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Mapping the Interaction of B Cell Leukemia 3 (BCL-3) and Nuclear Factor κB (NF-κB) p50 Identifies a BCL-3-mimetic Anti-inflammatory Peptide*

  • Patricia E. Collins
    Affiliations
    Centre for Immunobiology, Institute of Infection, Immunity and Inflammation, College of Medicine, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, United Kingdom
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  • Gianluca Grassia
    Footnotes
    Affiliations
    Centre for Immunobiology, Institute of Infection, Immunity and Inflammation, College of Medicine, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, United Kingdom
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  • Amy Colleran
    Affiliations
    Department of Biochemistry, University College Cork, Cork, Ireland
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  • Patrick A. Kiely
    Affiliations
    Department of Life Sciences, and Materials and Surface Science Institute, University of Limerick, Limerick, Ireland
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  • Armando Ialenti
    Affiliations
    Department of Pharmacy, University of Napoli Federico II, Naples 80131, Italy
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  • Pasquale Maffia
    Affiliations
    Centre for Immunobiology, Institute of Infection, Immunity and Inflammation, College of Medicine, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, United Kingdom

    Department of Pharmacy, University of Napoli Federico II, Naples 80131, Italy
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  • Ruaidhrí J. Carmody
    Correspondence
    To whom correspondence should be addressed
    Affiliations
    Centre for Immunobiology, Institute of Infection, Immunity and Inflammation, College of Medicine, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, United Kingdom
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  • Author Footnotes
    * This work was supported in part by Science Foundation Ireland Grant 08/IN.1/B1843, Biotechnology and Biological Sciences Research Council Grant BB/M003671/1 and the Institute of Infection, Immunity and Inflammation at the University of Glasgow. The authors declare that they have no conflicts of interest with the contents of this article.
    This article contains supplemental Table S1.
    1 Supported by the Engineering and Physical Sciences Research Council Research Grant EP/L014165/1.
Open AccessPublished:April 28, 2015DOI:https://doi.org/10.1074/jbc.M115.643700
      The NF-κB transcriptional response is tightly regulated by a number of processes including the phosphorylation, ubiquitination, and subsequent proteasomal degradation of NF-κB subunits. The IκB family protein BCL-3 stabilizes a NF-κB p50 homodimer·DNA complex through inhibition of p50 ubiquitination. This complex inhibits the binding of the transcriptionally active NF-κB subunits p65 and c-Rel on the promoters of NF-κB target genes and functions to suppress inflammatory gene expression. We have previously shown that the direct interaction between p50 and BCL-3 is required for BCL-3-mediated inhibition of pro-inflammatory gene expression. In this study we have used immobilized peptide array technology to define regions of BCl-3 that mediate interaction with p50 homodimers. Our data show that BCL-3 makes extensive contacts with p50 homodimers and in particular with ankyrin repeats (ANK) 1, 6, and 7, and the N-terminal region of Bcl-3. Using these data we have designed a BCL-3 mimetic peptide based on a region of the ANK1 of BCL-3 that interacts with p50 and shares low sequence similarity with other IκB proteins. When fused to a cargo carrying peptide sequence this BCL-3-derived peptide, but not a mutated peptide, inhibited Toll-like receptor-induced cytokine expression in vitro. The BCL-3 mimetic peptide was also effective in preventing inflammation in vivo in the carrageenan-induced paw edema mouse model. This study demonstrates that therapeutic strategies aimed at mimicking the functional activity of BCL-3 may be effective in the treatment of inflammatory disease.

      Background:

      BCL-3 is an essential negative regulator of inflammation.

      Results:

      A peptide derived from the ankyrin repeat 1 domain of BCL-3 has anti-inflammatory properties.

      Conclusion:

      The interaction of a short region of BCL-3 with p50 has significant functional consequences on inflammatory gene expression.

      Significance:

      Mimicking BCL-3 function has therapeutic potential.

