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The Molecular Chaperone HSP70 Binds to and Stabilizes NOD2, an Important Protein Involved in Crohn Disease*

  • Vishnu Mohanan
    Affiliations
    Department of Biological Sciences, University of Delaware, Newark, Delaware 19716
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  • Catherine Leimkuhler Grimes
    Correspondence
    To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716. Tel.: 302-831-2985; Fax: 302-831-6335
    Affiliations
    Department of Biological Sciences, University of Delaware, Newark, Delaware 19716

    Departments of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant P20GM103541 from NIGMS.
Open AccessPublished:April 30, 2014DOI:https://doi.org/10.1074/jbc.M114.557686
      Microbes are detected by the pathogen-associated molecular patterns through specific host pattern recognition receptors. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is an intracellular pattern recognition receptor that recognizes fragments of the bacterial cell wall. NOD2 is important to human biology; when it is mutated it loses the ability to respond properly to bacterial cell wall fragments. To determine the mechanisms of misactivation in the NOD2 Crohn mutants, we developed a cell-based system to screen for protein-protein interactors of NOD2. We identified heat shock protein 70 (HSP70) as a protein interactor of both wild type and Crohn mutant NOD2. HSP70 has previously been linked to inflammation, especially in the regulation of anti-inflammatory molecules. Induced HSP70 expression in cells increased the response of NOD2 to bacterial cell wall fragments. In addition, an HSP70 inhibitor, KNK437, was capable of decreasing NOD2-mediated NF-κB activation in response to bacterial cell wall stimulation. We found HSP70 to regulate the half-life of NOD2, as increasing the HSP70 level in cells increased the half-life of NOD2, and down-regulating HSP70 decreased the half-life of NOD2. The expression levels of the Crohn-associated NOD2 variants were less compared with wild type. The overexpression of HSP70 significantly increased NOD2 levels as well as the signaling capacity of the mutants. Thus, our study shows that restoring the stability of the NOD2 Crohn mutants is sufficient for rescuing the ability of these mutations to signal the presence of a bacterial cell wall ligand.

      Introduction

      The human innate immune system is charged with a tremendous task, i.e. to provide the first line of defense against invading pathogens. This task is even more challenging in the human gut where over a trillion commensal bacteria reside. Thus, this ancient system must overlook the signal from the “good” bacteria while properly responding to the “bad” bacteria (
      • Janeway Jr., C.A.
      • Medzhitov R.
      Innate immune recognition.
      ,
      • Akira S.
      • Uematsu S.
      • Takeuchi O.
      Pathogen recognition and innate immunity.
      ,
      • Medzhitov R.
      Recognition of microorganisms and activation of the immune response.
      ). To accomplish this daunting task, the system relies partially on a complex system of receptors known collectively as innate immune receptors or pattern recognition receptors (
      • Tosi M.F.
      Innate immune responses to infection.
      ). These receptors are made up of both Toll-like receptors (TLRs)
      The abbreviations used are:
      TLR
      Toll-like receptor
      HSP70
      heat shock protein 70
      HSP90
      heat shock protein 90
      GGA
      geranylgeranylacetone
      MDP
      muramyl dipeptide
      dox
      doxycycline
      co-IP
      co-immunoprecipitation.
      and Nod-like receptors (
      • Wright S.D.
      Toll, a new piece in the puzzle of innate immunity.
      ,
      • Imler J.L.
      • Hoffmann J.A.
      Toll receptors in innate immunity.
      ,
      • Brodsky I.
      • Medzhitov R.
      Two modes of ligand recognition by TLRs.
      ,
      • Martinon F.
      • Tschopp J.
      NLRs join TLRs as innate sensors of pathogens.
      ). It has recently been shown that these families of receptors work together to form an intricate set of feedback loops to ensure that the innate immune system properly responds to pathogenic and commensal bacteria (
      • Kinnebrew M.A.
      • Pamer E.G.
      Innate immune signaling in defense against intestinal microbes.
      ,
      • Abraham C.
