Isolation of Particles of Recombinant ASC and NLRP3

Abstract

NLRP3 inflammasome is a multiprotein complex responsible for the activation of inflammatory caspase-1, resulting in processing and release of pro-inflammatory cytoquines IL-1β and IL-18 (Schroder and Tschopp, 2010). This inflammasome is composed of the sensor protein NLRP3 connected to caspase-1 through the adaptor protein ASC (apoptosis-associated speck-like protein with a caspase-recruitment domain) (Schroder and Tschopp, 2010). We and others have reported that upon inflammasome activation functional oligomeric inflammasome particles of NLRP3 and ASC were released from cells, acting as danger signals to amplify inflammation by promoting the activation of caspase-1 extracellularly (Baroja-Mazo et al., 2014; Franklin et al., 2014). Studying the extracellular function of oligomeric ASC and NLRP3 inflammasome particles was possible by purification of recombinant particles of ASC or the constitutively activated NLRP3 mutant associated with cryopyrin-associated periodic syndromes (CAPS, mutation p.D303N), both tagged with the yellow fluorescent protein (YFP) and expressed in HEK293 cells. The purification process was facilitated by the fact that expression of recombinant ASC or mutant NLRP3 in HEK293 cells resulted in their spontaneous aggregation into specks (Baroja-Mazo et al., 2014) and the protocol was originally adapted from Fernandes-Alnemri and Alnemri (2008).

Figure 3. Representative image of ASC and NLRP3 (p.D303N) fluorescent particles after purification protocol

Figure 1. Representative scheme of ASC and NLRP3 (p.D303N) particles purification protocol illustrating steps from 1 to 8

Figure 2. Representative scheme of Percoll step (steps 8-10)