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: Homeostasis regulation of immune responses to cytosolic DNA is critical for limiting the risk of autoimmunity and survival of the host.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.

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 interacts with STING.(A) Luciferase activity of MEF cells transfected with the IFN-β luciferase reporter, together with STING, TBK1, MDA5 or VISA, and the empty vector (Vec) or the TRIM30α plasmid. (B) Luciferase activity of MEF cells transfected with the NF-κB luciferase reporter, together with STING or VISA, and the empty vector (Vec) or the TRIM30α plasmid. (C and D) Luciferase activity of MEF cells cotransfected with the IFN-β promoter or the NF-κB promoter and empty vector or increasing amounts of TRIM30α (C) or C35A (D) (0.2, 0.4 and 0.6 μg) together with STING. (E) Immunoblot analysis of lysates from HEK293T cells transfected with Flag-TRIM30α together with HA-tagged vector, STING or TAK1 plasmids, followed by immunoprecipitation (IP) with anti-Flag Ab and immunoblot analysis with anti-HA Ab. (F, G) Immunoblot analysis in lysates of D2SC cells mock treated or stimulated with poly(dA:dT) (1 μg/ml) (F); or with HSV-1 (MOI 10) (G) for 8 h, followed by immunoprecipitation with anti-TRIM30α Ab and immunoblot analysis with anti-STING Ab. (H) A schematic presentation of full-length TRIM30α and its mutants. RING, ring-finger domain; BBOX, B-box domain; CC, coiled-coil domain; SPRY, SPRY domain. (I) Immunoblot analysis of lysates from HEK293T cells transfected with the indicated plasmids, followed by immunoprecipitation (IP) with anti-HA Ab and immunoblot analysis with anti-Flag Ab. (J) A schematic presentation of full-length STING and its mutants. (Transmembrane) TM1, 21-41aa; TM2, 47-67aa; TM3, 87-106aa; TM4, 115-135aa; CTD, carboxy-terminal domain; CTT, carboxy-terminal tail. (K) Immunoblot analysis of lysates from HEK293T cells transfected with the indicated plasmids and then performed as in I. (L) Confocal microscopy of L929 cells transfected for 24 h with Flag-tagged STING and HA-tagged full-length TRIM30α, ΔRING or ΔSPRY mutant of TRIM30α. Immunofluorescence was performed using anti-HA (red) and anti-Flag (green). (M) Confocal microscopy of L929 cells transfected for 24 h with cyan fluorescent protein-labeled TRIM30a (CFP-TRIM30α) and yellow fluorescent protein-labeled STING (YFP-STING) and then mock treated or stimulated for 4 h with poly(dA:dT) (1 μg/ml) or ISD (1 μg/ml). Nuclei were stained with the DNA-intercalating dye DAPI. Staining of calnexin served as a marker of the endoplasmic reticulum (ER). The data are representative of three independent experiments and are presented as mean ± SEM. *p < 0.05 and **p < 0.01.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1005012.g005: TRIM30α interacts with STING.(A) Luciferase activity of MEF cells transfected with the IFN-β luciferase reporter, together with STING, TBK1, MDA5 or VISA, and the empty vector (Vec) or the TRIM30α plasmid. (B) Luciferase activity of MEF cells transfected with the NF-κB luciferase reporter, together with STING or VISA, and the empty vector (Vec) or the TRIM30α plasmid. (C and D) Luciferase activity of MEF cells cotransfected with the IFN-β promoter or the NF-κB promoter and empty vector or increasing amounts of TRIM30α (C) or C35A (D) (0.2, 0.4 and 0.6 μg) together with STING. (E) Immunoblot analysis of lysates from HEK293T cells transfected with Flag-TRIM30α together with HA-tagged vector, STING or TAK1 plasmids, followed by immunoprecipitation (IP) with anti-Flag Ab and immunoblot analysis with anti-HA Ab. (F, G) Immunoblot analysis in lysates of D2SC cells mock treated or stimulated with poly(dA:dT) (1 μg/ml) (F); or with HSV-1 (MOI 10) (G) for 8 h, followed by immunoprecipitation with anti-TRIM30α Ab and immunoblot analysis with anti-STING Ab. (H) A schematic presentation of full-length TRIM30α and its mutants. RING, ring-finger domain; BBOX, B-box domain; CC, coiled-coil domain; SPRY, SPRY domain. (I) Immunoblot analysis of lysates from HEK293T cells transfected with the indicated plasmids, followed by immunoprecipitation (IP) with anti-HA Ab and immunoblot analysis with anti-Flag Ab. (J) A schematic presentation of full-length STING and its mutants. (Transmembrane) TM1, 21-41aa; TM2, 47-67aa; TM3, 87-106aa; TM4, 115-135aa; CTD, carboxy-terminal domain; CTT, carboxy-terminal tail. (K) Immunoblot analysis of lysates from HEK293T cells transfected with the indicated plasmids and then performed as in I. (L) Confocal microscopy of L929 cells transfected for 24 h with Flag-tagged STING and HA-tagged full-length TRIM30α, ΔRING or ΔSPRY mutant of TRIM30α. Immunofluorescence was performed using anti-HA (red) and anti-Flag (green). (M) Confocal microscopy of L929 cells transfected for 24 h with cyan fluorescent protein-labeled TRIM30a (CFP-TRIM30α) and yellow fluorescent protein-labeled STING (YFP-STING) and then mock treated or stimulated for 4 h with poly(dA:dT) (1 μg/ml) or ISD (1 μg/ml). Nuclei were stained with the DNA-intercalating dye DAPI. Staining of calnexin served as a marker of the endoplasmic reticulum (ER). The data are representative of three independent experiments and are presented as mean ± SEM. *p < 0.05 and **p < 0.01.

