Sequential ubiquitination and deubiquitination enzymes synchronize the dual sensor and effector functions of TRIM21 Adam J. Fletcher1, Donna L. Mallery, Ruth E. Watkinson, Claire F. Dickson, and Leo C. James1 Division of Protein and Nucleic Acid Chemistry, Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom Edited by Jeremy Luban, University of Massachusetts Medical School, Worcester, Massachusetts, and accepted by the Editorial Board June 15, 2015 (received for review April 17, 2015) Tripartite motif (TRIM) 21 is a cytosolic antibody receptor that Ub ligase (E3), and the Ub-erasing deubiquitinase (DUB). Of neutralizes antibody-coated viruses that penetrate the cell and these, the E2 plays a central role in selecting the Ub chain simultaneously activates innate immunity. Here we show that the linkage topology (9–11). For example, the heterodimeric RING- conjugation of TRIM21 with K63-linked ubiquitin (Ub-63Ub) cata- domain E3 ligase BRCA1-BARD1 auto-monoubiquitinates us- lyzed by the sequential activity of nonredundant E2 Ub enzymes ing one of the E2s UbcH6, Ube2E2, UbcM2, or Ube2W, and is required for its dual antiviral functions. TRIM21 is first labeled the monoUb can be extended into Ub-63Ub or Ub-48Ub by with monoubiquitin (monoUb) by the E2 Ube2W. The monoUb is Ube2N/UbeV2 or Ube2K, respectively (12). PolyUb is typically a substrate for the heterodimeric E2 Ube2N/Ube2V2, resulting in attached to a substrate by the E3; however, with some proclivity, 63 63 TRIM21-anchored Ub- Ub. Depletion of either E2 abolishes Ub- Ub E3–E2 pairs can also generate unanchored polyUb chains 48 and Ub- Ub conjugation of TRIM21, NF-κB signaling, and virus neu- in vitro (13). Unanchored polyUb has been shown to activate 63 tralization. The formation of TRIM21-Ub- Ub precedes proteasome innate immune signaling (4, 5, 8, 14), but how it is generated and recruitment, and we identify an essential role for the 19S-resident regulated in cells remains poorly understood. and degradation-coupled deubiquitinase Poh1 in TRIM21 neutraliza- Once a Ub-modified substrate arrives at the proteasome, three tion, signaling, and cytokine induction. This study elucidates a com- DUBs in the 19S regulatory particle (RP), Usp14/Ubp6, Uch37/ plex mechanism of step-wise ubiquitination and deubiquitination UCHL5, and Rpn11/Poh1, trim or remove Ub modifications activities that allows contemporaneous innate immune signaling before substrate degradation (15). Although Ub-48Ub is a ca- and neutralization by TRIM21. nonical degradation signal, both Ub-48Ub and Ub-63Ub are hy- drolyzed by purified proteasomes in vitro (16, 17), and some TRIM21 | NF-kB | Ube2W | Ube2N/Ube2V2 | Poh1 studies have observed Ub-63Ub specificity in the polyUb ampu- tation activity of Poh1 (18, 19). Whether Ub-63Ub has a role at RIM21 is a cytoplasmic Ig receptor belonging to the tripartite the proteasome remains unclear; its presence at this location Tmotif family of E3 ubiquitin (Ub) ligases. Many TRIM pro- would seem incongruous with a model in which only Ub-48Ub- ∼ teins (there are 100 members in humans) have reported roles in labeled proteins are genuine substrates (20, 21). innate immunity (1). TRIM21 intercepts incoming, cytoplasmic In this report, we show that the effector and sensor functions antibody-coated pathogens and neutralizes them by mediating of TRIM21 are dependent on its sequential recruitment of the their VCP- and proteasome-dependent degradation (2, 3). In E2s Ube2W and Ube2N/Ube2V2, and the DUB Poh1. addition, TRIM21 initiates a signaling cascade, resulting in the transcriptional up-regulation of inflammatory cytokines and a Significance potent antiviral state in surrounding cells (4). Such antiviral du- ality has also been reported for a paralogue of TRIM21, TRIM5α (5), and more recently, TRIM19/PML (6), suggesting that an ability Tripartite motif (TRIM) proteins are a large family of E3 ubiq- to “sense” and neutralize viral infection is a conserved TRIM uitin (Ub) ligases, with many members having important roles function. The mechanism by which a TRIM protein coordinates in innate immunity. TRIM21 is a cytosolic antibody receptor sensor and effector functions remains unknown. that recognizes the Fc portion of antibodies bound to incoming For TRIM21, these dual functions are dependent upon its virions. On binding to these immune complexes, TRIM21 trig- activity as an E3 Ub ligase. In previous work, we have shown gers the catastrophic disassembly of viral capsids at the pro- that the TRIM21 really interesting new gene (RING) domain is teasome, terminating viral infection. Simultaneously, TRIM21 “ ” required for neutralization and catalyzes autoubiquitination senses the presence of the virus and provokes signaling κ with K48-linked polyubiquitin (polyUb) (Ub-48Ub, following cascades that activate the transcription factor NF- B, ultimately the nomenclature suggested by ref. 7) in vitro (3), suggesting alerting surrounding cells to the infection. Here, we