Fig 1: UAF1/USP1 interacts with NLRP3.a, b Confocal microscopy analysis of colocalization of NLRP3 with USP1 or UAF1. MEFs transfected with Myc-NLRP3, GFP-USP1 or Flag-UAF1 were stimulated with LPS for 2 h, then fixed and incubated with a secondary antibody conjugated to Alexa Fluor 637 or Alexa Fluor 488. Scale bar, 10 µm. c, d Extracts from HEK293T cells transiently transfected with Flag-USP1 and Myc-NLRP3 (c), and Flag-UAF1 and Myc-NLRP3 (d) were subjected to IP with anti-Flag, followed by western blot analysis with anti-Myc and anti-Flag. e PCMV6-USP1 and Myc-tagged NLRP3 were obtained by in vitro transcription and translation. Interaction between USP1 and NLRP3 was analyzed by mixing USP1 and NLRP3 together, followed by IP with USP1 antibody and immunoblot analysis with NLRP3 and USP1 antibody. f HA-tagged UAF1 and Myc-tagged NLRP3 were obtained by in vitro transcription and translation. Interaction between UAF1 and NLRP3 was assayed by mixing UAF1 and NLRP3 together, followed by IP with HA antibody and immunoblot analysis with NLRP3 and HA antibody. g, h Extracts of peritoneal macrophages stimulated with LPS for the indicated time periods were subjected to immunoprecipitation with anti-USP1 (g) and anti-UAF1 (h) followed by western blot analysis with indicated antibody. Proteins from a whole-cell lysate were used as positive control. i Schematic diagram of NLRP3 and its truncation mutants. j, k Myc-tagged NLRP3 or its mutants along with Flag-USP1 (j) or Flag-UAF1 (k) were individually transfected into HEK293T cells. The cell lysates were immunoprecipitated with Flag antibody and then immunoblotted with the indicated antibodies. US, unstimulated. Similar results were obtained from three independent experiments.
Fig 2: UAF1/USP1 removes NLRP3 ubiquitination and stabilizes NLRP3.a, b Western blot analysis of extracts obtained from HEK293T cells transfected with HA-Ub, Myc-NLRP3, Flag-USP1 WT, and USP1 C90S (a) or Flag-UAF1 (b), followed by IP with anti-Myc, and then probed with anti-HA. c, d Extracts obtained from HEK293T cells transiently cotransfected with NLRP3-Myc, HA-Ub, and Flag-USP1 (c) or Flag-UAF1 (d) were immunoprecipitated using Myc antibody. The immunoprecipitates were denatured and reimmunoprecipitated with Myc antibody (two-step immunoprecipitation, Re-IP), and then immunoblotted with the indicated antibodies. e, f Western blot analysis of extracts from HEK293T cells transfected with HA-K48-Ub or HA-K63-Ub, Myc-NLRP3 and Flag-USP1 (e) or Flag-UAF1 (f), followed by IP with anti-Myc and subsequently probed with anti-HA. g MEFs were pretreated with DMSO or ML323 for 4 h and then stimulated with LPS for 6 h. Cell lysates were immunoprecipitated with anti-NLRP3, followed by western blot analysis with anti-Ub. h Cell lysates from WT or Uaf1 deficient mouse peritoneal macrophages stimulated with LPS were immunoprecipitated with anti-NLRP3, followed by western blot analysis with anti-Ub. i, j Western blot analysis of NLRP3 and caspase-1 expression in mouse peritoneal macrophages pretreated with DMSO or ML323 for 4 h and then stimulated with LPS for 4 h, followed by treatment with cycloheximide (CHX) for the indicated time periods. NLRP3 expression level was quantitated by measuring band intensities using ‘ImageJ’ software (j). The values were normalized to actin (mean ± SEM, two-tailed t-test ML323 vs. DMSO, **P < 0.0001, respectively; n = 3 independent experiments). k Western blot analysis of NLRP3 expression in WT and Uaf1 deficient mouse peritoneal macrophages pretreated with LPS for 8 h, followed by treatment with MG132 for 4 h before harvesting the cells. Similar results were obtained from three independent experiments.