      • Medzhitov R.
      Interactions between the host innate immune system and microbes in inflammatory bowel disease.
      ). One can imagine the consequences if there is a breakdown of one of the receptors. NOD2 (nucleotide-binding oligomerization domain-containing protein 2) has been shown to be mutated in Crohn disease, a gastrointestinal disorder affecting the upper intestines (
      • Ogura Y.
      • Bonen D.K.
      • Inohara N.
      • Nicolae D.L.
      • Chen F.F.
      • Ramos R.
      • Britton H.
      • Moran T.
      • Karaliuskas R.
      • Duerr R.H.
      • Achkar J.P.
      • Brant S.R.
      • Bayless T.M.
      • Kirschner B.S.
      • Hanauer S.B.
      • Nuñez G.
      • Cho J.H.
      A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease.
      ). Inohara and co-workers (
      • Inohara N.
      • Ogura Y.
      • Fontalba A.
      • Gutierrez O.
      • Pons F.
      • Crespo J.
      • Fukase K.
      • Inamura S.
      • Kusumoto S.
      • Hashimoto M.
      • Foster S.J.
      • Moran A.P.
      • Fernandez-Luna J.L.
      • Nuñez G.
      Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn's disease.
      ) have shown that the Crohn mutants of NOD2 do not properly respond to their corresponding bacterial signals. More than 58 mutations in the NOD2 gene have been linked with various diseases, and 80% of these mutations are reported to be linked with Crohn disease (
      • Borzutzky A.
      • Fried A.
      • Chou J.
      • Bonilla F.A.
      • Kim S.
      • Dedeoglu F.
      NOD2-associated diseases: Bridging innate immunity and autoinflammation.
      ). The three highest point mutations with an association, R702W, G908R, and 1007fs, represent 32, 18, and 31%, respectively, of the total Crohn disease mutations (
      • Lesage S.
      • Zouali H.
      • Cézard J.P.
      • Colombel J.F.
      • Belaiche J.
      • Almer S.
      • Tysk C.
      • O'Morain C.
      • Gassull M.
      • Binder V.
      • Finkel Y.
      • Modigliani R.
      • Gower-Rousseau C.
      • Macry J.
      • Merlin F.
      • Chamaillard M.
      • Jannot A.S.
      • Thomas G.
      • Hugot J.P.
      • EPWG-IBD Group, EPIMAD Group, and GETAID Group
      CARD15/NOD2 mutational analysis and genotype-phenotype correlation in 612 patients with inflammatory bowel disease.
      ) Normally, NOD2 participates in the innate immune response by sensing fragments of bacterial peptidoglycan (
      • Ogura Y.
      • Inohara N.
      • Benito A.
      • Chen F.F.
      • Yamaoka S.
      • Nunez G.
      NOD2, a Nod1/Apaf-1 family member that is restricted to monocytes and activates NF-κB.
      ). The human protein, NOD2, is found in peripheral blood mononuclear cells such as macrophages, granulocytes, dendritic cells, and along the intestinal epithelial cells (
      • Ogura Y.
      • Inohara N.
      • Benito A.
      • Chen F.F.
      • Yamaoka S.
      • Nunez G.
      NOD2, a Nod1/Apaf-1 family member that is restricted to monocytes and activates NF-κB.
      ,
      • Gutierrez O.
      • Pipaon C.
      • Inohara N.
      • Fontalba A.
      • Ogura Y.
      • Prosper F.
      • Nunez G.
      • Fernandez-Luna J.L.
      Induction of NOD2 in myelomonocytic and intestinal epithelial cells via nuclear factor-κB activation.
      ,
      • Ogura Y.
      • Lala S.
      • Xin W.
      • Smith E.
      • Dowds T.A.
      • Chen F.F.
      • Zimmermann E.
      • Tretiakova M.
      • Cho J.H.
      • Hart J.
      • Greenson J.K.
      • Keshav S.
      • Nuñez G.
      Expression of NOD2 in Paneth cells: a possible link to Crohn's ileitis.
