiquitination The authors declare no conflict of interest. and/or the synthesis of free K63-linked polyubiquitin chains. These This article is a PNAS Direct Submission. polyubiquitinchains recruit the TAK1-TAB2/3 complex, leading 1To whom correspondence should be addressed. E-mail: [email protected]. κ to TAK1 activation. TAK1 activates NF- B and MAPKs, leading This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. to induction of various cytokines and chemokines and subsequent 1073/pnas.1318227111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1318227111 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 molecules or mechanisms responsible for the negative regulation AB C of TNFα and IL-1β signaling. TRIM38 belongs to the TRIM protein family that is involved in various biological processes, including cell proliferation and apoptosis, innate immunity, and inflammatory response (21). Previously, it has been shown that TRIM38 acts as a suppressor in TOLL-like receptor (TLR)-mediated IFN-β induction by promoting degradation of TRAF6 and NAP1 through the ubiquitin- D proteasome system in mouse macrophages (22, 23), whereas another study demonstrates that TRIM38 induces proteasomal degradation of TRIF, thereby inhibiting TLR3-mediated IFN-β induction (24). Whether and how TRIM38 plays a role in TNFα- and IL-1β–triggered signaling remains unknown. In this study, we identified TRIM38 as a negative regulator in TNFα- and IL-1β–triggered activation of NF-κB and MAPKs. EF TRIM38 promoted the degradation of TAB2/3 through a lysosomal-dependent pathway, which required its C-terminal PRY- SPRY but not the RING domain. In addition, TRIM38 deficiency resulted in increased recruitment of TAK1 to upstream adaptors RIP1 and TRAF6, which was associated with elevated activation of NF-κB and MAPKs and induction of downstream cytokines. Our findings thus identified a previously undiscovered mecha- α β– α β Fig. 1. Overexpression of TRIM38 inhibits TNF - and IL-1 triggered sig- nism for regulation of TNF and IL-1 signaling and a strategy naling. (A) Effects of TRIM38 on TNFα- and IL-1β–triggered NF-κB activation for the host to control excessive inflammatory responses induced in HEK293 cells. HEK293 cells (1 × 105) were transfected with the NF-κBlu- by TNFα or IL-1β. ciferase plasmid (0.01 μg) and an HA-TRIM38 plasmid (0.2 or 0.4 μg). Twenty hours after transfection, cells were treated with TNFα (10 ng/mL) or IL-1β Results (10 ng/mL) or left untreated for 10 h before luciferase assays were per- Overexpression of TRIM38 Inhibits TNFα-andIL-1β–Triggered Signaling. formed. Expression of transfected TRIM38 in each unstimulated sample was α examined by immunoblot analysis. (B) Effects of TRIM38 on TNFα- and IL-1β– In an attempt to identify additional proteins regulating TNF and κ IL-1β signaling, we screened ∼10,000 independent human cDNA triggered NF- B activation in HCT116 and HeLa cells. The experiments were performed as in A.(C) Effects of TRIM38 on IFNγ-induced activation of the expression plasmids for their abilities to regulate TNFα- and IL- β– κ IRF1 promoter. The experiments were performed as in A except that the IRF1 1 triggered activation of NF- B by reporter assays. This effort promoter reporter plasmid was used and transfected cells were treated with led to the identification of TRIM38, a member of the TRIM IFNγ (100 ng/mL). (D) Effects of TRIM38 on TNFα- and IL-1β–induced tran- family. To confirm that TRIM38 regulates TNFα-andIL-1β– scription of TNFA, IL-6, and IL-8 genes. HEK293 cells were transduced with triggered signaling, we constructed an HA-tagged TRIM38 ex- either an empty vector or an HA-TRIM38 plasmid to establish stable cell lines. pression plasmid and performed