Non-proteolytic ubiquitination of OTULIN regulates NF-κB signaling pathway

Abstract

NF-κB signaling regulates diverse processes such as cell death, inflammation, immunity, and cancer. The activity of NF-κB is controlled by methionine 1-linked linear polyubiquitin, which is assembled by the linear ubiquitin chain assembly complex (LUBAC) and the ubiquitin-conjugating enzyme UBE2L3. Recent studies found that the deubiquitinase OTULIN breaks the linear ubiquitin chain, thus inhibiting NF-κB signaling. Despite the essential role of OTULIN in NF-κB signaling has been established, the regulatory mechanism for OTULIN is not well elucidated. To discover the potential regulators of OTULIN, we analyzed the OTULIN protein complex by proteomics and revealed several OTULIN-binding proteins, including LUBAC and tripartite motif-containing protein 32 (TRIM32). TRIM32 is known to activate NF-κB signaling, but the mechanism is not clear. Genetic complement experiments found that TRIM32 is upstream of OTULIN and TRIM32-mediated NF-κB activation is dependent on OTULIN. Mutagenesis of the E3 ligase domain showed that the E3 ligase activity is essential for TRIM32-mediated NF-κB activation. Further experiments found that TRIM32 conjugates polyubiquitin onto OTULIN and the polyubiquitin blocks the interaction between HOIP and OTULIN, thereby activating NF-κB signaling. Taken together, we report a novel regulatory mechanism by which TRIM32-mediated non-proteolytic ubiquitination of OTULIN impedes the access of OTULIN to the LUBAC and promotes NF-κB activation.

Keywords: LUBAC; NF-κB; TNF; TRIM; linear ubiquitination; proteomics.

Figure 1

Proteomic analysis of the OTULIN protein complex. (A) Proteomic analysis pipeline. (B) The OTULIN protein interaction network. OTULIN and the HCIPs are shown as square and circles, respectively. The blue line indicates a previously known interaction and the red line indicates a new interaction. The arrow indicates TRIM32.

Figure 2

TRIM32 interacts and co-localizes with OTULIN. (A) Reciprocal immunoprecipitation between TRIM32 and OTULIN. Different tagged TRIM32 and OTULIN were co-transfected into HEK293 cells. After 48 h, cell lysates were immunoprecipitated and blotted with the indicated antibodies. (B) HEK293 cells were treated with 10 ng/ml TNFα for 30 min, and then the cell lysates were immunoprecipitated with anti-OTULIN antibody and blotted with anti-TRIM32 antibody to detect endogenous TRIM32. (C) A549 cells were transfected with FLAG-tagged TRIM32 or/and HA-tagged OTULIN. Cells were fixed and stained with the indicated antibody. Red: FLAG; green: HA; blue: DAPI, a nuclear stain. Right panel shows quantitated TRIM32–OTULIN colocalization data with three representative images. (D) A549 cells in the chamber slide were fixed with cold methanol and incubated with anti-TRIM32 and anti-OTULIN antibodies. Red: TRIM32; green: OTULIN; blue: DAPI, a nuclear stain. Right panel shows quantitated TRIM32–OTULIN colocalization data with three representative images. (E) In situ interaction between TRIM32 and OTULIN with vs. without 10 ng/ml TNFα stimulation in A549 cells determined by the PLA. The anti-GFP antibody was used as control antibody for PLA assays. *P < 0.05.

Figure 3

The domains required for the interaction between TRIM32 and OTULIN. (A) The schematics of TRIM32 mutants. RING, really interesting new gene; CC, coiled coil; NHL, NCL-1, HT2A, and Lin-41. (B) HA-tagged OTULIN (OTULIN-HA) was co-transfected with FLAG-tagged TRIM32 (TRIM32-FLAG) or the indicated mutant into HEK293 cells. After 48 h, cell lysates were immunoprecipitated and blotted as indicated. (C) The schematics of OTULIN mutants. (D) TRIM32-FLAG was co-transfected with OTULIN-HA or the indicated mutant into HEK293 cells. After 48 h, cell lysates were immunoprecipitated and blotted with the indicated antibodies. (E) The FLAG-tagged TRIM32 mutant (C356-FLAG) and the HA-tagged OTULIN mutant (C74-HA) were transfected into HEK293 cells. After 48 h, cell lysates were immunoprecipitated and blotted with the indicated antibodies. (F) OTULIN-HA was co-transfected with TRIM32-FLAG or the indicated point mutant into HEK293 cells. After 48 h, cell lysates were immunoprecipitated and blotted as indicated.

