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

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

Characterisation of the interaction between NOXO1 and NOXA1.

<p>(<b>A</b>) Upper part shows the raw calorimetric data for the interaction between human NOXA1 SH3 and NOXO1 SH3_AB–E. Lower part shows the integrated heat changes, corrected for heat of dilution, and fitted to a single site binding model. (▪) Human NOXA1 SH3 titrated into human NOXO1 SH3_AB–E (▴) Mouse NOXA1 SH3 titrated into mouse NOXO1 SH3_AB–E. (<b>B</b>) Upper part shows raw calorimetric data for the interaction of peptideA and human NOXA1 SH3. Lower part shows the integrated heat changes, corrected for heat of dilution, and fitted to a single site binding model. (▪) PeptideA titrated into human NOXA1 SH3 (▴) PeptideA titrated into mouse NOXA1 SH3.</p

Linear ubiquitin chains: enzymes, mechanisms and biology

Ubiquitination is a versatile post-translational modification that regulates a multitude of cellular processes. Its versatility is based on the ability of ubiquitin to form multiple types of polyubiquitin chains, which are recognized by specific ubiquitin receptors to induce the required cellular response. Linear ubiquitin chains are linked through Met 1 and have been established as important players of inflammatory signalling and apoptotic cell death. These chains are generated by a ubiquitin E3 ligase complex called the linear ubiquitin chain assembly complex (LUBAC) that is thus far the only E3 ligase capable of forming linear ubiquitin chains. The complex consists of three subunits, HOIP, HOIL-1L and SHARPIN, each of which have specific roles in the observed biological functions of LUBAC. Furthermore, LUBAC has been found to be associated with OTULIN and CYLD, deubiquitinases that disassemble linear chains and counterbalance the E3 ligase activity of LUBAC. Gene mutations in HOIP, HOIL-1L and OTULIN are found in human patients who suffer from autoimmune diseases, and HOIL-1L mutations are also found in myopathy patients. In this paper, we discuss the mechanisms of linear ubiquitin chain generation and disassembly by their respective enzymes and review our current understanding of their biological functions and association with human diseases

Thermodynamic parameters for the CARDa-CARDb interaction.

<p>Thermodynamic parameters for the CARDa-CARDb interaction.</p

Complex formation between NOD2 CARDab and RIP2-CARD.

<p>(A) NOD2 CARDab with a N-terminal GST-tag and RIP2-CARD equipped with a N-terminal GB1-tag and a C-terminal His₆-tag were co-expressed and pulled-down with glutathione sepharose beads. From left: Lane 1) Protein Marker, GE Healthcare. Lane 2) Soluble lysate. Lane 3) Bead eluate after 3C-protease cleavage. NOD2-CARDab 22.0 kDa and GB1-RIP2 CARD-His 18.6 kDa are indicated by green and red arrows, respectively. Lane 4) Supernatant bound to beads. GST-NOD2 CARDab 48.5 kDa (green) and GB1-RIP2 CARD-His 18.6 kDa (red) are indicated by arrows. (B) Effect of NOD2 CARDab single point mutations on RIP2 CARD binding. A representative cross section of the mutants tested are shown. GST-NOD2 CARDab 48.5 kDa and GB1-RIP2 CARD-His 18.6 kDa are indicated by green and red arrows, respectively. * = residual expression of GST. (C) Effect of RIP2 CARD single point mutations on NOD2 CARDab binding. A cross section of the mutants tested are shown. GST-NOD2 CARDab 48.5 kDa and GB1-RIP2 CARD-His 18.6 kDa are indicated by green and red arrows, respectively. (D) Effect of RIP2 CARD single point mutations on NOD1 CARD binding. A cross section of the mutants tested are shown. GST-NOD1 CARD 40.9 kDa (aa17–138) and GB1-RIP2 CARD-His 18.6 kDa are indicated by blue and red arrows, respectively.</p

Characterisation of the interaction between NOXO1 SH3_AB–E and p22^phox.

<p>Upper part shows the raw calorimetric data for the interaction of mouse p22^phoxC- NOXO1 SH3_AB–E. Lower part shows the integrated heat changes, corrected for heat of dilution, and fitted to a single site binding model. (▪) Human p22^phoxC titrated into human NOXO1 SH3_AB–E (▴) Mouse p22^phoxC titrated into mouse NOXO1 SH3_AB–E.</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

Characterisation of intermolecular interactions between NOXO1 and NOXA1.

<p>All measurements were performed at 18°C. The <i>K</i>_d is given in units of 10⁻⁶ M, Δ<i>H</i> and TΔ<i>S</i> are given in kcal mol⁻¹ (1 kcal/mol≡4.184 kJ/mol). The stoichiometry of complex formation for each binding site is N = 1.0±0.1.</p