A proximity-dependent biotinylation (BioID) approach flags the p62/sequestosome-1 protein as a caspase-1 substrate

Abstract

The inflammasome is a major component of the innate immune system, and its main function is to activate caspase-1, a cysteine protease that promotes inflammation by inducing interleukin-1β (IL-1β) maturation and release into the extracellular milieu. To prevent uncontrolled inflammation, this complex is highly regulated. When it is assembled, the inflammasome is insoluble, which has long precluded the analysis of its interactions with other proteins. Here we used the proximity-dependent biotinylation assay (BioID) to identify proteins associated with caspase-1 during inflammasome activation. Using the BioID in a cell-free system in which the inflammasome had been activated, we found that a caspase-1-biotin ligase fusion protein selectively labeled 111 candidates, including the p62/sequestosome-1 protein (p62). Using co-immunoprecipitation experiments, we demonstrated that p62 interacts with caspase-1. This interaction promoted caspase-1-mediated cleavage of p62 at Asp-329. Mechanistic and functional analyses revealed that caspase-1-mediated cleavage of p62 leads to loss of its interaction with the autophagosomal protein microtubule-associated protein 1 light chain 3 β (LC3B). Strikingly, overexpression of a p62 N-terminal fragment generated upon caspase-1 cleavage decreased IL-1β release, whereas overexpression of p62's C-terminal portion enhanced IL-1β release, by regulating pro-IL1β levels. Overall, the overexpression of both fragments together decreased IL-1β release. Taken together, our results indicate that caspase-1-mediated p62 cleavage plays a complex role in balancing caspase-1-induced inflammation.

Keywords: autophagy; caspase-1 (CASP1); inflammasome; inflammation; proteolytic enzyme; proteomics.

Figure 1.

Generation of HA-tagged BirA*–caspase-1–overexpressing macrophages. A, schematic showing caspase-1 WT (casp-1 WT) and the engineered Q281H-mutated caspase-1 fused to HA-tagged BirA* (casp-1*-BirA). B, immortalized bone marrow-derived macrophages from Caspase-1^−/− mice were reconstituted with a doxycycline (Dox)-inducible casp-1*-BirA (iCasp-1*-BirA). Reconstituted cells (KO-iCasp-1*-BirA) and their WT or caspase-1^−/− (KO) controls were plated in the presence or absence of doxycycline (1 μg/ml) and incubated for 24 h. Cells were harvested, centrifuged, and resuspended in a hypotonic low-potassium buffer at 4 °C. Cells were mechanically lysed, and nuclei and plasma membranes were removed. The cell-free preparation was then incubated at 37 °C for 6 h (T6) or not (T0). Proteins were analyzed by Western blotting after boiling and fractionating on a 10% SDS-polyacrylamide gel. C, ASC oligomerization was analyzed after cross-linking or not with DSS and analyzed by Western blotting under nonreducing conditions. D, ASC oligomerization was also verified in cells previously plated in the presence of doxycycline upon activation of the inflammasome by LPS (1 μg/liter, 3 h) + nigericin (5 μm, 45 min). ASC oligomers were cross-linked or not with DSS and analyzed by Western blotting under nonreducing conditions.

Figure 2.

Identification of caspase-1–neighboring proteins during inflammasome activation in a cell-free assay. A, schematic presenting the BioID approach adapted to the cell-free assay for inflammasome activation. B, reconstituted cells (KO-iCasp-1*-BirA) and their WT or Caspase-1^−/− (KO) controls were plated in presence or absence of doxycycline (Dox, 1 μg/ml) and incubated for 24 h. Cells were harvested, centrifuged, and resuspended in a hypotonic low-potassium buffer at 4 °C. Cells were mechanically lysed, and nuclei and plasma membranes were removed. The cell-free soup was then incubated at 37 °C for 6 h (T6) or not (T0). Supernatants were then incubated with streptavidin-coated beads overnight at 4 °C. Beads were collected and rigorously washed. Streptavidin-bound proteins were then analyzed by Western blotting after boiling and fractionating on a 15% SDS-polyacrylamide gel. ASC pulldown was selected as the readout for successful procedure before MS analysis. IP, immunoprecipitation. C, sorting process of the proteins identified by the caspase-1 BioID assay (1108 proteins were identified by MS, 628 were present in the uninduced sample, 112 proteins did not pass the 2× enrichment threshold between the 37 °C and the 4 °C samples, and 146 proteins were excluded based on CRAPome analysis, resulting in 111 proteins identified with confidence in the caspase-1 BioID assay). These candidates were analyzed for gene ontology (GO) enrichment according to biological process (PANTHER classification system).

Figure 3.

p62 interacts with caspase-1 upon inflammasome activation. A, HEK293T cells were transfected with plasmids encoding FLAG-tagged WT caspase-1 and HA-tagged p62 and incubated overnight. Cells were detached, lysed, and centrifuged. Supernatants were then incubated for 3 h at 4 °C with anti-FLAG– or anti-HA–agarose beads. Beads were collected and washed. Bound proteins were analyzed by Western blotting after boiling and fractionating on a 10% SDS-polyacrylamide gel. IP, immunoprecipitation. B, primary human monocyte-derived macrophages were treated as indicated. Cells were fixed, permeabilized, and incubated with the indicated antibodies, and images were collected using a confocal microscope.

