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Sequestosome-1/p62 Is the Key Intracellular Target of Innate Defense Regulator Peptide*

Open AccessPublished:October 22, 2009DOI:https://doi.org/10.1074/jbc.C109.073627
      Innate defense regulator-1 (IDR-1) is a synthetic peptide with no antimicrobial activity that enhances microbial infection control while suppressing inflammation. Previously, the effects of IDR-1 were postulated to impact several regulatory pathways including mitogen-activated protein kinase (MAPK) p38 and CCAAT-enhancer-binding protein, but how this was mediated was unknown. Using a combined stable isotope labeling by amino acids in cell culture-proteomics methodology, we identified the cytoplasmic scaffold protein p62 as the molecular target of IDR-1. Direct IDR-1 binding to p62 was confirmed by several biochemical binding experiments, and the p62 ZZ-type zinc finger domain was identified as the IDR-1 binding site. Co-immunoprecipitation analysis of p62 molecular complexes demonstrated that IDR-1 enhanced the tumor necrosis factor α-induced p62 receptor-interacting protein 1 (RIP1) complex formation but did not affect tumor necrosis factor α-induced p62-protein kinase ζ complex formation. In addition, IDR-1 induced p38 MAPK activity in a p62-dependent manner and increased CCAAT-enhancer-binding protein β activity, whereas NF-κB activity was unaffected. Collectively, these results demonstrate that IDR-1 binding to p62 specifically affects protein-protein interactions and subsequent downstream events. Our results implicate p62 in the molecular mechanisms governing innate immunity and identify p62 as a potential therapeutic target in both infectious and inflammatory diseases.

