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Dysfunctional Crohn's Disease-Associated NOD2 Polymorphisms Cannot be Reliably Predicted on the Basis of RIPK2 Binding or Membrane Association.

Parkhouse R, Monie TP - Front Immunol (2015)

Bottom Line: Loss of NOD2 signaling can result from a failure to detect ligand; alterations in cellular localization; and changes in protein interactions, such as an inability to interact with the downstream adaptor protein RIPK2.However, both these polymorphisms still associated with cellular membranes.Simply ascertaining whether or not NOD2 polymorphisms bind RIPK2 or associate with cellular membranes is not sufficient for determining their signaling competency.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge , Cambridge , UK.

ABSTRACT
Polymorphisms in NOD2 represent the single greatest genetic risk factor for the development of Crohn's disease. Three different non-synonomous NOD2 polymorphisms - R702W, G908R, and L1007fsincC - account for roughly 80% of all NOD2-associated cases of Crohn's disease and are reported to result in a loss of receptor function in response to muramyl dipeptide (MDP) stimulation. Loss of NOD2 signaling can result from a failure to detect ligand; alterations in cellular localization; and changes in protein interactions, such as an inability to interact with the downstream adaptor protein RIPK2. Using an overexpression system, we analyzed ~50 NOD2 polymorphisms reportedly connected to Crohn's disease to determine if they also displayed loss of function and if this could be related to alterations in protein localization and/or association with RIPK2. Just under half the polymorphisms displayed a significant reduction in signaling capacity following ligand stimulation, with nine of them showing near complete ablation. Only two polymorphisms, R38M and R138Q, lost the ability to interact with RIPK2. However, both these polymorphisms still associated with cellular membranes. In contrast, L248R, W355stop, L550V, N825K, L1007fsinC, L1007P, and R1019stop still bound RIPK2, but showed impaired membrane association and were unable to signal in response to MDP. This highlights the complex contributions of NOD2 polymorphisms to Crohn's disease and reiterates the importance of both RIPK2 binding and membrane association in NOD2 signaling. Simply ascertaining whether or not NOD2 polymorphisms bind RIPK2 or associate with cellular membranes is not sufficient for determining their signaling competency.

No MeSH data available.


Related in: MedlinePlus

Twenty-three NOD2 polymorphisms show a significant reduction in signaling following stimulation with MDP. The ability of NOD2 polymorphisms to signal via NFκB (black bars) or activate the IL-8 promoter (blue bars) was determined following stimulation with 100 ng/ml MDP. Signaling functionality is expressed as a percentage of wild-type receptor activity. All polymorphisms were tested in triplicate in a minimum of three separate experiments. Error bars show SEM. Data was log transformed and statistical significance determined using one-way ANOVA with Bonferroni post-test for multiple samples. Only polymorphisms showing a significant reduction in signaling are plotted (data for the other polymorphisms is provided in Figure S1 in Supplementary Material) – *p < 0.05, **p = < 0.01, ***p = < 0.001. A schematic of the NOD2 domain structure is provided with the location of polymorphisms showing <15% activity marked.
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Figure 1: Twenty-three NOD2 polymorphisms show a significant reduction in signaling following stimulation with MDP. The ability of NOD2 polymorphisms to signal via NFκB (black bars) or activate the IL-8 promoter (blue bars) was determined following stimulation with 100 ng/ml MDP. Signaling functionality is expressed as a percentage of wild-type receptor activity. All polymorphisms were tested in triplicate in a minimum of three separate experiments. Error bars show SEM. Data was log transformed and statistical significance determined using one-way ANOVA with Bonferroni post-test for multiple samples. Only polymorphisms showing a significant reduction in signaling are plotted (data for the other polymorphisms is provided in Figure S1 in Supplementary Material) – *p < 0.05, **p = < 0.01, ***p = < 0.001. A schematic of the NOD2 domain structure is provided with the location of polymorphisms showing <15% activity marked.

Mentions: To determine the effect of the NOD2 polymorphisms on receptor function, we assessed their ability to signal in response to MDP stimulation using NFκB and IL-8 reporter assays. Twenty-three of the polymorphisms, along with the Walker-B mutant D379A, showed a significant reduction in signaling (Figure 1). All polymorphisms that showed a significant reduction in signaling activity with the exception of A105T, D113N, D357A (IL-8 only), and P727C (NFκB only) were significantly impaired in both reporter systems. Nine of the polymorphisms (R38M, R138Q, L248R, W355stop, L550V, N825K, L1007fs, L1007P, and R1019stop) and the control D379A showed major functional impairment with signaling below 15% of wild-type NOD2 (Figure 1). None of the polymorphisms tested resulted in receptor hyperactivation in response to MDP (Figure S1 in Supplementary Material).


