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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 enhances the degradation of STING.(A) Immunoblot analysis of STING, TBK1, IRF3 and TRIM30α in lysates from wild type and Trim30α-/- BMDCs stimulated for 2–24 h with poly(dA:dT) (1 μg/ml). (B) Immunoblot analysis of STING in lysates from wild type BMDCs stimulated for 2–24 h with poly(dA:dT) (1 μg/ml) in the presence or absence of 20 mM MG132. (C) Confocal microscopy of L929 cells transfected for 24 h with Flag-tagged STING and then mock treated or stimulated for 12 h with ISD (1 μg/ml). Immunofluorescence was performed using anti-Flag (red) and anti-20S proteasome β1 (green). Nuclei were stained with the DNA-intercalating dye DAPI. (D) Immunoblot analysis of STING in lysates of L929 cells transfected with increasing doses of Myc-tagged TRIM30α (0, 0.8 and 1.2 μg) and then treated for 6 h with DMSO (negative control) or 20 mM MG132. Densitometry analysis to quantify ratio of STING to β-actin is shown on the below. (E) Immunoblot analysis of STING in lysates of L929 cells transfected with full-length TRIM30α, C35A or ΔR (1μg) and then treated as in D.
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ppat.1005012.g006: TRIM30α enhances the degradation of STING.(A) Immunoblot analysis of STING, TBK1, IRF3 and TRIM30α in lysates from wild type and Trim30α-/- BMDCs stimulated for 2–24 h with poly(dA:dT) (1 μg/ml). (B) Immunoblot analysis of STING in lysates from wild type BMDCs stimulated for 2–24 h with poly(dA:dT) (1 μg/ml) in the presence or absence of 20 mM MG132. (C) Confocal microscopy of L929 cells transfected for 24 h with Flag-tagged STING and then mock treated or stimulated for 12 h with ISD (1 μg/ml). Immunofluorescence was performed using anti-Flag (red) and anti-20S proteasome β1 (green). Nuclei were stained with the DNA-intercalating dye DAPI. (D) Immunoblot analysis of STING in lysates of L929 cells transfected with increasing doses of Myc-tagged TRIM30α (0, 0.8 and 1.2 μg) and then treated for 6 h with DMSO (negative control) or 20 mM MG132. Densitometry analysis to quantify ratio of STING to β-actin is shown on the below. (E) Immunoblot analysis of STING in lysates of L929 cells transfected with full-length TRIM30α, C35A or ΔR (1μg) and then treated as in D.

Mentions: It is very important to determine the mechanism by which interaction of TRIM30α with STING suppress the intracellular DNA-sensing pathway. As shown in Fig 6A, Trim30α-deficient BMDCs maintained a higher expression of STING than wild type BMDCs after poly(dA:dT) stimulations of different durations (Fig 6A). In contrast, the expression of TBK1 and IRF3, two adaptors downstream of STING, maintained unchanged in Trim30α-deficient BMDCs, suggesting that TRIM30α may suppress the stability of STING. We next treated wild type BMDCs with poly(dA:dT) in the presence or absence of MG132, the inhibitor of proteasome. We observed a high level expression of STING in BMDCs upon MG132 treatment than control, indicating that STING undergoes degradation after stimulation with intracellular DNA in a proteasome way (Fig 6B). Moreover, MG132 treatment significantly promoted much higher expression of phosphorylation of TBK1 and IRF3 than control cells, which mimics TRIM30α deficiency upon intracellular DNA stimulation. Confocal microscopy further demonstrated that STING was co-localized with proteasome during ISD stimulation, suggesting that STING may undergo degradation via proteasome in response to DNA stimulus (Fig 6C).


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 enhances the degradation of STING.(A) Immunoblot analysis of STING, TBK1, IRF3 and TRIM30α in lysates from wild type and Trim30α-/- BMDCs stimulated for 2–24 h with poly(dA:dT) (1 μg/ml). (B) Immunoblot analysis of STING in lysates from wild type BMDCs stimulated for 2–24 h with poly(dA:dT) (1 μg/ml) in the presence or absence of 20 mM MG132. (C) Confocal microscopy of L929 cells transfected for 24 h with Flag-tagged STING and then mock treated or stimulated for 12 h with ISD (1 μg/ml). Immunofluorescence was performed using anti-Flag (red) and anti-20S proteasome β1 (green). Nuclei were stained with the DNA-intercalating dye DAPI. (D) Immunoblot analysis of STING in lysates of L929 cells transfected with increasing doses of Myc-tagged TRIM30α (0, 0.8 and 1.2 μg) and then treated for 6 h with DMSO (negative control) or 20 mM MG132. Densitometry analysis to quantify ratio of STING to β-actin is shown on the below. (E) Immunoblot analysis of STING in lysates of L929 cells transfected with full-length TRIM30α, C35A or ΔR (1μg) and then treated as in D.
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ppat.1005012.g006: TRIM30α enhances the degradation of STING.(A) Immunoblot analysis of STING, TBK1, IRF3 and TRIM30α in lysates from wild type and Trim30α-/- BMDCs stimulated for 2–24 h with poly(dA:dT) (1 μg/ml). (B) Immunoblot analysis of STING in lysates from wild type BMDCs stimulated for 2–24 h with poly(dA:dT) (1 μg/ml) in the presence or absence of 20 mM MG132. (C) Confocal microscopy of L929 cells transfected for 24 h with Flag-tagged STING and then mock treated or stimulated for 12 h with ISD (1 μg/ml). Immunofluorescence was performed using anti-Flag (red) and anti-20S proteasome β1 (green). Nuclei were stained with the DNA-intercalating dye DAPI. (D) Immunoblot analysis of STING in lysates of L929 cells transfected with increasing doses of Myc-tagged TRIM30α (0, 0.8 and 1.2 μg) and then treated for 6 h with DMSO (negative control) or 20 mM MG132. Densitometry analysis to quantify ratio of STING to β-actin is shown on the below. (E) Immunoblot analysis of STING in lysates of L929 cells transfected with full-length TRIM30α, C35A or ΔR (1μg) and then treated as in D.
Mentions: It is very important to determine the mechanism by which interaction of TRIM30α with STING suppress the intracellular DNA-sensing pathway. As shown in Fig 6A, Trim30α-deficient BMDCs maintained a higher expression of STING than wild type BMDCs after poly(dA:dT) stimulations of different durations (Fig 6A). In contrast, the expression of TBK1 and IRF3, two adaptors downstream of STING, maintained unchanged in Trim30α-deficient BMDCs, suggesting that TRIM30α may suppress the stability of STING. We next treated wild type BMDCs with poly(dA:dT) in the presence or absence of MG132, the inhibitor of proteasome. We observed a high level expression of STING in BMDCs upon MG132 treatment than control, indicating that STING undergoes degradation after stimulation with intracellular DNA in a proteasome way (Fig 6B). Moreover, MG132 treatment significantly promoted much higher expression of phosphorylation of TBK1 and IRF3 than control cells, which mimics TRIM30α deficiency upon intracellular DNA stimulation. Confocal microscopy further demonstrated that STING was co-localized with proteasome during ISD stimulation, suggesting that STING may undergo degradation via proteasome in response to DNA stimulus (Fig 6C).

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