      ). It has been shown that NOD2 binds to muramyl dipeptide (MDP), a small degradation fragment of the bacterial cell wall (
      • Grimes C.L.
      • Ariyananda Lde Z.
      • Melnyk J.E.
      • O'Shea E.K.
      The innate immune protein NOD2 binds directly to MDP, a bacterial cell wall fragment.
      ,
      • Mo J.
      • Boyle J.P.
      • Howard C.B.
      • Monie T.P.
      • Davis B.K.
      • Duncan J.A.
      Pathogen sensing by nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is mediated by direct binding to muramyl dipeptide and ATP.
      ). Upon binding to its bacterial cell wall fragment, NOD2 interacts with RIP2 to activate the canonical NF-κB pathway, triggering an inflammatory response (
      • Abbott D.W.
      • Wilkins A.
      • Asara J.M.
      • Cantley L.C.
      The Crohn's disease protein, NOD2, requires RIP2 in order to induce ubiquitinylation of a novel site on NEMO.
      ). The Crohn-associated NOD2 mutants show a reduction in NF-κB signaling (
      • Inohara N.
      • Ogura Y.
      • Fontalba A.
      • Gutierrez O.
      • Pons F.
      • Crespo J.
      • Fukase K.
      • Inamura S.
      • Kusumoto S.
      • Hashimoto M.
      • Foster S.J.
      • Moran A.P.
      • Fernandez-Luna J.L.
      • Nuñez G.
      Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn's disease.
      ). NOD2 has a tri-domain structure consisting of two N-terminal caspase recruitment domains, a nucleotide binding domain, and a C-terminal leucine-rich repeat (
      • Strober W.
      • Murray P.J.
      • Kitani A.
      • Watanabe T.
      Signalling pathways and molecular interactions of NOD1 and NOD2.
      ). Mutations in the LRR region of NOD2 have been shown to increase the incidence of Crohn disease (
      • Ogura Y.
      • Bonen D.K.
      • Inohara N.
      • Nicolae D.L.
      • Chen F.F.
      • Ramos R.
      • Britton H.
      • Moran T.
      • Karaliuskas R.
      • Duerr R.H.
      • Achkar J.P.
      • Brant S.R.
      • Bayless T.M.
      • Kirschner B.S.
      • Hanauer S.B.
      • Nuñez G.
      • Cho J.H.
      A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease.
      ,
      • Bonen D.K.
      • Ogura Y.
      • Nicolae D.L.
      • Inohara N.
      • Saab L.
      • Tanabe T.
      • Chen F.F.
      • Foster S.J.
      • Duerr R.H.
      • Brant S.R.
      • Cho J.H.
      • Nuñez G.
      Crohn's disease-associated NOD2 variants share a signaling defect in response to lipopolysaccharide and peptidoglycan.
      ).
      Given the importance of NOD2 to human inflammatory disorders, there has been an urgency to understand the proper signaling mechanism of NOD2 in the innate immune response. With the proper signaling cascade understood, treatments for Crohn disease can be developed. Recently, three independent studies have shed light on the control mechanisms of NOD2 signaling. First, Nunez and co-workers (
      • McDonald C.
      • Chen F.F.
      • Ollendorff V.
      • Ogura Y.
      • Marchetto S.
      • Lécine P.
      • Borg J.P.
      • Nuñez G.
      A role for Erbin in the regulation of NOD2-dependent NF-κB signaling.
      ) demonstrated that NOD2 is negatively regulated by the protein Erbin. Erbin was shown to directly interact with NOD2, and its overexpression inhibited the ability of NOD2 to signal the presence of bacterial cell wall fragments (
      • McDonald C.
      • Chen F.F.
      • Ollendorff V.
      • Ogura Y.
      • Marchetto S.
      • Lécine P.
      • Borg J.P.
      • Nuñez G.
      A role for Erbin in the regulation of NOD2-dependent NF-κB signaling.
      ). Elegant work by Kobayashi and co-workers (
      • Lee K.H.
      • Biswas A.
      • Liu Y.J.
