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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 knockdown promotes immune signaling by cytoplasmic DNA and DNA virus infection in D2SC cells.(A) Immunoblot analysis of the knockdown of exogenous TRIM30α in D2SC cells treated with control siRNA (SC) or TRIM30α siRNA (T3) for 24 h and then stimulated for 16 h with poly(dA:dT) (1 μg/ml). β-actin served as a loading control throughout the experiment. (B, C) ELISA of type I IFN and IL-6 in D2SC cells treated with siRNA SC or T3 and then stimulated for 16 h with poly(dA:dT) (1 μg/ml), ISD (1 μg/ml), genomic DNA (2 μg/ml), c-di-GMP (8 μg/ml) or poly(I:C) (5 μg/ml) (C) or with HSV-1 (MOI 10) or vaccinia virus (VACV) (MOI 10). (D) Real-time PCR of IP-10 mRNA in D2SC cells treated with siRNA SC or T3 and then stimulated for 8 h with ISD (1 μg/ml) or poly(I:C) (5 μg/ml). (E) Immunoblot analysis of p-IRF3, p-p65 and TRIM30α in lysates of D2SC cells treated with siRNA SC or T3 and then stimulated for 1–5 h with poly(dA:dT) (1 μg/ml). Densitometry analysis to quantify ratio of p-IRF3 or p-p65 to β-actin is shown on the right. The data are representative of three independent experiments and are presented as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001.
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ppat.1005012.g001: TRIM30α knockdown promotes immune signaling by cytoplasmic DNA and DNA virus infection in D2SC cells.(A) Immunoblot analysis of the knockdown of exogenous TRIM30α in D2SC cells treated with control siRNA (SC) or TRIM30α siRNA (T3) for 24 h and then stimulated for 16 h with poly(dA:dT) (1 μg/ml). β-actin served as a loading control throughout the experiment. (B, C) ELISA of type I IFN and IL-6 in D2SC cells treated with siRNA SC or T3 and then stimulated for 16 h with poly(dA:dT) (1 μg/ml), ISD (1 μg/ml), genomic DNA (2 μg/ml), c-di-GMP (8 μg/ml) or poly(I:C) (5 μg/ml) (C) or with HSV-1 (MOI 10) or vaccinia virus (VACV) (MOI 10). (D) Real-time PCR of IP-10 mRNA in D2SC cells treated with siRNA SC or T3 and then stimulated for 8 h with ISD (1 μg/ml) or poly(I:C) (5 μg/ml). (E) Immunoblot analysis of p-IRF3, p-p65 and TRIM30α in lysates of D2SC cells treated with siRNA SC or T3 and then stimulated for 1–5 h with poly(dA:dT) (1 μg/ml). Densitometry analysis to quantify ratio of p-IRF3 or p-p65 to β-actin is shown on the right. 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: TRIM30α has been reported to be induced by TLR ligands in an NF-κB-dependent way and then negatively regulates TLR signaling by a ‘feedback’ mechanism. We found TRIM30α can also be induced upon intracellular poly(dA:dT) stimulation in bone marrow-derived dendritic cells (BMDCs) (S1A Fig). To address whether TRIM30α regulates the immune response to DNA-mediated signaling, we used TRIM30α siRNA (T3) to silence endogenous TRIM30α expression in D2SC cells, a mouse mDC cell line. The results showed that the T3 siRNA efficiently reduced the TRIM30α protein expression compared with control siRNA (SC) (Fig 1A). Various DNA ligands were used to stimulate D2SC cells, including poly(dA:dT), interferon stimulatory DNA (ISD), the genomic DNA of C57BL/6 mice and cyclic di-GMP (c-di-GMP). Knockdown of TRIM30α enhanced the production of type I IFN and IL-6 compared with controls (Fig 1B). In contrast, the induction of type I IFN by the synthetic analog of viral dsRNA poly (I:C) (the signaling of which is largely governed by MDA5) was reduced when TRIM30α was knocked down. To further confirm the function of TRIM30α in the anti-DNA viral response, we infected D2SC cells with the DNA viruses HSV-1 or vaccinia virus (VACV). The data showed that IFN-β and IL-6 production was dramatically increased in D2SC cells transfected with TRIM30α-specific siRNA (Fig 1C). TRIM30α knockdown also promoted the type I IFN and IL-6 expression at the mRNA level after stimulation with poly(dA:dT), ISD or HSV-1 (S1B and S1C Fig). We next examined the IFN-stimulated genes, such as the chemokine IP-10. Notably, TRIM30α knockdown enhanced the production of IP-10 mRNA upon ISD stimulation, but had no effect on the poly(I:C)-mediated signaling (Fig 1D).


