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Airway Inflammation Via Ubch7 Ndfip1 Regulates Itch Ligase Activity

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Airway Inflammation Via Ubch7 Ndfip1 Regulates Itch Ligase Activity The Journal of Immunology Ndfip1 Regulates Itch Ligase Activity and Airway Inflammation via UbcH7 Mahesh Kathania,* Minghui Zeng,* Viveka Nand Yadav,† Seyed Javad Moghaddam,‡ Baoli Yang,x and K Venuprasad* The ubiquitin-ligating enzyme (E3) Itch plays a crucial role in the regulation of inflammation, and Itch deficiency leads to severe airway inflammation. However, the molecular mechanisms by which Itch function is regulated remain elusive. In this study, we found that nontypeable Haemophilus influenzae induces the association of Itch with Ndfip1. Both Itch2/2 and Ndfip12/2 mice exhibited severe airway inflammation in response to nontypeable Haemophilus influenza, which was associated with elevated expression of proinflammatory cytokines. Ndfip1 enhanced Itch ligase activity and facilitated Itch-mediated Tak1 ubiquitination. Mechanistically, Ndfip1 facilitated recruitment of ubiquitin-conjugating enzyme (E2) UbcH7 to Itch. The N-terminal region of Ndfip1 binds to UbcH7, whereas the PY motif binds to Itch. Hence, Ndfip1 acts as an adaptor for UbcH7 and Itch. Reconstitution of full-length Ndfip1 but not the mutants that fail to interact with either UbcH7 or Itch, restored the defect in Tak1 ubiquitination and inhibited elevated proinflammatory cytokine expression by Ndfip12/2 cells. These results provide new mechanistic insights into how Itch function is regulated during inflammatory signaling, which could be exploited therapeutically in inflammatory diseases. The Journal of Immunology, 2015, 194: 2160–2167. nflammation is essential for host defense against invading valent attachment of Ub to a conserved cysteine residue in the pathogens, such as viruses and bacteria (1). The inflammatory HECT domain, and this Ub is transferred to the target protein (5). I response must be resolved after the pathogens are cleared, Itch belongs to the HECT family of E3 ligases, which contains because unchecked inflammation can cause host tissue damage, a PKC-related C2 domain, four WW domains, and the HECT ligase leading to pathological conditions, such as autoimmunity and ma- domain. Deficiency of Itch in mice results in severe multiorgan lignancy (2). However, the mechanism that controls resolution of inflammatory disease, including chronic pulmonary interstitial in- the inflammatory response is incompletely understood. flammation and alveolar proteinosis (6, 7). Further, a truncated Ubiquitin (Ub)-mediated degradation of signaling intermediates human Itch mutation results in severe lung inflammation in a group is an important means of termination of the inflammatory response of pediatric patients (8). (3). Ubiquitination is achieved via an enzyme cascade consisting of Originally, Ndfip1 was identified as a Nedd4-interacting protein (9). ubiquitin-activating, ubiquitin-conjugating (E2), and ubiquitin-li- It is composed of three highly conserved transmembrane domains and gating (E3) enzymes (4, 5). Ub is activated by the Ub-activating cytoplasmic PY motifs, and it is localized to the Golgi, endosomes, enzymes and is transferred to E2s. E2s, in turn, interact with E3s, and multivesicular bodies (10). Ndfip1 also was shown to interact which mediate conjugation of Ub to target proteins. There are two with Itch. Mice that are deficient in Ndfip1 develop inflammation in classes of E3 ligases: the RING domain class and the HECT domain the skin and lungs similar to Itch2/2 mice and die prematurely (11). class. RING domain E3s facilitate the direct transfer of Ub from E2 Nontypeable Haemophilus influenzae (NTHi) is a major cause to a substrate without the formation of a transient covalent E3-Ub of respiratory infections and is the most common colonizer of thioester intermediate. The HECT domain-containing E3 enzymes airways in patients with chronic obstructive pulmonary disease, act as intermediate acceptors of Ub. The catalytic activity of the leading to its exacerbation (12). NTHi was shown to induce an HECT domain ubiquitinates the target protein and catalyzes for- NF-kB–mediated inflammatory response through activation of mation of polyubiquitin chains. The E2s catalyze a thioester co- the kinase Tak1 (13). In this article, we demonstrate that Ndfip1 regulates Itch ligase activity toward Tak1 by promoting the binding *Baylor Institute for Immunology Research, Baylor Research Institute, Dallas, TX of the E2 UbcH7 and inhibits NTHi-induced airway inflammation. 