Cyclic Phosphodiesterase Activity Research Articles

Csx3 is a cyclic oligonucleotide phosphodiesterase associated with type III CRISPR–Cas that degrades the second messenger cA4

Cas10 is the signature gene for type III CRISPR-Cas surveillance complexes. Unlike type I and type II systems, type III systems do not require a protospacer adjacent motif and target nascent RNA associated with transcriptionally active DNA. Further, target RNA recognition activates the cyclase domain of Cas10, resulting in the synthesis of cyclic oligoadenylate second messengers. These second messengers are recognized by ancillary Cas proteins harboring CRISPR-associated Rossmann fold (CARF) domains and regulate the activities of these proteins in response to invading nucleic acid. Csx3 is a distant member of the CARF domain superfamily previously characterized as a Mn2+-dependent deadenylation exoribonuclease. However, its specific role in CRISPR-Cas defense remains to be determined. Here we show that Csx3 is strongly associated with type III systems and that Csx3 binds cyclic tetra-adenylate (cA4) second messenger with high affinity. Further, Csx3 harbors cyclic oligonucleotide phosphodiesterase activity that quickly degrades this cA4 signal. In addition, structural analysis identifies core elements that define the CARF domain fold, and the mechanistic basis for ring nuclease activity is discussed. Overall, the work suggests that Csx3 functions within CRISPR-Cas as a counterbalance to Cas10 to regulate the duration and amplitude of the cA4 signal, providing an off ramp from the programmed cell death pathway in cells that successfully cure viral infection.

An emerging role for cyclic dinucleotide phosphodiesterase and nanoRNase activities in Mycoplasma bovis: Securing survival in cell culture.

Mycoplasmas are host-restricted prokaryotes with a nearly minimal genome. To overcome their metabolic limitations, these wall-less bacteria establish intimate interactions with epithelial cells at mucosal surfaces. The alarming rate of antimicrobial resistance among pathogenic species is of particular concern in the medical and veterinary fields. Taking advantage of the reduced mycoplasma genome, random transposon mutagenesis was combined with high-throughput screening in order to identify key determinants of mycoplasma survival in the host-cell environment and potential targets for drug development. With the use of the ruminant pathogen Mycoplasma bovis as a model, three phosphodiesterases of the DHH superfamily were identified as essential for the proliferation of this species under cell culture conditions, while dispensable for axenic growth. Despite a similar domain architecture, recombinant Mbov_0327 and Mbov_0328 products displayed different substrate specificities. While rMbovP328 protein exhibited activity towards cyclic dinucleotides and nanoRNAs, rMbovP327 protein was only able to degrade nanoRNAs. The Mbov_0276 product was identified as a member of the membrane-associated GdpP family of phosphodiesterases that was found to participate in cyclic dinucleotide and nanoRNA degradation, an activity which might therefore be redundant in the genome-reduced M. bovis. Remarkably, all these enzymes were able to convert their substrates into mononucleotides, and medium supplementation with nucleoside monophosphates or nucleosides fully restored the capacity of a Mbov_0328/0327 knock-out mutant to grow under cell culture conditions. Since mycoplasmas are unable to synthesize DNA/RNA precursors de novo, cyclic dinucleotide and nanoRNA degradation are likely contributing to the survival of M. bovis by securing the recycling of purines and pyrimidines. These results point toward proteins of the DHH superfamily as promising targets for the development of new antimicrobials against multidrug-resistant pathogenic mycoplasma species.

The Effect of Nitroxyalkyl Succinimides on Activity of Cyclic Guanosine Monophosphate Phosphodiesterase.

The effect of N-nitroxymethyl succinimide (1), N-(2-nitroxyethyl) succinimide (2) and N-(3-nitroxypropyl) succinimide (3) on enzymatic activity of cyclic guanosine monophosphate (cGMP) phosphodiesterase was studied and crystal structure of compound (2) was determined. It was shown that all studied N-nitroxy succinimides inhibited cGMP phosphodiesterase in a concentration range of 0.1-0.001 mM. Compound (2) noncompetitively and reversibly inhibited hydrolytic function of enzyme with Ki=1.7×10-5 М. Inhibition constant for the reference compound N-(2-nitroethyl) nicotinamide (nicorandil) was 3×10-5 М.

