Abstract
The cyclic GMP-AMP synthase (cGAS) protein is a pattern-recognition receptor of the mammalian innate immune system that is recognized as a main cytosolic sensor of pathogenic or damaged DNA. cGAS DNA binding initiates catalytic production of the second messenger, cyclic GMP-AMP, which activates the STING-TBK1-IRF3 signaling axis to induce cytokine expression. Post-translational modification (PTM) has started to be recognized as a critical component of cGAS regulation, yet the extent of these modifications remains unclear. Here, we report the identification and functional analysis of cGAS phosphorylations and acetylations in several cell types under basal and immune-stimulated conditions. cGAS was enriched by immunoaffinity purification from human primary fibroblasts prior to and after infection with herpes simplex virus type 1 (HSV-1), as well as from immune-stimulated STING-HEK293T cells. Six phosphorylations and eight acetylations were detected, of which eight PTMs were not previously documented. PTMs were validated by parallel reaction monitoring (PRM) mass spectrometry in fibroblasts, HEK293T cells, and THP-1 macrophage-like cells. Primary sequence and structural analysis of cGAS highlighted a subset of PTM sites with elevated surface accessibility and high evolutionary sequence conservation. To assess the functional relevance of each PTM, we generated a series of single-point cGAS mutations. Stable cell lines were constructed to express cGAS with amino acid substitutions that prevented phosphorylation (Ser-to-Ala) and acetylation (Lys-to-Arg) or that mimicked the modification state (Ser-to-Asp and Lys-to-Gln). cGAS-dependent apoptotic and immune signaling activities were then assessed for each mutation. Our results show that acetyl-mimic mutations at Lys384 and Lys414 inhibit the ability of cGAS to induce apoptosis. In contrast, the Lys198 acetyl-mimic mutation increased cGAS-dependent interferon signaling when compared with the unmodified charge-mimic. Moreover, targeted PRM quantification showed that Lys198 acetylation is decreased upon infections with two herpesviruses-HSV-1 and human cytomegalovirus (HCMV), highlighting this residue as a regulatory point during virus infection.
Highlights
The cyclic GMP-AMP synthase protein is a pattern-recognition receptor of the mammalian innate immune system that is recognized as a main cytosolic sensor of pathogenic or damaged DNA. cGAS DNA binding initiates catalytic production of the second messenger, cyclic GMP-AMP, which activates the Stimulator of interferon genes (STING)-TANK binding kinase 1 (TBK1)-IFN regulatory factor-3 (IRF3) signaling axis to induce cytokine expression
CGAS Contains Multiple Acetylation and Phosphorylation Sites—posttranslational modifications (PTMs) are dynamically regulated by different external stimuli, and this regulation often occurs in a cell type dependent manner
To improve the likelihood of identifying cGAS PTM sites that have potential biological relevance, our initial analysis employed non-targeted, data-dependent mass spectrometry performed in two cell types that express GFP-tagged cGAS and under conditions that stimulate cGAS activity
Summary
The cyclic GMP-AMP synthase (cGAS) protein is a pattern-recognition receptor of the mammalian innate immune system that is recognized as a main cytosolic sensor of pathogenic or damaged DNA. cGAS DNA binding initiates catalytic production of the second messenger, cyclic GMP-AMP, which activates the STING-TBK1-IRF3 signaling axis to induce cytokine expression. The cyclic GMP-AMP synthase (cGAS) protein is a pattern-recognition receptor of the mammalian innate immune system that is recognized as a main cytosolic sensor of pathogenic or damaged DNA. CGAS DNA binding initiates catalytic production of the second messenger, cyclic GMP-AMP, which activates the STING-TBK1-IRF3 signaling axis to induce cytokine expression. DNA derived either from the genetic material of pathogens or from cellular DNA damage provides such a molecular pattern that can be sensed by PRRs. In recent years, cyclic GMP-AMP synthase (cGAS) has been characterized as a primary cytosolic DNA sensor during DNA transfection, bacterial infections, DNA virus or retrovirus infection, as well as self-DNA leakage (4 –12). STING activates IKK, which phosphorylates IB and releases the transcription factor NF-B into the nucleus, inducing cytokine expressions [18, 19] As this central cGAS-STING-TBK1-IRF3 immune signaling axis is well-defined, recent investigation has focused on understanding the regulation of cGAS activity. Interactions with viral proteins were found to underlie the ability of certain viruses to suppress host immune signaling, such as KSHV ORF52 and LANA [9, 34], dengue virus NS2B [35], HSV-1 pUL37 and VP22 [36, 37], and HCMV pUL31 and pUL83 [38, 39]
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