Figure 4.

Caspase-1 cleaves p62 at Asp-329. A, p62 cleavage in THP-1 cells was assayed using the cell-free system. The inflammasome was activated by incubating the cell-free soup at 37 °C during 1 h or 2 h in the presence or absence of pancaspase inhibitor (Z-VAD) or caspase-1 inhibitor (Z-YVAD). Caspase-1 (casp-1) and p62 were then analyzed by Western blotting after boiling and fractionating on a 10% SDS-polyacrylamide gel. Casp-1 p20 is the cleavage fragment, observed when caspase-1 is activated. B, primary human monocytes, obtained from whole blood of healthy donors, were incubated in the presence of the inflammasome activator (LPS and nigericin) with or without caspase-8 inhibitor (Z-IETD), calpain inhibitor, and caspase-1 inhibitor (Z-YVAD). Cells were then washed and lysed, and p62 cleavage was analyzed by Western blotting after boiling and fractionating on a 10% SDS-polyacrylamide gel. Ratios of cleaved/uncleaved p62 were calculated using ImageJ. C, HEK293T cells were co-transfected with plasmids encoding p62 and various caspases (casp-1 to 9) and incubated overnight. Cells were then washed and lysed, and p62 and IL-1β were analyzed by Western blotting after boiling and fractionating on a 10% SDS-polyacrylamide gel. D, HEK293T cells were co-transfected with plasmids encoding WT caspase-1 and WT or mutated p62 containing point mutations at the indicated amino acids and incubated overnight. Cells were then washed and lysed, and p62 and caspase-1 expression and cleavage were analyzed by Western blotting. E, HEK293T cells were co-transfected with plasmids encoding FLAG-tagged WT caspase-1 and WT or mutated p62. Cells were then washed, lysed, and incubated with anti-FLAG–agarose during 3 h at 4 °C. Beads were then rigorously washed, and bound proteins were analyzed by Western blotting. IP, immunoprecipitation.

Figure 5.

Caspase-1 cleavage disrupts p62 interaction with LC3B and modulates IL-1β secretion. A, representation of the p62 structure. The PB1 (Phox and Bem1) N-terminal domain allows p62 oligomerization and/or interactions with other autophagy adaptors. The LIR domain allows interaction with the LC3B-coated compartment (autophagosome), and the UBA domain binds ubiquitinated proteins. ZZ, ZZ-type zinc finger domain; TBS, TRAF6-binding domain; KIR, KEAP1-interacting region. Alignment of the protein sequence surrounding Asp-329 demonstrates conservation in numerous mammalian species (human, rat, bat (Myotis lucifugus), ferret (Mustela furo), cat (Felis catus), bovine) but not in mice. B, HEK293T cells were cotransfected with plasmids encoding HA-tagged WT p62 (WT-HA) or its uncleavable mutant (D329A-HA) along with the FLAG-tagged p62 N-terminal (N-FLAG) or C-terminal (C-FLAG) fragments and incubated overnight. Cells were then washed, lysed, and incubated with anti-HA–agarose during 3 h at 4 °C. Beads were then rigorously washed, and bound proteins were analyzed by Western blotting. IP, immunoprecipitation. C, HEK293T cells were cotransfected with plasmids encoding HA-tagged caspase-1 (casp1-HA) and FLAG-tagged p62 N-terminal or C-terminal fragments and incubated overnight. Cells were then washed, lysed, and incubated with anti-FLAG–agarose during 3 h at 4 °C. Beads were rigorously washed, and bound proteins were analyzed by Western blotting. D, schematic presenting the interactions between p62 fragments and p62 full-length, caspase-1, and LC3B. E and F, WT iBMDMs were transduced stably with (E) the pINDUCER21 doxycycline-inducible lentiviral vector encoding human p62 N-terminal or C-terminal fragments or transiently (F) with various ratios (1:1, 5:1, and 1:5) of the pINDUCER21 doxycycline-inducible lentiviral vector encoding human p62 N-terminal (N) and C-terminal (C) fragments as indicated. Cells were incubated with doxycycline for 24 h (1 μg/ml) to induce ectopic expression of p62 fragments. The inflammasome was then activated by addition of a priming signal (LPS, 1 μg/liter during 3 h) followed by either nigericin (5 μm, 45 min), MSU (50 μg/ml, 6 h), or ATP (1 mm, 60 min) as indicated. The supernatants were collected. IL-1β concentrations were measured by ELISA. *, p < 0.05; **, p < 0.001; ns, not significant.

Figure 6.

A proposed model for p62 regulation of the inflammasome. In the negative regulation model (left panel), caspase-1 cleavage of p62 and subsequent release of the N-terminal fragment (1) may favor full-length p62 self-oligomerization (2), interaction with the LC3B-coated compartment (3), and engulfment of the inflammasome into the autophagosome (4), allowing quelling of the inflammation. In the positive regulation model (right panel), caspase-1 cleavage of the entire p62 pool may alter p62 function and give rise to a dominant-negative C-terminal fragment (5) that competes with p62 for LC3B binding (6) to inhibit autophagic degradation of pro-IL-1β and the inflammasome (7), resulting in increased inflammasome activation, IL-1β release, and inflammatory cell death.