Structural basis for the evolution of cyclic phosphodiesterase activity in the U6 snRNA exoribonuclease Usb1.

U6 snRNA undergoes post-transcriptional 3′ end modification prior to incorporation into the active site of spliceosomes. The responsible exoribonuclease is Usb1, which removes nucleotides from the 3′ end of U6 and, in humans, leaves a 2′,3′ cyclic phosphate that is recognized by the Lsm2–8 complex. Saccharomycescerevisiae Usb1 has additional 2′,3′ cyclic phosphodiesterase (CPDase) activity, which converts the cyclic phosphate into a 3′ phosphate group. Here we investigate the molecular basis for the evolution of Usb1 CPDase activity. We examine the structure and function of Usb1 from Kluyveromyces marxianus, which shares 25 and 19% sequence identity to the S. cerevisiae and Homo sapiens orthologs of Usb1, respectively. We show that K. marxianus Usb1 enzyme has CPDase activity and determined its structure, free and bound to the substrate analog uridine 5′-monophosphate. We find that the origin of CPDase activity is related to a loop structure that is conserved in yeast and forms a distinct penultimate (n – 1) nucleotide binding site. These data provide structural and mechanistic insight into the evolutionary divergence of Usb1 catalysis.

Deletion and Characterization of Two Signaling Genes in Streptomyces

Streptomyces coelicolor is a filamentous, high G‐C (guanine‐cytosine), gram‐positive bacterium that produces an aerial mycelium. The genome consists of a 8,667,507 base pair linear chromosome for this organism and contains the largest number of genes so far discovered in a bacterium. It belongs to the phylum Actinobacteria. In addition, streptomycetes are used to produce the majority of antibiotics in the medical field. This study focuses on the effect of the deletion of Sco5218, which encodes a putative integral membrane protein and Sco5219, which encodes a putative lipoprotein in Streptomyces coelicolor. Both of these genes are found in a potential operon. The goal of this research is to construct the double mutant and discern differences in phenotype compared to the single deletion mutants of each gene. We hypothesize that the double mutant will have an enhanced effect on the sporulation defect of Streptomyces. Sco5218 is believed to contain an HD‐GYP domain through bioinformatics analysis and is highly conserved throughout the Streptomyces genus. The HD‐GYP domain is typically associated with cyclic di‐GMP specific phosphodiesterase activity. Following REDIRECT protocol, the Streptomyces coelicolor cosmid clone was introduced into E. coli via electroporation. PCR was then utilized for the deletion of Sco5218 and Sco5219. This PCR product was run onto a gel and purified. This pure PCR product was then transformed into the plasmid vector which will then be mated into Streptomyces coelicolor. The relevance of this research allows us to see the importance of the HD‐GYP domain in bacteria. We witnessed a slight delay in sporulation with the Sco5218 mutant deletion. With the added deletion of Sco5219 we hypothesize that there will be an enhanced delay in sporulation. If we witness a delay in the sporulation with the double mutant in Streptomyces we can deduce that both genes play a role in bacterial development. These findings will allow us to further delve into the study of other bacteria and have a basis for other orthologs containing the HD‐GYP domain.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Microevolution in response to transient heme-iron restriction enhances intracellular bacterial community development and persistence.