      • Kobayashi K.S.
      Proteasomal degradation of NOD2 protein mediates tolerance to bacterial cell wall components.
      ) went on to show that NOD2 activation via MDP is regulated by proteasomal degradation. Before stimulation, NOD2 forms a complex with heat shock protein 90 (HSP90). Upon binding MDP, NOD2 and HSP90 dissociate. Subsequently, NOD2 undergoes proteasomal degradation (
      • Lee K.H.
      • Biswas A.
      • Liu Y.J.
      • Kobayashi K.S.
      Proteasomal degradation of NOD2 protein mediates tolerance to bacterial cell wall components.
      ). This unique mechanism allows NOD2 to tolerate a large influx of MDP and avoid septic shock. In a subsequent study, McDonald and co-workers (
      • Richmond A.L.
      • Kabi A.
      • Homer C.R.
      • Marina-García N.
      • Nickerson K.P.
      • Nesvizhskii A.I.
      • Sreekumar A.
      • Chinnaiyan A.M.
      • Nuñez G.
      • McDonald C.
      The nucleotide synthesis enzyme CAD inhibits NOD2 antibacterial function in human intestinal epithelial cells.
      ) have shown that NOD2 is negatively regulated by the nucleotide synthesis enzyme carbamoyl-phosphate synthetase/aspartate transcarbamylase/dehydroorotase. Carbamoyl-phosphate synthetase/aspartate transcarbamylase/dehydroorotase was identified as a NOD2-interacting protein and was shown to inhibit the ability of NOD2 to signal the presence of bacterial cell wall ligands. Inhibition of carbamoyl-phosphate synthetase/aspartate transcarbamylase/dehydroorotase increased both the wild type and Crohn-associated NOD2 variants' ability to signal the presence of bacterial cell wall ligands (
      • Richmond A.L.
      • Kabi A.
      • Homer C.R.
      • Marina-García N.
      • Nickerson K.P.
      • Nesvizhskii A.I.
      • Sreekumar A.
      • Chinnaiyan A.M.
      • Nuñez G.
      • McDonald C.
      The nucleotide synthesis enzyme CAD inhibits NOD2 antibacterial function in human intestinal epithelial cells.
      ). Finally, independent genome-wide siRNA screens identified positive and negative regulators of the NOD2 or NF-κB pathways (
      • Lipinski S.
      • Grabe N.
      • Jacobs G.
      • Billmann-Born S.
      • Till A.
      • Häsler R.
      • Aden K.
      • Paulsen M.
      • Arlt A.
      • Kraemer L.
      • Hagemann N.
      • Erdmann K.S.
      • Schreiber S.
      • Rosenstiel P.
      RNAi screening identifies mediators of NOD2 signaling: implications for spatial specificity of MDP recognition.
      ,
      • Warner N.
      • Burberry A.
      • Franchi L.
      • Kim Y.G.
      • McDonald C.
      • Sartor M.A.
      • Núñez G.
      A genome-wide siRNA screen reveals positive and negative regulators of the NOD2 and NF-κB signaling pathways.
      ).
      In general, two different approaches have been used to identify protein-protein interactors involved in the NOD2-signaling cascade: 1) immunopurification (IP) followed by mass spectrometry and 2) siRNA screens (
      • Richmond A.L.
      • Kabi A.
      • Homer C.R.
      • Marina-García N.
      • Nickerson K.P.
      • Nesvizhskii A.I.
      • Sreekumar A.
      • Chinnaiyan A.M.
      • Nuñez G.
      • McDonald C.
      The nucleotide synthesis enzyme CAD inhibits NOD2 antibacterial function in human intestinal epithelial cells.
      ,
      • Warner N.
      • Burberry A.
      • Franchi L.
      • Kim Y.G.
      • McDonald C.
      • Sartor M.A.
      • Núñez G.
      A genome-wide siRNA screen reveals positive and negative regulators of the NOD2 and NF-κB signaling pathways.