Mentions: To identify the molecular mechanisms by which TRIM30α inhibits the DNA-triggered response, luciferase assays were performed to detect the target of TRIM30α. We found that TRIM30α inhibited the IFN-β reporter activation mediated by STING, a key adaptor protein for most DNA-sensing pathways [24,25]. However, TRIM30α did not affect the downstream kinase of STING, TBK1, and had no influence on MDA5- and virus-induced signaling adaptor (VISA)-induced signaling (Fig 5A). In addition, TRIM30α overexpression significantly inhibited STING-induced NF-κB reporter activation but did not affect VISA (Fig 5B). It is hypothesized that TRIM30α targets STING to inhibit DNA-mediated response. As shown in Fig 5C, exogenous expression of TRIM30α inhibited STING-induced IFN-β and NF-κB reporter activation in a dose dependent manner (Fig 5C). In contrast, TRIM30α (C35A), which contains an enzymatically inactive mutant of the RING domain had no effect on STING-induced IFN-β and NF-κB reporter activation (Fig 5D). All these suggest that TRIM30α suppresses STING signaling dependent on its RING domain. Furthermore, co-immunoprecipitation assays were performed and the results showed that TRIM30α interacted with STING when co-transfected into HEK293T cells (Fig 5E). TGF beta-activated kinase (TAK1) interacts with TRIM30α, which has been previously reported, was used as a positive control [22]. We next performed endogenous co-immunoprecipitation experiments and verified that endogenous TRIM30α could interact with STING in D2SC cells after poly(dA:dT) or HSV-1 stimulation (Fig 5F and 5G). To further demonstrate the interaction, TRIM30α and STING were quickly translated in vitro, and immunoprecipitation analysis indicated that TRIM30α could interact with STING directly (S5 Fig).


