IRGM governs the core autophagy machinery to conduct antimicrobial defense

Abstract

IRGM, encoded by a uniquely human gene conferring risk for inflammatory diseases, affects autophagy through an unknown mechanism. Here, we show how IRGM controls autophagy. IRGM interacts with ULK1 and Beclin 1 and promotes their co-assembly thus governing the formation of autophagy initiation complexes. We further show that IRGM interacts with pattern recognition receptors including NOD2. IRGM, NOD2, and ATG16L1, all of which are Crohn's disease risk factors, form a molecular complex to modulate autophagic responses to microbial products. NOD2 enhances K63-linked polyubiquitination of IRGM, which is required for interactions of IRGM with the core autophagy factors and for microbial clearance. Thus, IRGM plays a direct role in organizing the core autophagy machinery to endow it with antimicrobial and anti-inflammatory functions.

Figure 1. IRGM activates AMPK signaling and interacts with core autophagy machinery

(A) Lysates from HT-29 colon epithelial cells transfected with control and IRGM siRNA were subjected to Western blotting with antibodies to phospho-AMPK (Thr-172), AMPK, IRGM and actin. (B) Levels of phospho-AMPK (Thr-172) and phospho-Beclin 1 (Ser-93/96) in lysates from HEK293T cells co-expressing Flag-Beclin 1 and GFP or GFP-IRGM. (C) Levels of active phospho-ULK1 (Ser-555 and Ser-317) in lysates of HEK293T cells co-expressing Myc-ULK1 and either GFP or GFP-IRGM. Numbers beneath bands in B, C, quantification of phosphorylated proteins relative to the total abundance of the same protein. (D) Co-immunoprecipitation (Co-IP) analysis of interaction between IRGM and endogenous ULK1 and AMBRA1 in HEK293T lysates of cells expressing GFP or GFP-IRGM. (E) Top, confocal microscopy images of HEK293T cells expressing IRGM-V5 and Myc-ULK1 subjected to starvation for 2 h. Arrowheads, co-localization. Bottom, fluorescence intensity line tracing. (F) Co-IP analysis in lysates of HEK293T cells expressing indicated proteins. (G) Confocal microscopy images of HEK293T cells transiently expressing V5-IRGM and Flag-Beclin1 subjected to starvation for 2 h. Details as for panel E. (H) Lysates of HEK293T cells expressing GFP or GFP-IRGM with Myc-ULK1 subjected to immunoprecipitation with anti-GFP and blots probed with phospho-ULK1 Ser-317 or Ser-757 antibodies. (I) Lysates of cells expressing Myc-ULK1, Flag-Beclin-1 and increasing concentrations of GFP-IRGM subjected to immunoprecipitation with anti-Flag; blots probed as indicated. (J) HEK293T cell lysates co-expressing GFP-IRGM and Flag-Beclin 1 subjected to Western blotting with antibody to phospho-Beclin 1 (Ser-15) and antibodies as indicated. (K) Co-IP analysis of Flag-IRGM and endogenous ATG14. (L, M) Mapping of Beclin 1 regions interacting with IRGM. (L) Lysates of HEK293T cells co-expressing GFP-IRGM and Flag-Beclin 1 variants in panel M were subjected to immunoprecipitation with anti-Flag and blots probed as indicated. (M) Beclin 1 domain organization indicating its interacting proteins along with deletion constructs used in Co-IP analysis in panel L. (N) Co-IP analysis of the effects of IRGM overexpression on the interaction of Beclin 1 with its regulatory proteins. Lysates of HEK293T cells co-expressing GFP-IRGM and Flag-Beclin 1 were subjected to immunoprecipitation with anti-Flag and blots probed as indicated. (O) Model of IRGM-dependent autophagy induction based on the results obtained in Figure 1 and Figure S1. See also Figure S1.