Fig 3: UAF1 enhances NLRP3 expression.a, c Western blot analysis of NLRP3 (a), AIM2 and NLRC4 (c) in WT or Uaf1-deficient mouse peritoneal macrophages stimulated with LPS for the indicated time periods. b RT-PCR analysis of Nlrp3 mRNA expression in WT or Uaf1-deficient mouse peritoneal macrophages stimulated with LPS for 2 h (mean ± SEM, two-tailed t-test Uaf1CKO vs. WT, **P < 0.0001; n = 3 independent experiments). d, e Western blot analysis of p-p65 in WT or Uaf1-deficient mouse peritoneal macrophages stimulated with LPS (d) or poly(I:C) (e) for the indicated time periods. f Analysis of NF-?B reporter activation using a Luciferase assay in HEK293T cells transiently transfected with NF-?B reporter plasmid together with MyD88 or TRIF, and UAF1 expression plasmid or empty control plasmid (mean ± SEM, two-tailed t-test UAF1 plasmid vs. empty control plasmid, **P = 0.0024, <0.0001 in sequence; n = 6 independent experiments). Similar results were obtained from three independent experiments.
Fig 4: UAF1/USP12 and UAF1/USP46 complexes promote NLRP3 transcription.a RT-PCR analysis of Il1b, Il6 and Tnf mRNA expression in Ctrl siRNA-, Usp1 siRNA-, Usp12 siRNA-, or Usp46 siRNA-transfected mouse peritoneal macrophages, followed by stimulation with LPS for 2 h (mean ± SEM, two-tailed t-test Usp1 siRNA, Usp12 siRNA and Usp46 siRNA vs. Ctrl siRNA, left panel: ns = 0.8310, *P = 0.0135, **P < 0.0001 in sequence, middle panel: ns = 0.4945, **P = 0.0006, 0.0003 in sequence, right panel: ns = 0.8037, **P = 0.0005, 0.0007 in sequence; n = 3 independent experiments). b Analysis of NF-?B reporter activation using a Luciferase assay in HEK293T cells transiently transfected with the indicated plasmids (mean ± SEM, two-tailed t-test USP1, USP12 and USP46 plasmids vs. empty control plasmid, ns = 0.1774, *P = 0.0106, 0.0298 in sequence; n = 6 independent experiments). c, d Western blot analysis of p65 or p-p65 in Ctrl siRNA-, Usp12 siRNA-, or Usp46 siRNA-transfected mouse peritoneal macrophages followed by stimulation with LPS for the indicated time periods. e Western blot analysis of p65 in WT or Uaf1 deficient mouse peritoneal macrophages after stimulation with LPS for the indicated time periods. f Cell lysates of mouse peritoneal macrophages stimulated with LPS for the indicated time periods were subjected to immunoprecipitation (IP) with anti-p65, followed by western blot analysis with antibodies to UAF1, USP1, USP12, USP46, or p65. Proteins from a whole-cell lysate were used as a positive control (Input). g Cell lysates of HEK293T cells transiently transfected with Myc-p65, Flag-USP1, Flag-USP12, Flag-USP46, and Flag-UAF1 were subjected to IP using Myc antibody, followed by western blot analysis with anti-Myc or anti-Flag. h, j Cell lysates of HEK293T cells transiently transfected with Myc-p65, HA-Ub, or HA-tagged K48-linked ubiquitin (HA-K48-Ub), Flag-USP12, USP46, and Flag-UAF1 were subjected to IP with Myc antibody, followed by western blot analysis with anti-HA. i Western blot analysis of extracts from LPS-stimulated WT or Uaf1CKO macrophages, followed by IP with anti-p65, and subsequent probing with anti-Ub. US, unstimulated. Similar results were obtained from three independent experiments.
Fig 5: UAF1 facilitates NLRP3 inflammasome activation.a ELISA analysis of IL-1ß, IL-6 and TNF secretion in mouse peritoneal macrophages from wild-type (WT) or Uaf1CKO mice following priming with LPS for 8 h and subsequent stimulation with ATP or Nig for 40 min. US, unstimulated (mean ± SEM, two-tailed t-test Uaf1CKO vs. WT, left panel: **P = 0.0050, 0.0044 in sequence, middle panel: **P = 0.0070, 0.0052, 0.0009 in sequence, right panel: **P = 0.0035, 0.0014, 0.0079 in sequence; n = 3 independent experiments). b, c Western blot analysis of caspase-1 and IL-1ß cleavage in mouse peritoneal macrophages from WT or Uaf1CKO mice after priming with LPS for the indicated time periods and then stimulated with ATP (b) or Nig (c) for 40 min. d ELISA analysis of serum levels of IL-1ß, IL-6 and TNF from WT or Uaf1CKO mice after i.p. injection of LPS for 90 min (mean ± SEM, two-tailed t-test Uaf1CKO vs. WT, left panel: **P = 0.0005, middle panel: *P = 0.0321, right panel: **P < 0.0001; n = 6 independent experiments). US, unstimulated; SN, supernatants; CL, cell lysates. Similar results were obtained from three independent experiments.
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