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 interacts with STING.(A) Luciferase activity of MEF cells transfected with the IFN-β luciferase reporter, together with STING, TBK1, MDA5 or VISA, and the empty vector (Vec) or the TRIM30α plasmid. (B) Luciferase activity of MEF cells transfected with the NF-κB luciferase reporter, together with STING or VISA, and the empty vector (Vec) or the TRIM30α plasmid. (C and D) Luciferase activity of MEF cells cotransfected with the IFN-β promoter or the NF-κB promoter and empty vector or increasing amounts of TRIM30α (C) or C35A (D) (0.2, 0.4 and 0.6 μg) together with STING. (E) Immunoblot analysis of lysates from HEK293T cells transfected with Flag-TRIM30α together with HA-tagged vector, STING or TAK1 plasmids, followed by immunoprecipitation (IP) with anti-Flag Ab and immunoblot analysis with anti-HA Ab. (F, G) Immunoblot analysis in lysates of D2SC cells mock treated or stimulated with poly(dA:dT) (1 μg/ml) (F); or with HSV-1 (MOI 10) (G) for 8 h, followed by immunoprecipitation with anti-TRIM30α Ab and immunoblot analysis with anti-STING Ab. (H) A schematic presentation of full-length TRIM30α and its mutants. RING, ring-finger domain; BBOX, B-box domain; CC, coiled-coil domain; SPRY, SPRY domain. (I) Immunoblot analysis of lysates from HEK293T cells transfected with the indicated plasmids, followed by immunoprecipitation (IP) with anti-HA Ab and immunoblot analysis with anti-Flag Ab. (J) A schematic presentation of full-length STING and its mutants. (Transmembrane) TM1, 21-41aa; TM2, 47-67aa; TM3, 87-106aa; TM4, 115-135aa; CTD, carboxy-terminal domain; CTT, carboxy-terminal tail. (K) Immunoblot analysis of lysates from HEK293T cells transfected with the indicated plasmids and then performed as in I. (L) Confocal microscopy of L929 cells transfected for 24 h with Flag-tagged STING and HA-tagged full-length TRIM30α, ΔRING or ΔSPRY mutant of TRIM30α. Immunofluorescence was performed using anti-HA (red) and anti-Flag (green). (M) Confocal microscopy of L929 cells transfected for 24 h with cyan fluorescent protein-labeled TRIM30a (CFP-TRIM30α) and yellow fluorescent protein-labeled STING (YFP-STING) and then mock treated or stimulated for 4 h with poly(dA:dT) (1 μg/ml) or ISD (1 μg/ml). Nuclei were stained with the DNA-intercalating dye DAPI. Staining of calnexin served as a marker of the endoplasmic reticulum (ER). The data are representative of three independent experiments and are presented as mean ± SEM. *p < 0.05 and **p < 0.01.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1005012.g005: TRIM30α interacts with STING.(A) Luciferase activity of MEF cells transfected with the IFN-β luciferase reporter, together with STING, TBK1, MDA5 or VISA, and the empty vector (Vec) or the TRIM30α plasmid. (B) Luciferase activity of MEF cells transfected with the NF-κB luciferase reporter, together with STING or VISA, and the empty vector (Vec) or the TRIM30α plasmid. (C and D) Luciferase activity of MEF cells cotransfected with the IFN-β promoter or the NF-κB promoter and empty vector or increasing amounts of TRIM30α (C) or C35A (D) (0.2, 0.4 and 0.6 μg) together with STING. (E) Immunoblot analysis of lysates from HEK293T cells transfected with Flag-TRIM30α together with HA-tagged vector, STING or TAK1 plasmids, followed by immunoprecipitation (IP) with anti-Flag Ab and immunoblot analysis with anti-HA Ab. (F, G) Immunoblot analysis in lysates of D2SC cells mock treated or stimulated with poly(dA:dT) (1 μg/ml) (F); or with HSV-1 (MOI 10) (G) for 8 h, followed by immunoprecipitation with anti-TRIM30α Ab and immunoblot analysis with anti-STING Ab. (H) A schematic presentation of full-length TRIM30α and its mutants. RING, ring-finger domain; BBOX, B-box domain; CC, coiled-coil domain; SPRY, SPRY domain. (I) Immunoblot analysis of lysates from HEK293T cells transfected with the indicated plasmids, followed by immunoprecipitation (IP) with anti-HA Ab and immunoblot analysis with anti-Flag Ab. (J) A schematic presentation of full-length STING and its mutants. (Transmembrane) TM1, 21-41aa; TM2, 47-67aa; TM3, 87-106aa; TM4, 115-135aa; CTD, carboxy-terminal domain; CTT, carboxy-terminal tail. (K) Immunoblot analysis of lysates from HEK293T cells transfected with the indicated plasmids and then performed as in I. (L) Confocal microscopy of L929 cells transfected for 24 h with Flag-tagged STING and HA-tagged full-length TRIM30α, ΔRING or ΔSPRY mutant of TRIM30α. Immunofluorescence was performed using anti-HA (red) and anti-Flag (green). (M) Confocal microscopy of L929 cells transfected for 24 h with cyan fluorescent protein-labeled TRIM30a (CFP-TRIM30α) and yellow fluorescent protein-labeled STING (YFP-STING) and then mock treated or stimulated for 4 h with poly(dA:dT) (1 μg/ml) or ISD (1 μg/ml). Nuclei were stained with the DNA-intercalating dye DAPI. Staining of calnexin served as a marker of the endoplasmic reticulum (ER). The data are representative of three independent experiments and are presented as mean ± SEM. *p < 0.05 and **p < 0.01.
Mentions: To identify the molecular mechanisms by which TRIM30α inhibits the DNA-triggered response, luciferase assays were performed to detect the target of TRIM30α. We found that TRIM30α inhibited the IFN-β reporter activation mediated by STING, a key adaptor protein for most DNA-sensing pathways [24,25]. However, TRIM30α did not affect the downstream kinase of STING, TBK1, and had no influence on MDA5- and virus-induced signaling adaptor (VISA)-induced signaling (Fig 5A). In addition, TRIM30α overexpression significantly inhibited STING-induced NF-κB reporter activation but did not affect VISA (Fig 5B). It is hypothesized that TRIM30α targets STING to inhibit DNA-mediated response. As shown in Fig 5C, exogenous expression of TRIM30α inhibited STING-induced IFN-β and NF-κB reporter activation in a dose dependent manner (Fig 5C). In contrast, TRIM30α (C35A), which contains an enzymatically inactive mutant of the RING domain had no effect on STING-induced IFN-β and NF-κB reporter activation (Fig 5D). All these suggest that TRIM30α suppresses STING signaling dependent on its RING domain. Furthermore, co-immunoprecipitation assays were performed and the results showed that TRIM30α interacted with STING when co-transfected into HEK293T cells (Fig 5E). TGF beta-activated kinase (TAK1) interacts with TRIM30α, which has been previously reported, was used as a positive control [22]. We next performed endogenous co-immunoprecipitation experiments and verified that endogenous TRIM30α could interact with STING in D2SC cells after poly(dA:dT) or HSV-1 stimulation (Fig 5F and 5G). To further demonstrate the interaction, TRIM30α and STING were quickly translated in vitro, and immunoprecipitation analysis indicated that TRIM30α could interact with STING directly (S5 Fig).

Bottom Line: Homeostasis regulation of immune responses to cytosolic DNA is critical for limiting the risk of autoimmunity and survival of the host.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.

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