additional reporter assays. As Cells (4 × 105) from both stable cell lines were treated with TNFα or IL-1β for shown in Fig. 1A, overexpression of TRIM38 inhibited TNFα- the indicated times, and then total RNA was prepared for qPCR analysis. β– κ Expression of TRIM38 in the stable cell lines was examined by immunoblot and IL-1 triggered NF- B activation dose-dependently in α β– HEK293 cells. In addition, TRIM38 also inhibited TNFα- and analysis (Right). (E) Effects of TRIM38 on TNF - and IL-1 induced cytokine of TNFα, IL-6, and IL-8. HEK293 cells were transduced with either an empty IL-1β–triggered activation of NF-κBinHCT116andHeLacells × 5 B vector or an HA-TRIM38 plasmid to establish stable cell lines. Cells (4 10 ) (Fig. 1 ). In similar reporter assays, TRIM38 did not inhibit from both stable cell lines were treated with TNFα or IL-1β for the indicated IFNγ-triggered activation of IRF1 reporter (Fig. 1C). To confirm times, and then the medium was collected for ELISA analysis. (F) Effects of these results, we established stable cell lines that were trans- TRIM38 on IFNγ-induced transcription of IRF1 gene. Cells (4 × 105) were left duced with either an empty vector or the HA-TRIM38 plasmid untreated or treated with IFNγ (100 ng/mL) for the indicated times, and total and performed quantitative PCR (qPCR) and ELISA analysis RNA was extracted for qPCR analysis. Graphs show mean ± SD; n = 3. *P < with these cell lines. As shown in Fig. 1 D and E, ectopic ex- 0.05; **P < 0.01. pression of TRIM38 inhibited TNFα- and IL-1β–induced expression of cytokines including TNFα, IL-6, and IL-8 at both the production were correlated with the knockdown efficiencies of mRNA and the protein level. In contrast, IFNγ-induced expression of the IRF1 gene was comparable between the cell lines the respective RNAi plasmids, indicating critical negative regu- α β transduced with empty vector and HA-TRIM38 (Fig. 1F). These latory roles of TRIM38 in TNF and IL-1 signaling. data together suggest that overexpression of TIRM38 inhibits To further confirm these results, we generated TRIM38- TRIM38 NF-κB activation and cytokine induction after TNFα or IL-1β deficient HCT116 cells by deleting a part of exon 3 of the stimulation. gene in both alleles following a previously described gene knockout strategy (Fig. S2A). The deletion of TRIM38 was confirmed at Knockdown or Knockout of TRIM38 Potentiates TNFα- and IL-1β– both DNA and protein levels (Fig. S2B). In qPCR and ELISA Triggered Signaling. We next examined whether endogenous analysis, induction of TNFα, IL-6, and IL-8 was substantially α β − − + + TRIM38 was involved in negative regulation of TNF or IL-1 increased in TRIM38 / compared with TRIM38 / cells following signaling. We constructed two TRIM38-RNAi plasmids, both of TNFα or IL-1β stimulation (Fig. 2 D and E). Consistently, acti- which could inhibit expression of overexpressed or endogenous κ A vation of NF- B and MAPKs was significantly enhanced in TRIM38 in HEK293 cells (Fig. 2 ). In reporter assays, knock- TRIM38−/− TRIM38+/+ α α β– compared with cells following TNF down of TRIM38 potentiated TNF - and IL-1 triggered acti- β vation of NF-κB but not IFNγ-triggered activation of IRF1 or IL-1 stimulation, indicating that TRIM38 functioned up- F G promoter (Fig. 2 B and C). Consistent with these observations, stream of MAPKs and IKK (Fig. 2 and ). In contrast, γ IRF1 TNFα- and IL-1β–induced expression of TNFA, IL-6, and IL-8, IFN -induced expression of or phosphorylation of STAT1 −/− +/+ but not IFNγ-induced expression of IRF1, was increased by was comparable between TRIM38 and TRIM38 cells (Fig. knockdown of TRIM38 (Fig. S1). In these experiments, the S2 C and D). These results suggest that endogenous TRIM38 is degrees of positive regulation of NF-κB activation and cytokine required to negatively regulate TNFα and IL-1β signaling.