Fig 1: NALP7 forms an inflammasome to facilitate IL-1ß release in response to LPS and Pam3CSK4.(a) A representative z-stack confocal microscopy image obtained by immunofluoresence co-staining of NALP7 (green), ASC (red) and DAPI (blue) in PMA-differentiated THP-1 cells (5 × 105 per ml) following treatment for 4 h with LPS (100 ng ml-1). The 3D image is shown in XY, XZ and YZ coordinate planes. Scale bar, 10 µm. (b) Representative images obtained by immunofluorescence co-staining as above in PMA-differentiated THP-1 cells following treatment for 4 h with LPS (200 ng ml-1), Pam3CSK4 (100 ng ml-1) or vehicle control. Original magnification was × 100, Scale bar, 10 µm. (c) THP-1 cells transfected with NALP3, ASC, or NALP7 siRNA followed by treatment with LPS (200 ng ml-1) or Pam3CSK4 (100 ng ml-1) for 6 h secreted less IL-1ß as measured by ELISA compared to luciferase (LUC) siRNA control. Data shown as mean±s.d. (n=3). *P<0.05 compared to LUC control. (d,e) Immunoreactive NALP7 and NALP3 increased over time from lysates of THP-1 cells after (d) LPS (200 ng ml-1) or (e) Pam3CSK4 (100 ng ml-1) exposure for the indicated duration. Right, densitometric analysis of the NALP7 signal versus time, normalized to ß-actin. Data shown as mean±s.d. (n=3). *P<0.05 compared to 0 h. (c) Two-way analysis of variance (ANOVA) with post hoc Dunnett's multiple comparisons test. (d,e) One-sample t-test (hypothetical value=1).
Fig 2: LPS and Pam3CSK4 alter NALP7 protein stability and trafficking.(a) NALP7 mRNA expression analysed by qPCR in THP-1 cells exposed to LPS or Pam3CSK4 for 16 h was unchanged compared to untreated control, whereas pro-IL-1ß mRNA expression increased. Data shown as mean fold change±s.d. as determined by ??Cq analysis (n=3). (b) In cycloheximide (CHX) chase, NALP7 protein stability is increased in THP-1 cells exposed to LPS (200 ng ml-1) or Pam3CSK4 (100 ng ml-1) compared to vehicle control (CON) (c) NALP7 protein abundance increased over time when treated with leupeptin (50 µM) or bafilomycin (100 nM) but not MG132 (20 µM). (b,c) Densitometric analysis of the NALP7 signal versus time, normalized to ß-actin. Data shown as mean±s.d. (n=3). *P<0.05 compared to 0 h or **P<0.01 versus CON. (d) Immunofluorescence co-staining of NALP7 (green), Lysotracker (red) and DAPI (blue) in Beas2B cells exposed to LPS (5 µg ml-1), Pam3CSK4 (2 µg ml-1), leupeptin (100 µM), or vehicle control for 2 h. Lysotracker (1:2,000) was added for the final 30 min of incubation before fixing and staining. Original magnification was × 100, Scale bar, 5 µm. Images are representative of three or more images captured for each condition (n=2). (a) One-sample t-test (hypothetical value=1). (b,c) Two-way analysis of variance (ANOVA) with post hoc Dunnett's multiple comparisons test.
