The Tandem CARDs of NOD2: Intramolecular Interactions and Recognition of RIP2

Caspase recruitment domains (CARDs) are homotypic protein interaction modules that link the stimulus-dependent assembly of large signaling platforms such as inflammasomes to the activation of downstream effectors that often include caspases and kinases and thereby play an important role in the regulation of inflammatory and apoptotic signaling pathways. NOD2 belongs to the NOD-like (NLR) family of intracellular pattern recognition receptors (PRR) and induces activation of the NF-κB pathway in response to the recognition of bacterial components. This process requires the specific recognition of the CARD of the protein kinase RIP2 by the tandem CARDs of NOD2. Here we demonstrate that the tandem CARDs of NOD2 are engaged in an intramolecular interaction that is important for the structural stability of this region. Using a combination of ITC and pull-down experiments we identify distinct surface areas that are involved in the intramolecular tandem CARD interaction and the interaction with the downstream effector RIP2. Our findings indicate that while CARDa of NOD2 might be the primary binding partner of RIP2 the two CARDs of NOD2 do not act independently of one another but may cooperate to from a binding surface that is distinct from that of single CARDs

Det stora steget : Om elevers syn på hur förberedda de är att möta gymnasieskolans naturvetenskap

Övergången från grundskolan till gymnasieskolan är en stor händelse för många elever. Detställs högre och fler krav på eleverna när de börjar gymnasiet och deras förkunskaper harbetydelse för hur väl de klarar av utbildningen. I denna undersökning fick elever i årskurs ettpå det naturvetenskapliga programmet svara på frågor om de anser att de hade tillräckligakunskaper i de naturvetenskapliga ämnena från högstadiet. Undersökningen visar att de flestatycker att de hade tillräckliga kunskaper i biologi, kemi och matematik men inte i fysik.Kursplanen för fysik på gymnasiet skiljer sig från de andra kursplanerna, genom att det intestår lika tydligt att undervisningen ska bygga på elevernas kunskaper i grundskolan.Respondenterna i undersökningen fick även svara på frågor om vilket ämne som var svårt,roligt och om de läste i NO-block eller i separata ämnen på högstadiet. Undersökningen visaratt det finns en skillnad mellan flickor och pojkar när det gäller deras attityder och intresse

Surrogate potency assays: Comparison of binding profiles complements dose response curves for unambiguous assessment of relative potencies

Surface plasmon resonance (SPR) systems are widely used for detailed characterization of antibody activities including antigen and Fc-receptor binding. During the later stages of development, where the focus is to ensure that established critical quality attributes (CQAs) are maintained during cell culture, purification and formulation processes, analysis is simplified, and relative potencies are often determined. Here, simulation of binding data revealed that relative potency values, determined via parallel line analysis (PLA) and half maximal effective concentration (EC50) analysis accurately reflect changes in active concentration only if binding kinetics remain unchanged. Changes in the association rate constant shifted dose response curves, and therefore relative potencies, in the same way as changes in analyte concentration do. However, for interactions characterized by stable binding, changes in the dissociation rate constant did not result in any shift, suggesting that this type of change may go unnoticed in the dose response curve. Thus, EC50 and PLA analyses of dose response curves obtained with an anti-TNF-α antibody were complemented with the Biacore functionality for sensorgram comparison analysis, whereby changes in antigen and Fc-receptor binding profiles could be detected. Next, analysis of temperature stressed TNF-α antibody revealed that calibration free concentration analysis (CFCA) data correlated perfectly with relative potency values. Together, these results demonstrate that combinations of SPR based dose response curves, sensorgram comparison and CFCA can be used to strengthen the confidence in relative potency assessments, and suggest that SPR can potentially be used as a surrogate potency assay in the quality control of biotherapeutic medicines. Keywords: Surface plasmon resonance, EC50, Sensorgram comparison, Calibration free concentration analysis, Surrogate potency assay, TNF-

The interaction of NOD2 CARDab mutants with the CARD of RIP2.