      Introduction

      The NF-κB transcription factor is a critical factor for the normal development and homeostasis of the immune system and is essential for the inflammatory response (
      • Carmody R.J.
      • Chen Y.H.
      Nuclear factor-κB: activation and regulation during Toll-like receptor signaling.
      ). NF-κB controls the expression of hundreds of genes that encode pro-inflammatory effectors such as cytokines and chemokines, proteins involved in antigen presentation, and regulators of cell death and proliferation. Mice lacking specific NF-κB subunits or components of the NF-κB activation pathway are immunodefective and fail to develop the appropriate immunity to infection and have aberrant inflammatory responses (
      • Gerondakis S.
      • Grossmann M.
      • Nakamura Y.
      • Pohl T.
      • Grumont R.
      Genetic approaches in mice to understand Rel/NF-κB and IκB function: transgenics and knockouts.
      ). NF-κB is in fact a family of five related factors: p65/RelA, c-Rel, RelB, p50, and p52, all of which possess a Rel homology domain, which mediates subunit dimerization and DNA binding. Each subunit is capable of homodimerization or heterodimerization to yield a theoretical 15 possible dimers, although only 12 specific dimers have so far been demonstrated to exist in cells (
      • Huxford T.
      • Ghosh G.
      A structural guide to proteins of the NF-κB signaling module.
      ). The p50 and p52 subunits are generated from the limited proteasomal processing of the larger precursor proteins p105 and p100, respectively. Importantly both p50 and p52 lack a transactivation domain and when present as homodimers lack intrinsic transactivation activity (
      • Hayden M.S.
      • Ghosh S.
      NF-κB, the first quarter-century: remarkable progress and outstanding questions.
      ).
      The primary point of control of NF-κB transcriptional activity is the sequestration of NF-κB dimers in the cytoplasm by members of the IκB family of regulatory proteins, of which IκBα is the archetypal member. Upon receipt of a NF-κB activating stimulus, the inhibitor of κB kinase (IKK)
      The abbreviations used are: IKK
      IκB kinase
      ANK
      ankyrin repeat
      Dex
      dexamethasone
      BDP
      Bcl-3-derived peptide.
      complex, composed of the kinases IKKα and IKKβ, and the scaffold protein NEMO, phosphorylates IκBα, triggering its ubiquitination and subsequent proteasomal degradation. Free NF-κB dimers then translocate to the nucleus where they bind cognate sites on DNA to regulate the transcription of target genes (
      • Hayden M.S.
      • Ghosh S.
      NF-κB, the first quarter-century: remarkable progress and outstanding questions.
      ). More recently a number of studies have identified critical regulatory mechanisms in the nucleus that control NF-κB transcriptional activity. Most prominent among these is the regulation of NF-κB stability by the ubiquitin-proteasomal system (
      • Natoli G.
      • Chiocca S.
      Nuclear ubiquitin ligases, NF-κB degradation, and the control of inflammation.
      ). The ubiquitination of NF-κB is triggered by DNA binding and is also regulated by phosphorylation (
      • Bosisio D.
      • Marazzi I.
      • Agresti A.
      • Shimizu N.
      • Bianchi M.E.
      • Natoli G.
      A hyper-dynamic equilibrium between promoter-bound and nucleoplasmic dimers controls NF-κB-dependent gene activity.
      ,
      • Colleran A.
      • Collins P.E.
      • O'Carroll C.
      • Ahmed A.
      • Mao X.
      • McManus B.
      • Kiely P.A.
      • Burstein E.
      • Carmody R.J.
      Deubiquitination of NF-κB by ubiquitin-specific protease-7 promotes transcription.
      ). Ubiquitin-mediated proteasomal degradation of the NF-κB subunit p65/RelA is a major limiting factor in the transcription of target genes (
      • Colleran A.
      • Collins P.E.
      • O'Carroll C.
      • Ahmed A.
      • Mao X.
      • McManus B.
      • Kiely P.A.
      • Burstein E.
      • Carmody R.J.
      Deubiquitination of NF-κB by ubiquitin-specific protease-7 promotes transcription.
      ).
      The stability of one NF-κB dimer can also profoundly affect the transcriptional activity of other NF-κB dimers. The IκB protein BCL-3 inhibits the ubiquitination and subsequent proteasomal degradation of p50 homodimers to limit the expression of pro-inflammatory cytokines following activation of Toll-like receptors (
      • Carmody R.J.
