Establishing a novel biochemical platform to reconstitute innate immune signalling events

• Main Author: Koh, Chin Keat
• Other Authors: Wu Bin
• Format: Final Year Project
• Language: English
• Published: 2015
• Subjects: DRNTU::Science::Biological sciences::Molecular biology
• Online Access: http://hdl.handle.net/10356/65618
• Institution: Nanyang Technological University

NLR family, pyrin domain containing proteins (NLRPs) are a subset of intracellular pattern recognition receptors. NLRPs involved in inflammatory response are known to associate with the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) to initiate inflammasome assembly. The inflammasome functions as a caspase processing centre, catalyzing the activation of caspase-1 and caspase substrates, interleukin-1β/18. Mutations in NLRP1 and NLRP3 may lead to autoimmune and auto-inflammatory diseases. These malfunctioned receptors are also implicated in cancers, Alzheimer's and other metabolic diseases. With greater understanding of their molecular regulatory mechanisms, better treatment strategies could be developed. The lack of a convenient method to study induced oligomerization events prompted us to propose an in vitro oligomerization assay as a tool for investigating ASC interactions. This study reports preliminary establishment of this assay. ASC full-length oligomers were able to induce oligomerization of refolded ASC-pyrin monomers. Neither the pyrin nor CARD domains of NLRP1 and NLRP3 were active enough to produce similar effects, at the moment. Since it was previously reported that NLRP3-pyrin directly activates ASC-pyrin, this assay requires further optimization to be useful to recapitulate the full picture of the inflammasome activation events. Nevertheless, this study paves the way for the development of a convenient and useful molecular platform to investigate the molecular details of inflammasome regulation.