Limits...
TRIM30α Is a Negative-Feedback Regulator of the Intracellular DNA and DNA Virus-Triggered Response by Targeting STING.

Wang Y, Lian Q, Yang B, Yan S, Zhou H, He L, Lin G, Lian Z, Jiang Z, Sun B - PLoS Pathog. (2015)

Bottom Line: Biochemical analyses showed that TRIM30α interacted with the stimulator of interferon genes (STING), which is a critical regulator of the DNA-sensing response.Overexpression of TRIM30α promoted the degradation of STING via K48-linked ubiquitination at Lys275 through a proteasome-dependent pathway.These findings indicate that E3 ligase TRIM30α is an important negative-feedback regulator of innate immune responses to DNA viruses by targeting STING.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China; School of Life Sciences, University of Science and Technology of China, Hefei, China.

ABSTRACT
Uncontrolled immune responses to intracellular DNA have been shown to induce autoimmune diseases. Homeostasis regulation of immune responses to cytosolic DNA is critical for limiting the risk of autoimmunity and survival of the host. Here, we report that the E3 ubiquitin ligase tripartite motif protein 30α (TRIM30α) was induced by herpes simplex virus type 1 (HSV-1) infection in dendritic cells (DCs). Knockdown or genetic ablation of TRIM30α augmented the type I IFNs and interleukin-6 response to intracellular DNA and DNA viruses. Trim30α-deficient mice were more resistant to infection by DNA viruses. Biochemical analyses showed that TRIM30α interacted with the stimulator of interferon genes (STING), which is a critical regulator of the DNA-sensing response. Overexpression of TRIM30α promoted the degradation of STING via K48-linked ubiquitination at Lys275 through a proteasome-dependent pathway. These findings indicate that E3 ligase TRIM30α is an important negative-feedback regulator of innate immune responses to DNA viruses by targeting STING.

No MeSH data available.


Related in: MedlinePlus

TRIM30α is an E3 ubiquitin ligase and targets STING for K48-mediated ubiquitination at Lys275.(A) Immunoblot analysis of lysates from HEK293T cells transfected with plasmids for Flag-STING, HA-ubiquitin and increasing concentrations of Myc-TRIM30α (0, 1, 1.5 and 2 μg), followed by immunoprecipitation with anti-Flag, and analyzed via immunoblot with anti-HA Ab. (B) Immunoblot analysis of lysates from HEK293T cells transfected with various combinations of plasmids for Myc-TRIM30α, Flag-STING, HA-K48-ubi and HA-K63-ubi and then performed as in A. (C) Immunoblot analysis of lysates from HEK293T cells transfected with the indicated plasmids and Myc-tagged TRIM30α (C35A), ΔR and then performed as in B. (D) Immunoblot analysis of lysates in wild-type and Trim30α-/- BMDCs stimulated for 8 h with ISD (1 μg/ml), followed by immunoprecipitation with anti-STING Ab and analyzed with anti-Ub, anti-K48- and anti-K63-linked ubiquitin. (E) Immunoblot analysis of STING ubiquitination in vitro. TRIM30α and STING were quickly translated in vitro, and then, the biotin-ubiquitin E1 and indicated E2s were added for the ubiquitination assays. Ubiquitination was assessed by anti-ubi. (F) Immunoblot analysis of lysates from HEK293T cells transfected with Myc-tagged TRIM30α, Flag-tagged wild-type and mutant STING together with HA-tagged ubiquitin, then performed as in A. (G) Luciferase assays of MEF cells cotransfected with the IFN-β promoter and empty vector or TRIM30α together with wild-type and mutant STING plasmids. (H) Real-time PCR of IFN-β and IP-10 mRNA in L929 cells transfected with Flag-tagged vector, wild type STING and K275R mutant, 24 h after transfection, then infected the above cells with HSV-1 (MOI 10) for 10 h. The data are representative of three independent experiments and are presented as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4482643&req=5

ppat.1005012.g007: TRIM30α is an E3 ubiquitin ligase and targets STING for K48-mediated ubiquitination at Lys275.(A) Immunoblot analysis of lysates from HEK293T cells transfected with plasmids for Flag-STING, HA-ubiquitin and increasing concentrations of Myc-TRIM30α (0, 1, 1.5 and 2 μg), followed by immunoprecipitation with anti-Flag, and analyzed via immunoblot with anti-HA Ab. (B) Immunoblot analysis of lysates from HEK293T cells transfected with various combinations of plasmids for Myc-TRIM30α, Flag-STING, HA-K48-ubi and HA-K63-ubi and then performed as in A. (C) Immunoblot analysis of lysates from HEK293T cells transfected with the indicated plasmids and Myc-tagged TRIM30α (C35A), ΔR and then performed as in B. (D) Immunoblot analysis of lysates in wild-type and Trim30α-/- BMDCs stimulated for 8 h with ISD (1 μg/ml), followed by immunoprecipitation with anti-STING Ab and analyzed with anti-Ub, anti-K48- and anti-K63-linked ubiquitin. (E) Immunoblot analysis of STING ubiquitination in vitro. TRIM30α and STING were quickly translated in vitro, and then, the biotin-ubiquitin E1 and indicated E2s were added for the ubiquitination assays. Ubiquitination was assessed by anti-ubi. (F) Immunoblot analysis of lysates from HEK293T cells transfected with Myc-tagged TRIM30α, Flag-tagged wild-type and mutant STING together with HA-tagged ubiquitin, then performed as in A. (G) Luciferase assays of MEF cells cotransfected with the IFN-β promoter and empty vector or TRIM30α together with wild-type and mutant STING plasmids. (H) Real-time PCR of IFN-β and IP-10 mRNA in L929 cells transfected with Flag-tagged vector, wild type STING and K275R mutant, 24 h after transfection, then infected the above cells with HSV-1 (MOI 10) for 10 h. The data are representative of three independent experiments and are presented as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001.