Bacterial pathogens must sense, respond and adapt to a myriad of dynamic microenvironmental stressors to survive. Adaptation is key for colonization and long-term ability to endure fluctuations in nutrient availability and inflammatory processes. We hypothesize that strains adapted to survive nutrient deprivation are more adept for colonization and establishment of chronic infection. In this study, we detected microevolution in response to transient nutrient limitation through mutation of icc. The mutation results in decreased 3',5'-cyclic adenosine monophosphate phosphodiesterase activity in nontypeable Haemophilus influenzae (NTHI). In a preclinical model of NTHI-induced otitis media (OM), we observed a significant decrease in the recovery of effusion from ears infected with the icc mutant strain. Clinically, resolution of OM coincides with the clearance of middle ear fluid. In contrast to this clinical paradigm, we observed that the icc mutant strain formed significantly more intracellular bacterial communities (IBCs) than the parental strain early during experimental OM. Although the number of IBCs formed by the parental strain was low at early stages of OM, we observed a significant increase at later stages that coincided with absence of recoverable effusion, suggesting the presence of a mucosal reservoir following resolution of clinical disease. These data provide the first insight into NTHI microevolution during nutritional limitation and provide the first demonstration of IBCs in a preclinical model of chronic OM.

Deinococcus radiodurans HD-Pnk, a Nucleic Acid End-Healing Enzyme, Abets Resistance to Killing by Ionizing Radiation and Mitomycin C.

5'- and 3'-end healing are key steps in nucleic acid break repair in which 5'-OH and 3'-PO4 or 2',3'-cyclic-PO4 ends are converted to 5'-PO4 and 3'-OH termini suitable for sealing by polynucleotide ligases. Here, we characterize Deinococcus radiodurans HD-Pnk as a bifunctional end-healing enzyme composed of N-terminal HD (histidine-aspartate) phosphoesterase and C-terminal P-loop polynucleotide kinase (Pnk) domains. HD-Pnk phosphorylates 5'-OH DNA in the presence of ATP and magnesium. HD-Pnk has 3'-phosphatase and 2',3'-cyclic-phosphodiesterase activity in the presence of transition metals, optimally cobalt or copper, and catalyzes copper-dependent hydrolysis of p-nitrophenylphosphate. HD-Pnk is encoded by the LIG-PARG-HD-Pnk three-gene operon, which includes polynucleotide ligase and poly(ADP-ribose) glycohydrolase genes. We show that whereas HD-Pnk is inessential for Deinococcus growth, its absence sensitizes by 80-fold bacteria to killing by 9 kGy of ionizing radiation (IR). HD-Pnk protein is depleted during early stages of post-IR recovery and then replenished at 15 h, after reassembly of the genome from shattered fragments. ΔHD-Pnk mutant cells are competent for genome reassembly, as gauged by pulsed-field gel electrophoresis. Our findings suggest a role for HD-Pnk in repairing residual single-strand gaps or nicks in the reassembled genome. HD-Pnk-Ala mutations that ablate kinase or phosphoesterase activity sensitize Deinococcus to killing by mitomycin C.IMPORTANCE End healing is a process whereby nucleic acid breaks with "dirty" 3'-PO4 or 2',3'-cyclic-PO4 and 5'-OH ends are converted to 3'-OH and 5'-PO4 termini that are amenable to downstream repair reactions. Deinococcus radiodurans is resistant to massive doses of ionizing radiation (IR) that generate hundreds of dirty DNA double-strand breaks and thousands of single-strand breaks. This study highlights Deinococcus HD-Pnk as a bifunctional 3'- and 5'-end-healing enzyme that helps protect against killing by IR. HD-Pnk appears to act late in the process of post-IR recovery, subsequent to genome reassembly from shattered fragments. HD-Pnk also contributes to resistance to killing by mitomycin C. These findings are significant in that they establish a role for end-healing enzymes in bacterial DNA damage repair.

Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities

U6 small nuclear ribonucleoprotein (snRNP) biogenesis is essential for spliceosome assembly, but not well understood. Here, we report structures of the U6 RNA processing enzyme Usb1 from yeast and a substrate analog bound complex from humans. Unlike the human ortholog, we show that yeast Usb1 has cyclic phosphodiesterase activity that leaves a terminal 3′ phosphate which prevents overprocessing. Usb1 processing of U6 RNA dramatically alters its affinity for cognate RNA-binding proteins. We reconstitute the post-transcriptional assembly of yeast U6 snRNP in vitro, which occurs through a complex series of handoffs involving 10 proteins (Lhp1, Prp24, Usb1 and Lsm2–8) and anti-cooperative interactions between Prp24 and Lhp1. We propose a model for U6 snRNP assembly that explains how evolutionarily divergent and seemingly antagonistic proteins cooperate to protect and chaperone the nascent snRNA during its journey to the spliceosome.

