The deubiquitinating enzyme, ubiquitin‐specific peptidase 50, regulates inflammasome activation by targeting the ASC adaptor protein

NOD‐like receptor family protein 3 (NLRP3)‐mediated inflammasome activation promotes caspase‐1‐dependent production of interleukin‐1β (IL‐1β) and requires the adaptor protein ASC. Compared with the priming and activation mechanisms of the inflammasome signaling pathway, post‐translational ubiquitination/deubiquitination mechanisms controlling inflammasome activation have not been clearly addressed. We here demonstrate that the deubiquitinating enzyme USP50 binds to the ASC protein and subsequently regulates the inflammasome signaling pathway by deubiquitinating the lysine 63‐linked polyubiquitination of ASC. USP50 knockdown in human THP‐1 cells and mouse bone marrow‐derived macrophages shows a significant decrease in procaspase‐1 cleavage, resulting in a reduced secretion of IL‐1β and interleukin‐18 (IL‐18) upon treatment with NLRP3 stimuli and a reduction in ASC speck formation and oligomerization. Thus, we elucidate a novel regulatory mechanism of the inflammasome signaling pathway mediated by the USP50 deubiquitinating enzyme.


Immunofluorescence
Briefly, human THP-1 cells, stimulated on coverslips, were washed with PBS twice and fixed in cold methanol for 7 min, followed by blocking with 5 % BSA in PBS and incubation with primary antibodies at room temperature for 3 h. Anti-ASC (sc-22514R , Santa Cruz, 1:100) antibody was used to detect ASC specks. After five washes with PBS, coverslips were incubated at room temperature for 3 h with Alexa Fluor @ -488-conjugated goat anti-rabbit IgG (Invitrogen, 1:100). All coverslips were stained with DAPI (Santa Cruz) and mounted on glass slides. A laser-scanning confocal microscope (CarlZeiss LSM700) was used to image the fluorescent samples.
Immunocomplexes were washed twice with lysis buffer and separated from the beads by adding 2x sample buffer and boiling. Immunoblot analysis was performed using the indicated antibodies.

ASC oligomerization
ASC oligomerization was basically carried out as in the previously described protocol [15].
Pellets from whole-cell lysates were cross-linked with disuccinimidyl suberate (DSS, Sigma Aldrich) for 40 min at 37 ºC. The reaction was stopped by the addition of quenching solution at a final concentration of 20 mM Tris-HCl (pH 7.5) for 10 min at room temperature.
Samples were then fractionated by SDS-PAGE with 12 % polyacrylamide gel and immunoblotted with anti-ASC antibody.

Measurement of Cytokines
Cytokines were measured from cell culture supernatants using specific ELISA kits according to the manufacturer's instructions. Secretions of human and mouse IL-1β were analyzed using ELISA kits purchased from BD Biosciences. ELISA kits for human and mouse TNF-α and mouse IL-18 were purchased from eBiosciences and R&D systems, respectively. To examine the effects of USP50 on AIM2-mediated inflammasome activation, secretion of IL-1 and TNF- into culture media were analyzed by ELISA 16 h after transfection of 1 g/ml poly(dA:dT) into human THP-1 cells. All reactions were independently repeated at least three times to ensure reproducibility and values were calculated on the basis of a standard curve constructed for each assay.

RNA extraction and quantitative real-time RT-PCR (qRT-PCR)
Total RNA was extracted from cells using the TRIzol reagent (Invitrogen). Oligo-dT and PrimeScript Reverse Transcriptase (TaKaRa) was used for reverse transcription. For qRT-PCR of USP50 mRNA, an iCycler real-time PCR machine and iQ SYBR Green Supermix (Bio-Rad) were used for real-time detection of PCR products under the following conditions: 40 cycles of 95 °C for 15 sec and 60 °C for 45 sec. The analysis of the expression of USP50 and Gapdh genes was performed using the software provided by Bio-Rad. Primer sequences of the USP50 and Gapdh genes are described in Supplementary Table 3. All qRT-PCR reactions were independently repeated at least three times to ensure reproducibility.

Pull-down and ubiquitination assay by Ni-NTA columns
Ni-NTA-mediated pull-down assays were performed as previously described [14]. 0.1 % Triton X-100). The resulting Ni-NTA agarose resin was suspended in elution buffer and loaded onto SDS-PAGE gels. Immunoblotting was performed using the indicated antibodies. Expression of the β-actin protein was used as a loading control.

