Apart from its vital function as a redox cofactor, nicotinamide adenine dinucleotide (NAD+) has emerged as a crucial substrate for NAD+‐consuming enzymes, including poly(ADP‐ribosyl)transferase 1 (PARP1) and CD38/CD157. Their association with severe diseases, such as cancer, Alzheimer's disease, and depressions, necessitates the development of new analytical tools based on traceable NAD+ surrogates. Here, the synthesis, photophysics and biochemical utilization of an emissive, thieno[3,4‐d]pyrimidine‐based NAD+ surrogate, termed NthAD+, are described. Its preparation was accomplished by enzymatic conversion of synthetic thATP by nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1). The new NAD+ analogue possesses useful photophysical features including redshifted absorption and emission maxima as well as a relatively high quantum yield. Serving as a versatile substrate, NthAD+ was reduced by alcohol dehydrogenase (ADH) to NthADH and afforded thADP‐ribose (thADPr) upon hydrolysis by NAD+‐nucleosidase (NADase). Furthermore, NthAD+ was engaged in cholera toxin A (CTA)‐catalyzed mono(thADP‐ribosyl)ation, but was found incapable in promoting PARP1‐mediated poly(thADP‐ribosyl)ation. Due to its high photophysical responsiveness, NthAD+ is suited for spectroscopic real‐time monitoring. Intriguingly, and as an N7‐lacking NAD+ surrogate, the thieno‐based cofactor showed reduced compatibility (i.e., functional similarity compared to native NAD+) relative to its isothiazolo‐based analogue. The distinct tolerance, displayed by diverse NAD+ producing and consuming enzymes, suggests unique biological recognition features and dependency on the purine N7 moiety, which is found to be of importance, if not essential, for PARP1‐mediated reactions.
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.
Bacterial cyclic dinucleotides (CDNs) play important roles in regulating biofilm formation, motility and virulence. In eukaryotic cells, theses bacterial CDNs are recognized as pathogen‐associated molecular patterns (PAMPs) and trigger an innate immune response. We report the photophysical analyses of a novel group of enzymatically synthesized emissive CDN analogues comprised of two families of isomorphic ribonucleotides. The highly favorable photophysical features of the CDN analogues, when compared to their non‐emissive natural counterparts, are used to monitor in real time the dinucleotide cyclase‐mediated synthesis and phosphodiesterase (PDE)‐mediated hydrolysis of homodimeric and mixed CDNs, providing effective means to probe the activities of two classes of bacterial enzymes and insight into their biomolecular recognition and catalytic features.
We evaluated the effect of Tween 80 permeabilization on ginsenoside secretion in Panax ginseng hairy roots. Tween 80 (1.2%, w/v) had no significant effect on hairy root vitality. After a 25-day treatment with Tween 80, approximately 76% of the total ginsenosides was released into the surrounding medium. In the case of control, the ginsenosides secreted into the medium were negligible. Furthermore, when compared with control, the level of total ginsenosides was enhanced by approximately threefold under Tween treatment. Additionally, secretion of the typical ginsenoside monomers including Rb1 , Rg1 , and Re was analyzed, indicating that the most of them were released into the medium. Moreover, it was observed that dammarenediol synthase, a key enzyme involved in ginsenoside biosynthesis, was upregulated at both gene expression and enzyme activity levels. The expression of genes CYP716A47 and CYP716A53v2 encoding Cyt P450 enzymes catalyzing the formation of protopanaxadiol from dammarenediol and protopanaxatriol from protopanaxadiol, respectively, was slightly upregulated. These results clearly demonstrated that Tween 80 could act not only as an efficient permeabilizer to enhance ginsenoside secretion from the hairy roots, but also as an elicitor to promote the biosynthesis of ginsenoside.
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