Research ArticleCell Biology

The parkin-coregulated gene product PACRG promotes TNF signaling by stabilizing LUBAC

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Science Signaling  04 Feb 2020:
Vol. 13, Issue 617, eaav1256
DOI: 10.1126/scisignal.aav1256
  • Fig. 1 PACRG does not influence mitophagy.

    (A) Schematic representation of the PRKN and PACRG locus. The genes encoding Parkin and PACRG are linked in a head-to-head arrangement on opposite DNA strands and share a 5′ core bidirectional promoter of 204 base pairs. (B) Representative immunofluorescence images of HeLa cells transiently expressing HA-tagged PACRG or Parkin and treated with CCCP to induce mitochondrial depolarization and subsequent degradation. Fixed cells were analyzed by indirect immunofluorescence using either the Parkin-specific antibody PRK8 or an HA antibody to detect PACRG and an Hsp60-specific antibody to visualize mitochondria. Scale bar, 100 μm. (C) Quantification of CCCP-induced mitochondrial clearance in HeLa cells expressing Parkin, PACRG, or both Parkin and PACRG. (D) Quantification of CCCP-induced mitochondrial clearance in SH-SY5Y cells transfected with control or PACRG siRNAs together with Parkin cDNA. PACRG knockdown efficiency was determined by real-time reverse transcription (RT)–PCR using exon-flanking PACRG-specific primers. Data represent the means ± SEM of at least three independent experiments, each performed in triplicate. At least 300 transfected cells were counted per condition. For statistical analysis Mann-Whitney U test was performed.

  • Fig. 2 PACRG deficiency impairs TNF-induced NF-κB activation.

    (A) HEK293T cells were transfected with PACRG, Parkin, or EGFP (negative control) and an NF-κB luciferase reporter construct. Luciferase activity was quantified in lysates of untreated and TNF-treated cells, with luciferase activity in untreated EGFP-expressing cells set to 1. Quantifications were based on at least three independent experiments, each performed in triplicate. Expression of proteins from the transfected plasmids was verified by Western blotting for Parkin and PACRG. β-Actin is a loading control. Data represent the means ± SEM of 7 to 12 independent experiments. For statistical analysis, ANOVA followed by Tukey’s multiple comparison test was performed. (B) Parkin KO MEFs were transfected with either PACRG or Parkin and an NF-κB luciferase reporter construct. Luciferase activity was measured in lysates of untreated and TNF-treated cells as in (A). Data represent the means ± SEM of four to five independent experiments. For statistical analysis, one-tailed Mann-Whitney U test was performed. (C) Representative immunofluorescence images showing p65 localization in SH-SY5Y cells transfected with control or PACRG-specific siRNAs and treated with TNF. Nuclei were labeled with DAPI. Scale bar, 100 μm. (D) Quantification of TNF-induced nuclear translocation of p65 in control and PACRG knockdown SH-SY5Y cells based on three independent experiments, each performed in duplicate. PACRG knockdown efficiency was determined using real-time RT-PCR. Data represent the means ± SEM of three to five independent experiments. For statistical analysis, one-tailed Mann-Whitney U test was performed. (E) HEK293T cells were transfected with either control or PACRG-specific siRNAs and an NF-κB luciferase reporter construct and then treated with TNF or left untreated. Luciferase activity in cell lysates was measured. Data represent the means ± SEM of three independent experiments, each performed in duplicate. For statistical analysis, one-tailed Mann-Whitney U test was performed. PACRG knockdown efficiency was determined using real-time RT-PCR. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

  • Fig. 3 PACRG increases TNF-induced linear ubiquitylation.

    (A) HEK293T cells transfected with control or PACRG-specific siRNA were treated with TNF 1 day after transfection for the indicated times. Degradation of IκBα was determined by Western blotting (WB) for IκBα. Numbers below the IκBα-reactive bands indicate the mean value of three independent experiments normalized to β-actin levels ± SEM. Graph shows PACRG knockdown efficiency as determined by real-time RT-PCR. (B) HEK293T cells were transiently transfected with plasmids encoding the indicated proteins. One day after transfection, cells were treated with TNF and lysed. Lysates generated under denaturing conditions were subjected to pulldown with the Strep-tagged UBAN domain of NEMO, and proteins affinity purified by Strep-Tactin beads were analyzed by immunoblotting with an antibody specific for M1-linked ubiquitin. The input was immunoblotted for HOIP, PACRG, EGFP, and β-actin. Signal intensities of three independent experiments were quantified and normalized to the amount of linear ubiquitin chains in samples with EGFP overexpression and TNF treatment. Data represent the means ± SEM of three independent experiments. For statistical analysis one-tailed Mann-Whitney U test was performed. (C) HEK293T cells were transiently transfected with the indicated siRNAs. TNF treatment, lysis, and pulldown were performed as in (B). Signal intensities of three independent experiments were quantified and normalized to the amount of linear ubiquitin chains in samples treated with control siRNA and TNF. PACRG knockdown efficiency was determined by real-time RT-PCR. Data represent the means ± SEM of three independent experiments. For statistical analysis, one-tailed Mann-Whitney U test was performed. *P ≤ 0.05

  • Fig. 4 PACRG binds to LUBAC.

