Proteomic analysis of RAW macrophages treated with cGAMP or c-di-GMP reveals differentially activated cellular pathways

Abstract

<p>Global and quantitative analysis of the proteome help to reveal how host cells sense invading bacteria and<br />respond to bacterial signaling molecules. Here, we performed label free quantitative proteomic analysis of<br />RAW macrophages treated with host-derived cGAMP and bacterial-derived c-di-GMP, in an attempt to<br />identify cellular pathways impacted by these dinucleotides and determine if the host responds<br />differentially to these two cyclic dinucleotides. We identified a total of 3811 proteins of which<br />abundances of 404 proteins in cGAMP and 236 proteins in c-di-GMP treated cells were significantly<br />different compared to the control. Many of the proteins that were strongly and commonly upregulated,<br />such as interferon-induced proteins 47, 202 and 204 (Ifi47, Ifi202, Ifi204), ubiquitin-activating enzyme E7<br />(Uba7), interferon-induced protein with tetratricopeptide repeats 1, 2 or 3 (Ifit1, Ifit2, Ifit3), ubiquitin-like<br />protein ISG15 (ISG15), might be due to the fact that both dinucleotides promote the production of<br />interferons, which induce the expression of many proteins. However, there were also other proteins that<br />were differentially affected by cGAMP or c-di-GMP treatment, including probable ATP-dependent RNA<br />helicase DHX58 (Dhx58), nuclear autoantigen Sp-100 (Sp100), MARCKS-related protein (Marcksl1) and<br />antigen peptide transporter 2 (Tap2). This is probably due to the differential levels of IFNs produced by<br />the dinucleotides or may indicate that non-STING activation might also contribute to the host's response<br />to c-di-GMP and cGAMP. Interestingly Trex1, a nuclease that degrades DNA (an activator of cGAS to<br />produce cGAMP), was upregulated (3.22 fold) upon cGAMP treatment, hinting at a possible feedback<br />loop to regulate cGAMP synthesis. These results lay a foundation for future studies to better characterize<br />and understand the complex c-di-GMP and cGAMP signaling network.<br /></p>