cGLRs are a diverse family of pattern recognition receptors in innate immunity.

Cyclic GMP-AMP synthase (cGAS) is an enzyme in human cells that controls an immune response to cytosolic DNA. Upon binding DNA, cGAS synthesizes a nucleotide signal 2'3'-cGAMP that activates STING-dependent downstream immunity. Here, we discover that cGAS-like receptors (cGLRs) constitute a major family of pattern recognition receptors in innate immunity. Building on recent analysis in Drosophila, we identify >3,000 cGLRs present in nearly all metazoan phyla. A forward biochemical screening of 150 animal cGLRs reveals a conserved mechanism of signaling including response to dsDNA and dsRNA ligands and synthesis of isomers of the nucleotide signals cGAMP, c-UMP-AMP, and c-di-AMP. Combining structural biology and in vivo analysis in coral and oyster animals, we explain how synthesis of distinct nucleotide signals enables cells to control discrete cGLR-STING signaling pathways. Our results reveal cGLRs as a widespread family of pattern recognition receptors and establish molecular rules that govern nucleotide signaling in animal immunity.

CBASS Immunity Uses CARF-Related Effectors to Sense 3'-5'- and 2'-5'-Linked Cyclic Oligonucleotide Signals and Protect Bacteria from Phage Infection.

cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes are immune sensors that synthesize nucleotide second messengers and initiate antiviral responses in bacterial and animal cells. Here, we discover Enterobacter cloacae CD-NTase-associated protein 4 (Cap4) as a founding member of a diverse family of >2,000 bacterial receptors that respond to CD-NTase signals. Structures of Cap4 reveal a promiscuous DNA endonuclease domain activated through ligand-induced oligomerization. Oligonucleotide recognition occurs through an appended SAVED domain that is an unexpected fusion of two CRISPR-associated Rossman fold (CARF) subunits co-opted from type III CRISPR immunity. Like a lock and key, SAVED effectors exquisitely discriminate 2'-5'- and 3'-5'-linked bacterial cyclic oligonucleotide signals and enable specific recognition of at least 180 potential nucleotide second messenger species. Our results reveal SAVED CARF family proteins as major nucleotide second messenger receptors in CBASS and CRISPR immune defense and extend the importance of linkage specificity beyond mammalian cGAS-STING signaling.

Cyclic dinucleotides bind the C-linker of HCN4 to control channel cAMP responsiveness.

cAMP mediates autonomic regulation of heart rate by means of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which underlie the pacemaker current If. cAMP binding to the C-terminal cyclic nucleotide binding domain enhances HCN open probability through a conformational change that reaches the pore via the C-linker. Using structural and functional analysis, we identified a binding pocket in the C-linker of HCN4. Cyclic dinucleotides, an emerging class of second messengers in mammals, bind the C-linker pocket (CLP) and antagonize cAMP regulation of the channel. Accordingly, cyclic dinucleotides prevent cAMP regulation of If in sinoatrial node myocytes, reducing heart rate by 30%. Occupancy of the CLP hence constitutes an efficient mechanism to hinder ß-adrenergic stimulation on If. Our results highlight the regulative role of the C-linker and identify a potential drug target in HCN4. Furthermore, these data extend the signaling scope of cyclic dinucleotides in mammals beyond their first reported role in innate immune system.

cGLRs are a diverse family of pattern recognition receptors in animal innate immunity.

cGAS (cyclic GMP-AMP synthase) is an enzyme in human cells that controls an immune response to cytosolic DNA. Upon binding DNA, cGAS synthesizes a nucleotide signal 2'3'-cGAMP that activates the protein STING and downstream immunity. Here we discover cGAS-like receptors (cGLRs) constitute a major family of pattern recognition receptors in animal innate immunity. Building on recent analysis in Drosophila , we use a bioinformatic approach to identify >3,000 cGLRs present in nearly all metazoan phyla. A forward biochemical screen of 140 animal cGLRs reveals a conserved mechanism of signaling including response to dsDNA and dsRNA ligands and synthesis of alternative nucleotide signals including isomers of cGAMP and cUMP-AMP. Using structural biology, we explain how synthesis of distinct nucleotide signals enables cells to control discrete cGLR-STING signaling pathways. Together our results reveal cGLRs as a widespread family of pattern recognition receptors and establish molecular rules that govern nucleotide signaling in animal immunity.

Small molecule AKAP-protein kinase A (PKA) interaction disruptors that activate PKA interfere with compartmentalized cAMP signaling in cardiac myocytes.

A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3'-diamino-4,4'-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating ß-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure.

Characterizing the Epothilone Binding Site on ß-Tubulin by Photoaffinity Labeling: Identification of ß-Tubulin Peptides TARGSQQY and TSRGSQQY as Targets of an Epothilone Photoprobe for Polymerized Tubulin.

Photoaffinity labeling with an epothilone A photoprobe led to the identification of the ß-tubulin peptides TARGSQQY and TSRGSQQY as targets of the photoprobe for polymerized tubulin. These peptides represent residues 274-281 in different ß-tubulin isotypes. Placing the carbene producing 21-diazo/triazolo moiety of the photoprobe in the vicinity of the TARGSQQY peptide in a homology model of TBB3 predicted a binding pose and conformation of the photoprobe that are very similar to the ones reported for 1) the high resolution cocrystal structure of epothilone A with an α,ß-tubulin complex and for 2) a saturation transfer difference NMR and transferred NOESY NMR study of dimeric and polymerized tubulin. Our findings thus provide additional support for these models as physiologically the most relevant among several modes of binding that have been proposed for epothilone A in the taxane pocket of ß-tubulin.