      Introduction

      Innate defense regulator-1 (IDR-1)
      The abbreviations used are: IDR
      innate defense regulator
      C/EBP
      CCAAT-enhancer-binding protein
      NF-κB
      nuclear factor κ-light-chain-enhancer of activated B cells
      PKC
      protein kinase C
      SILAC
      stable isotope labeling of amino acids in cell culture
      TRAF6
      tumor necrosis factor receptor-associated factor 6
      RIP1
      receptor-interacting protein 1
      XIAP-4
      X-linked inhibitor-of-apoptosis proteins
      ZZ
      ZZ-type zinc finger
      MAPK
      mitogen-activated protein kinase
      TNF
      tumor necrosis factor
      Sp
      specific
      nSP
      nonspecific.
      (KSRIVPAIPVSLL-NH2) is a synthetic peptide with no antimicrobial activity that enhances host bacterial infection control while suppressing harmful inflammation. Treatment of mice with IDR-1 provides protection from otherwise lethal infections with Gram-positive and Gram-negative pathogens, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enteroccocus, and Salmonella enterica (
      • Scott M.G.
      • Dullaghan E.
      • Mookherjee N.
      • Glavas N.
      • Waldbrook M.
      • Thompson A.
      • Wang A.
      • Lee K.
      • Doria S.
      • Hamill P.
      • Yu J.J.
      • Li Y.
      • Donini O.
      • Guarna M.M.
      • Finlay B.B.
      • North J.R.
      • Hancock R.E.
      ). In addition, IDR-1 enhances production of some monocyte-produced chemokines, including MCP-1 and RANTES (regulated on activation normal T cell expressed and secreted) and the anti-inflammatory cytokine interleukin-10. IDR-1 also suppresses production of Toll-like receptor-induced proinflammatory cytokines, including interleukin-6 and TNFα (
      • Scott M.G.
      • Dullaghan E.
      • Mookherjee N.
      • Glavas N.
      • Waldbrook M.
      • Thompson A.
      • Wang A.
      • Lee K.
      • Doria S.
      • Hamill P.
      • Yu J.J.
      • Li Y.
      • Donini O.
      • Guarna M.M.
      • Finlay B.B.
      • North J.R.
      • Hancock R.E.
      ). Previously, the effects of IDR-1 were postulated to impact several intracellular pathways, including MAPK p38 and C/EBP, but the preceding molecular events remained unknown.
      In this study, the cytoplasmic protein sequestosome-1 (p62) was identified as a molecular target of IDR-1. p62 is a multidomain scaffold (adaptor) protein, with many known interacting partners, including PKCζ (
      • Moscat J.
      • Diaz-Meco M.T.
      • Albert A.
      • Campuzano S.
      ,
      • Puls A.
      • Schmidt S.
      • Grawe F.
      • Stabel S.
      ), p38 (
      • Kawai K.
      • Saito A.
      • Sudo T.
      • Osada H.
      ), RIP1 (
      • Sanz L.
      • Sanchez P.
      • Lallena M.J.
      • Diaz-Meco M.T.
      • Moscat J.
      ), and TRAF6 (
      • Sanz L.
      • Diaz-Meco M.T.
      • Nakano H.
      • Moscat J.
      ). p62 comprises an N-terminal PB1 domain that is primarily important for atypical PKC binding (
      • Moscat J.
      • Diaz-Meco M.T.
      • Albert A.
      • Campuzano S.
      ), a ZZ-type zinc finger (ZZ) domain that interacts with RIP1 (
      • Sanz L.
      • Sanchez P.
      • Lallena M.J.
      • Diaz-Meco M.T.
      • Moscat J.
      ), and a TRAF6 binding sequence domain recognized by TRAF6 (
      • Sanz L.
      • Diaz-Meco M.T.
      • Nakano H.
      • Moscat J.
      ). Additionally, a C-terminal ubiquitin-associated domain binds to polyubiquitin (
      • Vadlamudi R.K.
      • Joung I.
      • Strominger J.L.
      • Shin J.
      ), a function recently demonstrated to facilitate the efficient activation of prosurvival and proapoptotic pathways by binding polyubiquitinated signaling proteins (
      • Jin Z.
      • Li Y.
      • Pitti R.
      • Lawrence D.
      • Pham V.C.
      • Lill J.R.
      • Ashkenazi A.
      ). The ubiquitin-associated domain is also considered the basis for the association between p62 and protein trafficking to the proteasome (
      • Seibenhener M.L.
      • Geetha T.
      • Wooten M.W.
      ,
      • Moscat J.
      • Diaz-Meco M.T.
      • Wooten M.W.
      ). Thus, p62 functions as a nodal point in cellular signaling pathways, particularly in the regulation of NF-κB (
      • Duran A.
      • Linares J.F.
      • Galvez A.S.
      • Wikenheiser K.
      • Flores J.M.
      • Diaz-Meco M.T.
      • Moscat J.
      ,
      • Durán A.
      • Serrano M.
      • Leitges M.
      • Flores J.M.
      • Picard S.
      • Brown J.P.
      • Moscat J.
      • Diaz-Meco M.T.
      ) and cellular differentiation (
      • Rodriguez A.
      • Durán A.
      • Selloum M.
      • Champy M.F.
      • Diez-Guerra F.J.
      • Flores J.M.
      • Serrano M.
      • Auwerx J.
      • Diaz-Meco M.T.
      • Moscat J.
      ).
      Here we provide evidence that IDR-1 specifically binds to the ZZ domain of p62. This binding event selectively stabilized TNFα-induced p62-RIP1 complex formation, but not TNFα-induced p62-PKCξ complex formation, and specifically modulated the downstream signaling pathways by activating MAPK p38 and C/EBPβ but not NF-κB. These studies provide new evidence for p62 as an important component of the immune system and demonstrate that IDR-1 can be used for the interrogation of the molecular events governing these innate responses.

      EXPERIMENTAL PROCEDURES

      Experimental Procedures are detailed in the supplemental material.

