Functional and biochemical characterization of Nod2 and Hsp70, important proteins involved in Crohn's disease
Date
2015
Authors
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Publisher
University of Delaware
Abstract
Microbes are detected by specific host pattern recognition receptors (PRR) through the pathogen-associated molecular patterns (PAMPs). Nucleotide binding oligomerization domain-containing protein 2 (Nod2) is an intracellular PRR that recognizes fragments of bacterial cell wall. Nod2 is important to human biology, as when it is mutated it loses the ability to respond properly to bacterial cell wall fragments and subsequently mutations in Nod2 correlated to the development of a number of human diseases, including Crohn’s disease. In order to determine the mechanisms of misactivation in the Nod2 Crohn’s associated Nod2 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’s associated Nod2 mutations. Hsp70 is a highly ubiquitous protein that is responsible for stabilizing many proteins within the cell. Recently, Hsp70 has been linked with inflammation, especially in the regulation of anti-inflammatory molecules. This thesis uses a combination of molecular cellular biology and biochemistry to dissect the role of Hsp70 in Nod2 signaling. ☐ The preliminary identification of the Hsp70:Nod2 interaction was confirmed using a series of co-immunoprecipitation experiments in which the domains of Hsp70 and Nod2 that are necessary and sufficient for the interaction were identified. We identified that the nucleotide binding domain (NBD) and the leucine rich repeat (LRR) domain of Nod2 interact with Hsp70, whereas the substrate binding domain (SBD) of Hsp70 interacts with Nod2. ☐ Limited proteolysis experiments were performed to probe if the mechanism of Hsp70 stabilization of Nod2 was dependent of the ATPase activity of Hsp70. For these experiments, a mutant of Hsp70 that is capable of binding to ADP but not ATP was utilized. Our data suggest that the ATPase activity is dispensable for Nod2 stabilization. Additional biochemical experiments imply that a co-chaperone, Hsp40, plays a role in the Nod2-Hsp70 interaction. Thus, these biochemical studies have identified that interaction of Hsp70-Hsp40 with Nod2 is sufficient for stabilization. ☐ In order to determine the effect of Hsp70 in cells on Nod2 signaling, a series of experiments were performed in which the levels of Hsp70 was modulated. If the levels of Hsp70 were increased, Nod2’s cellular response to bacterial cell wall fragments increased. Alternatively, if Hsp70 levels were decreased, using a Hsp70 inhibitor, KNK437, Nod2 mediated NF-κB activation in response to bacterial cell wall stimulation decreased. We found Hsp70 to regulate Nod2’s half-life, as increasing the Hsp70 level in cells increased Nod2’s half-life, and down-regulating Hsp70 decreased Nod2’s half-life. ☐ The Hsp70:Nod2 interaction also was present with the Crohn’s associated Nod2 variants. We found that the expression level of the Crohn’s associated Nod2 variants was lower compared to that of wild-type. 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’s mutants is sufficient for rescuing the ability of these mutations to signal in the presence of a bacterial cell wall ligand. Thus, by identifying Hsp70 as a Nod2 interactor, this thesis has: (1) allowed for the development of two Nod2 stabilization assays (in vitro proteolysis and cellular half-life) to be developed, (2) accelerated the endogenous expression and purification of both wild type and Crohn’s associated Nod2 mutants and (3) identified a novel mechanism, stabilization of Nod2, to treat Crohn’s disease.
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Keywords
Chaperone, Crohn's disease, Hsp70, Innate immunity, Nod2, Protein stability