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1318227111 Hu et al. Downloaded by guest on October 1, 2021 not TAK1-, IKKβ-, or p65-mediated activation of NF-κB, was AB C − − substantially enhanced in TRIM38 / cells compared with that + + in TRIM38 / cells in reporter assays (Fig. S2E). In addition, results from transient transfection and coimmunoprecipitation experiments suggested that TRIM38 interacted with TAB2 and TAB3 but not with TAB1 (Fig. 3A), supporting the notion that TRIM38 functioned at the level of TAB2/3. Furthermore, we D found that TRIM38 interacted with TAB2 and TAB3 constitutively, and the association was enhanced following TNFα or IL-1β treatment (Fig. 3B). Because TAB2 shares a highly homologous amino acid sequence and functions redundantly with TAB3, we next fo- cused on the regulation of TAB2 by TRIM38. Domain mapping analysis indicated that the C-terminal PRY-SPRY domain of TRIM38 (amino acids 290–645) was associated with TAB2, E whereas the middle fragment of TAB2 (amino acids 361–553) was responsible for its association with TRIM38 (Fig. S3 A and B).

FG IMMUNOLOGY

Fig. 2. Knockdown or knockout of TRIM38 potentiates TNFα- and IL-1β– triggered signaling. (A) Efficiencies of TRIM38-RNAi plasmids on TRIM38 levels. (Upper) HEK293 cells (4 × 105) were transfected with expression plasmids for TRIM38-Flag and HA-β-actin (0.1 μg each) and the indicated RNAi plasmids (1 μg each). Twenty-four hours after transfection, cell lysates were analyzed by immunoblot with anti-Flag or anti-HA. (Lower) HEK293 cells (1 × 107) were transfected with control or the indicated TRIM38-RNAi plasmids (10 μg each) for 36 h. Cell lysates were analyzed by immunoblot with anti-TRIM38 or anti–β-actin. (B) Effects of TRIM38-RNAi on TNFα- and IL-1β–triggered NF-κB activation in HEK293 and HeLa cells. The cells (1 × 105) were transfected with RNAi plasmids (1 μg each) along with the NF-κB reporter plasmid (0.01 μg). Thirty-six hours after transfection, cells were left untreated or treated with TNFα (10 ng/mL) or IL-1β (10 ng/mL) for 10 h before luciferase assays were performed. (C) Effects of TRIM38-RNAi on IFNγ-induced IRF1 promoter activation. Reporter assays were performed as in B except that cells were transfected with IRF1 promoter reporter plasmid and treated with IFNγ (100 ng/mL). (D) Effects of TRIM38 deficiency on TNFα and IL-1β–induced transcription of TNFA, IL-6, and IL-8 genes. The indicated × 5 α β cells (4 10 ) were treated with TNF (10 ng/mL) or IL-1 (10 ng/mL) for the Fig. 3. TRIM38 interacts with and destabilizes TAB2 through its C-terminal PRY- indicated times, and then total RNA was extracted for qPCR analysis. (E) SPRY domain. (A) TRIM38 interacts with TAB2 and TAB3 in mammalian α β– Effects of TRIM38 deficiency on TNF and IL-1 induced cytokine pro- overexpression system. HEK293 cells (1 × 107) were transfected with the in- × 5 α duction. The indicated cells (4 10 ) were treated with TNF (10 ng/mL) or dicated plasmids for 24 h. Coimmunoprecipitation and immunoblots were β IL-1 (10 ng/mL) for the indicated times, and then the medium was collected performed with the indicated antibodies. (B) Endogenous TRIM38 interacts α for ELISA analysis. (F) TRIM38 deficiency potentiates TNF -triggered MAPK with TAB2/3. HEK293 cells (3 × 107) were left untreated or treated with TNFα × 7 α activation. The indicated cells (1 10 ) were left untreated or treated with TNF (Left) or IL-1β (Right) for the indicated times. Endogenous coimmunopre- (10 ng/mL) for the indicated times. Cells were lyzed and immunoblot analysis cipitation and immunoblots were