The molecular chaperone HSP70 binds to and stabilizes NOD2, an important protein involved in Crohn disease.
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.
From the Departments of Biological Sciences and.
Vishnu
V
Mohanan
From the Departments of Biological Sciences and Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716 cgrimes@udel.edu.
Catherine Leimkuhler
CL
Grimes
D000119:Acetylmuramyl-Alanyl-Isoglutamine / Q000494:pharmacology; D000818:Animals; D002473:Cell Wall / Q000378:metabolism; D003424:Crohn Disease / Q000235:genetics / Q000378:metabolism* / Q000382:microbiology / Q000473:pathology; D005786:Gene Expression Regulation / Q000187:drug effects; D045325:HCT116 Cells; D057809:HEK293 Cells; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006207:Half-Life; D006801:Humans; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D053473:Nod2 Signaling Adaptor Protein / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D055550:Protein Stability / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D013752:Tetracycline / Q000494:pharmacology
D018840:HSP70 Heat-Shock Proteins; D016328:NF-kappa B; D053473:Nod2 Signaling Adaptor Protein; D000119:Acetylmuramyl-Alanyl-Isoglutamine; D013752:Tetracycline
Cell Wall; HSP70; Innate Immunity; Muramyl Dipeptide; NF-κB; NOD2; Nod-like Receptor; Peptidoglycan; Signal Transduction