      ). Both approaches have been successful in identifying new protein-protein interactors of NOD2. However, the mechanism for rescuing the Crohn phenotype remains unresolved. To determine novel protein-protein interactors of NOD2, we designed a cell-based system that would allow for identification of interactors that used normal endogenous NOD2 levels in the cell and would allow for essential proteins to be identified. We were intrigued by the tetracycline-inducible promoter system that was developed by Blau and co-workers (
      • Rossi F.M.
      • Guicherit O.M.
      • Spicher A.
      • Kringstein A.M.
      • Fatyol K.
      • Blakely B.T.
      • Blau H.M.
      Tetracycline-regulatable factors with distinct dimerization domains allow reversible growth inhibition by p16.
      ) and has successfully elucidated many signaling mechanisms (
      • Onesto C.
      • Berra E.
      • Grépin R.
      • Pagès G.
      Poly(A)-binding protein-interacting protein 2, a strong regulator of vascular endothelial growth factor mRNA.
      ). Tetracycline-regulated expression enabled purification and functional analysis of recombinant connexin channels from mammalian cells (
      • Meylan E.
      • Curran J.
      • Hofmann K.
      • Moradpour D.
      • Binder M.
      • Bartenschlager R.
      • Tschopp J.
      Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus.
      ). We envisioned that this sophisticated system would give us a method to control the expression of an epitope-tagged version of NOD2 in human cells.
      Using the tetracycline-regulated expression system, we have identified heat shock protein 70 (HSP70) as a bona fide interactor of NOD2. We show that HSP70 increases the half-life of both wild type and the Crohn mutant, NOD2. More importantly, we demonstrate that overexpression of HSP70 is able to rescue the Crohn mutant response to MDP, the bacterial cell wall fragment recognized by NOD2. Our data suggest that the Crohn mutants are inherently unstable and unable to properly respond to the bacterial cell wall ligands. Thus, a novel therapy providing stability to Crohn disease-associated NOD2 variants could be extremely useful.

      DISCUSSION

      NOD2 is an important innate immune receptor that is responsible for sensing the occurrence of bacterial cell wall fragments within mammalian cells. It has been known for over 10 years that mutations in NOD2 correlate to the development of Crohn disease (
      • Ogura Y.
      • Bonen D.K.
      • Inohara N.
      • Nicolae D.L.
      • Chen F.F.
      • Ramos R.
      • Britton H.
      • Moran T.
      • Karaliuskas R.
      • Duerr R.H.
      • Achkar J.P.
      • Brant S.R.
      • Bayless T.M.
      • Kirschner B.S.
      • Hanauer S.B.
      • Nuñez G.
      • Cho J.H.
      A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease.
      ). Moreover, these mutations do not properly respond to bacterial cell wall components (
      • Inohara N.
      • Ogura Y.
      • Fontalba A.
      • Gutierrez O.
      • Pons F.
      • Crespo J.
      • Fukase K.
      • Inamura S.
      • Kusumoto S.
      • Hashimoto M.
      • Foster S.J.
      • Moran A.P.
      • Fernandez-Luna J.L.
      • Nuñez G.
      Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn's disease.
      ). Studies have shown that when cells containing the NOD2 mutations are treated with the bacterial cell wall fragments, a lower NF-κB response is obtained (
      • Inohara N.
      • Ogura Y.
      • Fontalba A.
      • Gutierrez O.
      • Pons F.
      • Crespo J.
      • Fukase K.
      • Inamura S.
      • Kusumoto S.
      • Hashimoto M.
      • Foster S.J.
      • Moran A.P.
      • Fernandez-Luna J.L.
      • Nuñez G.
      Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn's disease.
      ,
      • Bonen D.K.
      • Ogura Y.
      • Nicolae D.L.
      • Inohara N.
      • Saab L.
      • Tanabe T.
      • Chen F.F.
      • Foster S.J.
      • Duerr R.H.
      • Brant S.R.
      • Cho J.H.
      • Nuñez G.
      Crohn's disease-associated NOD2 variants share a signaling defect in response to lipopolysaccharide and peptidoglycan.
