The DNA Sensor cGAS is Decorated by Acetylation and Phosphorylation Modifications in the Context of Immune Signaling

Abstract

<jats:p>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.</jats:p>

The clinical potential of prm-PASEF mass spectrometry

“…The performance of the mass spectrometer is independent of biological products like antibodies making the technique independent of antigen interference, production variations, storage problems, and commercial availability. At the same time, it is a method, which can measure changes to protein abundance or detect specific mutants or posttranslational modifications with high specificity [34].…”

Role of Post-Translational Modifications of cGAS in Innate Immunity

“…It is predicted that more PTMs will be discovered under versatile contexts using powerful omics tools, such as proteomics. For example, a recent proteomic study found five phosphorylation and six acetylation sites that have not been previously documented [ 62 ]. The role of new PTMs will help elucidate the regulatory mechanisms for cGAS, and the PTM sites will be ideal drug targets for therapeutics in autoimmune diseases and cancers.…”

Cytosolic DNA sensing by cGAS: regulation, function, and human diseases

“…Notably, a recent proteomics effort in examining cGAS post-translational modifications upon HSV-1 infection from both human primary fibroblasts and HEK293T cells revealed new PTMs occurring on cGAS, including phosphorylation at Ser37, Ser116, Ser201, Ser221, Ser263, and acetylation at Lys198, Lys285, Lys355, and Lys414 in hcGAS. 127 Further functional validation suggests that acetylation at Lys414 suppresses, while acetylation at Lys198 promotes hcGAS activation. Interestingly, hcGAS-Lys198 acetylation was found to be decreased by quantitative proteomics upon infection by either HSV-1 or HCMV (human cytomegalovirus), suggesting that these DNA viruses might hijack this acetylation regulation to targetedly inactivate cGAS to evade innate-immune surveillance.…”

“…Interestingly, hcGAS-Lys198 acetylation was found to be decreased by quantitative proteomics upon infection by either HSV-1 or HCMV (human cytomegalovirus), suggesting that these DNA viruses might hijack this acetylation regulation to targetedly inactivate cGAS to evade innate-immune surveillance. 127 The detailed mechanism(s) mediating acetylation-dependent cGAS activity control on these sites remain unclear.…”