Stand-Alone EAL Domain Proteins Form a Distinct Subclass of EAL Proteins Involved in Regulation of Cell Motility and Biofilm Formation in Enterobacteria.

The second messenger cyclic dimeric GMP (c-di-GMP) is almost ubiquitous among bacteria as are the c-di-GMP turnover proteins, which mediate the transition between motility and sessility. EAL domain proteins have been characterized as c-di-GMP-specific phosphodiesterases. While most EAL domain proteins contain additional, usually N-terminal, domains, there is a distinct family of proteins with stand-alone EAL domains, exemplified by Salmonella enterica serovar Typhimurium proteins STM3611 (YhjH/PdeH), a c-di-GMP-specific phosphodiesterase, and the enzymatically inactive STM1344 (YdiV/CdgR) and STM1697, which regulate bacterial motility through interaction with the flagellar master regulator, FlhDC. We have analyzed the phylogenetic distribution of EAL-only proteins and their potential functions. Genes encoding EAL-only proteins were found in various bacterial phyla, although most of them were seen in proteobacteria, particularly enterobacteria. Based on the conservation of the active site residues, nearly all stand-alone EAL domains encoded by genomes from phyla other than proteobacteria appear to represent functional phosphodiesterases. Within enterobacteria, EAL-only proteins were found to cluster either with YhjH or with one of the subfamilies of YdiV-related proteins. EAL-only proteins from Shigella flexneri, Klebsiella pneumoniae, and Yersinia enterocolitica were tested for their ability to regulate swimming and swarming motility and formation of the red, dry, and rough (rdar) biofilm morphotype. In these tests, YhjH-related proteins S4210, KPN_01159, KPN_03274, and YE4063 displayed properties typical of enzymatically active phosphodiesterases, whereas S1641 and YE1324 behaved like members of the YdiV/STM1697 subfamily, with Yersinia enterocolitica protein YE1324 shown to downregulate motility in its native host. Of two closely related EAL-only proteins, YE2225 is an active phosphodiesterase, while YE1324 appears to interact with FlhD. These results suggest that in FlhDC-harboring beta- and gammaproteobacteria, some EAL-only proteins evolved to become catalytically inactive and regulate motility and biofilm formation by interacting with FlhDC.IMPORTANCE The EAL domain superfamily consists mainly of proteins with cyclic dimeric GMP-specific phosphodiesterase activity, but individual domains have been classified in three classes according to their functions and conserved amino acid signatures. Proteins that consist solely of stand-alone EAL domains cannot rely on other domains to form catalytically active dimers, and most of them fall into one of two distinct classes: catalytically active phosphodiesterases with well-conserved residues of the active site and the dimerization loop, and catalytically inactive YdiV/CdgR-like proteins that regulate bacterial motility by binding to the flagellar master regulator, FlhDC, and are found primarily in enterobacteria. The presence of apparently inactive EAL-only proteins in the bacteria that do not express FlhD suggests the existence of additional EAL interaction partners.

Structural similarities and functional differences clarify evolutionary relationships between tRNA healing enzymes and the myelin enzyme CNPase