Figure 4

TRIM32 mediates NF-κB activation via OTULIN. (A–C) HEK293 and TRIM32 knockout cells were stimulated with 10 ng/ml TNFα for the designated times. (A) Cell lysates were blotted as indicated. (B and C) Real-time PCR was performed to determine A20 (B) and VCAM-1 (C) mRNA levels. (D) HEK293 cells were transfected with TRIM32 or the indicated mutant with the NF-κB luciferase reporter. After 48 h, luciferase activities were measured. (E) The lysates of wild-type (WT) HEK293 and the indicated OTULIN knockout (KO) generated by CRISPR were blotted with the indicated antibodies. (F) The indicated OTULIN WT and KO cells were transfected with TRIM32 together with the NF-κB luciferase reporter and pRL-SV40. After 48 h, luciferase activities were measured. Data represent mean ± SD of three independent experiments. The P-value was calculated (two-tailed Student’s t-test). *P < 0.05.

Figure 5

TRIM32 ubiquitinates OTULIN. (A) HEK293 cells were transfected with TRIM32 or the RING deletion mutant with the NF-κB luciferase reporter and pRL-SV40. After 48 h, luciferase activities were measured. The right panel shows protein expression levels of TRIM32 and the mutant by western blotting. (B) HEK293 cells were transfected with TRIM32 or the C44S/C47S mutant with the NF-κB luciferase reporter and pRL-SV40. After 48 h, luciferase activities were measured. Data represent mean ± SD of three independent experiments. The P-value was calculated (two-tailed Student’s t-test). *P < 0.05. The lower panel shows protein expression levels of TRIM32 and the mutant by western blotting. (C) HEK293 cells were stimulated with 10 ng/ml TNFα for the designated times. Cell lysates were immunoprecipitated with anti-OTULIN antibody and blotted as indicated. (D) OTULIN-FLAG was co-transfected with vector, TRIM32-HA, or the indicated mutant into HEK293 cells. Cell lysates were immunoprecipitated with anti-FLAG antibody and blotted with the indicated antibodies. (E) In vitro ubiquitination of OTULIN by TRIM32. FLAG-tagged OTULIN, HA-tagged TRIM32, or the indicated mutant, plus E1, E2 (UBCH5A), ATP, and ubiquitin were added as indicated and incubated at 30°C for 2 h. The reaction mixtures were immunoprecipitated with anti-FLAG antibody. After washing with 1 M urea, the OTULIN protein was eluted with FLAG peptide. The eluates were blotted with the indicated antibodies. (F) In vitro ubiquitination of OTULIN by TRIM32 and the NHL mutants. HA-tagged OTULIN, FLAG-tagged TRIM32, or the indicated mutant, plus E1, E2 (UBCH5A), ATP, and ubiquitin were added as indicated and incubated at 30°C for 2 h. The reaction mixtures were immunoprecipitated with anti-FLAG antibody. After washing with 1 M urea, the OTULIN protein was eluted with FLAG peptide. The eluates were blotted with the indicated antibodies.

Figure 6

TRIM32 mediates non-proteolytic ubiquitination of OTULIN. (A) HEK293 cells were transfected with different doses of TRIM32. After 48 h, cell lysates were blotted with the indicated antibodies. (B) OTULIN-FLAG was transfected with wild-type ubiquitin or the indicated mutant into HEK293 cells. After 48 h, cell lysates were immunoprecipitated with anti-FLAG antibody and blotted as indicated.

Figure 7

K64 and K66 are the major ubiquitination sites of OTULIN. (A and B) HEK293 cells were transfected with TRIM32 and OTULIN or the indicated OTULIN mutant. After 48 h, cell lysates were immunoprecipitated and blotted with the indicated antibodies. (C) OTULIN or the indicated mutant was transfected into HEK293 cells along with LUBAC, NF-κB luciferase reporter, and pRL-SV40. After 48 h, luciferase activities were measured. *P < 0.05. The protein expression levels of LUBAC and OTULIN were determined by western blotting.

Figure 8

The non-proteolytic ubiquitination perturbs OTULIN–HOIP interaction. (A) HOIP, TRIM32, and OTULIN or the K64R/K66R mutant were transfected into HEK293 cells in the indicated combination. After 48 h, cell lysates were immunoprecipitated and blotted with the indicated antibodies. (B) OTULIN-FLAG was transfected with TRIM32 or the RING deletion mutant (Mut) into HEK293 cells. After 48 h, cell lysates were immunoprecipitated with anti-FLAG antibody and blotted as indicated. (C) OTULIN-FLAG was transfected into HEK293 and the TRIM32 knockout cells. After 48 h, cell lysates were immunoprecipitated with anti-FLAG antibody and blotted as indicated. (D) TRIM32 or the RING deletion mutant was transfected into HEK293 cells along with OTULIN, LUBAC, NF-κB luciferase reporter, and pRL-SV40 in the indicated combination. After 48 h, luciferase activities were measured. *P < 0.05. The protein expression levels of TRIM32, LUBAC, and OTULIN were determined by western blotting. The non-specific band (n.s.) is indicated. (E) The working model for TRIM32-mediated OTULIN ubiquitination and NF-κB activation.