Figure 6.
Figure 6. ULK1 is recruited to NLRP3 complexes by wild-type TRIM20 but not by FMF disease-associate TRIM20 mutants. (A) Coimmunoprecipitation analysis of ULK1 in NLRP3 complexes in HEK293T cells expressing GFP-TRIM20 or GFP alone. IP, immunoprecipitation; WB, Western blot. (B) The effect of NLRP3 expression on the presence of phospho-ULK1 in TRIM20 complexes. Lysates from HEK293 cells transiently expressing Myc-ULK1, GFP-TRIM20 (or GFP alone), and Flag-NLRP3 (or not) were immunoprecipitated with anti-GFP and immunoblots were probed as indicated. (C) Model of TRIM20’s function in autophagy as a regulator-receptor: TRIM20 assembles autophagy machinery (ULK1, Beclin 1, ATG16L1, and mAtg8s) and recognizes substrates (NLRP3, pro–caspase 1, and NLRP1) delivering them for autophagic degradation. The recognition of substrate enriches active p-ULK1 on the TRIM20 platform. (D) The levels of IL-1β were determined in supernatants of THP-1 cells that had been subjected to knockdown of ULK1 or TRIM20, treated with IFN-γ and LPS, and stimulated with nigericin for 30 min. (E) FLICA-positive cells were quantified using THP-1 cells that had been subjected to knockdown of TRIM20, treated with IFN-γ, and then treated with or without LPS (2 h) and nigericin (10 min), and stained for active caspase-1 (with FLICA); >150 cells per experiment were analyzed for quantification. Scr, scrambled. (F) Predominant FMF-associated point mutations of TRIM20 reside in the PRY/SPRY domain. (G) Levels of NLRP3 were determined in lysates of HEK293 cell expressing GFP-TRIM20 (wild-type or FMF-associated variants) or GFP and induced for autophagy by starvation in EBSS for 3 h. (H) Effects of FMF-associated variants on ULK1 presence in TRIM20 complexes. HEK293 cells were transiently transfected with Myc-ULK1, and either GFP-TRIM20 (wild-type or FMF-associated variants) or GFP alone. Lysates were immunoprecipitated with anti-GFP, and immunoblots were probed as indicated. Numbers indicate relative intensity of the band above. (I) Role of FMF-associated mutation in NLRP3 degradation. The presence of NLRP3 promotes phosphorylation of ULK1 in TRIM20 complex, leading to autophagic degradation of NLRP3. TRIM20 mutants recruited less ULK1 and phospho-ULK1, which results in lower autophagic activity and diminishes degradation of inflammasome components. Asterisks denote common FMF-associated point mutations in TRIM20. Data, means ± SE; n ≥ 3. *, P < 0.05 (ANOVA).

ULK1 is recruited to NLRP3 complexes by wild-type TRIM20 but not by FMF disease-associate TRIM20 mutants. (A) Coimmunoprecipitation analysis of ULK1 in NLRP3 complexes in HEK293T cells expressing GFP-TRIM20 or GFP alone. IP, immunoprecipitation; WB, Western blot. (B) The effect of NLRP3 expression on the presence of phospho-ULK1 in TRIM20 complexes. Lysates from HEK293 cells transiently expressing Myc-ULK1, GFP-TRIM20 (or GFP alone), and Flag-NLRP3 (or not) were immunoprecipitated with anti-GFP and immunoblots were probed as indicated. (C) Model of TRIM20’s function in autophagy as a regulator-receptor: TRIM20 assembles autophagy machinery (ULK1, Beclin 1, ATG16L1, and mAtg8s) and recognizes substrates (NLRP3, pro–caspase 1, and NLRP1) delivering them for autophagic degradation. The recognition of substrate enriches active p-ULK1 on the TRIM20 platform. (D) The levels of IL-1β were determined in supernatants of THP-1 cells that had been subjected to knockdown of ULK1 or TRIM20, treated with IFN-γ and LPS, and stimulated with nigericin for 30 min. (E) FLICA-positive cells were quantified using THP-1 cells that had been subjected to knockdown of TRIM20, treated with IFN-γ, and then treated with or without LPS (2 h) and nigericin (10 min), and stained for active caspase-1 (with FLICA); >150 cells per experiment were analyzed for quantification. Scr, scrambled. (F) Predominant FMF-associated point mutations of TRIM20 reside in the PRY/SPRY domain. (G) Levels of NLRP3 were determined in lysates of HEK293 cell expressing GFP-TRIM20 (wild-type or FMF-associated variants) or GFP and induced for autophagy by starvation in EBSS for 3 h. (H) Effects of FMF-associated variants on ULK1 presence in TRIM20 complexes. HEK293 cells were transiently transfected with Myc-ULK1, and either GFP-TRIM20 (wild-type or FMF-associated variants) or GFP alone. Lysates were immunoprecipitated with anti-GFP, and immunoblots were probed as indicated. Numbers indicate relative intensity of the band above. (I) Role of FMF-associated mutation in NLRP3 degradation. The presence of NLRP3 promotes phosphorylation of ULK1 in TRIM20 complex, leading to autophagic degradation of NLRP3. TRIM20 mutants recruited less ULK1 and phospho-ULK1, which results in lower autophagic activity and diminishes degradation of inflammasome components. Asterisks denote common FMF-associated point mutations in TRIM20. Data, means ± SE; n ≥ 3. *, P < 0.05 (ANOVA).

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