Mentions: Most TRIM proteins contain RING domains, which allow them to mediate ubiquitylation events [28]. Therefore, we hypothesized that TRIM30α is an E3 ubiquitin ligase for STING. To address this, TRIM30α and STING were co-transfected in HEK293T cells. Immunoprecipitation and immunoblot analysis showed that TRIM30α could ubiquitinate STING in a dose-dependent manner (Fig 7A). Moreover, we observed that TRIM30α overexpression enhanced K48-linked ubiquitination of STING but not K63-linked ubiquitination (Fig 7B). K48-linked ubiquitination is normally linked to proteasomes-mediated degradation of proteins, which is in line with our early data (Fig 6D and 6E). As shown in Fig 7C, TRIM30α (C35A) and ΔR dramatically attenuated the ubiquitination of STING compared with full-length TRIM30α, suggesting that TRIM30α ubiquitinates STING dependent upon its RING domain activity (Fig 7C). Next, we examined the ubiquitination of STING in primary cells and observed that wild type and K48-linked ubiquitination of STING in wild type BMDCs was increased in comparison to Trim30α-deficient BMDCs stimulated with ISD for 8 h (Fig 7D). Collectively, these findings indicate that TRIM30α is an E3 ubiquitin ligase for STING and mediates STING degradation via the proteasome pathway. To further investigate whether TRIM30α directly ubiquitinates STING, TRIM30α and STING were quickly translated in vitro. In vitro ubiquitination showed that the TRIM30α protein directly mediated ubiquitination of STING (Fig 7E).


TRIM30α Is a Negative-Feedback Regulator of the Intracellular DNA and DNA Virus-Triggered Response by Targeting STING.

Wang Y, Lian Q, Yang B, Yan S, Zhou H, He L, Lin G, Lian Z, Jiang Z, Sun B - PLoS Pathog. (2015)

TRIM30α is an E3 ubiquitin ligase and targets STING for K48-mediated ubiquitination at Lys275.(A) Immunoblot analysis of lysates from HEK293T cells transfected with plasmids for Flag-STING, HA-ubiquitin and increasing concentrations of Myc-TRIM30α (0, 1, 1.5 and 2 μg), followed by immunoprecipitation with anti-Flag, and analyzed via immunoblot with anti-HA Ab. (B) Immunoblot analysis of lysates from HEK293T cells transfected with various combinations of plasmids for Myc-TRIM30α, Flag-STING, HA-K48-ubi and HA-K63-ubi and then performed as in A. (C) Immunoblot analysis of lysates from HEK293T cells transfected with the indicated plasmids and Myc-tagged TRIM30α (C35A), ΔR and then performed as in B. (D) Immunoblot analysis of lysates in wild-type and Trim30α-/- BMDCs stimulated for 8 h with ISD (1 μg/ml), followed by immunoprecipitation with anti-STING Ab and analyzed with anti-Ub, anti-K48- and anti-K63-linked ubiquitin. (E) Immunoblot analysis of STING ubiquitination in vitro. TRIM30α and STING were quickly translated in vitro, and then, the biotin-ubiquitin E1 and indicated E2s were added for the ubiquitination assays. Ubiquitination was assessed by anti-ubi. (F) Immunoblot analysis of lysates from HEK293T cells transfected with Myc-tagged TRIM30α, Flag-tagged wild-type and mutant STING together with HA-tagged ubiquitin, then performed as in A. (G) Luciferase assays of MEF cells cotransfected with the IFN-β promoter and empty vector or TRIM30α together with wild-type and mutant STING plasmids. (H) Real-time PCR of IFN-β and IP-10 mRNA in L929 cells transfected with Flag-tagged vector, wild type STING and K275R mutant, 24 h after transfection, then infected the above cells with HSV-1 (MOI 10) for 10 h. The data are representative of three independent experiments and are presented as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482643&req=5