BackgroundEukaryotic tRNA splicing is an essential process in the transformation of a primary tRNA transcript into a mature functional tRNA molecule. 5′-phosphate ligation involves two steps: a healing reaction catalyzed by polynucleotide kinase (PNK) in association with cyclic phosphodiesterase (CPDase), and a sealing reaction catalyzed by an RNA ligase. The enzymes that catalyze tRNA healing in yeast and higher eukaryotes are homologous to the members of the 2H phosphoesterase superfamily, in particular to the vertebrate myelin enzyme 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase).ResultsWe employed different biophysical and biochemical methods to elucidate the overall structural and functional features of the tRNA healing enzymes yeast Trl1 PNK/CPDase and lancelet PNK/CPDase and compared them with vertebrate CNPase. The yeast and the lancelet enzymes have cyclic phosphodiesterase and polynucleotide kinase activity, while vertebrate CNPase lacks PNK activity. In addition, we also show that the healing enzymes are structurally similar to the vertebrate CNPase by applying synchrotron radiation circular dichroism spectroscopy and small-angle X-ray scattering.ConclusionsWe provide a structural analysis of the tRNA healing enzyme PNK and CPDase domains together. Our results support evolution of vertebrate CNPase from tRNA healing enzymes with a loss of function at its N-terminal PNK-like domain.

The Characterization of Escherichia coli CpdB as a Recombinant Protein Reveals that, besides Having the Expected 3´-Nucleotidase and 2´,3´-Cyclic Mononucleotide Phosphodiesterase Activities, It Is Also Active as Cyclic Dinucleotide Phosphodiesterase

Endogenous cyclic diadenylate phosphodiesterase activity was accidentally detected in lysates of Escherichia coli BL21. Since this kind of activity is uncommon in Gram-negative bacteria, its identification was undertaken. After partial purification and analysis by denaturing gel electrophoresis, renatured activity correlated with a protein identified by fingerprinting as CpdB (cpdB gene product), which is annotated as 3´-nucleotidase / 2´,3´-cyclic-mononucleotide phosphodiesterase, and it is synthesized as a precursor protein with a signal sequence removable upon export to the periplasm. It has never been studied as a recombinant protein. The coding sequence of mature CpdB was cloned and expressed as a GST fusion protein. The study of the purified recombinant protein, separated from GST, confirmed CpdB annotation. The assay of catalytic efficiencies (kcat/Km) for a large substrate set revealed novel CpdB features, including very high efficiencies for 3´-AMP and 2´,3´-cyclic mononucleotides, and previously unknown activities on cyclic and linear dinucleotides. The catalytic efficiencies of the latter activities, though low in relative terms when compared to the major ones, are far from negligible. Actually, they are perfectly comparable to those of the ‘average’ enzyme and the known, bona fide cyclic dinucleotide phosphodiesterases. On the other hand, CpdB differs from these enzymes in its extracytoplasmic location and in the absence of EAL, HD and DHH domains. Instead, it contains the domains of the 5´-nucleotidase family pertaining to the metallophosphoesterase superfamily, although CpdB lacks 5´-nucleotidase activity. The possibility that the extracytoplasmic activity of CpdB on cyclic dinucleotides could have physiological meaning is discussed.

Immunomodulatory effects of Lippia sidoides extract: induction of IL-10 through cAMP and p38 MAPK-dependent mechanisms.

Lippia sidoides is an aromatic shrub that grows wild in the northeastern region of Brazil. In local traditional medicine, the aerial portions of this species are used as anti-infectives, antiseptics, spasmolytics, sedatives, hypotensives, and anti-inflammatory agents. In this research, we evaluate the potential immunological properties of Lippia extract through in vitro analysis of its ability to modulate intracellular cyclic adenosine monophosphate (cAMP) levels and interleukin-10 (IL-10) production. These results show that Lippia extract increases intracellular cAMP through the inhibition of phosphodiesterase activity. They also demonstrate that Lippia extract increases IL-10 production in THP-1 monocytes through both an increase in intracellular cAMP and the activation of p38 MAPK. These results suggest that the Lippia-mediated inhibition of phosphodiesterase activity and the subsequent increase in intracellular cAMP may explain some of the biological activities associated with L. sidoides. In addition, the anti-inflammatory activity of L. sidoides may also be due, in part, to its ability to induce IL-10 production through the inhibition of cyclic nucleotide-dependent phosphodiesterase activity and by its activation of the p38 MAPK pathway.

