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Functional and Evolutionary Analyses Identify Proteolysis as a General Mechanism for NLRP1 Inflammasome Activation

Fig 3

Human NLRP1 is activated by proteolysis that removes the N-terminal PYD.

(A) For detection of NLRP1 expression, 293T cells were co-transfected for 24h with 100ng human full-length pcDNA3.1-NLRP1-MYC (CMV promoter) (FL), or with constructs in which the leucine rich repeat or pyrin domains had been deleted (ΔLRR, ΔPYD). Cells were also co-transfected with 500ng empty pcDNA3.1 vectors and with plasmids encoding human ASC (1ng), hCASP1 (1ng), and IL-1β–V5 (200ng). After 36h, cells were analyzed by immunoblot (IB) using an antibody against MYC. (B) Cells were transfected as in A, but to avoid spontaneous NLRP1 activation, the amount of hNLRP1 plasmid was reduced to 10ng and adjusted with 90ng of empty vector. Lysates were blotted with an anti-V5 antibody. (C) Cleavage sites for TEV protease were introduced into pcDNA3.1-hNLRP1-MYC at the indicated positions (see S4 Fig for details) to generate TEV T1, TEV T2, and TEV T3 expression constructs. 293T cells were co-transfected with 10g of these NLRP1 constructs along with 50ng of a vector encoding TEV-protease (pTEV) or a control empty vector (V), and all supplemented with 200ng empty pcDNA3.1. After 24h, lysates were analyzed by immunoblot using anti-Myc antibody to detect full-length NLRP1, the FIIND-processed (30kDa) C-terminal product, or the products that result from TEV cleavage (144kDa for T1, 155kDa for T2, and 133kDa for T3). (D) 293T cells were co-transfected with 5ng of NLRP1 constructs along with 50ng of vector encoding TEV-protease (pTEV) or a control empty vector (V), and plasmids encoding human ASC (1ng), CASP1 (1ng), and IL-1β-V5 (200ng), supplemented with 500ng empty pcDNA3.1. After 24h, lysates were analyzed by immunoblot using anti-V5 antibody to detect CASP1-dependent processing of IL-1β into the mature p17 fragment. (E) A panel of constructs for expressing full-length or truncated GFP-fused human NLRP1 in pMSCV (LTR promoter) was generated as depicted. The TEV T1 and T2 sites were introduced into two of the constructs as indicated (also see S4 Fig). 293T cells were transfected with 100ng these constructs along with 50ng empty vector or a plasmid encoding the TEV protease (pTEV). Cell lysates were analyzed by immunoblot (IB) with anti-GFP. (F) 293T cells were transfected for 24h with 12.5ng plasmids encoding full-length or truncated GFP-fused human NLRP1 in pMSCV (LTR promoter), along with plasmids encoding human ASC (1ng), CASP1 (1ng), V5-tagged IL-1β (200ng) and TEV protease (50ng, pTEV). After 24h, lysates were analyzed by immunoblot using anti-V5 antibody to detect CASP1-dependent processing of IL-1β into the mature p17 fragment. For all panels, data shown are representative of at least three similar experiments.

Fig 3

doi: https://doi.org/10.1371/journal.ppat.1006052.g003