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 knockdown promotes immune signaling by cytoplasmic DNA and DNA virus infection in D2SC cells.(A) Immunoblot analysis of the knockdown of exogenous TRIM30α in D2SC cells treated with control siRNA (SC) or TRIM30α siRNA (T3) for 24 h and then stimulated for 16 h with poly(dA:dT) (1 μg/ml). β-actin served as a loading control throughout the experiment. (B, C) ELISA of type I IFN and IL-6 in D2SC cells treated with siRNA SC or T3 and then stimulated for 16 h with poly(dA:dT) (1 μg/ml), ISD (1 μg/ml), genomic DNA (2 μg/ml), c-di-GMP (8 μg/ml) or poly(I:C) (5 μg/ml) (C) or with HSV-1 (MOI 10) or vaccinia virus (VACV) (MOI 10). (D) Real-time PCR of IP-10 mRNA in D2SC cells treated with siRNA SC or T3 and then stimulated for 8 h with ISD (1 μg/ml) or poly(I:C) (5 μg/ml). (E) Immunoblot analysis of p-IRF3, p-p65 and TRIM30α in lysates of D2SC cells treated with siRNA SC or T3 and then stimulated for 1–5 h with poly(dA:dT) (1 μg/ml). Densitometry analysis to quantify ratio of p-IRF3 or p-p65 to β-actin is shown on the right. The data are representative of three independent experiments and are presented as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001.
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ppat.1005012.g001: TRIM30α knockdown promotes immune signaling by cytoplasmic DNA and DNA virus infection in D2SC cells.(A) Immunoblot analysis of the knockdown of exogenous TRIM30α in D2SC cells treated with control siRNA (SC) or TRIM30α siRNA (T3) for 24 h and then stimulated for 16 h with poly(dA:dT) (1 μg/ml). β-actin served as a loading control throughout the experiment. (B, C) ELISA of type I IFN and IL-6 in D2SC cells treated with siRNA SC or T3 and then stimulated for 16 h with poly(dA:dT) (1 μg/ml), ISD (1 μg/ml), genomic DNA (2 μg/ml), c-di-GMP (8 μg/ml) or poly(I:C) (5 μg/ml) (C) or with HSV-1 (MOI 10) or vaccinia virus (VACV) (MOI 10). (D) Real-time PCR of IP-10 mRNA in D2SC cells treated with siRNA SC or T3 and then stimulated for 8 h with ISD (1 μg/ml) or poly(I:C) (5 μg/ml). (E) Immunoblot analysis of p-IRF3, p-p65 and TRIM30α in lysates of D2SC cells treated with siRNA SC or T3 and then stimulated for 1–5 h with poly(dA:dT) (1 μg/ml). Densitometry analysis to quantify ratio of p-IRF3 or p-p65 to β-actin is shown on the right. 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: TRIM30α has been reported to be induced by TLR ligands in an NF-κB-dependent way and then negatively regulates TLR signaling by a ‘feedback’ mechanism. We found TRIM30α can also be induced upon intracellular poly(dA:dT) stimulation in bone marrow-derived dendritic cells (BMDCs) (S1A Fig). To address whether TRIM30α regulates the immune response to DNA-mediated signaling, we used TRIM30α siRNA (T3) to silence endogenous TRIM30α expression in D2SC cells, a mouse mDC cell line. The results showed that the T3 siRNA efficiently reduced the TRIM30α protein expression compared with control siRNA (SC) (Fig 1A). Various DNA ligands were used to stimulate D2SC cells, including poly(dA:dT), interferon stimulatory DNA (ISD), the genomic DNA of C57BL/6 mice and cyclic di-GMP (c-di-GMP). Knockdown of TRIM30α enhanced the production of type I IFN and IL-6 compared with controls (Fig 1B). In contrast, the induction of type I IFN by the synthetic analog of viral dsRNA poly (I:C) (the signaling of which is largely governed by MDA5) was reduced when TRIM30α was knocked down. To further confirm the function of TRIM30α in the anti-DNA viral response, we infected D2SC cells with the DNA viruses HSV-1 or vaccinia virus (VACV). The data showed that IFN-β and IL-6 production was dramatically increased in D2SC cells transfected with TRIM30α-specific siRNA (Fig 1C). TRIM30α knockdown also promoted the type I IFN and IL-6 expression at the mRNA level after stimulation with poly(dA:dT), ISD or HSV-1 (S1B and S1C Fig). We next examined the IFN-stimulated genes, such as the chemokine IP-10. Notably, TRIM30α knockdown enhanced the production of IP-10 mRNA upon ISD stimulation, but had no effect on the poly(I:C)-mediated signaling (Fig 1D).

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