      ). It has been suggested that a basal level of NOD2 activation is needed in the cell to control other innate immune receptors. Without the ability to activate NF-κB, an uncontrollable amount of inflammation ensues, and Crohn disease develops (
      • Fava F.
      • Danese S.
      Crohn's disease: bacterial clearance in Crohn's disease pathogenesis.
      ,
      • Noguchi E.
      • Homma Y.
      • Kang X.
      • Netea M.G.
      • Ma X.
      A Crohn's disease-associated NOD2 mutation suppresses transcription of human IL10 by inhibiting activity of the nuclear ribonucleoprotein hnRNP-A1.
      ). Much effort has been focused on trying to identify the mechanism by which NOD2 signals to the NF-κB response. Many protein interactors have been found. However, none explain the Crohn NOD2 phenotype, i.e. a lack of response to bacterial cell wall ligand.
      To understand the mechanism of NOD2 signaling, we developed a novel system to screen for NOD2 protein-protein interactors. Our approach has two main advantages over previous NOD2 studies. First, a tetracycline-inducible promoter system allowed a controllable, low level expression of NOD2 to be obtained (Fig. 1A). Second, the expression of an epitope-tagged NOD2 allowed for the efficient purification of NOD2 and the interacting proteins (Fig. 2B). The interacting proteins could then be identified using mass spectrometry. Our small screen identified HSP70 as a protein that interacts with NOD2. Initially, we were skeptical of the significance of this finding, as HSP70 is ubiquitously expressed in the cells, and we believed that this finding could be an artifact. However, recently HSP70 has been shown to regulate a number of innate immune receptors. Our study went on to validate HSP70 as a genuine NOD2 protein-protein interactor (Fig. 1, A and C). We showed that NOD2 interacts with the substrate binding domain of HSP70 (Fig. 2E). We investigated the role of HSP70 in NOD2 signaling using established cell-based assays to monitor NF-κB activation (Fig. 3, A and B). We observed that overexpression of HSP70 yielded an increase in NOD2's ability to signal when stimulated with MDP. The literature has reported that overexpression of HSP70 blocks the denaturation of IκB kinase thereby increasing the nuclear translocation of NF-κB (
      • Lee K.H.
      • Lee C.T.
      • Kim Y.W.
      • Han S.K.
      • Shim Y.S.
      • Yoo C.G.
      Heat shock protein 70 negatively regulates the heat-shock-induced suppression of the IκB/NF-κB cascade by facilitating IκB kinase renaturation and blocking its further denaturation.
      ). This could explain the increase in TNFα-induced NF-κB signaling upon HSP70 overexpression. However, the degree of NOD2 activation via MDP upon HSP70 overexpression was higher, compared with the TNFα-induced activation (Fig. 3A), implying that more than just IκB kinase stabilization was necessary for the increase. We went on to show that HSP70 increases the half-life of NOD2 (Fig. 5, A and B). At this point, we wondered whether the half-life of the Crohn mutants was relative to the wild-type NOD2. We found that the Crohn mutants are inherently unstable, and their half-life is less than that of the wild-type protein (Fig. 6, A–C). This fact intrigued us, and we investigated whether the overexpression of HSP70 could provide a rescue mechanism for the NOD2 Crohn mutants, as the Crohn mutants also interact with HSP70 (Fig. 7A). We found that overexpression of HSP70 was able to restore the ability of the Crohn mutants to signal upon stimulation with MDP (Fig. 7C).
      Overexpression of HSP70 to rescue a protein's function is not a new phenomenon. There are many examples in the literature supporting this idea. For example, p53 and mutants of this protein are protected from degradation in the presence of excess HSP70 (
      • Wiech M.
      • Olszewski M.B.
      • Tracz-Gaszewska Z.
      • Wawrzynow B.
      • Zylicz M.
      • Zylicz A.
      Molecular mechanism of mutant p53 stabilization: the role of HSP70 and MDM2.