performed with the indicated antibodies. was performed with the indicated antibodies. (G) TRIM38 deficiency (C) TRIM38 specifically destabilizes TAB2/3. HEK293 (4 × 105) cells were β– potentiates IL-1 triggered MAPK activation. The experiments were performed transfected with the indicated plasmids for 24 h, and then immunoblots β as in E, except that cells were treated with IL-1 (10 ng/mL). Graphs show were performed with the indicated antibodies. (D) Effects of TRIM38 ± = < < mean SD; n 3. *P 0.05; **P 0.01. truncation mutants on destabilization of TAB2. HEK293 (4 × 105) cells were transfected with the indicated plasmids for 24 h before immunoblots were performed with the indicated antibodies. (E) Analysis of TRIM38 expression − − TRIM38 Interacts with TAB2/3 Through Its C-Terminal PRY-SPRY Domain. in TRIM38 / cells stably transduced with an empty vector (II), TRIM38-Flag The results above indicate that TRIM38 functions upstream of (III), TRIM38(63-465)-Flag (IV), or TRIM38(290-465)-Flag (V), respectively and + + MAPKs and NF-κBinbothTNFα-andIL-1β–triggered signaling in TRIM38 / cells stably transduced with an empty vector (I). Cells (1 × 107) pathways. Considering that TNFα-andIL-1β–triggered signaling (I, II, III, IV, V) were harvested and lysed. Immunoblot analysis was performed with the indicated antibodies. (F) Reconstitution of TRIM38 or TRIM38 pathways converge at the TAK1-TAB1-TAB2/3 complex, which is mutant (63–465) into TRIM38-deficient cells leads to down-regulation of upstream of MAPKs and IKK (4), we thus hypothesized that TAB2. Cells (1 × 107) (I, II, III, IV, V) were left untreated or treated with TNFα or TRIM38 functioned at the leveloftheTAK1-TAB1-TAB2/3 IL-1β for the indicated times. Immunoblot analysis was performed with the complex. Interestingly, TAB2-, TAB3-, TRAF6-, and RIP1-, but indicated antibodies.

Hu et al. PNAS Early Edition | 3of6 Downloaded by guest on October 1, 2021 These data collectively suggest that TRIM38 interacts with TAB2 through its C-terminal PRY-SPRY domain.

TRIM38 Destabilizes TAB2/3 Through Its C-Terminal PRY-SPRY Domain. When we examined TRIM38-TAB2/3 association in transient transfection and coimmunoprecipitation experiments, we routinely found that TRIM38 markedly down-regulated the expression level of TAB2/3, but not of TAK1 and TAB1 (Fig. 3 A and C). Inter- estingly, the C-terminal PRY-SPRY domain of TRIM38 was sufficient to down-regulate the expression level of TAB2 (Fig. 3D) as well as to inhibit TNFα- and IL-1β–triggered activation of NF-κB(Fig. S3C), indicating that TRIM38 down-regulated the expression level of TAB2 independently of its RING domain or its E3 ubiquitin ligase activity. Furthermore, TRIM38 promoted down-regulation of endogenous TAB2 at the protein level without altering the mRNA expression level of TAB2 (Fig. S3D), indicating that TRIM38 destabilized TAB2 at the protein level. − − To further confirm the results, we reconstituted TRIM38 / cells with empty vector (II), Flag-tagged TRIM38 (III), TRIM38 (63–465) (IV), or TRIM38(290–465) (V) by retroviral trans- duction and examined TNFα- or IL-1β–triggered signaling in + + these cells. In these experiments, TRIM38 / cells reconstituted with empty vector (I) were used as a control (Fig. 3E). The results indicated that reconstitution of TRIM38, TRIM38(63– 465), or TRIM38(290–465) could potently inhibit TNFα- or IL- 1β–induced transcription of TNFA, IL-6, and IL-8 (Fig. S4A), but had no effect on IFNγ-induced expression of IRF1 (Fig. S4B). In Fig. 4. TRIM38 mediates lysosomal degradation of TAB2. (A) Effects of × addition, the protein level of TAB2 was significantly higher in inhibitors on TRIM38-mediated destabilization of TAB2. HEK293 cells (4 − − + + 105) were