Pulsed electromagnetic fields dosing impacts postoperative pain in breast reduction patients

BackgroundPulsed electromagnetic fields (PEMF) reduce postoperative pain and narcotic requirements in breast augmentation, reduction, and reconstruction patients. PEMF enhances both calmodulin-dependent nitric oxide and/or cyclic guanosine monophosphate signaling and phosphodiesterase activity, which blocks cyclic guanosine monophosphate. The clinical effect of these competing responses on PEMF dosing is not known. MethodsTwo prospective, nonrandomized, active cohorts of breast reduction patients, with 15 min PEMF per 2 h; “Q2 (active)”, and 5 min PEMF per 20 min; “5/20 (active)”, dosing regimens were added to a previously reported double-blind clinical study wherein 20 min PEMF per 4 h, “Q4 (active)”, dosing significantly accelerated postoperative pain reduction compared with Q4 shams. Postoperative visual analog scale pain scores and narcotic use were compared with results from the previous study. ResultsVisual analog scale scores at 24 h were 43% and 35% of pain at 1 h in the Q4 (active) and Q2 (active) cohorts, respectively (P < 0.01). Pain at 24 h in the 5/20 (active) cohort was 87% of pain at 1 h, compared with 74% in the Q4 (sham) cohort (P = 0.451). Concomitantly, narcotic usage in the 5/20 (active) and Q4 (sham) cohorts was not different (P = 0.478), and 2-fold higher than the Q4 (active) and Q2 (active) cohorts (P < 0.02). ConclusionsThis prospective study shows Q4/Q2, but not 5/20 PEMF dosing, accelerated postoperative pain reduction compared with historical shams. The 5/20 (active) regimen increases NO 4-fold faster than the Q4 (active) regimen, possibly accelerating phosphodiesterase inhibition of cyclic guanosine monophosphate sufficiently to block the PEMF effect. This study helps define the dosing limits of clinically useful PEMF signals.

Effects of Water-Soluble Polysubstituted Fullerene Derivatives on Sarcoplasmic Reticulum Ca2+-ATPase and Cyclic Guanosine Monophosphate Phosphodiesterase Activities

The effects of new fullerene derivatives on the functioning of the hydrolases sarcoplasmic reticulum (SR) Ca2+-ATPase and cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) were studied. Most of the fullerene derivatives studied were found to inhibit the activity of both enzymes. Thus, compounds PSF-1, PSF-II, and PSF-III completely inhibited Ca2+ transport at a concentration of 0.009 mM but inhibited ATP hydrolysis by only 55 ± 6%, 42 ± 8%, and 60 ± 6% respectively, thus uncoupling the hydrolytic and transport functions of the enzyme. This induced changes in the extra- and intracellular Ca2+ ion levels and affected adhesion of metastatic cells to the capillary endothelium. Compound PSF-III had noncompetitive, reversible effects on the hydrolytic function of SR Ca2+-ATPase with K i = 1.6 × 10–6 M. All the fullerene derivatives studied inhibited cGMP PDE function and could induce an antiaggregation effect which resulted from accumulation of the cGMP PDE substrate, i.e., cGMP, which is a second messenger in living organisms. These results can be used to predict the potential therapeutic effects of the fullerene derivatives studied.

Two DHH Subfamily 1 Proteins in Streptococcus pneumoniae Possess Cyclic Di-AMP Phosphodiesterase Activity and Affect Bacterial Growth and Virulence

Cyclic di-AMP (c-di-AMP) and cyclic di-GMP (c-di-GMP) are signaling molecules that play important roles in bacterial biology and pathogenesis. However, these nucleotides have not been explored in Streptococcus pneumoniae, an important bacterial pathogen. In this study, we characterized the c-di-AMP-associated genes of S. pneumoniae. The results showed that SPD_1392 (DacA) is a diadenylate cyclase that converts ATP to c-di-AMP. Both SPD_2032 (Pde1) and SPD_1153 (Pde2), which belong to the DHH subfamily 1 proteins, displayed c-di-AMP phosphodiesterase activity. Pde1 cleaved c-di-AMP into phosphoadenylyl adenosine (pApA), whereas Pde2 directly hydrolyzed c-di-AMP into AMP. Additionally, Pde2, but not Pde1, degraded pApA into AMP. Our results also demonstrated that both Pde1 and Pde2 played roles in bacterial growth, resistance to UV treatment, and virulence in a mouse pneumonia model. These results indicate that c-di-AMP homeostasis is essential for pneumococcal biology and disease.