In vitro deubiquitination assay
To perform in vitro deubiquitination assay, HA-ubiquitin conjugated Flag-ASC proteins was generated from HEK293 cells, which were transiently expressing Flag-ASC and HAubiquitin plasmids, by immunoprecipitation with anti-Flag ® M2 affinity Gel (Sigma Aldrich, A2220) and elution with Flag ® peptide (Sigma Aldrich, F3290). Flag-USP50 proteins were also obtained from HEK293 cells through the same protocol and subsequently used as the deubiquitinating enzyme. The eluted Flag-ASC was added to a deubiquitination reaction (40 mM Tris-HCl pH 7.6, 50 mM NaCl, 5 mM MgCl 2 , 5 mM DTT, 2 mM ATP) in the absence or presence of eluted Flag-USP50. Reactions were incubated at 37 °C for 6 hours, terminated by the addition of 4X SDS sample buffer followed by a heat inactivation with boiling.
Immunoblotting was performed using anti-HA antibody to detect HA-ubiquitin-conjugated ASC proteins.

In vivo ubiquitination assay of endogenous ASC protein
Differentiated human THP-1 cells were knockdowned by control siRNA and an USP50specific siRNA pool (siUSP50s). USP50-knockdown and control THP-1 cells were primed with 1 g/ml LPS for 4 h and subsequently stimulated by 10 μM nigericin for 1 h. Cells were harvested in 1 ml PBS containing 5 mM N-ethyl maleimide (NEM). Dissociation of noncovalent protein interactions and dilution of samples were previously described [14]. For immunoprecipitation, cell lysates were precleared with 1 μg IgG antibody bound to protein G agarose beads (GenDEPOT) for 12 h at 4 °C. The precleared lysates were immunoprecipitated with 1 μg anti-ASC antibody (AG-25B-0006-C100, Adipogen) or control IgG antibody bound to protein G agarose beads. The beads were washed three times with lysis buffer and immunoprecipitates were separated from the beads by adding 2X sample buffer. After boiling, the immunoprecipitates were fractioned by SDS-PAGE. The separated immunoprecipitates were transferred onto PVDF membranes. The membranes were denaturated by 6 M guanidine buffer (TBS contacting 6 M guanidine chloride, 20 mM Tris-HCl pH 7.5 and 5mM -mercaptoethanol) at 4 °C for 30 min. After denaturation, membranes were washed by TBST three times. The membranes were blocked with 5 % BSA and incubated with anti-FK2-HRP antibody (BML-PW8810, Enzo lifesciences) and anti-K63linkage-specific-HRP antibody (BML-PW0605, Enzo lifesciences).

Statistical analysis
All experiments have at least three biological replicates. Results are expressed as mean ± SD.
Statistical analyses were made with an unpaired t-test for two groups using Prism software (GraphPad). A P value <0.05 was considered as a statistically significant difference.
Differences with P<0.05 are annotated as *, P<0.01 are annotated as ** and P<0.001 as ***. (A) Mouse BMDMs were infected with concentrated lentiviruses expressing two independent shRNAs targeting mouse USP50 mRNA (sh-mUSP50 #3 and sh-mUSP50 #4). USP50 depletion was analyzed by real-time RT-PCR for USP50 transcripts. Lentiviruses expressing green fluorescence protein (GFP)-specific shRNA (shGFP) were used as a negative control. The data were statistically analyzed by a t-test and show the mean ± SD of three independent experiments. **P<0.01 compared to control shGFP-expressing mouse BMDMs. (B-E) USP50knockdowned BMDMs were treated with LPS (1 g/ml) for 4 h, followed by Alum (300 g/ml) for 6 h or NIG treatment (10 M) for 1 h. Secretion of extracellular IL-1β (B, C) and IL-18 (D, E) into culture supernatants was analyzed by ELISA. (F) USP50-knockdowned or control BMDMs were treated with or without LPS. Secretion of extracellular TNF-α was analyzed by ELISA. All data in (B-F) were statistically analyzed by a t-test and show the mean ± SD. *P<0.05, **P<0.01, ***P<0.001 compared to the shGFP-expressing control BMDMs. n.s., not significant. The data are representative of three independent replicates, statistically analyzed by a t-test and the mean ±SD is shown. **P<0.01compared to control THP-1 cells treated with AIM2 stimuli. n.s., not significant.