transfected with the indicated plasmids. Fourteen hours after TRIM38 / than in TRIM38 / cells without stimulation, and TAB2 α β– transfection, the cells were treated with the indicated inhibitors for 6 h protein was more resistant to TNF -orIL-1 induced degra- before immunoblot analysis was performed. (B) Effects of NH Cl and MG132 −/− +/+ 4 dation in TRIM38 cells compared with TRIM38 cells (Fig. 3F). on down-regulation of TAB2 triggered by TNFα and IL-1β. HEK293 (1 × 107) Reconstitution of TRIM38(1–465, 63–465, or 290–465) into cells were treated with NH Cl or MG132 for 4 h and then further treated − − 4 TRIM38 / cells markedly down-regulated the protein levels of with TNFα and IL-1β for 2 h before immunoblot analysis was performed. (C) TAB2 and accelerated TNFα- or IL-1β–induced down-regulation TRIM38 promotes translocation of TAB2 to the lysosome. HEK293 cells (1 × 105) of TAB2 (Fig. 3F). Taken together, these data suggest that were transfected with Cherry-TAB2 and GFP-LAMP1 (Left)orCFP-TRIM38 TRIM38 mediates destabilization of TAB2 in rest cells as well (Right). Twenty hours after transfection, cells were fixed with 4% (wt/vol) as after TNFα or IL-1β stimulation, which depends on the PRY- paraformaldehyde and subjected for confocal microscopy. (D)EffectofTRIM38 deficiency on TNFα-orIL-1β–induced colocalization of TAB2 with the lysosomes. + + − − − − SPRY domain and is independent of its RING domain. TRIM38 / , TRIM38 / ,orTRIM38/ cells reconstituted with the PRY-SPRY domain (1 × 105) were transfected with GFP-TAB2. Twenty hours after transfection, TRIM38 Mediates Lysosome-Dependent Degradation of TAB2. Protein cells were stained with Red Lysotracker (200 nM) for 2 h and treated with TNFα degradation is one of the main strategies involved for turning off (20 ng/mL) or IL-1β (20 ng/mL) for 1 h and then fixed with 4% (wt/vol) para- protein functions in biological processes. At least three systems formaldehyde and subjected to confocal microscopy. A random 10 cells in each exist for protein degradation, including the ubiquitin-proteasome, sample were used for calculating the colocalization dots that were normalized lysosomal, and autophagosome pathways. Because TRIM38 me- to the total lysosome-red dots. Graphs show mean ± SD; n = 3. **P < 0.01. diated degradation of TAB2 independently of its RING domain, we speculated that TRIM38 might induce proteolysis of TAB2 in TRIM38−/− D a manner independent of the ubiquitin-proteasome system. completely absent in cells (Fig. 4 ). As mentioned Consistent with our hypothesis, TRIM38 did not catalyze ubiq- above, the PRY-SPRY domain of TRIM38 is sufficient and nec- uitination of TAB2 (Fig. S5). In addition, TRIM38-mediated essary for inhibition of TAB2. Consistently, we found that the PRY-SPRY domain was also sufficient to rescue TAB2 recruit- degradation of TAB2 was completely inhibited by the lysosomal − − ment to lysosomes in TRIM38 / cells following TNFα or IL-1β inhibitor NH4Cl but not MG132 or 3MA, which are inhibitors for the proteasome and autophagosome pathways, respectively stimulation. These results demonstrate that the TRIM38-mediated (Fig. 4A). These results indicate that TRIM38 mediated degra- lysosomal degradation pathway is involved in TNFα- and IL-1β– dation of TAB2 through the lysosomal pathway. Actually, we induced proteolysis of TAB2. found that TNFα- or IL-1β–induced down-regulation of TAB2 TRIM38 Promotes Translocation of TAB2 to Endosomes/Lysosomes. To was partially restored by NH4Cl or MG132 alone but completely explore the detail of the mechanism of how TAB2 is delivered to restored by a combination of NH4Cl and MG132 (Fig. 4B), indicating that TNFα- or IL-1β–induced down-regulation of TAB2 the lysosome for