Volume 52, Issue 6, Pages 794-804 (December 2013) Endogenous Nitrated Nucleotide Is a Key Mediator of Autophagy and Innate Defense against Bacteria  Chiaki Ito, Yohei Saito, Takashi Nozawa, Shigemoto Fujii, Tomohiro Sawa, Hirofumi Inoue, Tetsuro Matsunaga, Shahzada Khan, Soichiro Akashi, Ryota Hashimoto, Chihiro Aikawa, Eriko Takahashi, Hiroshi Sagara, Masaaki Komatsu, Keiji Tanaka, Takaaki Akaike, Ichiro Nakagawa, Hirokazu Arimoto  Molecular Cell  Volume 52, Issue 6, Pages 794-804 (December 2013) DOI: 10.1016/j.molcel.2013.10.024 Copyright © 2013 Elsevier Inc. Terms and Conditions

Molecular Cell 2013 52, 794-804DOI: (10.1016/j.molcel.2013.10.024) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 Induction of Autophagy by 8-Nitro-cGMP (A) Chemical structure of 8-nitro-cGMP. (B) Modification of a cysteine residue by 8-nitro-cGMP (protein S-guanylation). (C) Immunocytochemical analysis of autophagosomes in RAW 264.7 cells with 8-bromo-cGMP (100 μM), 8-nitro-cGMP (100 μM), and rapamycin (200 nM) treatments for 24 hr. The cells were stained with DAPI (blue) and LC3 antibody (green). Scale bar, 10 μm. (D) Average number of autophagosomes per cell subjected to immunocytochemical staining shown in (C). At least 100 cells were counted in each experiment. Data are presented as the means ± SD for three independent experiments. ∗∗∗p < 0.001. (E) Immunoblotting analysis showing LC3-II accumulation in RAW 264.7 cells treated with 8-nitro-cGMP (100 μM, 24 hr) with (+) or without (−) 200 nM bafilomycin A1 for the last 2 hr. Relative band intensity of LC3-II versus actin is given at the bottom. LC3-II increased in bafilomycin A1-treated cells upon addition of 8-nitro-cGMP. (F) Autophagosome formation in wild-type (Atg5+/+) and Atg5-deficient (Atg5−/−) MEFs with or without 8-nitro-cGMP (100 μM, 24 hr). The cells were stained with DAPI (blue) and LC3 antibody (green). Scale bar, 10 μm. (G) Average number of autophagosomes per cell subjected to immunocytochemical staining shown in (F). At least 100 cells were counted in each experiment. Data are presented as the means ± SEM for five independent experiments. ∗∗∗p < 0.001. See also Figure S1. Molecular Cell 2013 52, 794-804DOI: (10.1016/j.molcel.2013.10.024) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Effects of 8-Nitro-cGMP, NOS Inhibitor L-NMMA, NaHS, and Streptolysin O Knockout on GAS Viability and Formation of GAS-Containing Autophagosome-like Vacuoles in RAW 264.7 Cells (A) Viability of intracellular GAS at 6 hr after infection in RAW 264.7 cells treated with 8-nitro-cGMP (100 μM) in the presence or absence of the lysosomal enzyme inhibitors E64d (10 μM) and leupeptin (10 μg/ml). Data are presented as the means ± SD for three independent experiments. ∗∗∗p < 0.001; NS, not significant. (B) Viability of intracellular SLO-deficient (ΔSLO) GAS mutant at 6 hr after infection in RAW 264.7 cells treated with 8-nitro-cGMP (100 μM). Data are presented as the means ± SD for three independent experiments. NS, not significant. (C) Electron micrographs of intracellular GAS surrounded by a double membrane (GcAV) in RAW 264.7 cells at 2 hr after infection in the presence of 8-nitro-cGMP (100 μM). Arrowheads indicate double-membrane-bound compartments. Scale bar, 1 μm. (D) Viability of intracellular GAS at 6 hr after infection in RAW 264.7 cells treated with L-NMMA (10 mM) or NaHS (1 mM). Data are presented as the means ± SD for three independent experiments. ∗∗∗p < 0.001. (E and F) Levels of GcAV-containing RAW 264.7 cells. RAW 264.7 cells expressing EmGFP-LC3 were infected with GAS in the presence or absence of 8-nitro-cGMP (100 μM), L-NMMA (10 mM), and NaHS (1 mM). After infection, the cells were stained with DAPI to visualize GAS. (E) Percentage of cells containing GcAVs. The number of cells containing GcAVs is counted and presented as the percentage of the total number of GAS-infected cells. At least 100 GAS-infected cells were counted in each experiment. Data are presented as the means ± SD for three independent experiments. ∗∗p < 0.01; ∗∗∗p < 0.001; NS, not significant. (F) Confocal micrographs of RAW 264.7 cells at 2 hr after infection. Arrowheads show intracellular GcAVs. Scale bar, 10 μm. See also Figure S2. Molecular Cell 2013 52, 794-804DOI: (10.1016/j.molcel.2013.10.024) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 Modification of GAS Surface by Endogenous 8-Nitro-cGMP in Autophagic Vacuoles in RAW 264.7 and Peritoneal Macrophage Cells (A) RAW 264.7 cells infected with GAS were stained with DAPI (blue), LC3 antibody (green), and RS-cGMP antibody that recognizes protein S-guanylation (red). The cells were observed 2 hr after GAS infection. DAPI was used to visualize GAS along with nuclei. Scale bar, 10 μm. (B) Intensity of fluorescence signals along the white line in the frame of the merged image in (A). (C) Percentage of GAS cells modified at the surface by 8-nitro-cGMP (S-guanylation) in RAW 264.7 cells containing GcAVs. At least 200 GAS cells were