NLRP7 inter-domain interactions: the NACHT-associated domain is the physical mediator for oligomeric assembly

Abstract

Mutations in NLRP7 (NOD-like-receptor family, pyrin domain containing 7) are responsible for a type of recurrent pregnancy loss known as recurrent hydatidiform mole (HYDM1). This condition is characterized by abnormal growth of the placenta, a lack of proper embryonic development and abnormal methylation patterns at multiple imprinted loci in diploid biparental molar tissues. The role of NLRP7 protein in the disease manifestation is currently not clear. In order to better understand how the effects of HYDM1 are associated with mutations on the structure of NLRP7, we performed an inter-domain interaction screen using a yeast two-hybrid system. Additionally, we generated in silico structural models of NLRP7 in its non-activated and activated forms. Our observations from the yeast two-hybrid screen and modeling suggest that the NACHT-associated domain (NAD) of the NLRP7 protein is central to its oligomeric assembly. Upon activation, the NAD and a small part of the leucine rich repeat (LRR) of one molecule emerged out of the protective LRR domain and interact with the NACHT domain of the second molecule to form an oligomer. Furthermore, we investigated the molecular basis for the pathophysiological effect of four missense mutations, three HYDM1-causing and one rare non-synonymous variant, on the protein using confocal microscopy of transiently transfected NLRP7 in HEK293T cells and in silico structural analysis. We found that with the two clinically severe missense mutations, L398R and R693W, the normal molecule to molecule interaction was apparently affected thus decreasing their oligomerization potential while aggresome formation was increased; these changes could disturb the normal downstream functions of NLRP7 and therefore be a possible molecular effect underlying their pathophysiological impact.

ATP binding by NLRP7 is required for inflammasome activation in response to bacterial lipopeptides

“…We previously reported that several HM-linked mutations yield gain-of-function phenotypes in a reconstituted inflammasome system (Khare et al, 2012). In agreement, a recent study indicated that certain HM-linked mutations within the NACHT increase NLRP7 oligomerization by destabilizing the closed NACHT conformation (Singer et al, 2014). However, HM-linked mutations have not been identified within the Walker A motif (Touitou et al, 2004), which according to our results would yield in a loss-of-function phenotype.…”

“…Here we demonstrate that NLRP7 binds and hydrolyzes ATP through its predicted NBD and that this activity is necessary for NLRP7 oligomerization and inflammasome activation, reminiscent of NLRP3 (Duncan et al, 2007). During the preparation of our manuscript, El-Maarri and colleagues demonstrated that the NLRP7 NAD is required for NLPR7 dimerization (Singer et al, 2014). We extend this finding to demonstrate that ATP binding and hydrolysis is essential for oligomerization and inflammasome activation in response to acLPs and S. aureus , thus demonstrating a functional consequence of ATP binding.…”

“…It has also been reported to co-localize with the Golgi and microtubules and inhibit IL-1β (Messaed et al, 2011a,b; Kinoshita et al, 2005). NLRP7 contains a predicted NBD domain and has recently been shown to self-associate through its NAD domain (Singer et al, 2014). However, it is currently unknown, if the NLRP7 NBD has ATPase activity and if this is involved in inflammasome activation.…”

NLRP7 contributes to in vitro decidualization of endometrial stromal cells

“…NLRP7 siRNA also only down-regulated the small isoform, and interfered with the decidualization process, while preserving the large isoform. Because the LRR domain of NLRP7 is responsible for the recognition of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) or protein-protein interaction [ 46 , 47 ], the two isoforms that differ in the number of LRR domains may have distinct functions in the endometrial stromal cells. Data supporting the aforementioned hypothesis comes from two previous studies, in which NLRP7 proteins carrying mutations in the LRR regions caused dysregulated IL-1β expression in the LPS-stimulated monocytes or reconstituted NLRP7 inflammasome [ 3 , 5 ].…”

ATP-Binding and Hydrolysis in Inflammasome Activation

“…The three-dimensional structure of the isolated NLRP7 pyrin domain was obtained by NMR spectroscopy [ 143 ]. Furthermore, the intermolecular interfaces for multimeric NLRP7 complexes were explored using a combination of yeast two-hybrid and co-immunoprecipitation with subsequent application to a homology model of the NLRP7 inflammasome superstructure generated with the APAF1 backbone [ 144 ]. When modelling of the activated and non-activated NLRP7 protein was completed, several important structural changes were proposed.…”