Figure 2. IRGM is required for stable levels of the autophagy initiation proteins

(A,C,E) U937 cells transfected with control or IRGM siRNAs, untreated or treated with LPS (500 ng/ml for 4 h) were lysed and subjected to Western blotting with antibody to (A) ULK1, (C) ATG14L, AMBRA1 and ATG5, and (E) ATG16L1. IRGM knock down efficiency and quantifications are shown in Supplementary Figure S2A,B. (B,D,F) Left, confocal images of U937 cells transfected with control or IRGM siRNA treated with LPS (500 ng/ml for 4 h), Immunofluorescence analysis was performed with (B) phopho-ULK1 (Ser-317), (D) ATG5, and (F) ATG16L1. Graphs, means ± SD (corrected total cell fluorescence of cells; > 30 cells from 5 fields measured using Image J). *, p < 0.05 (Student’s unpaired t test). (G) Lysates from HEK293T cells expressing GFP or GFP-IRGM were subjected to immunoprecipitation with anti-GFP and blot probed with indicated antibodies. (H) Schematic of ATG16L1 domain structure indicating IRGM interacting regions mapped in panels I. (I) Lysates of HEK293T cells co-expressing GFP-IRGM and the indicated Flag-ATG16L1 variants in panel H were subjected to immunoprecipitation with anti-Flag and blots probed as indicated. Results, representative of three independent experiments. See also Figure S2.

Figure 3. IRGM is required for PAMPs induced autophagy

(A) Abundance of IRGM mRNA (relative to GAPDH) in THP-1 cells (control or infected with invasive E. coli LF82) determined by quantitative real-time PCR (qRT-PCR). (B) Effect of LPS (30 min) or (C) MDP exposure (16 h) on IRGM mRNA levels in U937 cells. Gene expression (qRT-PCR) was normalized relative to GAPDH. Data, means ± SD (n>3); *, p< 0.05 (Student’s unpaired t test). (D) Schematic summary of the physiological signals activating IRGM expression based on data in panels A–C and in Figure S3A–H. (E, F) Left, Western blot analysis of LC3-II abundance in U937 cells transfected with control or IRGM siRNA: (E) treated or not with LPS (500 ng/ml; 4 h); (F) treated or not with MDP (5 µg/ml for 8 h). Right, densitometric analysis of Western blots using ImageJ software. (G, H) Left, confocal images of LC3 puncta in LPS treated (500 ng/ml; 4 h) (G) or MDP-treated (5 µg/ml; 8 h), (H) U937cells transfected with control or IRGM siRNA. Graphs (right of panels G and H), represent mean corrected total cell fluorescence ± SE (25–35 cells from 10–15 fields measured using ImageJ. *, p<0.05 (ANOVA). (I) Analysis of endogenous interactions (Co-IP) using THP-1 lysates infected with invasive E. coli LF82 (1 h) or stimulated with LPS (2 µg/ml, 2 h) or MDP (10 µg/ml, 2 h). Lysates were subjected to immunoprecipitation with IRGM antibody or control IgG and probed as indicated. (J) Schematic summary of the results obtained in Figure 3E–I. See also Figure S3.

Figure 4. IRGM interacts and co-localizes with ATG16L1 and NOD2

(A, B) Co-IP analysis of endogenous (A) or overexpressed (B) IRGM, with NOD2 and ATG16L1 in (A) starved HT29 cells and (B) HEK293T cells. (C) Top, confocal microscopy images of HEK293T cells transiently expressing GFP-IRGM and Flag-NOD2. Bottom, fluorescence intensity line tracing corresponding to dashed line. (D) Schematic of NOD2 domain organization along with deletion constructs used in Co-IP analysis in panel E. (E) Left panel, lysates of HEK293T cells coexpressing GFP-IRGM and the Flag-NOD2 variants shown in panel D subjected to immunoprecipitation with anti-Flag and blot probed with antibodies as indicated. Right panel, densitometric analysis of Western blots (IP blot/Input blot). (F) Flag tag pull-down assays performed with affinity purified NOD2 variants from 293T cell lysates and purified recombinant GST-IRGM shown in the schematic (left panel). (G) Top, confocal microscopy images showing co-localization of GFP-IRGM and Flag-NOD2 and Rhodamine-MDP in HEK293T cells. Bottom, fluorescence intensity line tracing corresponding to red line. (H) Effect of MDP (10 µg/ml, 8 h) on GFP-IRGM and Flag-NOD2 interactions in HCT116 cells. (I) Model of IRGM-NOD2 interactions. See also Figure S4.