degradation by TRIM38, we analyzed the is mediated through both the proteasome- and lysosome-dependent subcellular localizations of these two proteins by cell fraction- degradation pathways. In this context, it has been reported that ation assays. In these experiments, a small fraction of TRIM38 RBCK1 induces proteasomal degradation of TAB2 after TNFα was found in the membrane fraction in unstimulated cells, and or IL-1β treatment (10). Confocal microcopy analysis indicated TNFα or IL-1β stimulation promoted translocation of TRIM38 that a fraction of TAB2 did localize in lysosomes in unstimulated from the cytosol to the membrane fraction. Interestingly, the cells, and an increased amount of TAB2 was found to localize in majority of TAB2 was found in the membrane fraction in the lysosomes when cotransfected with TRIM38 (Fig. 4C), indicating absence of TNFα or IL-1β and was dissociated from the mem- that TRIM38 promoted translocation of TAB2 to the lysosomes. brane fraction into the cytosol upon TNFα or IL-1β stimulation In addition, TNFα or IL-1β treatment induced colocalization of (Fig. S6 A and B). In this context, it has been reported that TAB2 with the LysoTracker in wild-type cells, which was almost dissociation of TAB2 from the membrane to the cytosol is

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1318227111 Hu et al. Downloaded by guest on October 1, 2021 − − + + required for the recruitment of TAK1 to the upstream adaptors was enhanced in TRIM38 / compared with TRIM38 / cells (25). Interestingly, however, the association of TAB2 and TRIM38 following TNFα or IL-1β stimulation (Fig. 5 A and B). was markedly increased both in the cytosol and the membrane fractions following TNFα or IL-1β stimulation (Fig. S6B). The Discussion simplest explanation for this is that TAB2 undergoes a stepwise It has been well documented that the TAK1-TAB2/3 complex plays regulation after TNFα or IL-1β stimulation. First, upon TNFα crucial roles in TNFα and IL-1β signaling. In this study, we found or IL-1β stimulation, TAB2 is disassociated from the plasma that TRIM38 negatively regulated TNFα-andIL-1β–triggered membrane to the cytosol to form the TAK1-TAB1-TAB2 com- signaling by promoting TAB2 proteolysis in a lysosome-depen- plex, which is recruited to polyubiquitinated RIP1 or TRAF6, dent manner. Deficiency of TRIM38 resulted in increased re- leading to activation of TAK1. Second, TAB2 in the TAK1- cruitment of TAK1 to the upstream adaptors RIP1 and TRAF6 TAB1-TAB2 complex is immediately captured and translocated and potentiated activation of TAK1 following TNFα or IL-1β from the cytosol to certain membrane fractions for proteolysis by treatment. Consistently, TNFα- or IL-1β–induced activation of TRIM38 to prevent persistent activation of TAK1. IKKα/β and MAPKs and the subsequent expression of cytokines To determine to which membrane TRIM38 and TAB2 may were increased in TRIM38-deficient cells compared with wild- attach, we performed confocal microscopy experiments. Results type cells following TNFα or IL-1β treatment. These results to- from confocal microscopy analysis indicated that a fraction of gether suggest that TRIM38 is required for the restriction of TRIM38 was colocalized with EEA1 (endosome marker), but TNFα- and IL-1β–triggered signaling. not with BID (mitochondrial marker), LC3 (autophagesome It has been reported that TRIM38 negatively regulates TLR- marker), Sec61β (ER marker), or LAMP1 (lysosome marker) triggered NF-κB activation and type I IFN induction by medi- (Fig. S7A). Furthermore, a small fraction of TAB2 protein was ating degradation of TRAF6, NAP1, and TRIF via the ubiquitin- also found to colocalize with EEA1 (Fig. S7B). Coexpression of proteasome–dependent