    (A) HEK293T cells were transfected with PACRG and then treated with TNF for the indicated times. Cells were lysed under native conditions, and endogenous HOIP was immunoprecipitated (IP). Immunoprecipitated proteins were detected by Western blotting for PACRG and HOIP. The input was immunoblotted for PACRG and β-actin. (B) Schematic presentation of the HOIP constructs used for immunoprecipitation. All constructs included an N-terminal HA tag. PUB, peptide N-glycosidase/ubiquitin–associated domain; ZF, zinc finger domain; NZF, nuclear protein localization 4-type zinc finger domain; UBA, ubiquitin-associated domain; RING, really interesting new gene; IBR, in-between RING domain; LDD, linear ubiquitin chain–determining domain. (C) HEK293T cells were transfected with either N-terminal (amino acids 1 to 697), C-terminal (amino acids 697 to 1072), or full-length (amino acids 1 to 1072) HA-HOIP together with PACRG. After cell lysis, HOIP was immunoprecipitated and affinity purified. Copurified PACRG was detected by Western blotting using an antibody recognizing PACRG. c-Myc is a negative control. Asterisk notes a nonspecific immunoreactive band. The HOIP IP control and the HOIP input were probed with an antibody specific for HA. (D) HEK293T cells were transfected with PACRG and the indicated HOIP constructs. Coimmunoprecipitation experiments were performed as in (C). (E) HEK293T cells were transfected with either ΔUBA or full-length HA-HOIP (amino acids 1 to 1072) and PACRG. Coimmunoprecipitation experiments were performed as in (C). (F and G) HEK293T cells were transfected with the indicated HOIP constructs and either HOIL-1L (F) or SHARPIN (G). Coimmunoprecipitation experiments were performed as in (C). HOIP input levels were detected with an antibody specific for HA. HOIL-1L, SHARPIN, and β-actin were detected with antibodies against the respective proteins. (H) HEK293T cells were transfected with PACRG and then treated with FLAG-TNF for the indicated times. After cell lysis, FLAG-TNF was immunoprecipitated and affinity purified. Copurified PACRG and HA-HOIP were detected by Western blotting for PACRG and HA, respectively. All blots are representative of at least three independent experiments.

  • Fig. 5 PACRG restores defective NF-κB activation and prevents increased cell death in SHARPIN-deficient cells.

    (A) HEK293T cells were transfected with control, SHARPIN-, or PACRG-specific siRNAs with or without PACRG or SHARPIN plasmids as indicated, and then the cells were treated with TNF and lysed under denaturing conditions. Linear ubiquitination was analyzed by UBAN affinity purification by Strep-Tag II beads followed by immunoblotting for M1-linked ubiquitin. PACRG knockdown efficiency was determined by real-time RT-PCR. Blot is representative of three independent experiments. (B) HEK293T cells were transfected with HA-NEMO and either PACRG or SHARPIN, and then lysed under denaturing conditions followed by immunoprecipitation of NEMO by anti-HA agarose. Anti–c-Myc agarose was used as a control for nonspecific binding. Immunoprecipitated NEMO was immunoblotted for M1-linked ubiquitin and NEMO. The input was immunoblotted for NEMO, PACRG, SHARPIN, and β-actin. Blot is representative of three independent experiments. (C) Wild-type (WT) and cpdm MEFs were transfected with SHARPIN, PACRG, or EGFP (negative control), and then the cells were treated with TNF. The fraction of cells showing nuclear translocation of p65 was determined for each condition. Data represent the means ± SEM of three to four independent experiments. For statistical analysis, one-tailed Mann-Whitney U test was performed. At least 100 cells were analyzed per condition. (D) Cpdm MEFs were transfected with either SHARPIN or PACRG. Transfection of EGFP was used as a control. After TNF treatment, apoptotic cell death was quantified by counting transfected cells positive for active caspase-3. Data represent the means ± SEM of three independent experiments. For statistical analysis one-tailed Mann-Whitney U test was performed. At least 100 cells were analyzed per condition. (E) MEFs were transiently transfected with the indicated siRNAs and lysed after 48 hours. Lysates were analyzed by immunoblotting for HOIP, SHARPIN, and β-actin. PACRG knockdown efficiency was determined by real-time RT-PCR. Data represent the means ± SEM of five independent experiments; intensity normalized to β-actin is presented as ratio to total intensity of all three bands. For statistical analysis, ANOVA followed by Tukey’s multiple comparison test was performed. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/13/617/eaav1256/DC1

    Fig. S1. PACRG coelutes with endogenous LUBAC components.

    Fig. S2. PACRG restores defective p65 translocation in SHARPIN-deficient cells.

    Fig. S3. PACRG stabilizes LUBAC.

    Fig. S4. PACRG protects against STS- and MPP+-induced apoptotic cell death.

    Fig. S5. TNF treatment of PACRG-silenced MEFs does not activate caspase-3.

    Table S1. Antibodies used in this study.

  • This PDF file includes:

    • Fig. S1. PACRG coelutes with endogenous LUBAC components.
    • Fig. S2. PACRG restores defective p65 translocation in SHARPIN-deficient cells.
    • Fig. S3. PACRG stabilizes LUBAC.
    • Fig. S4. PACRG protects against STS- and MPP+-induced apoptotic cell death.
    • Fig. S5. TNF treatment of PACRG-silenced MEFs does not activate caspase-3.
    • Table S1. Antibodies used in this study.

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