      DISCUSSION

      The intracellular signaling network in host cells allows for efficient communication between the sensing of an incoming pathogen and the initiation of anti-infective responses. The p62 protein is emerging as one of the central signaling hubs of the cell, controlling this process (
      • Jin Z.
      • Li Y.
      • Pitti R.
      • Lawrence D.
      • Pham V.C.
      • Lill J.R.
      • Ashkenazi A.
      ,
      • Seibenhener M.L.
      • Geetha T.
      • Wooten M.W.
      ,
      • Moscat J.
      • Diaz-Meco M.T.
      • Wooten M.W.
      ,
      • Kim J.Y.
      • Ozato K.
      ).
      The data presented here demonstrate direct binding of IDR-1 to p62 and provide evidence that this binding has specific consequences on protein-protein interactions, as well as on downstream signaling pathways. p62 was discovered as the intracellular target of IDR-1 by an unbiased, non-hypothesis-driven proteomics approach. Due to high affinity of IDR-1 for p62, it is likely that p62 is the most physiologically relevant target. Although we identified XIAP-4 in the SILAC experiments, strong direct binding of IDR-1 to XIAP was not detected in human cell lysates, nor was binding to XIAP detected with other related IDR peptides that did bind p62. Another IDR-1 weak binding partner, glyceraldehyde 3-phosphate dehydrogenase, was also recently described (
      • Mookherjee N.
      • Lippert D.N.
      • Hamill P.
      • Falsafi R.
      • Nijnik A.
      • Kindrachuk J.
      • Pistolic J.
      • Gardy J.
      • Miri P.
      • Naseer M.
      • Foster L.J.
      • Hancock R.E.
      ).
      Human p62 was confirmed as the IDR-1 target using binding assays and pulldown experiments from both endogenous and p62-overexpressing cell lysates. IDR-1 binding to both human and mouse p62, which share 99% amino acid sequence identity, was demonstrated. Through the investigation of p62 deletion mutants, we discovered that IDR-1 binds to the ZZ domain of p62, a zinc-binding protein interaction domain. Most compounds that act on protein-protein interactions antagonize or inhibit complex formation, but IDR-1 addition to cells led to a surprisingly specific effect; the TNFα-dependent receptor signaling complex of RIP1 and p62, which occurs at the ZZ domain, was stabilized, whereas the p62-PKCζ complex, which occurs at the PB1 domain proximal to the RIP1 binding region, was not affected.
      The specific modulation of p62 signaling complexes by IDR-1 appears to have a corresponding effect on the downstream signaling. On one hand, p62 has been described as an activator of NF-κB due to its receptor-induced signaling complex formation with PKCζ (
      • Duran A.
      • Linares J.F.
      • Galvez A.S.
      • Wikenheiser K.
      • Flores J.M.
      • Diaz-Meco M.T.
      • Moscat J.
      ,
      • Durán A.
      • Serrano M.
      • Leitges M.
      • Flores J.M.
      • Picard S.
      • Brown J.P.
      • Moscat J.
      • Diaz-Meco M.T.
      ), but IDR-1 treatment did not modulate NF-κB activity downstream of TNFα, consistently with the p62-PKCζ complex formation being unaffected. On the other hand, p38 MAPK activation downstream of TNFα is regulated by RIP1 (
      • Lee T.H.
      • Huang Q.
      • Oikemus S.
      • Shank J.
      • Ventura J.J.
      • Cusson N.
      • Vaillancourt R.R.
      • Su B.
      • Davis R.J.
      • Kelliher M.A.
      ), and the increase of RIP1-p62 interaction by IDR-1 may explain the previously reported role of IDR-1 in p38-mediated signaling pathways (
      • Scott M.G.
      • Dullaghan E.
      • Mookherjee N.
      • Glavas N.
      • Waldbrook M.
      • Thompson A.
      • Wang A.
      • Lee K.
      • Doria S.
      • Hamill P.
      • Yu J.J.
      • Li Y.
      • Donini O.
      • Guarna M.M.
      • Finlay B.B.
      • North J.R.
      • Hancock R.E.
      ). This is further strengthened by the observation that IDR-1-mediated activation of p38 is dependent on p62. The IDR-1-induced activation of C/EBPβ, downstream of p38, is also consistent with these and previous findings. Thus, we propose that the specific effects on intracellular signaling presented here contribute to the previously described IDR-1-modulated cytokine profile (Fig. 3F).
      IDR-1 has been demonstrated to prevent and treat infections and reduce associated inflammation (
      • Scott M.G.
      • Dullaghan E.
      • Mookherjee N.
      • Glavas N.
      • Waldbrook M.
      • Thompson A.
      • Wang A.
      • Lee K.
      • Doria S.
      • Hamill P.
      • Yu J.J.
      • Li Y.
      • Donini O.
      • Guarna M.M.
      • Finlay B.B.
      • North J.R.
      • Hancock R.E.
      ). In addition, recent studies report that p62 expression contributes to regulating macrophage-mediated (
      • Kim J.Y.
      • Ozato K.
      ) and cancer-associated inflammation (
      • Duran A.
      • Linares J.F.
      • Galvez A.S.
      • Wikenheiser K.
      • Flores J.M.
      • Diaz-Meco M.T.
      • Moscat J.
      ,
      • Moscat J.
      • Diaz-Meco M.T.
      ,
      • Hiruma Y.
      • Honjo T.
      • Jelinek D.F.
      • Windle J.J.
      • Shin J.
      • Roodman G.D.
      • Kurihara N.
      ), raising the question as to whether IDR-1 might also affect inflammatory responses in the absence of pathogen stimulation. The discovery that p62 is the target of IDR-1 highlights the importance of p62 in innate immunity and reveals p62 as a potential therapeutic target for anti-infective therapy without induction of harmful inflammation, and conversely, potential anti-inflammatory therapy with added anti-infective function.

      Acknowledgments

      We gratefully acknowledge the technical expertise of Mira Jovanovic.

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