counted in each experiment. Data are presented as the means ± SD for three independent experiments. (D) Peritoneal macrophages infected with GAS were stained with DAPI (blue), LC3 antibody (green), and RS-cGMP antibody that recognizes protein S-guanylation (red). The cells were observed 2 hr after GAS infection. DAPI was used to visualize GAS along with nuclei. Scale bar, 10 μm. (E) Percentage of S-guanylation-positive GAS at 2 hr after infection in peritoneal macrophages containing GcAVs. At least 200 GAS cells were counted in each experiment. Data are presented as the means ± SD for three independent experiments. ∗∗∗p < 0.001. See also Figure S3. Molecular Cell 2013 52, 794-804DOI: (10.1016/j.molcel.2013.10.024) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 Lys63-Linked Polyubiquitination Is Essential for 8-Nitro-cGMP-Induced Autophagy (A) Immunocytochemical analysis of autophagosomes in A549 cells transfected with various ubiquitin-RFP fusion constructs in the presence of bafilomycin A1 (200 nM). The cells were treated or untreated with 8-nitro-cGMP (100 μM, 24 hr). Autophagy was not induced in K63R and K48 mutants, where Lys63 was replaced with arginine. At least 100 cells were counted in each experiment. Data are presented as the means ± SD for three independent experiments. (B) Immunoblotting analysis of Lys63- and Lys48-linked polyubiquitinated proteins in RAW 264.7 cells treated (right panel) or not (left panel) with bafilomycin A1 (200 nM). Culture medium was exchanged at 0 hr with new medium with or without 8-nitro-cGMP (100 μM) and incubated for the indicated period. Bafilomycin was added to the media in the last 2 hr of the incubation period. Over time, 8-nitro-cGMP decreased Lys63-linked polyubiquitination level, but not Lys48-linked polyubiquitination level (left). Bafilomycin treatment caused recovery of Lys63-linked polyubiquitination level in 8-nitro-cGMP-treated cells (right). (C) Immunofluorescence staining of Lys63-linked polyubiquitin (red) and its colocalization with LC3 puncta (green) in EGFP-LC3 expressing RAW 264.7 cells. The cells were treated or not with 8-nitro-cGMP (100 μM, 24 hr). Scale bar, 5 μm. (D) Average number of LC3 puncta colocalized with Lys63-linked polyubiquitin in EGFP-LC3 expressing RAW 264.7 cells treated or not with 8-nitro-cGMP (100 μM, 24 hr). At least 50 cells were counted in each experiment. Data are presented as the means ± SD for three independent experiments. ∗∗∗p < 0.001. Molecular Cell 2013 52, 794-804DOI: (10.1016/j.molcel.2013.10.024) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 Polyubiquitination of S-Guanylated GAS Surface (A) Confocal micrographs of RAW 264.7 cells at 3 hr after infection with GAS. The cells were stained with DAPI (blue), multiubiquitination antibody (FK2, green), and RS-cGMP antibody (red). Scale bar, 5 μm. (B) Percentage of GAS-infected RAW 264.7 cells containing S-guanylated and ubiquitinated GAS in the presence or absence of NaHS (1 mM). Ubiquitination was detected with a multiubiquitination-recognizing antibody (FK2). At least 200 GAS-infected cells were counted in each experiment. Data are presented as the means ± SD for three independent experiments. (C) Confocal micrographs of GAS-infected RAW 264.7 cells at 3 hr after infection. The cells were stained with DAPI (blue), Lys63-linked polyubiquitin antibody (green), and RS-cGMP antibody (red). Scale bar, 5 μm. (D) Percentage of GAS-infected RAW 264.7 cells containing Lys63-linked polyubiquitination-positive GAS in the presence or absence of NaHS (1 mM). At least 200 GAS-infected cells were counted in each experiment. Data are presented as the means ± SD for three independent experiments. (E) Percentage of Lys63-linked polyubiquitination in S-guanylated (S-guanylation-positive) or unmodified (S-guanylation-negative) GAS in RAW 264.7 cells at 3 hr after infection. At least 200 GAS cells were counted in each experiment. Data are presented as the means ± SD for three independent experiments. ∗∗∗p < 0.001. See also Figure S4. Molecular Cell 2013 52, 794-804DOI: (10.1016/j.molcel.2013.10.024) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 6 Proteomic Analysis of GAS-Infected RAW 264.7 Cells (A) 2D western blot analysis of S-guanylated proteins using RS-cGMP antibody. (B) LC-MS analysis of S-guanylated protein spots excised from 2D gel. See the Supplemental Experimental Procedures for details. Molecular Cell 2013 52, 794-804DOI: (10.1016/j.molcel.2013.10.024) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 7 S-Guanylation Promotes Ubiquitination of GAS Surface After GAS escape from phagosome into the cytosol, their surface is modified by 8-nitro-cGMP via S-guanylation, which promotes subsequent Lys63-linked polyubiquitination. The ubiquitin chain is a known molecular tag for selective transport to autophagosomes. See also Figure S5. Molecular Cell 2013 52, 794-804DOI: (10.1016/j.molcel.2013.10.024) Copyright © 2013 Elsevier Inc. Terms and Conditions