Dysfunctional Crohn's Disease-Associated NOD2 Polymorphisms Cannot be Reliably Predicted on the Basis of RIPK2 Binding or Membrane Association.

Parkhouse R, Monie TP - Front Immunol (2015)

Twenty-three NOD2 polymorphisms show a significant reduction in signaling following stimulation with MDP. The ability of NOD2 polymorphisms to signal via NFκB (black bars) or activate the IL-8 promoter (blue bars) was determined following stimulation with 100 ng/ml MDP. Signaling functionality is expressed as a percentage of wild-type receptor activity. All polymorphisms were tested in triplicate in a minimum of three separate experiments. Error bars show SEM. Data was log transformed and statistical significance determined using one-way ANOVA with Bonferroni post-test for multiple samples. Only polymorphisms showing a significant reduction in signaling are plotted (data for the other polymorphisms is provided in Figure S1 in Supplementary Material) – *p < 0.05, **p = < 0.01, ***p = < 0.001. A schematic of the NOD2 domain structure is provided with the location of polymorphisms showing <15% activity marked.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4597273&req=5

Figure 1: Twenty-three NOD2 polymorphisms show a significant reduction in signaling following stimulation with MDP. The ability of NOD2 polymorphisms to signal via NFκB (black bars) or activate the IL-8 promoter (blue bars) was determined following stimulation with 100 ng/ml MDP. Signaling functionality is expressed as a percentage of wild-type receptor activity. All polymorphisms were tested in triplicate in a minimum of three separate experiments. Error bars show SEM. Data was log transformed and statistical significance determined using one-way ANOVA with Bonferroni post-test for multiple samples. Only polymorphisms showing a significant reduction in signaling are plotted (data for the other polymorphisms is provided in Figure S1 in Supplementary Material) – *p < 0.05, **p = < 0.01, ***p = < 0.001. A schematic of the NOD2 domain structure is provided with the location of polymorphisms showing <15% activity marked.
Mentions: To determine the effect of the NOD2 polymorphisms on receptor function, we assessed their ability to signal in response to MDP stimulation using NFκB and IL-8 reporter assays. Twenty-three of the polymorphisms, along with the Walker-B mutant D379A, showed a significant reduction in signaling (Figure 1). All polymorphisms that showed a significant reduction in signaling activity with the exception of A105T, D113N, D357A (IL-8 only), and P727C (NFκB only) were significantly impaired in both reporter systems. Nine of the polymorphisms (R38M, R138Q, L248R, W355stop, L550V, N825K, L1007fs, L1007P, and R1019stop) and the control D379A showed major functional impairment with signaling below 15% of wild-type NOD2 (Figure 1). None of the polymorphisms tested resulted in receptor hyperactivation in response to MDP (Figure S1 in Supplementary Material).

Bottom Line: Loss of NOD2 signaling can result from a failure to detect ligand; alterations in cellular localization; and changes in protein interactions, such as an inability to interact with the downstream adaptor protein RIPK2.However, both these polymorphisms still associated with cellular membranes.Simply ascertaining whether or not NOD2 polymorphisms bind RIPK2 or associate with cellular membranes is not sufficient for determining their signaling competency.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge , Cambridge , UK.

ABSTRACT
Polymorphisms in NOD2 represent the single greatest genetic risk factor for the development of Crohn's disease. Three different non-synonomous NOD2 polymorphisms - R702W, G908R, and L1007fsincC - account for roughly 80% of all NOD2-associated cases of Crohn's disease and are reported to result in a loss of receptor function in response to muramyl dipeptide (MDP) stimulation. Loss of NOD2 signaling can result from a failure to detect ligand; alterations in cellular localization; and changes in protein interactions, such as an inability to interact with the downstream adaptor protein RIPK2. Using an overexpression system, we analyzed ~50 NOD2 polymorphisms reportedly connected to Crohn's disease to determine if they also displayed loss of function and if this could be related to alterations in protein localization and/or association with RIPK2. Just under half the polymorphisms displayed a significant reduction in signaling capacity following ligand stimulation, with nine of them showing near complete ablation. Only two polymorphisms, R38M and R138Q, lost the ability to interact with RIPK2. However, both these polymorphisms still associated with cellular membranes. In contrast, L248R, W355stop, L550V, N825K, L1007fsinC, L1007P, and R1019stop still bound RIPK2, but showed impaired membrane association and were unable to signal in response to MDP. This highlights the complex contributions of NOD2 polymorphisms to Crohn's disease and reiterates the importance of both RIPK2 binding and membrane association in NOD2 signaling. Simply ascertaining whether or not NOD2 polymorphisms bind RIPK2 or associate with cellular membranes is not sufficient for determining their signaling competency.

No MeSH data available.


Related in: MedlinePlus