      ). In addition, human ether-a-go-go-related gene, a subunit of the potassium current I protein, is stabilized when HSP70 is overexpressed (
      • Li P.
      • Ninomiya H.
      • Kurata Y.
      • Kato M.
      • Miake J.
      • Yamamoto Y.
      • Igawa O.
      • Nakai A.
      • Higaki K.
      • Toyoda F.
      • Wu J.
      • Horie M.
      • Matsuura H.
      • Yoshida A.
      • Shirayoshi Y.
      • Hiraoka M.
      • Hisatome I.
      Reciprocal control of hERG stability by HSP70 and Hsc70 with implication for restoration of LQT2 mutant stability.
      ). Moreover, this study shows another protein (human ether-a-go-go-related gene) that HSP70 is capable of rescuing in its mutated form. An exciting outcome of our findings is that it suggests a potential method to treat Crohn disease, a small molecule that can specifically stabilize NOD2, by acting as a surrogate for the HSP70 protein. Our study complements the finding by Kobayahsi and co-workers (
      • Lee K.H.
      • Biswas A.
      • Liu Y.J.
      • Kobayashi K.S.
      Proteasomal degradation of NOD2 protein mediates tolerance to bacterial cell wall components.
      ) that NOD2 is rapidly degraded in the presence of MDP, as HSP90 is released. We found that NOD2 interacts with HSP70 independent of the presence of MDP, its bacterial cell wall ligand. Thus, HSP70 may provide a mechanism to reinitiate the ability of NOD2 to signal when there is a new burst of bacterial cell wall ligands. If, however, NOD2 is mutated, it is unable to signal the presence of bacterial cell wall fragments as its half-life in the cell is decreased. We found that overexpression of HSP70 is able to rescue NOD2's response to bacterial cell wall ligands. We propose that HSP90 could play a similar role, and we are currently working to determine whether HSP90 interacts with the Crohn mutants. This mechanism is different from HSP70's ability to alter the ability of TLR to signal. HSP70 (
      • Afrazi A.
      • Sodhi C.P.
      • Good M.
      • Jia H.
      • Siggers R.
      • Yazji I.
      • Ma C.
      • Neal M.D.
      • Prindle T.
      • Grant Z.S.
      • Branca M.F.
      • Ozolek J.
      • Chang E.B.
      • Hackam D.J.
      Intracellular heat shock protein-70 negatively regulates TLR4 signaling in the newborn intestinal epithelium.
      ) has been shown to specifically down-regulate the TLR4 response to lipopolysaccharide (
      • Afrazi A.
      • Sodhi C.P.
      • Good M.
      • Jia H.
      • Siggers R.
      • Yazji I.
      • Ma C.
      • Neal M.D.
      • Prindle T.
      • Grant Z.S.
      • Branca M.F.
      • Ozolek J.
      • Chang E.B.
      • Hackam D.J.
      Intracellular heat shock protein-70 negatively regulates TLR4 signaling in the newborn intestinal epithelium.
      ). Thus, the cell has designed HSP70 to be both an activator and an inhibitor of innate immune signaling.
      In conclusion, we have developed a tetracycline-inducible NOD2 expression system that has allowed us to control the expression of NOD2 and purify interacting proteins. We have identified HSP70 as a novel NOD2 protein interactor. HSP70 is able to increase the half-life of NOD2 in the cell, and it also restores the response of the Crohn-associated NOD2 mutants. We propose that small molecules that can specifically stabilize the NOD2 Crohn mutants may be useful in the treatment of Crohn disease.

      Acknowledgments

      We acknowledge Dr. John C. Kappes (University of Alabama, Birmingham) for kindly providing lentiviral vector K2605. We acknowledge Leila Choe and Professor Kelvin Lee at the University of Delaware Proteomics and Mass Spectrometry Facility, which is supported in part by the Delaware IDeA Network of Biomedical Research Excellence, National Institutes of Health Grant 8 P20 GM103446-13, for the mass spectrometry and protein identification. We thank Dr. Brian Bahnson and Amy Schaefer for critical reading of the manuscript.

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