Sequential ubiquitination and deubiquitination enzymes synchronize the dual sensor and effector functions of TRIM21

Abstract

Tripartite motif (TRIM) 21 is a cytosolic antibody receptor that neutralizes antibody-coated viruses that penetrate the cell and simultaneously activates innate immunity. Here we show that the conjugation of TRIM21 with K63-linked ubiquitin (Ub-63 Ub) catalyzed by the sequential activity of nonredundant E2 Ub enzymes is required for its dual antiviral functions. TRIM21 is first labeled with monoubiquitin (monoUb) by the E2 Ube2W. The monoUb is a substrate for the heterodimeric E2 Ube2N/Ube2V2, resulting in TRIM21-anchored Ub-63 Ub. Depletion of either E2 abolishes Ub-63 Ub and Ub-48 Ub conjugation of TRIM21, NF-κB signaling, and virus neutralization. The formation of TRIM21-Ub-63 Ub precedes proteasome recruitment, and we identify an essential role for the 19S-resident and degradation-coupled deubiquitinase Poh1 in TRIM21 neutralization, signaling, and cytokine induction. This study elucidates a complex mechanism of step-wise ubiquitination and deubiquitination activities that allows contemporaneous innate immune signaling and neutralization by TRIM21.

Sequential Poly-ubiquitylation by Specialized Conjugating Enzymes Expands the Versatility of a Quality Control Ubiquitin Ligase

“…We further show that for efficient poly-ubiquitylation, both E2 enzymes must functionally interact with the Doa10 RING domain (Figure 3). Poly-ubiquitylation processes involving the consecutive activity of distinct E2 enzymes were previously postulated from in vitro reconstitution studies on the APC and SCF βTrCP2 UB ligase complexes, on the autoubiquitylation of the RING-finger ligase BRCA1, as well as on the function of TRIM21 at the immune system (Christensen et al, 2007;Fletcher et al, 2015;Rodrigo-Brenni and Morgan, 2007;Wu et al, 2010). Still, to our knowledge this study is the first to show the biological relevance of such tandem ubiquitylation by demonstrating its requirement for the sequential activity of Ubc6 and Ubc7 during processing of Doa10 PQC client proteins in yeast cells.…”

“…Consequently, conjugating enzymes of the priming type must be able to identify promiscuous Ub ligation sites in large set of heterogeneous peptide environments, whereas the elongating enzymes should merely synthesize homogenous poly-Ub chains on a primary Ub. Indeed, several in vitro studies suggest that individual Ub conjugating enzymes of the same E3 ligase complex assume distinct functions in the course of poly-Ub chain formation (Fletcher et al, 2015;Rodrigo-Brenni and Morgan, 2007;Wu et al, 2010).…”

“…Tandem E2 activities, operating with a single E3 Ub ligase, probably contribute to efficient substrates ubiquitylation in other cellular processes, and hence represent a common functional adaptation of the Ub conjugation system to target highly diverse protein populations (Fletcher et al, 2015;Rodrigo-Brenni and Morgan, 2007;Wu et al, 2010).…”

Delicate coordination of TRIM21’s dual activity in virus neutralization and signaling

“…In conclusion, the work of Fletcher et al (3) proposes an intriguing model of TRIM21 activation through stepwise autoubiquitination and Poh1-mediated deubiquitination. Interestingly, a similar activation mechanism has been recently reported for TRIM5α (11).…”

“…In a series of elegant in vitro experiments, Fletcher et al (3) demonstrate that Ube2W first facilitates TRIM21 auto-monoubiquitination, and that TRIM21 monoubiquitination then serves as a substrate for the covalent attachment of K63-polyubiquitin chains to TRIM21 catalyzed by Ube2N/Ube2V2. Without monoubiquitination of TRIM21 by Ube2W, TRIM21 was still capable of catalyzing unanchored K63-linked ubiquitin chains; however, unexpectedly, these unanchored K63-linked chains did not trigger NF-κB activation in the cell.…”