pathway (22–24). However, it is unlikely TRIM38 markedly increased translocation of TAB2 to the that TRIM38 inhibits TNFα or IL-1β signaling via a similar EEA1-labeled subcellular compartments (Fig. S7C). In addition, mechanism for the following reasons. First, TRIM38 did not TRIM38 and TAB2 were found to colocalize with EEA1 (Fig. mediate ubiquitination of TAB2 when coexpressed in HEK293 S7C), but not with LAMP1 (Fig. 4C). These observations together cells, indicating that TRIM38 was not an E3 for TAB2. Second, suggest that TRIM38 interacts with TAB2 in both the cytosol and the C-terminal PRY-SPRY domain of TRIM38, which lacks the the endosome membrane fractions and promotes the trans- N-terminal RING domain, was sufficient to interact with TAB2 location of TAB2 to the endosomes/lysosomes for proteolysis. and promote TAB2 degradation, indicating that the E3 ubiquitin IMMUNOLOGY ligase activity was not required for TRIM38-mediated degrada- TRIM38 Deficiency Leads to Increased Recruitment of TAK1 to Upstream tion of TAB2. Third, TRIM38-mediated proteolysis of TAB2 Adaptors. TAB2 is a chaperone protein critical for TAK1 recruit- was dramatically inhibited by the lysosomal inhibitor NH4Cl but ment to upstream adaptors such as RIP1 and TRAF6 following not the proteasome inhibitor MG132, supporting the notion that α β TNF or IL-1 stimulation, respectively. So we examined whether TRIM38 promoted lysosomal proteolysis of TAB2. Fourth, TNFα TAK1 recruitment would be affected in TRIM38-deficient cells or IL-1β stimulation resulted in the translocation of TAB2 to the α β after TNF or IL-1 stimulation. In coimmunoprecipitation as- lysosomes, a process depending on TRIM38, indicating that says, TAK1-RIP1 and TAK1-TRAF6 associations were sub- TAB2 could be degraded through the lysosomal pathway. Al- TRIM38−/− stantially increased in cells compared with those though it has been shown that overexpression of TRIM38 alone TRIM38+/+ α β in cells following TNF or IL-1 stimulation, re- could activate NF-κB in reporter assays (21), our studies suggest α β– spectively. In contrast, TNF - or IL-1 induced TRAF2-RIP1 or that endogenous TRIM38 suppresses TNFα- or IL-1β–triggered TRIM38+/+ IRAK1-TRAF6 association was comparable between signaling via the lysosomal degradation pathway. TRIM38−/− A B and cells (Fig. 5 and ). These data suggest that In cell fractionation assays and confocal microscopy experi- TRIM38 deficiency results in increased recruitment of TAK1 to ments, we found that TRIM38 promoted translocation of TAB2 the upstream adaptors, which is probably due to increased cellular to the endosomes/lysosomes for degradation. How endosome- TRIM38−/− abundance of TAB2 in cells. In support of this located TAB2 was degraded in lysosomes is by far less clear. α β notion, we observed that phosphorylation of TAK1 and IKK / The simplest hypothesis is that the TAB2-containing endosomes are fused with prelysosomes or undergo acidization to form acid lysosomes, where TRIM38 is disassociated and TAB2 remains to be degraded. It is possible that other proteins are involved in regulating this process in addition to TRIM38. In this context, it has been shown that TRIM5α promotes TAB2 proteolysis in a lysosome-dependent manner (11). Whether and how TRIM38 and TRIM5α function redundantly or cooperatively in mediating lysosomal degradation of TAB2 requires further investigations. TNFα and IL-1β are two proinflammatory cytokines that are involved in many human diseases, including rheumatoid arthritis, infectious and inflammatory diseases, and cancers. Thus, our identification of TRIM38 as a negative regulator of TNFα and IL-1β signaling provides a therapeutic target for disease control or treatment in the future.