<p>Presence (+)/absence (−) of RIP2 CARD on the beads following coexpression and GST-pull downs as analyzed by SDS-PAGE. N/D = not determined (no or too low expression for evaluation).</p

Oligomeric state of the CARDs of NOD2.

<p>(A) Schematic representation of the domain structure of human NOD2 and the domain boundaries of the constructs (tandem CARD, CARDa and CARDb) used in this study. (B) AUC Sedimenation Equilibrium resulted in MW of 22 kDa±0.5 kDa for CARDab (calculated MW = 22043 Da). The sample was run at three different concentrations and three different speeds (18, 22, 26 kprm). 11 scans were collected over 4 days. (C) AUC Sedimentation Velocity resulted in MW of 10–11 kDa for CARDa (blue line, calculated MW = 11155 kDa) and CARDb (red line, calculated MW = 11403 kDa), respectively. Samples were run at OD₆₀₀∼0.5. SedFit was used for data analysis. A sample containing equal molar amounts of CARDa and CARDb displayed a MW of ∼18 kDa.</p

Residues involved in the NOD2-RIP2 interaction.

<p>(A) Structure of the CARD-CARD complex between Apaf-1 (light blue) and procaspase-9 (yellow), pdb ID 3YGS. The relative location of residues that were identified to disrupt the NOD2-RIP2 interaction has been mapped onto the CARD-CARD structure, based on the alignment shown in (B). These include R38 and R86 (shown in dark blue) located in CARDa of NOD2 that are shown mapped onto the CARD of procaspase-9 and D461, E472, D473, E475 and D492 in RIP2 (shown in red), mapped onto the CARD of Apaf-1. (B) The featured residues are highly conserved in CARDs. CARDa R38 and R86 correspond to two (R13 and R56) of the three basic residues in procaspase-9 (shown in blue) that are crucial for the interaction with Apaf-1. CARDa has no equivalent to the third residue, R52. Conversely, RIP2 CARD D461 corresponds to Apaf-1 D27 and RIP2 E472, D473 and E475 are located in the region of Apaf-1 E40. Apaf-1 D27 and E40 (shown in red) are both crucial for the interaction with caspase-9.</p

The interaction of RIP2 CARD mutants with NOD2 CARDab and NOD1 CARD.

<p>Presence (+)/absence (−) of RIP2 CARD on the beads following coexpression and GST-pull downs as analyzed by SDS-PAGE.</p

Thermodynamics of the NOD2 CARDa-CARDb interaction.

<p>(A) ITC measurement of complex formation between CARDa in the syringe (475 µM) and CARDb in the cell (45 µM). T = 25°C. The binding isotherm was fitted to a one-site binding model with a K_d of 1.1 µM. A control experiment of CARDa into buffer is shown. (B) Determination of the heat capacity, ΔC_p. Enthalpies, ΔH, from CARDa-CARDb titrations at different temperatures were plotted against the temperatures. Linear regression analysis gave ΔC_p = dΔH/dT = −450 cal/(mole °C). (C) Effect of CARDa point mutations as monitored by ITC at 25°C. Titration of CARDa E69K (345 µM) into CARDb (40 µM) is shown in blue, CARDa E72K (205 µM) into CARDb (26 µM) in green and CARDa R86A (504 µM) into CARDb (62 µM) in red. The titrations were performed in the same buffer as in (A).</p

Salt dependence of the CARDa-CARDb interaction.

<p>ITC measurements were performed at 30°C in 50 mM Tris-HCl, 2 mM DTT, pH 7.5 with the ionic strength ranging from 50–1000 mM NaCl. Sample concentrations were 430–594 µM in the syringe (CARDa) and 38–52 µM in the cell (CARDb). The low n-value (N = 0.6) obtained at 50 mM NaCl may reflect that CARDb is partially unfolded at this NaCl concentration.</p