Volume 50, Issue 6, Pages 818-830 (June 2013) OTULIN Restricts Met1-Linked Ubiquitination to Control Innate Immune Signaling  Berthe Katrine Fiil, Rune Busk Damgaard, Sebastian Alexander Wagner, Kirstin Keusekotten, Melanie Fritsch, Simon Bekker-Jensen, Niels Mailand, Chunaram Choudhary, David Komander, Mads Gyrd-Hansen  Molecular Cell  Volume 50, Issue 6, Pages 818-830 (June 2013) DOI: 10.1016/j.molcel.2013.06.004 Copyright © 2013 Elsevier Inc. Terms and Conditions

Molecular Cell 2013 50, 818-830DOI: (10.1016/j.molcel.2013.06.004) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 OTULIN Regulates Signaling in Response to NOD2 Stimulation (A) Schematic depiction of human OTULIN. Circles illustrate residues required for ubiquitin binding, and stars denote residues required for cleavage of Met1-Ub. (B) NF-κB activity in HEK293T cell lysates transfected with HA-NOD2 alone or with OTULINWT or the indicated point mutants. (C) Immunofluorescence analysis of nuclear translocation of the NF-κB subunit RelA/p65 (green) in response to L18-MDP stimulation (1 μg/ml) in U2OS/NOD2 cells transfected with empty vector (pcDNA3-3xHA; HA-vector), OTULINWT or OTULINWA/CA. Scale bar, 10 μm. (D) Quantification of nuclear NF-κB translocation after L18-MDP stimulation of cells treated as in (C). (E) Relative levels of TNF, IL8, and IL6 transcripts measured by qRT-PCR on cDNA from U2OS/NOD2 control and OTULIN-depleted cells treated with L18-MDP (200 ng/ml). Immunoblot of OTULIN levels in control (MM) and siOTULIN-treated cells. (F) Immunoblotting of IκBα degradation and phosphorylation of signaling components in response to stimulation with L18-MDP (200 ng/ml) in control and OTULIN-depleted cells. Data in (B), (D), and (E) represent the mean ±SEM of at least three independent experiments, each performed in duplicate. In (D) at least 150 cells were counted per condition in each experiment. ∗∗p < 0.01. See also Figure S1. Molecular Cell 2013 50, 818-830DOI: (10.1016/j.molcel.2013.06.004) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 OTULIN Is Part of the NOD2 Receptor Complex and Antagonizes Met1-Ub-Dependent Signaling (A) Immunoprecipitation with anti-HA in U2OS/NOD2 cells. HA-NOD2 expression was induced for 24 hr with doxycycline (DOX). Immunoprecipitates were examined for copurification of OTULIN and known members of the NOD2 receptor complex. (B–D) NF-κB activity in HEK293T cell lysates transfected with luciferase reporters and OTULIN, LUBAC (HOIP, HOIL-1), DN-LUBAC (RING-mutated HOIP, HOIL-1), or XIAP as indicated. (E) Schematic depiction of the engineered AVPI-Ub4GS protein, which binds to XIAP BIR2 and BIR3 domains via an N-terminal IBM to activate NF-κB. DUB, deubiquitinase. (F and G) NF-κB activity in HEK293T cell lysates transfected luciferase reporters and XIAPF/A, AVPI-Ub4GS, DN-LUBAC, or OTULIN as indicated. Data in (B)–(D), (F), and (G) represent the mean ±SEM of at least three independent experiments (except “OTULIN” in B, where n = 2), each performed in duplicate. ∗∗p < 0.01. n.s., not significant. See also Figure S2. Molecular Cell 2013 50, 818-830DOI: (10.1016/j.molcel.2013.06.004) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 Proteome-wide Quantification of NOD2-Regulated Met1-Ub (A) Immunofluorescence analysis of nuclear translocation of the NF-κB subunit RelA/p65 (red) in response to L18-MDP stimulation (1 μg/ml) in U2OS/NOD2 cells transfected with GFP vector, GFP-M1-SUB, or a Ub-binding mutant GFP-M1-SUBmut. Scale bar, 10 μm. (B) Quantification of nuclear NF-κB translocation after L18-MDP stimulation of cells treated as in (A). (C) Purification of endogenous Ub conjugates using GST-coupled M1-SUB (Met1-Ub) or TUBE (all Ub-linkages). Pull-down with glutathione Sepharose beads on HEK293T cell lysates transfected with LUBAC (HOIP and HOIL) and the indicated OTULIN mutants. Purified material and lysate were examined by immunoblotting. (D) Outline of the SILAC-based proteomics approach for quantification of protein ubiquitination in response to NOD2 stimulation. (E) MS data from