Fig. 5. TRIM38 deficiency leads to increased recruitment of TAK1 to the up- Materials and Methods stream adaptors. (A) TRIM38 deficiency potentiates TAK1 recruitment to RIP1 Reagents and Antibodies. Recombinant human TNFα, IL-1β, and IFNγ (R&D + + − − upon TNFα stimulation. TRIM38 / and TRIM38 / cells (3 × 107)wereleft Systems); mouse monoclonal antibodies against Flag (Sigma), HA (Covance), untreated or treated with TNFα (20 ng/mL) for the indicated times. Coim- and β-actin (Sigma); mouse anti-TAK1, p-TAK1, p-IKKα/β; rabbit anti-JNK, munoprecipitation and immnoblot analysis were performed with the in- p-JNK, p38, p-p38, Erk1/2, p-Erk1/2 (CST); rabbit anti-TRAF6, RIP1, IRAK1, dicated antibodies. (B) TRIM38 deficiency potentiates TAK1 recruitment to TRAF2 (Santa Cruz Biotechnology); rabbit anti-TAB3 (Epitomics); LysoTracker TRAF6 upon IL-1β stimulation. The experiment was performed as in A,except (Invitrogene); and ELISA kits for human TNFα, IL-6, and IL-8 (BOSTER) were that IL-1β (20 ng/mL) and the TRAF6 antibody were used. purchased from the indicated manufacturers. Mouse anti-TRIM38 and rabbit

Hu et al. PNAS Early Edition | 5of6 Downloaded by guest on October 1, 2021 anti-TRIM38 were raised against human TRIM38. Mouse anti-IκBα antibodies Fluorescent Confocal Microscopy. HEK293, HeLa, and HCT116 cells were were raised against human IκBα. Mouse anti-TAB2 was previously described (10). transfected with the indicated plasmids by lipofectamine 2000 (Invitrogen). At 24 h after transfection, the cells were treated with TNFα or IL-1β for the Constructs. Mammalian expression plasmids for Flag- or HA-tagged RIP-1, indicated time points followed by fixation with 4% (wt/vol) paraformaldehyde TRAF6, TAB2 and its mutants, TAB3, TRIM38 and its mutants, GFP-tagged for 15 min at 4 °C. The cells were then observed with an Olympus confocal TRIM38 and TAB2, and Cherry-tagged TRIM38 and TAB2 were constructed by microscope under a 100× oil objective. standard molecular biology techniques. NF-κB luciferase reporter plasmid was kindly provided by Gary Johnson (University of Colorado Health Other Materials and Methods. Other materials and methods used in this study Sciences Center, Denver). Organelle markers are kindly provided by Li Yu are described in SI Materials and Methods. (Tsinghua University, Beijing, China). ACKNOWLEDGMENTS. We thank Dr. Shu Li, Shuai Wang, and Shang-Ze Li RNAi Experiments. Double-strand oligonucleotides corresponding to the (Wuhan University) for technical help and stimulating discussions. This study target sequences were cloned into the pSuper.Retro RNAi plasmid (Oligoengine was supported by the Ministry of Science and Technology of China Inc.). The following sequences were targeted for human TRIM38 cDNA: #1— (2012CB910201, 2014CB542600) and the National Natural Science Founda- 5′-GGATCCAGATACAGCTCAT-3′;#2—5′-GGAAAGAGAAGGTACAGAT-3. tion of China (31221061, 31130020, 3171427, and 91029302).

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