An E3 ubiquitin ligase is essential for immune homeostasis

Author: Brandon Eden

TRIM29, an E3 ubiquitin ligase as depicted in the above figure, tags STING for proteasome-dependent degradation to downregulate the antiviral cystolic DNA immune response.

https://www.nature.com/articles/s41421-018-0010-9

 

It is commonly thought that the innate immune response is completely non-specific. However, relatively recent studies have shown that viral DNA is recognized by germline-encoded pattern recognition receptors (PRRs) on innate immune cells, which then trigger an immune response (Akira et al 2006). Viral DNA can be sensed by Toll-like receptors which are found on macrophages and dendritic cells (Takeda et al 2003). Activation of these receptors leads to downstream pathways that result in increased type I interferon-I (IFN-1) and pro-inflammatory cytokines (Li et al 2018). Pathogenic self-DNA that escaped from a cell’s nucleus or mitochondria can also result in increased IFN-1 (Takeda et al 2003). The recognition of such cytosolic DNA triggers the synthesis of cGAMP from cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS), and then cGAMP binds directly to the stimulator of interferon genes (STING) (Ishikawa et al 2009). The STING protein can then phosphorylate interferon regulator factor 3 (IRF3), a transcription factor that enters the nucleus and promotes expression of interferon and cytokine genes (Li et al 2018).

Studies have shown that STING is the central signaling molecule in the innate immune response to cytosolic nucleic acids, which can indicate infection or cancer (Burdette et al 2013). Once activated, STING must have the ability to be deactivated to prevent chronic inflammation and inflammatory related disease. In a recent study presented by Li et al, TRIM29, an E3 ubiquitin ligase, has been shown to ubiquitinate STING and activate it for degradation and therefore downregulate the immune response (Li et al 2018). As studied in BCM441, ubiquitination is a critical process that enables misfolded proteins to be tagged for degradation. In this study, a key regulatory protein in the innate immune response to viral cytosolic DNA is shown to downregulate the immune response by being targeted for degradation by ubiquitination.

It has been previously reported that tripartite motif-containing protein 29 (TRIM29) is a key negative regulator of alveolar macrophages and downregulates the expression of type 1 interferons and pro-inflammatory cytokines in the lungs (Xing et al 2016). In order to explore the specific mechanism of TRIM29, Li et al first confirmed that TRIM29 mRNA levels in macrophages and dendritic cells were increased in the presence of cytosolic DNA from herpes simplex virus type 1 (Li et al 2018). The role of TRIM29 as a negative feedback inducer was then explored, and authors found that knocking down TRIM29 expression in macrophages led to significantly higher levels of interferons, cytokines, and interleukins (Li et al 2018). In fact, TRIM29 knockout mice infected with herpes simplex virus-1 (HSV-1) survived longer than WT mice infected with HSV-1, suggesting that TRIM29 knockout mice are more resistant to the virus due to heightened immune responses (Li et al 2018). TRIM29 knockout mice had higher levels of chemokines that recruit immune cells to the site of infection in addition to higher MX1 expression, an antiviral host protein that disrupts viral DNA and RNA replication (Li et al 2018). These experiments confirm that TRIM29 expression is necessary to suppress the immune system in response to pathogenic cytosolic DNA to prohibit a prolonged and exacerbated immune response.

After establishing TRIM29 as a negative regulator of the immune response to cytosolic DNA, the authors’ next goals were to determine which signaling pathway is affected, and where in the pathway TRIM29 exerts its effect. During antiviral immune responses, cytosolic DNA sensor cGAS synthesizes cGAMP which binds to STING, allowing it to phosphorylate transcription factor IRF3 to promote expression of inflammatory genes (Li et al 2018). Authors stimulated WT and TRIM29 knockout cells with increased STING ligand cGAMP and then infected the cells with HSV-60 (Li et al 2018). Authors detected increased amounts of phosphorylated IRF3 in TRIM29 knockout cells, suggesting that TRIM29 is downregulates the STING-IRF3 pathway (Li et al 2018). Reconstitution of TRIM29 in TRIM29 knockout cells abrogated the enhanced activation of the STING-IRF3 pathway (Li et al 2018).

Previous studies claim that STING is the central signaling molecule in response to pathogenic cytosolic DNA, and the authors’ observation of increased levels of IRF3 downstream of STING in TRIM29 deficient cells aligns with this assessment (Burdette et al 2013). Li et al hypothesized that TRIM29 exerts its negative feedback effect at the regulation point involving STING. Additional experiments determined that STING levels were indeed decreased in WT immune cells infected with HSV-60, while TRIM29 knockout cells did not have the ability to degrade STING (Li et al 2018). To investigate how TRIM29 reduces levels of STING, authors performed an immunoprecipitation experiment with an antibody to TRIM29, and determined that TRIM29 binds to STING (Li et al 2018). TRIM29 formed a complex with STING in cells infected with HSV-60, indicating that TRIM29 suppresses inflammation by directly interacting with STING (Li et al 2018).

TRIM29 is an E3 ubiquitin ligase, and authors were fairly certain that TRIM29 ubiquitinates STING and marks it for degradation by the proteasome. To confirm this, authors introduced MG132, a proteasome inhibitor, into WT HSV-60 infected cells and rescued low levels of STING induced by TRIM29 (Li et al 2018). Authors then overexpressed ubiquitin with lysine residues in different locations, and discovered that K48 ubiquitin polyubiquitinates STING to mediate proteasome degradation. Lastly, authors further investigated the binding interaction of STING and TRIM29, and performed experiments with different truncated versions of TRIM29 to determine that TRIM29 interacts with STING through its C-terminal domain.

The authors were able to determine that TRIM29 binds and ubiquitinates STING to downregulate the inflammatory immune response and prevent excessive stimulation. This study provides unique information one strategy of antiviral immune response, and can aid in vaccine development (Li et al 2018). The STING-IF3 pathway can be stimulated by both viral DNA and pathogenic host DNA that escaped into the cytosol. These findings can provide insight into strategies to prevent self-DNA triggered autoimmunity, and constitutive expression of TRIM29 is required for immune homeostasis (Li et al 2018).

 

 

 

References

Akira, Shizuo, Satoshi Uematsu, and Osamu Takeuchi. “Pathogen Recognition and Innate Immunity.” Cell 124, no. 4 (February 24, 2006): 783–801. https://doi.org/10.1016/j.cell.2006.02.015.

Burdette, Dara L., and Russell E. Vance. “STING and the Innate Immune Response to Nucleic Acids in the Cytosol.” Nature Immunology 14, no. 1 (January 2013): 19–26. https://doi.org/10.1038/ni.2491.

Ishikawa, Hiroki, Zhe Ma, and Glen N. Barber. “STING Regulates Intracellular DNA-Mediated, Type I Interferon-Dependent Innate Immunity.” Nature 461, no. 7265 (October 8, 2009): 788–92. https://doi.org/10.1038/nature08476.

Li, Qijie, Liangbin Lin, Yanli Tong, Yantong Liu, Jun Mou, Xiaodong Wang, Xiuxuan Wang, et al. “TRIM29 Negatively Controls Antiviral Immune Response through Targeting STING for Degradation.” Cell Discovery 4, no. 1 (March 20, 2018): 13. https://doi.org/10.1038/s41421-018-0010-9.

Takeda, Kiyoshi, Tsuneyasu Kaisho, and Shizuo Akira. “Toll-like Receptors.” Annual Review of Immunology 21, no. 1 (April 2003): 335–76. https://doi.org/10.1146/annurev.immunol.21.120601.141126.

 

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