TRIM-mediated precision autophagy targets cytoplasmic regulators of innate immunity

Abstract

The present paradigms of selective autophagy in mammalian cells cannot fully explain the specificity and selectivity of autophagic degradation. In this paper, we report that a subset of tripartite motif (TRIM) proteins act as specialized receptors for highly specific autophagy (precision autophagy) of key components of the inflammasome and type I interferon response systems. TRIM20 targets the inflammasome components, including NLRP3, NLRP1, and pro-caspase 1, for autophagic degradation, whereas TRIM21 targets IRF3. TRIM20 and TRIM21 directly bind their respective cargo and recruit autophagic machinery to execute degradation. The autophagic function of TRIM20 is affected by mutations associated with familial Mediterranean fever. These findings broaden the concept of TRIMs acting as autophagic receptor regulators executing precision autophagy of specific cytoplasmic targets. In the case of TRIM20 and TRIM21, precision autophagy controls the hub signaling machineries and key factors, inflammasome and type I interferon, directing cardinal innate immunity response systems in humans.

Figure 1.

TRIMs regulate IFN-γ–induced autophagy. (A) THP-1 cells were subjected to TRIM knockdown and treated with 1,000 U/ml IFN-γ for 4 h, and high content (HC) analysis was performed using a Cellomics HCS scanner (epifluorescence) and iDEV software. HC (magenta, endogenous LC3B immunofluorescence; blue, nuclei stained with Hoechst). Mask overlay, software-defined objects (primary objects, cell outlines; internal secondary objects, LC3 puncta). (B) Average count of LC3 puncta per cell from cells treated as in A (data from two 96-well plates with identical siRNA arrangements; the corresponding data are shown in Fig. S1 C). Encircled are IFN-γ–treated wells (right) and wells with vehicle controls (bottom left). TRIM knockdowns that reduced LC3 puncta readout in both of the two experiments by 3 SDs (horizontal dot lines) from the average of IFN-γ–treated controls (horizontal solid line) are indicated by corresponding TRIM numbers (open circle). TRIMs that were chosen in follow up experiments in Fig. 1 C are also indicated with number. (C) Similar to B, except that THP-1 cells were subjected to specific TRIM or scrambled (Scr; control) knockdowns and were analyzed in quadruplicates or more repeats. (D) Model of TRIMs-mediated IFN-γ–induced autophagy based on the results obtained in Fig. 1 and Fig. S1 thus far. (E) THP-1 cells were treated with TRIM20 or scrambled siRNAs, incubated with or without IFN-γ for 4 h in the presence of bafilomycin A1 (Baf A₁), and LC3-II conversion was determined by immunoblots. RI, relative intensity. (F) HeLa cells were transfected with GFP or GFP-TRIM20, and HC analysis was performed. Data, means ± SE; n ≥ 3. *, P < 0.05 (ANOVA, C and E, or t test in F). Bars, 5 µm.

Figure 2.

TRIM20 interacts with ULK1 and Beclin 1. (A and B) Coimmunoprecipitation analysis of GFP-TRIM20 (T20) with Myc-ULK1 (A) or Flag-Beclin 1 (B) in HEK293 cells extracts. IP, immunoprecipitation; WB, Western blot. (C) Confocal microscopy of HeLa cells coexpressing mCherry-TRIM20 with GFP-ULK1. Line tracing corresponds to arrow. White outline, cell boundary defined by background fluorescence. Insets, enlarged areas as indicated by white outlined squares. Bars, 10 µm. (D) Coimmunoprecipitation analysis of TRIM20 complexes with p-ULK1 (Ser-317) in HEK293 cells. (E) TRIM20 domains and deletion constructs used. Dotted lines, deleted regions. (F) Coimmunoprecipitation analysis of interactions between deletion variants of TRIM20 (as GFP fusions) with Myc-ULK1 in HEK293 cells. (G) GST pull-down analysis of radiolabeled Myc-ULK1 with GST-tagged deletion variants of TRIM20. (top) Autoradiogram of pull-down products. (bottom) Coomassie brilliant blue (CBB)–stained SDS-polyacrylamide gel with GST ­deletion variants of TRIM20. Data are representative of three or more experiments.

Figure 3.

TRIM20 assembles ULK1 and Beclin 1 in a complex and interacts with ATG16L1. (A) TRIM20 domains and deletion constructs used. (B) Coimmunoprecipitation analysis of interaction between deletion variants of TRIM20 (as GFP fusions; asterisks denote fusion products on the bottom blot) with Flag-Beclin 1 in HEK293 cells. (C) Coimmunoprecipitation analysis of ULK1 in Beclin 1 complexes in the presence and absence of TRIM20 in HEK293T cell lysates. (D) Coimmunoprecipitation analysis of GFP-TRIM20 with endogenous ATG16L1. (E) TRIM20 domains and deletion constructs used. (F) Coimmunoprecipitation analysis of interaction between deletion variants of TRIM20 with Flag-ATG16L1 in HEK293 cells. (G) ATG16L1 domains and deletion constructs used. (H) Coimmunoprecipitation analysis of interactions between deletion variants of Flag-ATG16L1 and GFP-TRIM20 in HEK293 cells. (I) Model of TRIM20-dependent autophagy induction based on Fig. 2, Fig. 3, and Fig. S2. Data are representative of three or more experiments.

Figure 4.