Abstract 3659: Next generation sulindac.

Abstract Sulindac displays promising antineoplastic activity, but toxicity from cyclooxygenase (COX) inhibition limits its long-term use for chemoprevention. Previous studies have concluded that the tumor cell growth inhibitory activity of sulindac is COX-independent and suggest that safer and more efficacious drugs can be developed by targeting the underlying mechanism. Here we report that sulindac sulfide (SS) inhibits cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) activity, increases intracellular cGMP levels and activates protein kinase G at concentrations that suppress proliferation and induce apoptosis of colon tumor cells. Normal colonocytes were refractory to the anti-proliferative and pro-apoptotic effects of SS as well as its ability to induce cGMP signaling. PDE5-specific inhibitors such as tadalafil also inhibited the growth of colon tumor cells that expressed high levels of the cGMP-specific PDE5 isozyme compared with colonocytes. RNAi knockdown of PDE5 selectively inhibited proliferation and induced apoptosis of colon tumor cells as did SS. SS, 8-bromo-cGMP, and PDE5 siRNA reduced β-catenin levels, as well as the expression of cyclin D and survivin that are regulated by β-catenin-dependent TCF transcriptional activity by a mechanism involving the transcriptional repression of β-catenin. The significance of these findings are highlighted by studies in the FCCC Min mouse model of intestinal tumorigenesis showing that a novel non-COX inhibitory derivative of SS can strongly inhibit colon tumor formation by a mechanism that appears to involve cGMP PDE inhibition. These observations suggest that safer and more efficacious drugs can be developed for colorectal cancer chemoprevention by targeting PDE5 and possibly other cGMP degrading isozymes. Citation Format: Nan Li, Yaguang Xi, Heather N. Tinsley, Evrim Gurpinar, Bernard D. Gary, Bing Zhu, Yonghe Li, Adam B. Keeton, William E. Grizzle, Gary A. Piazza. Next generation sulindac. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3659. doi:10.1158/1538-7445.AM2013-3659

Cyclic nucleotide phosphodiesterase activity in stem cells of human periodontal ligament (PDL-MSCs) before and after osteogenic induction

The aim of this work was to evaluate both the level of endogenous cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cAMP) and phosphodiesterase activity in mesenchymal stem cells (MSCs) before and during the osteogenic induction. Samples were organized into control (nondifferentiated) and test groups which were analyzed at 3 different time points: 1, 2, and 4 weeks. Periodontal ligament MSCs were isolated and then expanded in an MSCM medium while cyclic nucleotide levels and phosphodiesterase activity were assessed. cAMP and cGMP levels were markedly higher in the first week than in the following stages. Similarly, PDE activity increased during the first week and reached the peak in the second week. This work validates that cAMP, cGMP, and PDE activities are important factors in the first phase of the osteogenic induction of a human stem cell.

The sequential 2',3'-cyclic phosphodiesterase and 3'-phosphate/5'-OH ligation steps of the RtcB RNA splicing pathway are GTP-dependent