      • Ruan Q.
      • Palmer S.
      • Hilliard B.
      • Chen Y.H.
      Negative regulation of Toll-like receptor signaling by NF-κB p50 ubiquitination blockade.
      ). BCL-3 is an atypical IκB protein that, in contrast to IκBα, is predominantly nuclear in localization and is not degraded following activation of the IKK complex (
      • Carmody R.J.
      • Ruan Q.
      • Palmer S.
      • Hilliard B.
      • Chen Y.H.
      Negative regulation of Toll-like receptor signaling by NF-κB p50 ubiquitination blockade.
      ). BCL-3-stabilized p50 homodimers form a stable repressor complex at NF-κB binding sites that competes with transcriptionally active NF-κB dimers composed of p65 or c-Rel to inhibit target gene expression. BCL-3 is important for establishing TLR tolerance, a state of altered responsiveness to Toll-like receptor stimulation in macrophage characterized by a block in pro-inflammatory cytokine expression (
      • Carmody R.J.
      • Ruan Q.
      • Palmer S.
      • Hilliard B.
      • Chen Y.H.
      Negative regulation of Toll-like receptor signaling by NF-κB p50 ubiquitination blockade.
      ). Mice deficient in Bcl3 lack Toll-like receptor tolerance (
      • Carmody R.J.
      • Ruan Q.
      • Palmer S.
      • Hilliard B.
      • Chen Y.H.
      Negative regulation of Toll-like receptor signaling by NF-κB p50 ubiquitination blockade.
      ), fail to clear infection (
      • Pene F.
      • Paun A.
      • Sønder S.U.
      • Rikhi N.
      • Wang H.
      • Claudio E.
      • Siebenlist U.
      The IκB family member Bcl-3 coordinates the pulmonary defense against Klebsiella pneumoniae infection.
      ), are more sensitive to the development of type I diabetes (
      • Ruan Q.
      • Zheng S.J.
      • Palmer S.
      • Carmody R.J.
      • Chen Y.H.
      Roles of Bcl-3 in the pathogenesis of murine type 1 diabetes.
      ), and undergo increased granulopoiesis under inflammatory conditions (
      • Kreisel D.
      • Sugimoto S.
      • Tietjens J.
      • Zhu J.
      • Yamamoto S.
      • Krupnick A.S.
      • Carmody R.J.
      • Gelman A.E.
      Bcl3 prevents acute inflammatory lung injury in mice by restraining emergency granulopoiesis.
      ). More recently BCL-3 has been identified as an important enforcer of T cell differentiation states (
      • Tang W.
      • Wang H.
      • Claudio E.
      • Tassi I.
      • Ha H.L.
      • Saret S.
      • Siebenlist U.
      The oncoprotein and transcriptional regulator Bcl-3 governs plasticity and pathogenicity of autoimmune T cells.
      ) and a key factor in promoting dendritic cell priming of T cells (
      • Tassi I.
      • Claudio E.
      • Wang H.
      • Tang W.
      • Ha H.L.
      • Saret S.
      • Ramaswamy M.
      • Siegel R.
      • Siebenlist U.
      The NF-κB regulator Bcl-3 governs dendritic cell antigen presentation functions in adaptive immunity.
      ). Thus BCL-3 is an important regulator of inflammation and immune responses.
      We recently employed peptide array techniques to identify critical amino acids of p50, which mediate the interaction with BCL-3 (
      • Collins P.E.
      • Kiely P.A.
      • Carmody R.J.
      Inhibition of transcription by B cell leukemia 3 (Bcl-3) protein requires interaction with nuclear factor κB (NF-κB) p50.
      ). In this study we employ similar techniques to identify residues of BCL-3, which mediate interaction with p50. This approach confirmed previously indicated sites of interaction from in silico modeling studies (
      • Manavalan B.
      • Basith S.
      • Choi Y.M.
      • Lee G.
      • Choi S.
      Structure-function relationship of cytoplasmic and nuclear IκB proteins: an in silico analysis.
      ,
      • Carmody R.J.
      • Ruan Q.
      • Liou H.C.
      • Chen Y.H.
      Essential roles of c-Rel in TLR-induced IL-23 p19 gene expression in dendritic cells.
      ) but also identified additional sites of interaction not predicted by computational methods. Our findings also provide important information on the recognition of NF-κB dimers by IκB proteins. We used the results of our peptide array analysis to generate a short peptide of BCL-3, which mimicked the inhibitory effect of full-length BCL-3 on NF-κB in vitro. Moreover, the BCL-3 mimetic peptide significantly inhibited carrageenan-induced paw inflammation in mice. Our study demonstrates that mimicking BCL-3 function may represent an effective strategy for the inhibition of inflammation.