uncover TRIM21 might synthesize Ub-48Ub to recruit the proteasome. the stepwise ubiquitination mechanism catalyzed by TRIM21, We also demonstrated that TRIM21, with the heterodimeric E2 as well as the various cofactors required, that allows these two enzyme Ube2N/Ube2V1, catalyses the formation of unanchored antiviral activities to occur synchronously at the proteasome. K63-linked polyUb (Ub-63Ub) in vitro (4). Ube2N and Ub-63Ub were essential for TRIM21 to mount an innate signaling re- Author contributions: A.J.F., D.L.M., R.E.W., and L.C.J. designed research; A.J.F., D.L.M., 63 and R.E.W. performed research; A.J.F., D.L.M., R.E.W., and C.F.D. contributed new re- sponse, consistent with a role for unanchored Ub- Ub in NF-κB agents/analytic tools; A.J.F., D.L.M., R.E.W., C.F.D., and L.C.J. analyzed data; and A.J.F. signaling (8). Thus, the ability of TRIM21 to mediate multiple and L.C.J. wrote the paper. functions seems associated with its ability to synthesize mul- The authors declare no conflict of interest. tiple Ub chain types. Moreover, because TRIM21 only initiates This article is a PNAS Direct Submission. J.L. is a Guest Editor invited by the Editorial Board. innate signaling upon recognition of antibody-bound virus (4), See Commentary on page 9797. this suggests that the effector and sensor functions of TRIM21 1To whom correspondence may be addressed. Email: [email protected] or lcj@ are mechanistically connected. mrc-lmb.cam.ac.uk. Four enzymes are involved in the ubiquitination cycle: the Ub This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. activating (E1), the Ub conjugating (E2), the substrate-specifying 1073/pnas.1507534112/-/DCSupplemental. 10014–10019 | PNAS | August 11, 2015 | vol. 112 | no. 32 www.pnas.org/cgi/doi/10.1073/pnas.1507534112 Downloaded by guest on September 25, 2021 Results substrates with anchored Ub-63Ub chains (12, 22), we depleted Ube2W and Ube2N Are Required for TRIM21 Dual Effector and Sensor Ube2W, using shRNA, to ask whether it too has a role in virus SEE COMMENTARY Functions. Previously, we have shown that infection of non- neutralization. Interestingly, Ube2W depletion also reversed professional cells with antibody-coated adenovirus (AdV:Ab) neutralization of adenovirus by 9C12 (Fig. 1C), suggesting both results in TRIM21-dependent NF-κB activation and proteasome- Ube2W and Ube2N/Ube2V2 are cofactors for TRIM21-medi- mediated virus neutralization (4). To investigate the functional ated virus neutralization. Furthermore, we observed that Ube2W relationship between these two antiviral outcomes, we first asked depletion abolished potent activation of NF-κB-Luc on challenge whether the generation of an innate immune response via NF-κB with Adv:Ab (Fig. 1D). In contrast, we measured no decrease in κ α activation precedes virus destruction by the proteasome. We NF- B-Luc activation in response to TNF in Ube2W-depleted treated human TE671 cells with chemical inhibitors of the pro- cells (Fig. 1E). Western blots confirmed depletions of Ube2N (Fig. teasome (MG132, bortezomib) or NF-κB signaling (5Z-7-Oxo- 1F), and RT-qPCR confirmed depletions of Ube2W (Fig. 1G). zeaenol, IKK Inhibitor VII). As shown in Fig. 1A, 7-h treatment of Together, these experiments suggest Ube2N and Ube2W are human TE671 cells with MG132, bortezomib, IKK VII, or 5Z-7- nonredundant cofactors for both sensing and effector responses of κ TRIM21, an unexpected observation because Ube2W is reported Oxozeaenol inhibited the stimulation of a model NF- Bpromoter 63 that drives luciferase expression (NF-κB-Luc) after infection with to anchor polyUb to substrates (22, 23) and Ub- Ub chains are AdV:Ab. However, although both MG132 and bortezomib not thought to recruit proteins to the proteasome (17). inhibited virus neutralization by the monoclonal antihexon anti- body 9C12 (Fig. 1B), IKK inhibitor VII or 5Z-7-Oxozeaenol did Ube2W Monoubiquitinates TRIM21. We next investigated whether not. Thus, TRIM21-dependent NF-κB activation is not a requisite TRIM21 can act as an E3 enzyme for Ube2W. We expressed for virus neutralization, suggesting signaling and neutralization full-length TRIM21 bearing an N-terminal MBP tag that im- might be synchronous, rather than sequential. proved protein solubility. We incubated purified MBP-TRIM21 To test this idea, we considered whether TRIM21 ubiquiti- with E1, Ube2W, and Ub. As increasing amounts of Ube2W nation, as required for innate signaling, was also required for were titrated into the reactions, we observed an increase in modified MBP-TRIM21 (Fig. 2A, Top). Probing with an anti-Ub virus neutralization. In vitro, TRIM21 synthesizes unanchored 63 63 antibody revealed a band of the same size as modified MBP- Ub- Ub in the presence of Ube2N/Ube2V1, and both Ub- Ub TRIM21, suggesting the adduct was a monoUb (Fig. 2A, Bot- and Ube2N are necessary for TRIM21 to provoke an innate tom). There were additional bands in the Ub blot with sizes immune signal (4).