Figure 5. NOD2 promotes K63-linked polyubiquitination of IRGM, enhancing its interactions with autophagy initiation factors

(A–C) Effects of NOD2 expression on IRGM self-association (A), and IRGM’s interaction with Beclin 1 (B) or with ULK1 (C) in HEK293T cells. (D, E) Analysis of IRGM ubiquitination in HEK293T cells. Cells co-expressing GFP or GFP-IRGM and (D) HA-tagged Ubiquitin C or (E) HA-tagged Ubiquitin C mutated for all lysines except lysine 48 (HA-K48) or Lysine 63 (HA-K63) and Flag-NOD2 were subjected to immunoprecipitation with GFP antibody and blots probed with indicated antibodies. Blot in (E) was processed to remove irrelevant lanes (dashed vertical line). (F) Cells co-expressing GFP-IRGM, HA-K63 and Flag-NOD2 deletion variants as in Figure 4D were subjected to immunoprecipitation analysis with anti-GFP and blot probed with indicated antibodies. (G) Cells co-expressing GFP or GFP-IRGM or GFP-IRGM-K^mut (IRGM variant with all lysine residues mutated to alanine) and HA-K63 were subjected to immunoprecipitation analysis with anti-GFP and blot was probed with indicated antibodies. Blot was processed (dashed vertical line) to remove irrelevant lanes. (H) Lysates of cells co-expressing GFP or GFP-IRGM or GFP-IRGM-K^mut and Flag-IRGM were subjected to immunoprecipitation with anti-GFP and blot probed with indicated antibodies. (I) Lysates of cells expressing GFP or GFP-IRGM or GFP-IRGM-K^mut were subjected to immunoprecipitation with anti-GFP and blots probed with indicated antibodies. Results representative of three independent experiments. See also Figure S5.

Figure 6. Ubiquitination of IRGM is required for NOD2 degradation and ULK1 stability

(A) Effects of IRGM expression on NOD2 levels in transfected HEK293T cells. Data, means ± SE; *, p< 0.05 (Student’s unpaired t test). (B) Lysates of HEK293T cell co-expressing GFP or GFP-IRGM and Flag-NOD2, untreated/treated with Bafilomycin A1 (100 nM for 8 h) were subjected to Western blotting. (C) Lysates of cells co-expressing Flag-NOD2 and GFP, GFP-IRGM, or GFP-IRGM-K^mut were subjected to Western blotting. (D, E) Lysates from HEK293T cells co-expressing Myc-ULK1 and either GFP or increasing amounts of GFP-IRGM were subjected to Western blotting as in (D) with the relative abundance of Myc-ULK1 shown in (E). Blot was processed (dashed vertical line) to remove irrelevant lanes. (F) HEK293T cells transfected with plasmids encoding GFP, GFP-IRGM, or GFP-IRGM-K^mut and either Myc-ULK1 or Flag-Beclin 1 were lysed and subjected to Western blotting. Data from densitometric analyses of Western blots (B, C, E), means ± SE, n=3 *, p< 0.05 (ANOVA). (G) Depiction of the role of IRGM ubiquitination in NOD2 degradation and ULK1 stabilization. See also Figure S6.

Figure 7. Ubiquitination of IRGM is important for preventing inflammation

(A) Effect of IRGM (WT and K^mut) expression with and without NOD2 on the nuclear localization of NF-κB-p65 in HeLa cells upon E. coli LF82 infection. (B) Graph, mean % cells with NFκB-p65 nuclear localization (from 10 microscopic fields) ± SD; *, p< 0.05 (ANOVA). (C) Effect of E. coli infection on IL-1β mRNA expression in THP-1 cells subjected to IRGM knockdown (qRT-PCR normalized to GAPDH). Data, means ± SD (n>3); *, p< 0.05 (ANOVA). (D, E, F) Lysates of cells co-expressing either GFP or GFP-IRGM and (D) Flag-NOD1, (E) Flag-Rig-I, or (F) Flag-TLR3, subjected to immunoprecipitation with anti-GFP (D, E) or anti-Flag (F); blots were probed with indicated antibodies. (G) Effect of FLAG-tagged NOD1, RIG-I, or TLR3 expression on IRGM ubiquitination (K63-linked) in HEK293T cells. (H) Model of IRGM-mediated xenophagy. IRGM expression is induced by physiological cues including starvation, microbes, or microbial products (PAMPs). IRGM protein increases the abundance of active AMPK, which subsequently promotes autophagy by activating ULK1 and Beclin 1. Not only does IRGM amplify this fundamental autophagy signaling but it also assembles the core autophagy machinery. Association of IRGM with NOD2, which is enhanced in the presence of MDP, promotes IRGM ubiquitination and the assembly of autophagy initiation factors. Together, these molecular events promote antimicrobial autophagy and suppress excessive inflammatory responses. See also Figure S7.