      Discussion

      BCL-3 mediates inhibition of p50 homodimer ubiquitination, leading to the formation of a stable p50·BCL-3 repressor complex bound to the promoters of NF-κB target genes (
      • Carmody R.J.
      • Ruan Q.
      • Palmer S.
      • Hilliard B.
      • Chen Y.H.
      Negative regulation of Toll-like receptor signaling by NF-κB p50 ubiquitination blockade.
      ). Interaction with p50 is necessary and sufficient for this anti-inflammatory function of BCL-3 (
      • Collins P.E.
      • Kiely P.A.
      • Carmody R.J.
      Inhibition of transcription by B cell leukemia 3 (Bcl-3) protein requires interaction with nuclear factor κB (NF-κB) p50.
      ). To further investigate the role of this complex in the regulation of NF-κB-mediated gene transcription we employed a BCL-3 peptomimetic strategy. A peptide array approach identified short peptides of BCL-3 with p50 binding activity representing the N terminus, ANK1, ANK6, and ANK7. Following alanine substitution analysis, critical residues within these peptides were identified. There was considerable overlap between the individual residues identified by peptide array and those predicted from computation modeling particularly in ANK6 and ANK7, further supporting the peptide array as a method to identify critical p50 binding regions.
      Current computational models of BCL-3/p50 are constrained by the available crystal structures, which are limited to the independent crystal structures of DNA bound p50 homodimer and the central ankyrin repeat domain of BCL-3 (
      • Manavalan B.
      • Basith S.
      • Choi Y.M.
      • Lee G.
      • Choi S.
      Structure-function relationship of cytoplasmic and nuclear IκB proteins: an in silico analysis.
      ,
      • Michel F.
      • Soler-Lopez M.
      • Petosa C.
      • Cramer P.
      • Siebenlist U.
      • Müller C.W.
      Crystal structure of the ankyrin repeat domain of Bcl-3: a unique member of the IκB protein family.
      ). In both cases the extreme N- and C-terminal regions are unstructured and so are not represented in either the resolved structures or in silico models. The data from the experiments performed here along with previous studies on the interaction of BCL-3 and p50 (
      • Collins P.E.
      • Kiely P.A.
      • Carmody R.J.
      Inhibition of transcription by B cell leukemia 3 (Bcl-3) protein requires interaction with nuclear factor κB (NF-κB) p50.
      ,
      • Manavalan B.
      • Basith S.
      • Choi Y.M.
      • Lee G.
      • Choi S.
      Structure-function relationship of cytoplasmic and nuclear IκB proteins: an in silico analysis.
      ,
      • Michel F.
      • Soler-Lopez M.
      • Petosa C.
      • Cramer P.
      • Siebenlist U.
      • Müller C.W.
      Crystal structure of the ankyrin repeat domain of Bcl-3: a unique member of the IκB protein family.
      ) suggests that the N-terminal domain of BCL-3 most likely interacts with the extreme C terminus of p50.
      Our peptide array data have identified amino acids of the N-terminal and ANK1 domain of BCL-3, which interact with p50. These data are supported by earlier studies indicating that the first ANK domain is required for interaction with p50 (
      • Zhang Q.
      • Didonato J.A.
      • Karin M.
      • McKeithan T.W.
      BCL3 encodes a nuclear protein which can alter the subcellular location of NF-κB proteins.
      ). Multiple sequence alignment of this region of BCL-3-(144–158) with the homologous regions of the other IκB proteins identifies it as an area of low sequence similarity between IκB proteins. This region has also been suggested to contribute to the functional divergence of IκB factors and has also been implicated in BCL-3-mediated survival of activated T and B cells (
