Screening Of NLRP3Interacting Proteins By Yeast Two-Hybrid

Abstract:Multiple recognition systems have coevolved in mammals so as to maintain the normal interactions with the commensal flora and to initiate immune responses to pathogens and stimulations in order to maintain the tissue homeostasis. Unlike the endosomal and extracellularToll-like receptors which mainly recognize pathogen-associated molecular patterns in microbes, a multitude of cytosolic receptors recognize not only intracellular pathogen-associated molecular patterns but also the host-derived signals known as’damage-associated molecular patterns’, such as the endogenous danger signals that are generated by stress, tissue damage and cell death.Nod-like receptors (NLRs) are cytosolic pattern-recognition receptors. The initial concept of "inflammasome" was a molecule platform composed of NLRP1, ASC, caspase1and caspase5. This large complex, was proved to be necessary for the activation of pro-inflammatory cytokine IL-1β. After that, more and more sensor proteins from NLRs family and PYHINs family were proved to be able to form inflammasome with the presence of DAMPs and PAMPs responses. Until now, inflammasome is a notable landmark, by common consent, in the field of cytosolic immune surveillance system.Targeting therapies based upon inflammasome are currently being studied. Therefore, the further exploration of molecule mechanisms of NLRP3signalling pathway regulation may provide important clues for revealing the relationship between inflammation, immunity and diseases and meanwhile provide new potential targets for diverse therapies.The research upon NLRP3inflammasome and its interacting proteins in recent decades have deepened our understanding in the regulation of NLRP3function. To further explore the signalling mechanisms and biological function of inflammasome, yeast two-hybrid assays was used in this experiment to screen the interaction proteins of NLRP3from the human leukocyte cDNA library using three basic domains of NLRP3:PYD, NACHT and LRR as baits. Through repeated confirmations, we got4 NLRP3interaction proteins from87candidates, respectively as:ECHS1(Homo sapiens enoyl CoA hydratase, short chain,1), UBE2I (ubiquitin-conjugating enzyme E2I), RPL34(Homo sapiens ribosomal protein L34) and PXN (Homo sapiens paxillin). To further investigate the interaction between these proteins, Co-IP was performed using HEK293T to examine the interaction between NLRP3and ECHS and confirmed this interaction. Our discovery of the NLRP3and ECHS complex might be a novel NLRP3regulator and further studies of this complex might lead to the discovery of a new mechanism concerning regulation of NLRP3function. There are15figures,3tables,58references...