Ubiquitin Ligase TRIM62 Regulates CARD9-Mediated Anti-fungal Immunity and Intestinal Inflammation

Abstract

CARD9 is a central component of anti-fungal innate immune signaling via C-type lectin receptors, and several immune-related disorders are associated with CARD9 alterations. Here, we used a rare CARD9 variant that confers protection against inflammatory bowel disease as an entry point to investigating CARD9 regulation. We showed that the protective variant of CARD9, which is C-terminally truncated, acted in a dominant-negative manner for CARD9-mediated cytokine production, indicating an important role for the C terminus in CARD9 signaling. We identified TRIM62 as a CARD9 binding partner and showed that TRIM62 facilitated K27-linked poly-ubiquitination of CARD9. We identified K125 as the ubiquitinated residue on CARD9 and demonstrated that this ubiquitination was essential for CARD9 activity. Furthermore, we showed that similar to Card9-deficient mice, Trim62-deficient mice had increased susceptibility to fungal infection. In this study, we utilized a rare protective allele to uncover a TRIM62-mediated mechanism for regulation of CARD9 activation.

Figure 1. C-terminal CARD9 Truncations Impair Depleted Zymosan- and TDM-induced Cytokine Production

(A) Schematic of CARD9 variants used. All indicated variants were found in a human immune cDNA panel with the exception of CARD9 416-536, which was designed for experimental use. CC: coiled-coil domain. (B–D) Card9^−/− murine BMDCs transduced with indicated CARD9 variants were stimulated with either depleted zymosan, TDM or LPS and cytokine levels were assessed by ELISA. Data were obtained from three independent experiments performed in duplicate (n = 3). (E) Expression of CARD9 variants in total lysates from (B–D) as detected by Western blot. Bars represent means ± s.d. *P < 0.05, **P < 0.01. Comparisons in (B–D) are relative to stimulated CARD9 WT. See also Figure S1.

Figure 2. CARD9 Δ11 Is a Dominant Negative Variant

(A) THP-1 cells were transduced with FLAG-CARD9 WT or FLAG-CARD9 Δ11, stimulated with depleted zymosan, and NF-κB levels were assessed by NF-κB luciferase assay. (B) Western blot showing expression of CARD9 as detected by antibodies against endogenous CARD9 (top) or FLAG (bottom) in lysates from (A). Note that the antibody detecting endogenous CARD9 has an N-terminal epitope and therefore does not recognize CARD9 Δ11. (C) WT BMDCs were transduced with FLAG-StrepII empty vector or FLAG-StrepII-tagged CARD9 Δ11 followed by immunoprecipitation of tagged CARD9 Δ11. (D) Purified FLAG-tagged CARD9 WT or CARD9 Δ11 proteins were incubated with immobilized GST-CARD9; interactions were assessed by ELISA. Coomassie gel at right shows purified proteins used in ELISA. See also Figure S2.

Figure 3. TRIM62 Interacts with Full-length CARD9, but not CARD9 C-terminal Truncations

(A) HEK293T cells were transfected with indicated constructs followed by immunoprecipitation of tagged CARD9. (B) WT BMDCs were stimulated with depleted zymosan and stained for endogenous CARD9 and TRIM62. Scale bars, 5 μm. (C) Quantification of results shown in (B). (D) CARD9^−/− THP-1 cells were transduced with V5-tagged CARD9 WT or CARD9 Δ11 and stimulated with depleted zymosan. Cells were stained for endogenous TRIM62 and tagged CARD9. (E) Quantification of results shown in (D). See also Figure S3.

Figure 4. TRIM62 Promotes the Ubiquitination of Full-length CARD9, but not CARD9 Truncations

(A) HEK293T cells were transfected with indicated constructs followed by immunoprecipitation of tagged CARD9, then blotted for tagged ubiquitin, SUMO1, SUMO2, SUMO3, and ISG15. (B) WT BDMCs were stimulated with depleted zymosan and immunoprecipitated for CARD9, then blotted for endogenous CARD9 and ubiquitin. (C) HEK293T cells were transfected with indicated constructs followed by immunoprecipitation of tagged CARD9, then blotted for ubiquitin in the presence of either WT or a ligase-dead (C11A,C14A) version of TRIM62. (D) HEK293T cells were transfected with indicated constructs followed by immunoprecipitation of tagged CARD9, then blotted for tagged ubiquitin. (E–F) HEK293T cells were transfected with indicated constructs followed by immunoprecipitation of tagged CARD9, then blotted for indicated ubiquitin mutants. In (E), transfected ubiquitin constructs have single point mutations to indicated lysine residues. In (F), transfected ubiquitin constructs have point mutations to multiple lysines, leaving only the indicated lysine residue intact. FS, FLAG-StrepII. See also Figure S4.

Figure 5. TRIM62-mediated CARD9 Ubiquitination at K125 is Critical for CARD9 Activation

(A) HEK293T cells were transfected with TRIM62, ubiquitin (K27 only), and indicated CARD9 point mutants, followed by immunoprecipitation of CARD9. (B) Indicated proteins were purified and incubated in vitro to assess ubiquitination of CARD9. (C) CARD9^−/− THP-1 cells were reconstituted with CARD9 WT, CARD9 Δ11, or CARD9 K125R, stimulated with depleted zymosan, and NF-κB levels were assessed by NF-κB luciferase assay. (D) Western blot showing expression levels of CARD9 in cells used in (C). See also Figure S5.

Figure 6. TRIM62 Regulates Immune Responses and Susceptibility to Candida albicans Infection In Vivo

(A) WT and Trim62^−/− mice were injected i.v. with heat-killed C. albicans and serum IL-6 levels were quantified 4 h later by ELISA (n = 5). Bars display IL-6 relative to WT levels. (B) WT and Trim62^−/− mice were injected with C. albicans and fungal loads in kidney, spleen, and liver were assessed at 3 and 6 days post-infection. (C) WT and Trim62^−/− BMDMs were incubated with pHrodo-labeled C. albicans, and phagocytosed C. albicans was quantified by intracellular fluorescence via flow cytometry. (D) Survival curve showing percent survival over time in WT and Trim62^−/− mice infected with C. albicans. Bars represent means ± s.d. *P < 0.05. See also Figure S6.