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Identification of a pharmaceutical therapeutic for Nod2, a protein mutated in Crohn's Disease, through development of a screen using split GFP complementation
Author(s) -
Wastyk Hannah Constance,
Hou Ching Wen,
Grimes Catherine
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.604.19
Subject(s) - nod2 , muramyl dipeptide , innate immune system , complementation , peptide , microbiology and biotechnology , biology , chemistry , green fluorescent protein , immune system , mutant , receptor , biochemistry , genetics , gene
The human body houses over 10 trillion bacterial cells to make up our microbiome. These bacteria can be commensal or pathogenic, and it is the role of our innate immune system to delineate between the two. Innate immune receptors, such as Nod2, are responsible for binding to bacterial cell wall fragments. Nod2 binds to bacterial cell wall fragment, muramyl dipeptide (MDP), which leads to subsequent downstream activation of the NF‐κB pathway and a protective inflammatory response. Mutations in Nod2 are correlated with an increased susceptibility to Crohn's disease. Nod2 mutations that decrease its stability disrupt the downstream signaling response. Initial work identifies a chaperone protein, Hsp70, which binds to Nod2 and assists its stability and activity. The interaction of Nod2 and Hsp70 was characterized to suggest that Hsp70's ATPase activity is dispensable for Nod2 stabilization. A truncated 71 amino acid domain of Hsp70 was shown to bind and stabilize Nod2 (wild type and Crohn's variant). Here, we go a step further by expressing the minimized peptide chain in cells and measuring the relative signaling response via a NF‐kB luciferase assay. The 71 minimized peptide chain was also used as a pharmacological tool through the principle of split Green Fluorescent Protein (GFP) complementation for development of a screen for pharmaceutical therapeutics. The peptide and Nod2 were tagged and detected with engineered self‐assembling fragments of GFP. When the peptide and Nod2 are bound, fluorescence is detected. Each pharmacoperone was individually incubated with the fluorescent protein complex. Effective pharmacoperones should competitively bind to Nod2, and thus disrupt the binding of the peptide and lead to lower fluorescence. The most promising pharmacoperones from the screen will be further analyzed for Nod2 stability and signaling in cell based assays. Successful identification of a pharmacoperone that mimics the natural stabilization of Hsp70 can be applied to restore function of a number of disease‐relevant mutated proteins. Support or Funding Information The authors would like to thank the following sources of funding: the Chemistry‐Biology Interface program of NIH, the Howard Hughes Medical Institute, and the Hofmann Fund through Nucleus.