Fig 3: STAMBP modulates NALP7 protein stability and NALP7 inflammasome activity.(a) DUB screening in HeLa cells. Overexpression of plasmid-encoded STAMBP, but not other DUBs, increased endogenous NALP7 protein abundance. Note: V5 signal from BRCC3 is in the same location as actin causing actin signal distortion. (b) Densitometric analysis of NALP7 signal. Data shown as mean fold change±s.d. (n=2). *P<0.05 compared to empty vector control. (c) Overexpression of STAMBP plasmid increases NALP7 levels, but not other inflammasome constituents in Beas2B cells. (d) STAMBP knockdown with three separate siRNAs decreased endogenous NALP7 abundance compared to non-targeting control (NTC) siRNA in THP-1 cells. Relative abundance compared to control noted below each blot, as determined by densitometry using ImageJ software. (e) STAMBP knockdown with siRNA decreased endogenous NALP7 abundance compared to NTC siRNA after LPS exposure (500 ng ml-1 for 6 h) in THP-1 cells. Relative abundance noted as described previously. (f) STAMBP (SBP) expression by lentiviral transfection rescues NALP7 protein abundance in THP-1 cells treated with STAMBP siRNA. Below, densitometric analysis of relative NALP7 protein abundance as determined by ImageJ software. Data shown as mean±s.d. (n=4). **P<0.05 compared to STAMBP knockdown alone and STAMBP knockdown with GFP lentivirus rescue. (g) Endogenous NALP7 half-life is decreased with STAMBP knockdown by siRNA compared to NTC siRNA in Beas2B cells. (h) In vitro DUB assay with NALP7 substrate. Immunopurified Ub-NALP7 substrate abundance decreased over time when incubated at 37 °C with purified recombinant STAMBP (200 nM) but not STAM (400 nM) or vehicle control. V5 is shown as a loading control. (i) IL-1ß secretion following exposure to LPS (200 ng ml-1) or Pam3CSK4 (100 ng ml-1) for 6 h decreased in THP-1 cells transfected with STAMBP siRNA compared to control (luciferase, LUC) siRNA, as measured by ELISA. Data shown as mean±s.d. (n=3). *P< 0.05 compared to LUC control. (b) One-sample t-test (hypothetical value=1). (i) Two-way analysis of variance (ANOVA) with post hoc Dunnett's multiple comparisons test.
Fig 4: Identification of NALP7 ubiquitin acceptor sites.(a) TUBEs pulldown of ubiquitinated NALP7-V5 from denatured Beas2B cell lysates increased with leupeptin (LEU) (50 µM) treatment compared to untreated control. (b) Ubiquitinated NALP7-V5 was identified by immunoblotting after pulldown with non-selective TUBEs reagent and anti-K63 TUBEs from denatured Beas2B cell lysates. (c) UbiCREST assay. Immunopurified Ub-NALP7-V5 abundance decreased when incubated at 37 °C for 30 min with purified, recombinant USP2, Trabid, OTUD3 and STAMBP, but not OTULIN, OTUB1, Yod1 or Cezanne in vitro. (d) Mapping of NALP7 using truncation mutants. (e) Half life analysis of transfected wild-type and truncation mutant NALP7-V5 and (f) transfected K?R point mutant NALP7-V5 in Beas2B cells. (g,h) Densitometric analysis of (g) NALP7 truncation mutant and (h) NALP7 K?R point mutant signal versus time, normalized to ß-actin. Data shown as mean±s.d. (n=2). *P<0.05 compared to wild-type using a two-way analysis of variance (ANOVA) with post hoc Dunnett's multiple comparisons test. (i) Immunopurified NALP7-V5 KRK288RRR (KRK) mutant showed decreased ubiquitination compared to wild-type or K374R mutant in Beas2B cells.
Fig 5: NALP7 inflammasome activity requires STAMBP DUB activity that is antagonized by BC-1471.In the absence of inflammatory stimuli, NALP7 is constitutively ubiquitinated (1) and bound by STAM as cargo for endosomal trafficking (2). Ub-NALP7 is shuttled to the lysosome for degradation (3). STAMBP deubiquitinates Ub-NALP7 facilitating NALP7 recue from degradation (4). With LPS or Pam3CSK4 exposure, NALP7 protein abundance increases (5) by decreasing endolysosomal trafficking in a process dependent on the DUB activity of STAMBP. The NALP7 inflammasome assembles in response to LPS or Pam3CSK4 (6) with subsequent IL-1ß cleavage to its active form and inflammatory signalling. BC-1471 antagonizes STAMBP DUB activity (7), inhibiting NALP7 rescue, downstream of inflammasome assembly and IL-1ß maturation.
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