Formation and structure of a NAIP5-NLRC4 inflammasome induced by direct interactions with conserved N- and C-terminal regions of flagellin

The NOD-like receptors NAIP5 and NLRC4 play an essential role in the innate immune response to the bacterial tail protein flagellin. Upon flagellin detection, NAIP5 and NLRC4 form a hetero-oligomeric inflammasome that induces caspase-1-dependent cell death. So far, both the mechanism of formation of the NAIP5-NLRC4 inflammasome and its structure are poorly understood. In this study we combine inflammasome reconstitution in HEK293 cells, purification of inflammasome components, and negative stain electron microscopy to address these issues. We find that a Salmonella typhimurium flagellin fragment comprising the D0 domain and the neighboring spoke region is able to co-precipitate NAIP5 and induce formation of the NAIP5-NLRC4 inflammasome. Comparison with smaller fragments indicates that flagellin recognition is mediated by its C-terminal residues as well as the spoke region. We reconstitute the inflammasome from purified flagellin, NAIP5, and NLRC4, thus proving that no other cellular components are required for its formation. Electron micrographs of the purified inflammasome provide unprecedented insight into its architecture, revealing disk-like complexes consisting of 11 or 12 protomers in which NAIP5 and NLRC4 appear to occupy equivalent positions. On the basis of our data, we propose a model for inflammasome formation wherein direct interaction of flagellin with a single NAIP5 induces the recruitment and progressive incorporation of NLRC4, resulting in the formation of a hetero-oligomeric inflammasome

The comparative responsiveness of Hospital Universitario Princesa Index and other composite indices for assessing rheumatoid arthritis activity

Objective To evaluate the responsiveness in terms of correlation of the Hospital Universitario La Princesa Index (HUPI) comparatively to the traditional composite indices used to assess disease activity in rheumatoid arthritis (RA), and to compare the performance of HUPI-based response criteria with that of the EULAR response criteria. Methods Secondary data analysis from the following studies: ACT-RAY (clinical trial), PROAR (early RA cohort) and EMECAR (pre-biologic era long term RA cohort). Responsiveness was evaluated by: 1) comparing change from baseline (Delta) of HUPI with Delta in other scores by calculating correlation coefficients; 2) calculating standardised effect sizes. The accuracy of response by HUPI and by EULAR criteria was analyzed using linear regressions in which the dependent variable was change in global assessment by physician (Delta GDA-Phy). Results Delta HUPI correlation with change in all other indices ranged from 0.387 to 0.791); HUPI's standardized effect size was larger than those from the other indices in each database used. In ACT-RAY, depending on visit, between 65 and 80% of patients were equally classified by HUPI and EULAR response criteria. However, HUPI criteria were slightly more stringent, with higher percentage of patients classified as non-responder, especially at early visits. HUPI response criteria showed a slightly higher accuracy than EULAR response criteria when using Delta GDA-Phy as gold standard. Conclusion HUPI shows good responsiveness in terms of correlation in each studied scenario (clinical trial, early RA cohort, and established RA cohort). Response criteria by HUPI seem more stringent than EULAR''s

Implications for collagen I chain registry from the structure of the collagen von Willebrand factor A3 domain complex

Fibrillar collagens, the most abundant proteins in the vertebrate body, are involved in a plethora of biological interactions. Plasma protein von Willebrand factor (VWF) mediates adhesion of blood platelets to fibrillar collagen types I, II, and III, which is essential for normal haemostasis. High affinity VWF-binding sequences have been identified in the homotrimeric collagen types II and III, however, it is unclear how VWF recognizes the heterotrimeric collagen type I, the superstructure of which is unknown. Here we present the crystal structure of VWF domain A3 bound to a collagen type III-derived homotrimeric peptide. Our structure reveals that VWF-A3 interacts with all three collagen chains and binds through conformational selection to a sequence that is one triplet longer than was previously appreciated from platelet and VWF binding studies. The VWF-binding site overlaps those of SPARC (also known as osteonectin) and discodin domain receptor 2, but is more extended and shifted toward the collagen amino terminus. The observed collagen-binding mode of VWF-A3 provides direct structural constraints on collagen I chain registry. A VWF-binding site can be generated from the sequences RGQAGVMF, present in the two α1(I) chains, and RGEOGNIGF, in the unique α2(I) chain, provided that α2(I) is in the middle or trailing position. Combining these data with previous structural data on integrin binding to collagen yields strong support for the trailing position of the α2(I) chain, shedding light on the fundamental and long-standing question of the collagen I chain registry

Implications for collagen I chain registry from the structure of the collagen von Willebrand factor A3 domain complex

Fibrillar collagens, the most abundant proteins in the vertebrate body, are involved in a plethora of biological interactions. Plasma protein von Willebrand factor (VWF) mediates adhesion of blood platelets to fibrillar collagen types I, II, and III, which is essential for normal haemostasis. High affinity VWF-binding sequences have been identified in the homotrimeric collagen types II and III, however, it is unclear how VWF recognizes the heterotrimeric collagen type I, the superstructure of which is unknown. Here we present the crystal structure of VWF domain A3 bound to a collagen type III-derived homotrimeric peptide. Our structure reveals that VWF-A3 interacts with all three collagen chains and binds through conformational selection to a sequence that is one triplet longer than was previously appreciated from platelet and VWF binding studies. The VWF-binding site overlaps those of SPARC (also known as osteonectin) and discodin domain receptor 2, but is more extended and shifted toward the collagen amino terminus. The observed collagen-binding mode of VWF-A3 provides direct structural constraints on collagen I chain registry. A VWF-binding site can be generated from the sequences RGQAGVMF, present in the two α1(I) chains, and RGEOGNIGF, in the unique α2(I) chain, provided that α2(I) is in the middle or trailing position. Combining these data with previous structural data on integrin binding to collagen yields strong support for the trailing position of the α2(I) chain, shedding light on the fundamental and long-standing question of the collagen I chain registry

Cryoelectron tomography of the NAIP5/NLRC4 inflammasome: implications for NLR activation

Inflammasomes are high molecular weight protein complexes that play a crucial role in innate immunity by activating caspase-1. Inflammasome formation is initiated when molecules originating from invading microorganisms activate nucleotide-binding domain and leucine-rich repeat-containing receptors (NLRs) and induce NLR multimerization. Little is known about the conformational changes involved in NLR activation and the structural organization of NLR multimers. Here, we show by cryoelectron tomography that flagellin-induced NAIP5/NLRC4 multimers form right- and left-handed helical polymers with a diameter of 28 nm and a pitch of 6.5 nm. Subtomogram averaging produced an electron density map at 4 nm resolution, which was used for rigid body fitting of NLR subdomains derived from the crystal structure of dormant NLRC4. The resulting structural model of inflammasome-incorporated NLRC4 indicates that a prominent rotation of the LRR domain of NLRC4 is necessary for multimer formation, providing unprecedented insight into the conformational changes that accompany NLR activation

Formation and Structure of a NAIP5-NLRC4 Inflammasome Induced by Direct Interactions with Conserved N- and C-terminal Regions of Flagellin

Pathophysiology and treatment of rheumatic disease