TRIM20 interacts with mammalian Atg8 paralogues (mAtg8s). (A) GST pull-down analysis of interactions between radiolabeled Myc-TRIM20 and GST-tagged mAtg8s. (top) Autoradiogram of pull-down products. (bottom) CBB-stained SDS-polyacrylamide gel with GST-mAtg8s. (B) TRIM20 domains and deletion constructs used. (C) GST pull-down analysis of binding between radiolabeled Myc-TRIM20 deletion variants and GST-GABARAP and GST-LC3A. (D) Identification of GABARAP-interacting regions on TRIM20 by peptide array. Three series of TRIM20 peptides (regions of primary sequence staggered by three amino acid residues), with either three or four positive consecutive binding signals, were identified. The peptide sequences corresponding to the positive binding signals (encompassed spots; defined as regions I, II, and III) were mutated as indicated and were subjected to the GST pull-down experiments in E and Fig. S2 F. (E) GST pull-down analysis of interaction between radiolabeled Myc-TRIM20 triple mutants and GST-GABARAP. Data are representative of three or more experiments.

Figure 5.

TRIM20 degrades inflammasome components through autophagy. (A) Levels of NLRP3 were determine in lysates from THP-1 cells subjected to TRIM20 or scrambled (Scr) siRNA were activated with 1,000 U/ml IFN-γ for 3 h, and 2.5 µg/ml LPS for 2 h (for optimal TRIM20 expression; Fig. S3 B). RI, relative intensity. (B) Levels of NLRP3 were determined in THP-1 subjected to TRIM20 or control knockdowns and treated or not with bafilomycin A1 (Baf A₁). (C) The abundance of NLRP3 protein was determined in THP-1 cells subjected to TRIM20 or control knockdowns and exposed to Escherichia coli strain LF82 and IFN-γ in the presence or absence of bafilomycin A1. (D) The abundance of NLRP3 protein was determined in primary human MDMs subjected to TRIM20 or control knockdowns and exposed to LPS and IFN-γ in the presence or absence of bafilomycin A1. (E) Levels of NLRP3 were determined in THP-1 cells subjected to ULK1, Beclin 1, or scrambled (Scr) siRNA and treated with IFN-γ and LPS. (F–H) Levels of NLRP3 (F), NLRP1 (G), or pro–caspase 1 (H) were determined in cells expressing GFP or GFP-TRIM20 after autophagy induction (Earle's balanced salt solution , 3 h) in the presence or absence of bafilomycin A1. Data, means ± SE; n ≥ 3. *, P < 0.05; ^†, P ≥ 0.05 (ANOVA).

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).

Figure 7.

TRIM21 interacts with autophagy regulators and effectors. (A and B) Coimmunoprecipitation analyses of GFP-TRIM21 (T21) with Myc-ULK1 (A) and Flag-Beclin 1 (B) in HEK293 cells extracts. (C) GST pull-down analysis of binding between radiolabeled Myc-TRIM21 and GST-mAtg8s. (top) Autoradiogram of pull-down products. (bottom) CBB-stained SDS-polyacrylamide gel with GST-mAtg8s. (D) TRIM21 domains and deletion constructs used. (E) GST pull-down analysis of binding between radiolabeled Myc-TRIM21 deletion mutants and GST-GABARAP and GST-p62. Asterisks and squares denote presence or absence of Myc-TRIM21, respectively. (F) p62 domains and deletion constructs used. (G) GST pull-down analysis of interaction between radiolabeled Myc-TRIM21 and GST-tagged p62. Data representative of three or more experiments. IP, immunoprecipitation; WB, Western blotting.

Figure 8.

TRIM21 promotes autophagic degradation of IRF3 dimers and attenuates type I IFN production. (A) Confocal microscopy of HeLa cells coexpressing mCherry-TRIM21, Flag-IRF3, and GFP-LC3B in the presence of bafilomycin A1. White outline, cell boundary. Arrows; colocalization. (B) Confocal microscopy of HEK293 cells coexpressing mCherry-TRIM21, Flag-IRF3, and GFP-ULK1. Arrows, colocalization. (C) Co-immunoprecipitation analysis of IRF3-ULK1 complexes in the presence and absence of TRIM21. Lysates from HEK293 cells transiently expressing Myc-ULK1, Flag-IRF3, and either GFP-TRIM20 or GFP were immunoprecipitated with anti-Myc, and immunoblots were probed as indicated. (D) Levels of dimerized IRF3 were assessed by native PAGE of extracts from THP-1 cells subjected to TRIM21 or control knockdowns and stimulated for 12h by herring testis DNA (HT-DNA) transfected into the cells in the presence of 200 U/ml IFN-γ. (E) Effects of autophagy inhibition (bafilomycin A1) on TRIM21-dependent IRF3 dimer degradation in THP-1 cells. (F) Effects of TRIM21 knockdown on IFN-β mRNA levels after stimulation of THP-1 cells with IFN-γ and HT-DNA. (G) Model of TRIMs’ roles in regulation of inflammation by precision autophagy. TRIM20 targets the inflammasome components for autophagic degradation, whereas TRIM21 targets IRF3 dimer, to suppress inflammasome activity and type I IFN response, respectively. TRIM20 and TRIM21, responding to IFN-γ, directly bind their respective cargo and recruit autophagic machinery to execute degradation. Dashed arrow, cooperation between TRIM20 and TRIM21 may play a role in autophagic responses to IFN-γ. Bars, 10 µm. Data, means ± SE; n ≥ 3; *, P < 0.05; ^†, P ≥ 0.05 (ANOVA). IP, immunoprecipitation; Scr, scrambled; WB, Western blot.