TUBE purification of ubiquitinated proteins from THP-1 cells treated or not with L18-MDP (200 ng/ml) for 60 min. The circle representing RIPK2 is marked in red. The mass spectrum shows the relative abundance of the RIPK2 peptide TQNILLDNEFHVK in L18-MDP-treated (SILAC heavy) cells compared with untreated (SILAC light). (F) Same as in (E) but after purification of ubiquitinated proteins with M1-SUB. RIPK2 is marked in red. (G) SILAC H/L ratios of proteins identified in (E) and (F) with reported function in the NOD2 signaling complex. NA, not available. (H) Time course analysis of RIPK2 ubiquitination with TUBE and M1-SUB in lysates of U2OS/NOD2 cells treated with L18-MDP (200 ng/ml). Purified material and lysate were examined by immunoblotting. Data in (B) represent the mean ±SEM of three independent experiments. At least 140 cells were counted per condition in each experiment. ∗p < 0.05. See also Figure S3 and Table S1. Molecular Cell 2013 50, 818-830DOI: (10.1016/j.molcel.2013.06.004) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 OTULIN Limits Met1-Linked Ubiquitination of RIPK2 after NOD2 Stimulation (A and B) Purification of endogenous Ub conjugates with M1-SUB from (A) OTULIN-depleted U2OS/NOD2 cells treated with either 200 ng/ml L18-MDP for 1 hr or 5 ng/ml L18-MDP for 2 hr. (B) OTULIN-overexpressing U2OS/NOD2 cells treated with L18-MDP (200 ng/ml) for the indicated times. Purified material and lysate from (A) and (B) were examined by immunoblotting. (C) Ub-chain restriction analysis of ubiquitinated RIPK2 isolated with M1-SUB from L18-MDP treated and siMM or siOTULIN-transfected cells. Purified Ub conjugates were incubated with the indicated deubiquitinases (DUBs) for 1 hr, and samples were examined by immunoblotting. (D) Coomassie staining of the recombinant DUBs used in (C). (E) Immunoprecipitation of endogenous NEMO from OTULIN-depleted cells treated with 200 ng/ml L18-MDP for 1 hr. Purified material and lysate were examined by immunoblotting. (F) Immunoprecipitation of endogenous NEMO from OTULIN-overexpressing cells treated with 200 ng/ml L18-MDP for 1 hr or 5 ng/ml L18-MDP for 2 hr. Purified material and lysate were examined by immunoblotting. Molecular Cell 2013 50, 818-830DOI: (10.1016/j.molcel.2013.06.004) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 OTULIN Regulates Early NOD2 Signaling and Sensitivity to L18-MDP (A) Relative levels of OTULIN transcripts measured by qRT-PCR with two different primer sets on cDNA from U2OS/NOD2 control cells treated with 200 ng/ml L18-MDP for the times indicated. (B) Purification of endogenous Ub conjugates with M1-SUB from OTULIN-depleted cells treated with L18-MDP for up to 4 hr. Purified material and lysate were examined by immunoblotting. (C) Immunoblotting of IκBα degradation and phosphorylation of signaling components in response to stimulation with 5 ng/ml of L18-MDP in control and OTULIN-depleted U2OS/NOD2 cells. (D) Relative levels of TNF, IL8, and IL6 transcripts measured by qRT-PCR on cDNA from U2OS/NOD2 control and OTULIN-depleted cells treated with 5 ng/ml of L18-MDP. Data in (A) and (D) represent the mean ±SEM of at least three independent experiments. See also Figure S4. Molecular Cell 2013 50, 818-830DOI: (10.1016/j.molcel.2013.06.004) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 6 Decreased OTULIN Function Leads to Promiscuous Met1-Ub Accumulation (A–C) Samples used in Figure 3C (A), Figure 4A (B), and Figure 4B (C) were immunoblotted for LUBAC components as indicated. (D) Purification of endogenous Ub conjugates with M1-SUB in U2OS/NOD2 control and OTULIN-depleted cells treated with TNF (10 ng/ml). Purified material and lysate were examined by immunoblotting. (E) TNF-RSC purification by FLAG-TNF (100 ng/ml) immunoprecipitation from TREx293 control or OTULIN-depleted cells at different times after FLAG-TNF stimulation. Purified material was analyzed by immunoblotting. See also Figure S5. Molecular Cell 2013 50, 818-830DOI: (10.1016/j.molcel.2013.06.004) Copyright © 2013 Elsevier Inc. Terms and Conditions