8 research outputs found
Strength of selected interactions in terms of HIS3 reporter gene activation.
<p>The maximum concentration of 3-aminotrizol supporting visible growth of transformants is indicated.</p
The interaction matrix.
<p>In an βeach against allβ approach an overall number of 676 (26Γ26) effector domain combinations were analyzed. A β+β indicates an interaction between a particular pair, whereas βββ symbolizes no interaction. In total, the approach yielded 25 distinct associations, which actually corresponded to 7 unique pairs of interacting proteins, as well as 5 homodimerizations.</p
Cloning of constructs.
<p>GenBank <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004931#pone.0004931-Benson1" target="_blank">[66]</a> accession numbers, featured domains as well as corresponding amino acid residues are indicated, respectively.</p>*<p>NOD2 tripled mutation β E69K, D70K, D71K; CARD: caspase activation and recruitment domain, PYD: pyrin domain.</p
NOD2 directly interacts with NLRP1, NLRP3, and NLRP12.
<p><i>Left panel</i>, Yeast two-hybrid analysis of NOD2 CARDs (residues 1β267) showed interaction with NLRP1,-3 and -12, but not with other NLRP proteins (NLRP2, -7, -10, or -11; column 1). Lam c was used as a negative control (column 2). Interestingly, NOD2 mutant E69K maintained the binding to NLRP1, -3, and -12 (column 3), whereas NOD2 D70K (column 4) as well as the NOD2 triple mutant (3xmut, column 5) did not. An unrelated mutation not located within the prospective interaction interface had no effect (column 6). <i>Right panel</i>, Physical interaction of NOD2 and NLRP3 in human cells. Western analysis of lysates (IN) and immunoprecipitated complexes (IP) from HEK293T cells, transiently transfected with expression plasmid encoding human HA-NOD2 and FLAG-NLRP3. NLRP3 was immunoprecipitated from cell lysates using a FLAG-epitope specific antibody. Proteins were detected using anti-HA and anti-FLAG antibodies, respectively. As negative control, proteins were immunoprecipitated with FLAG-epitope specific antibody from lysates of HEK293T cells transiently transfected with HA-NOD2, but not FLAG-NLRP3.</p
RIPK2 CARD forms homodimers/-oligomers.
<p><i>Left panel</i>, Yeast two-hybrid analysis revealed that RIPK2 CARD (residues 427β527) forms homodimers/-oligomers (column 1 on SD/-4). Lamin c (Lam c) was used as control (column 5). <i>Right panel</i>, GST pull down assay. Specific binding of <sup>35</sup>S-labeled RIPK2 CARD (residues 427β527) was observed to recombinant expressed GST-RIPK2 CARD (residues 427β527), whereas binding to GST-Lam c was not detected. SD/-2: SD/-Leu/-Trp, SD/-4: SD/-Ade/-His/-Leu/-Trp.</p
Interaction of distinct NOD2 mutants with RIPK2.
<p><i>Left panel</i>, Yeast two-hybrid analysis. NOD2 CARD1+2 interacts with RIPK2 CARD (column 1). No interactions were observed for NOD2 variants harboring a disrupted acidic patch (transformation 2, 3, and 4). An unrelated mutation not affecting the interaction interface had no effect (column 5). <i>Right panel</i>, Autoradiography of an <i>in vitro</i> transcription/translation of NOD2 proteins. NOD2 constructs were expressed using similar amounts of DNA and lysates.</p
NLR protein-protein interactions.
<p>Respective protein-protein interactions were mined from the scientific literature or retrieved from MiMI <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004931#pone.0004931-Jayapandian1" target="_blank">[65]</a>. Results represent data from our yeast two-hybrid analysis (interaction observed or not observed). Baits and preys indicate individual domains tested in our analysis with interacting constructs in bold. References to reported interactions are specified.</p>Β§<p>Initially not observed by βeach against allβ approach, but observed subsequently with refined constructs.</p>*<p>No data on a direct interaction.</p
NOD2 interaction with distinct NLRP members.
<p><i>Upper panel</i>, A β+β indicates an interaction, βββ symbolizes no interaction (n.d.: not done). A short isoform of NOD2 (NOD2-S, residues 1β180), maintains the interaction with NLRP1, -3, and -12. Furthermore, a linker region within NLRP1 (residues 92β341) proved sufficient for interaction with NOD2. <i>Lower panel</i>, Schematic illustration of particular NOD2/NLRP1 constructs and their respective interactions. FIIND: Function to find domain.</p