ppat.1005012.g007: TRIM30α is an E3 ubiquitin ligase and targets STING for K48-mediated ubiquitination at Lys275.(A) Immunoblot analysis of lysates from HEK293T cells transfected with plasmids for Flag-STING, HA-ubiquitin and increasing concentrations of Myc-TRIM30α (0, 1, 1.5 and 2 μg), followed by immunoprecipitation with anti-Flag, and analyzed via immunoblot with anti-HA Ab. (B) Immunoblot analysis of lysates from HEK293T cells transfected with various combinations of plasmids for Myc-TRIM30α, Flag-STING, HA-K48-ubi and HA-K63-ubi and then performed as in A. (C) Immunoblot analysis of lysates from HEK293T cells transfected with the indicated plasmids and Myc-tagged TRIM30α (C35A), ΔR and then performed as in B. (D) Immunoblot analysis of lysates in wild-type and Trim30α-/- BMDCs stimulated for 8 h with ISD (1 μg/ml), followed by immunoprecipitation with anti-STING Ab and analyzed with anti-Ub, anti-K48- and anti-K63-linked ubiquitin. (E) Immunoblot analysis of STING ubiquitination in vitro. TRIM30α and STING were quickly translated in vitro, and then, the biotin-ubiquitin E1 and indicated E2s were added for the ubiquitination assays. Ubiquitination was assessed by anti-ubi. (F) Immunoblot analysis of lysates from HEK293T cells transfected with Myc-tagged TRIM30α, Flag-tagged wild-type and mutant STING together with HA-tagged ubiquitin, then performed as in A. (G) Luciferase assays of MEF cells cotransfected with the IFN-β promoter and empty vector or TRIM30α together with wild-type and mutant STING plasmids. (H) Real-time PCR of IFN-β and IP-10 mRNA in L929 cells transfected with Flag-tagged vector, wild type STING and K275R mutant, 24 h after transfection, then infected the above cells with HSV-1 (MOI 10) for 10 h. The data are representative of three independent experiments and are presented as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001.
Mentions: Most TRIM proteins contain RING domains, which allow them to mediate ubiquitylation events [28]. Therefore, we hypothesized that TRIM30α is an E3 ubiquitin ligase for STING. To address this, TRIM30α and STING were co-transfected in HEK293T cells. Immunoprecipitation and immunoblot analysis showed that TRIM30α could ubiquitinate STING in a dose-dependent manner (Fig 7A). Moreover, we observed that TRIM30α overexpression enhanced K48-linked ubiquitination of STING but not K63-linked ubiquitination (Fig 7B). K48-linked ubiquitination is normally linked to proteasomes-mediated degradation of proteins, which is in line with our early data (Fig 6D and 6E). As shown in Fig 7C, TRIM30α (C35A) and ΔR dramatically attenuated the ubiquitination of STING compared with full-length TRIM30α, suggesting that TRIM30α ubiquitinates STING dependent upon its RING domain activity (Fig 7C). Next, we examined the ubiquitination of STING in primary cells and observed that wild type and K48-linked ubiquitination of STING in wild type BMDCs was increased in comparison to Trim30α-deficient BMDCs stimulated with ISD for 8 h (Fig 7D). Collectively, these findings indicate that TRIM30α is an E3 ubiquitin ligase for STING and mediates STING degradation via the proteasome pathway. To further investigate whether TRIM30α directly ubiquitinates STING, TRIM30α and STING were quickly translated in vitro. In vitro ubiquitination showed that the TRIM30α protein directly mediated ubiquitination of STING (Fig 7E).

Bottom Line: Biochemical analyses showed that TRIM30α interacted with the stimulator of interferon genes (STING), which is a critical regulator of the DNA-sensing response.Overexpression of TRIM30α promoted the degradation of STING via K48-linked ubiquitination at Lys275 through a proteasome-dependent pathway.These findings indicate that E3 ligase TRIM30α is an important negative-feedback regulator of innate immune responses to DNA viruses by targeting STING.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China; School of Life Sciences, University of Science and Technology of China, Hefei, China.

ABSTRACT
Uncontrolled immune responses to intracellular DNA have been shown to induce autoimmune diseases. Homeostasis regulation of immune responses to cytosolic DNA is critical for limiting the risk of autoimmunity and survival of the host. Here, we report that the E3 ubiquitin ligase tripartite motif protein 30α (TRIM30α) was induced by herpes simplex virus type 1 (HSV-1) infection in dendritic cells (DCs). Knockdown or genetic ablation of TRIM30α augmented the type I IFNs and interleukin-6 response to intracellular DNA and DNA viruses. Trim30α-deficient mice were more resistant to infection by DNA viruses. Biochemical analyses showed that TRIM30α interacted with the stimulator of interferon genes (STING), which is a critical regulator of the DNA-sensing response. Overexpression of TRIM30α promoted the degradation of STING via K48-linked ubiquitination at Lys275 through a proteasome-dependent pathway. These findings indicate that E3 ligase TRIM30α is an important negative-feedback regulator of innate immune responses to DNA viruses by targeting STING.

No MeSH data available.


Related in: MedlinePlus