Limits...
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

The relationship between residue conservation and functional impact. (A) Scatter plot of percentage residue identity and percentage NFκB signaling activity for all polymorphisms studied. Polymorphisms that cause a significant reduction in signaling are colored red and labeled. The points for R38M and R138Q overlap on the graph. (B) WebLogo representation of residue conservation for those polymorphisms producing a significant reduction in signaling. Polymorphism position is denoted on the x-axis and colored by level of NFκB signaling compared to WT NOD2 – red <15%, blue 15–45%, gray 46–75%, and black >75%. Amino acid residues are colored as follows: basic residues – blue; acidic residues – red; hydrophobic residues – black; polar acidic residues – green; and polar basic residues – pink.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4597273&req=5

Figure 2: The relationship between residue conservation and functional impact. (A) Scatter plot of percentage residue identity and percentage NFκB signaling activity for all polymorphisms studied. Polymorphisms that cause a significant reduction in signaling are colored red and labeled. The points for R38M and R138Q overlap on the graph. (B) WebLogo representation of residue conservation for those polymorphisms producing a significant reduction in signaling. Polymorphism position is denoted on the x-axis and colored by level of NFκB signaling compared to WT NOD2 – red <15%, blue 15–45%, gray 46–75%, and black >75%. Amino acid residues are colored as follows: basic residues – blue; acidic residues – red; hydrophobic residues – black; polar acidic residues – green; and polar basic residues – pink.

Mentions: Amino acids with important functional roles regularly show higher levels of evolutionary conservation. We updated our previous cross-species alignment of NOD2 (22) and compared the level of evolutionary conservation for each of our polymorphic NOD2 variants (Figure 2; Figures S1 and S2 in Supplementary Material). Consistent with a key functional role, nine of the polymorphisms that showed a significant reduction in signaling were completely conserved across all aligned species, and a further two (E825 and L1007) were conserved in 29/30 species (Figure 2). However, high levels of residue conservation could not be used as a direct predictor of functional impact as seven completely conserved residues showed no impact on signaling (Figure 2A). Interestingly, V162 is only conserved in five of the 30 species and despite all of the substitutions being conservative hydrophobic ones, the polymorphism V162I signals at <25% of the wild-type protein (Figures 1 and 2B). In fact 10 species, including members of the bovine, porcine, caprine, and canine families, all possess an isoleucine in this position and therefore may show impaired NOD2 functionality.


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)

The relationship between residue conservation and functional impact. (A) Scatter plot of percentage residue identity and percentage NFκB signaling activity for all polymorphisms studied. Polymorphisms that cause a significant reduction in signaling are colored red and labeled. The points for R38M and R138Q overlap on the graph. (B) WebLogo representation of residue conservation for those polymorphisms producing a significant reduction in signaling. Polymorphism position is denoted on the x-axis and colored by level of NFκB signaling compared to WT NOD2 – red <15%, blue 15–45%, gray 46–75%, and black >75%. Amino acid residues are colored as follows: basic residues – blue; acidic residues – red; hydrophobic residues – black; polar acidic residues – green; and polar basic residues – pink.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: The relationship between residue conservation and functional impact. (A) Scatter plot of percentage residue identity and percentage NFκB signaling activity for all polymorphisms studied. Polymorphisms that cause a significant reduction in signaling are colored red and labeled. The points for R38M and R138Q overlap on the graph. (B) WebLogo representation of residue conservation for those polymorphisms producing a significant reduction in signaling. Polymorphism position is denoted on the x-axis and colored by level of NFκB signaling compared to WT NOD2 – red <15%, blue 15–45%, gray 46–75%, and black >75%. Amino acid residues are colored as follows: basic residues – blue; acidic residues – red; hydrophobic residues – black; polar acidic residues – green; and polar basic residues – pink.
Mentions: Amino acids with important functional roles regularly show higher levels of evolutionary conservation. We updated our previous cross-species alignment of NOD2 (22) and compared the level of evolutionary conservation for each of our polymorphic NOD2 variants (Figure 2; Figures S1 and S2 in Supplementary Material). Consistent with a key functional role, nine of the polymorphisms that showed a significant reduction in signaling were completely conserved across all aligned species, and a further two (E825 and L1007) were conserved in 29/30 species (Figure 2). However, high levels of residue conservation could not be used as a direct predictor of functional impact as seven completely conserved residues showed no impact on signaling (Figure 2A). Interestingly, V162 is only conserved in five of the 30 species and despite all of the substitutions being conservative hydrophobic ones, the polymorphism V162I signals at <25% of the wild-type protein (Figures 1 and 2B). In fact 10 species, including members of the bovine, porcine, caprine, and canine families, all possess an isoleucine in this position and therefore may show impaired NOD2 functionality.

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