Figure 4. TRIM22 Mediates K63-linked NOD2 Polyubiquitination.

A) TRIM22 enhances NOD2 polyubiquitination in HEK293 cells.

HEK293T cells were transiently co-transfected with HA-NOD2, GFP-ubiquitin, and either wildtype (WT) or variant (R442C) FLAG-TRIM22. Twenty-four hours post-transfection, cells were lysed followed by immunoprecipitation of HA-NOD2. Ubiquitination of NOD2 was evaluated by immunoblotting for GFP (ubiquitin). Co-transfection with wildtype TRIM22 enhanced ubiquitination of NOD2, which was abrogated with the R442C variant TRIM22. All experiments were carried out in triplicate, independent experiments.

B) Transgenic TRIM22 rescues NOD2 polyubiquitination in TRIM22-shRNA-HT29 cells.

HT29 cells stably transduced with control (scramble) or TRIM22 shRNA were transiently co-transfected with HA-NOD2 and either wildtype (WT) or variant (R442C) FLAG-TRIM22. Twenty-four hours post-transfection, cells were lysed followed by immunoprecipitation of the HA-NOD2. Evaluation of NOD2 poly-ubiquitination by GFP immunoblotting demonstrated rescued ubiquitination following transfection of WT-TRIM22 in the TRIM22 shRNA cell line that was not observed following transfection of the variant R442C. All experiments were carried out in triplicate, independent experiments.

C) Variants in TRIM22 abrogates K63-linked NOD2 polyubiquitination.

HEK293T cells were transiently co-transfected with FLAG-NOD2, HA-(K63-specific) ubiquitin, and either wildtype (WT) or variant (R150T, S244L, R321K, or R442C) V5-TRIM22. Twenty-four hours post-transfection, cells were lysed followed by immunoprecipitation of the FLAG-NOD2. Evaluation of NOD2 K63 specific-ubiquitination by HA immunoblotting demonstrated significantly reduced ubiquitination in cells co-transfected with TRIM22 variants compared to wildtype. All experiments were carried out in triplicate, independent experiments.

D and E) TRIM22-shRNA reduces MDP- and RSV-induced K63-linked NOD2 polyubiquitination.

HEK293T cells stably transduced with control (scramble) or TRIM22 shRNA were transfected with HA-tagged K63-specific ubiquitin and FLAG-NOD2. Forty-eight hours after transfection, cell lysates were stimulated by MDP (D) or RSV (E) for indicated the time points followed by lysis and immunoprecipitation using anti-FLAG-agarose. K63-poly-ubiquination of NOD2 was evaluated by immunoblotting using anti-HA antibody. Following stimulation by either MDP or RSV, NOD2 K63-specific ubiquitination increased over treatment time. However, in TRIM22 knockdown cells, this ubiquitination was significantly reduced or absent. All experiments were carried out in triplicate independent experiments and blots are representative.

F) Schematic overview of the proposed impact of TRIM22 and its identified variants on the NOD2 signaling axis.

Wildtype TRIM22 binds and mediates the K63-specific polyubiquitination of NOD2, potentiating downstream signaling through the MAVS and RIPK2 signaling axes depending on antigen. TRIM22 variants disrupt binding to NOD2, abrogating this polyubiquination thus attenuating downstream pro-inflammatory signaling