75204; †Department of Pathology, University of Michigan, Ann Arbor, MI 48109; ‡Pulmonary Medicine, The University of Texas MD Anderson Cancer Center, Hous- ton, TX 77030; and xDepartment of Obstetrics and Gynecology, Carver College of Materials and Methods Medicine, University of Iowa, Iowa City, IA 52242 Mice Received for publication October 29, 2014. Accepted for publication December 29, 2/2 2/2 2014. Itch and Ndfip1 mice were described previously (11, 14). C57BL/6 mice were purchased from Charles River Laboratory. All mice were This work was supported by an American Cancer Society Research Scholar grant housed in microisolator cages in the barrier facility of the Baylor Institute (122713-RSG-12-260-01-LIB) and a Baylor Charles A. Sammons pilot project (to KV.). for Immunology Research. All experiments were performed in accordance Address correspondence and reprint requests to Dr. K Venuprasad, Baylor Institute with the guidelines of the Institutional Animal Care and Use Committee for Immunology Research, Baylor Research Institute, 3410 Worth Street, Suite 600, of Baylor Research Institute. Dallas, TX 75204. E-mail address: [email protected] The online version of this article contains supplemental material. Plasmid construction and cell transfection Abbreviations used in this article: BMDM, bone marrow–derived macrophage; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-ligating enzyme; HA, hemagglutinin; Flag-Itch, Flag-Tak1, hemagglutinin (HA)-Ub (wild-type [WT]), HX-Itch NTHi, nontypeable Haemophilus influenzae; Ub, ubiquitin; WT, wild-type. (C830A), and Myc-Ndfip1 were described previously (14, 15). HA-Ube2L3 (UbcH7) was obtained from Addgene (cat. no. 27561). Myc–Ndfip1-DN, Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 Myc–Ndfip1-DM, and Myc–Ndfip1-DC were created from Myc-Ndfip1 by www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402742 The Journal of Immunology 2161 subcloning into pcDNA3.1/myc-His (-) A (Invitrogen) between the XhoI and Lentiviral transduction HindIII restriction sites. Flag–Ndfip1-D1–41 and Flag–Ndfip1-DPY were created from Myc-Ndfip1 by subcloning into pCMVtag2B between HindIII Lentivirus transfection was performed as described earlier (14). Expression and XhoI sites. All clone sequences were confirmed. Transient transfection of clones were created by Gateway cloning technology, according to the manu- 293T cells was performed using Lipofectamine 2000 (Invitrogen), according facturer’s instructions (Invitrogen). The full-length Ndfip1 expression clones to the manufacturer’s instructions (14). were constructed first by PCR subcloning of Ndfip1 into the pENTR-3C entry vector (Invitrogen) between EcoRI and XhoI, followed by recombination by Protein identification by liquid chromatography-tandem mass Gateway LR cloning into pLenti6.2/N-Lumio/V5-DEST (Invitrogen). Ex- D D spectrometry pression clones for Ndfip1- 1–41 and Ndfip1- PY deletion mutants were similarly prepared first by PCR subcloning into pENTR-3C entry vector Immunoprecipitated proteins were separated by SDS-PAGE. In-gel di- (Invitrogen), followed by recombination by LR into pLenti6.2/N-Lumio/V5- gestion with trypsin, followed by protein identification using liquid DEST (Invitrogen). The ViraPower lentiviral expression system (Invitrogen) chromatography-tandem mass spectrometry, was performed as described was used for lentiviral transduction of bone marrow–derived macrophages elsewhere (16). Briefly, tryptic peptides were resolved on a nano-LC (BMDMs). First, 293FT cells were used for packaging and production of column (Magic AQ C18; Michrom Bioresources) and introduced into an viruses. BMDMs were incubated with the virus-containing supernatants Orbitrap mass spectrometer (Thermo Scientific). The Orbitrap was set to overnight at 37˚C in a 5% CO2 incubator. collect a high-resolution MS1 (FWHM 30,000@400 m/z), followed by NTHi lysate preparation and exposure data-dependent collision-induced dissociation spectra on the “top 9” ions in the linear ion trap. Spectra were searched against a human protein da- NTHi lysates were prepared as described earlier (18). In brief, bacteria were tabase (UniProt release 2011_05) using the X!Tandem/TPP software suite grown on chocolate agar plates at 37˚C in the presence of 5% CO2 and (17). Proteins identified with a ProteinProphet probability $ 0.9 false inoculated in brain heart infusion broth supplemented with 3.5 mg/ml NAD discovery rate , 2% were considered for further analysis. and hemin. The bacterial lysate was prepared by sonication. The final protein concentration was adjusted to 2.5 mg/ml in PBS, and aliquots were Abs and reagents frozen at 280˚C. Mouse inhalation of NTHi lysate was performed with an The following Abs were used in this study: anti–c-Myc (sc-40; Santa Cruz), anti- Aeromist CA-209 nebulizer (CIS-US, Bedford, MA). In brief, a thawed Flag (F1804, F7425; Sigma), anti-Itch (611198; BD), anti–b-actin (A5441; NTHi lysate was placed in the nebulizer driven by 10 l/min of room air Sigma), anti-Ub (sc-8017; Santa Cruz), anti-Xpress (R910-25; Invitrogen), anti- supplemented with 5% CO2 for 20 min. HA (sc-805; Santa Cruz), anti-UBE2G2 (sc-100613; Santa Cruz), anti-V5
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