      • Mitchell T.C.
      • Teague T.K.
      • Hildeman D.A.
      • Bender J.
      • Rees W.A.
      • Kedl R.M.
      • Swanson B.
      • Kappler J.W.
      • Marrack P.
      Stronger correlation of bcl-3 than bcl-2, bcl-xL, costimulation, or antioxidants with adjuvant-induced T cell survival.
      ,
      • Mitchell T.C.
      • Thompson B.S.
      • Trent J.O.
      • Casella C.R.
      A short domain within Bcl-3 is responsible for its lymphocyte survival activity.
      ). These previous studies and the data presented here suggested that this region of BCL-3 may contain important functional activity.
      To explore the functional activity of peptides comprising the 144–148 sequence of BCL-3 we utilized the HIV tat cargo carrying peptide sequence. Because BCL-3 is a predominantly nuclear protein that regulates NF-κB activity at gene promoters we reasoned that the tat peptide sequence was most suited to delivering the BCL-3-derived peptide to the site of BCL-3 activity as it rapidly translocates through the plasma membrane and accumulates in the nucleus (
      • Vivès E.
      • Brodin P.
      • Lebleu B.
      A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus.
      ). Our initial analyses demonstrated potent inhibitory activity of the BDP peptide in both luciferase-based reporter and ELISA-based assays of Toll-like receptor-induced expression of the Il23 and TNFα cytokines, respectively. Of note, both of these genes have previously been established as targets of BCL-3 inhibitory activity (
      • Carmody R.J.
      • Ruan Q.
      • Palmer S.
      • Hilliard B.
      • Chen Y.H.
      Negative regulation of Toll-like receptor signaling by NF-κB p50 ubiquitination blockade.
      ,
      • Carmody R.J.
      • Ruan Q.
      • Liou H.C.
      • Chen Y.H.
      Essential roles of c-Rel in TLR-induced IL-23 p19 gene expression in dendritic cells.
      ). The BDP peptide was also a potent inhibitor of cytokine gene expression and inflammation in vivo. Treatment of mice with the BDP2 peptide was effective in preventing carageenan-induced cytokine expression and paw edema. By mutating the amino acids identified as important for peptide interaction with p50 we were able to demonstrate that the functional effects of the BDP peptide, both in vitro and in vivo, were dependent on interaction with p50.
      NF-κB regulates the transcription of a number of genes critical for the inflammatory response and is considered a potential therapeutic target in a range of human diseases where inflammation plays a role. Hundreds of inhibitors of NF-κB activation have been described but are limited by broad specificity (
      • Gilmore T.D.
      • Herscovitch M.
      Inhibitors of NF-κB signaling: 785 and counting.
      ). The BCL-3-derived peptide described here, through mimicking BCL-3 function, represents a novel class of anti-inflammatory agents that target a nuclear regulatory event in the NF-κB pathway. This BCL-3-derived peptide provides an important proof of principle that targeting transcriptional regulators of inflammation is a valid strategy for developing novel anti-inflammatory compounds of therapeutic value. The BDP peptide is also a valuable tool for further research on BCL-3 function and for the development of BCL-3-based therapeutic agents for inflammatory disease.

      Acknowledgments

      We thank Karen Keeshan for critical evaluation of the manuscript.

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