The RNA ligase RtcB splices broken RNAs with 5′-OH and either 2′,3′-cyclic phosphate or 3′-phosphate ends. The 3′-phosphate ligase activity requires GTP and entails the formation of covalent RtcB-(histidinyl)-GMP and polynucleotide-(3′)pp(5′)G intermediates. There are currently two models for how RtcB executes the strand sealing step. Scheme 1 holds that the RNA 5′-OH end attacks the 3′-phosphorus of the N(3′)pp(5′)G end to form a 3′,5′-phosphodiester and release GMP. Scheme 2 posits that the N(3′)pp(5′)G end is converted to a 2′,3′-cyclic phosphodiester, which is then attacked directly by the 5′-OH RNA end to form a 3′,5′-phosphodiester. Here we show that the sealing of a 2′,3′-cyclic phosphate end by RtcB requires GTP, is contingent on formation of the RtcB–GMP adduct, and involves a kinetically valid RNA(3′)pp(5′)G intermediate. Moreover, we find that RtcB catalyzes the hydrolysis of a 2′,3′-cyclic phosphate to a 3′-phosphate at a rate that is at least as fast as the rate of ligation. These results weigh in favor of scheme 1. The cyclic phosphodiesterase activity of RtcB depends on GTP and the formation of the RtcB–GMP adduct, signifying that RtcB guanylylation precedes the cyclic phosphodiesterase and 3′-phosphate ligase steps of the RNA splicing pathway.

Novel Mechanism of RNA Repair by RtcB via Sequential 2′,3′-Cyclic Phosphodiesterase and 3′-Phosphate/5′-Hydroxyl Ligation Reactions

RtcB enzymes are a newly discovered family of RNA ligases, implicated in tRNA splicing and other RNA repair reactions, that seal broken RNAs with 2',3'-cyclic phosphate and 5'-OH ends. Parsimony and energetics would suggest a one-step mechanism for RtcB sealing via attack by the O5' nucleophile on the cyclic phosphate, with expulsion of the ribose O2' and generation of a 3',5'-phosphodiester at the splice junction. Yet we find that RtcB violates Occam's razor, insofar as (i) it is adept at ligating 3'-monophosphate and 5'-OH ends; (ii) it has an intrinsic 2',3'-cyclic phosphodiesterase activity. The 2',3'-cyclic phosphodiesterase and ligase reactions both require manganese and are abolished by mutation of the RtcB active site. Thus, RtcB executes a unique two-step pathway of strand joining whereby the 2',3'-cyclic phosphodiester end is hydrolyzed to a 3'-monophosphate, which is then linked to the 5'-OH end to form the splice junction. The energy for the 3'-phosphate ligase activity is provided by GTP, which reacts with RtcB in the presence of manganese to form a covalent RtcB-guanylate adduct. This adduct is sensitive to acid and hydroxylamine but resistant to alkali, consistent with a phosphoramidate bond.

The exopolyphosphatase TbrPPX1 of Trypanosoma brucei

BackgroundExopolyphosphatases and pyrophosphatases play important but still incompletely understood roles in energy metabolism, and also in other aspects of cell biology such as osmoregulation or signal transduction. Earlier work has suggested that a human exopolyphosphatase, Prune, might exhibit cyclic nucleotide phosphodiesterase activity.ResultsThe kinetoplastida, a large order of unicellular eukaryotes that contains many important pathogens such as Trypanosoma brucei (human sleeping sickness), Trypanosoma cruzi (Chagas disease) or Leishmania ssp (several clinically dinstinct leishmaniases) all contain several exo- and pyrophosphatases. The current study provides a systematic classification of these enzymes, which now allows to situate the information that is already available on some of these enzymes. It then analyses the exopolyphosphatase TbrPPX1 of T. brucei in detail, using RNA interference and genetic knockouts in an attempt to define its function, and immunofluorescence microscopy to study its subcellular localization.TbrPPX1 is an exopolyphosphatase that does hydrolyze pentasodium triphosphate, but not organic triphosphates such as ATP, pyrophosphate or long-chain polyphosphates. Finally, the study investigates the potential cyclic nucleotide phosphodiesterase activity of TbrPPX1.ConclusionsAll kinetoplastid genomes that are currently available contain genes for an exopolyphosphatase and two classes of pyrophosphatases, one associated with the acidocalcisomes and one cytoplasmic. TbrPPX1 represents the T. brucei exopolyphosphatase. It is located throughout the cytoplasm, and its genetic ablation does not produce a dramatic phenotype. Importantly, TbrPPX1 does not exhibit any cyclic nucleotide specific phosphodiesterase activity, which definitively eliminates it as an additional player in cAMP signalling of the kinetoplastida.