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Query: UNIPROT:P06889 (Mol)
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Trypanosoma cruzi, the causative agent of Chagas disease, encodes a number of different cAMP-specific PDE (phosphodiesterase) families. Here we report the identification and characterization of TcrPDEB1 and its comparison with the previously identified TcrPDEB2 (formerly known as TcPDE1). These are two different PDE enzymes of the TcrPDEB family, named in accordance with the recent recommendations of the Nomenclature Committee for Kinetoplast PDEs [Kunz, Beavo, D'Angelo, Flawia, Francis, Johner, Laxman, Oberholzer, Rascon, Shakur et al. (2006) Mol. Biochem. Parasitol. 145, 133-135]. Both enzymes show resistance to inhibition by many mammalian PDE inhibitors, and those that do inhibit do so with appreciable differences in their inhibitor profiles for the two enzymes. Both enzymes contain two GAF (cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA) domains and a catalytic domain highly homologous with that of the T. brucei TbPDE2/TbrPDEB2 family. The N-terminus+GAF-A domains of both enzymes showed significant differences in their affinities for cyclic nucleotide binding. Using a calorimetric technique that allows accurate measurements of low-affinity binding sites, the TcrPDEB2 N-terminus+GAF-A domain was found to bind cAMP with an affinity of approximately 500 nM. The TcrPDEB1 N-terminus+GAF-A domain bound cAMP with a slightly lower affinity of approximately 1 muM. The N-terminus+GAF-A domain of TcrPDEB1 did not bind cGMP, whereas the N-terminus+GAF-A domain of TcrPDEB2 bound cGMP with a low affinity of approximately 3 muM. GAF domains homologous with those found in these proteins were also identified in related trypanosomatid parasites. Finally, a fluorescent cAMP analogue, MANT-cAMP [2'-O-(N-methylanthraniloyl)adenosine-3',5'-cyclic monophosphate], was found to be a substrate for the TcPDEB1 catalytic domain, opening the possibility of using this molecule as a substrate in non-radioactive, fluorescence-based PDE assays, including screening for trypanosome PDE inhibitors.
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PMID:Characterization of a novel cAMP-binding, cAMP-specific cyclic nucleotide phosphodiesterase (TcrPDEB1) from Trypanosoma cruzi. 1677 50

Benzo[alpha]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), the major metabolite of benzo[a]pyrene (B[a]P), shows an ultimate complete carcinogen in various animals and is a causative agent for human cancers. However, its effects on the activation of signal pathways and the expression of genes involved in its carcinogenic effect remain largely unknown. In this study, the effects of B[a]PDE on induction of cyclooxygenase (COX)-2 and the signal pathways leading to the induction were investigated. Treatment of mouse epidermal Cl41 cells with B[a]PDE caused an increase in the expression of COX-2 at both transcription and protein levels, while its parental compound B[a]P did not show significant inductive effect. The COX-2 induction by B[a]PDE was dependent on the activation of mitogen-activated protein kinases (MAPK)s/activation protein (AP)-1 pathway, because inhibition of AP-1 by either overexpression of TAM67 (dominant negative mutant of c-jun), or pretreatment of cells with PD98059 (MEK1/2-ERKs pathway inhibitor) or SB202190 (p38K inhibitor), markedly inhibited B[a]PDE-induced COX-2 expression. In addition, impairment of NF-kappaB pathway by either NEMO-BDBP (an NF-kappaB specific inhibitor) or IkappaB kinase (IKK)beta-KM (dominant negative mutant of IKKbeta) also caused marked reduction of COX-2 induction by B[a]PDE. In contrast, inhibition of nuclear factor of activated T cells (NFAT) with FK506, did not show any effect on B[a]PDE-induced COX-2 expression. Collectively, these data indicate that exposure of Cl41 cells to B[a]PDE can induce COX-2 expression by increasing its transcription, which requires the activation of MAPKs/AP-1 and IKKbeta/NF-kappaB pathways, but not NFAT pathway. In view of the importance of COX-2 in carcinogenesis, we anticipate that the induction of COX-2 by B[a]PDE may coordinate its mutagenic effects to facilitate the development of skin cancer.
Mol Carcinog 2007 Jan
PMID:Benzo[a]pyrene diol-epoxide (B[a]PDE) upregulates COX-2 expression through MAPKs/AP-1 and IKKbeta/NF-kappaB in mouse epidermal Cl41 cells. 1692 90

Bis-(3'-5')-cyclic-di-guanosine monophosphate (c-di-GMP) is a bacterial signalling molecule produced by diguanylate cyclases (DGC, carrying GGDEF domains) and degraded by specific phosphodiesterases (PDE, carrying EAL domains). Neither its full physiological impact nor its effector mechanisms are currently understood. Also, the existence of multiple GGDEF/EAL genes in the genomes of most species raises questions about output specificity and robustness of c-di-GMP signalling. Using microarray and gene fusion analyses, we demonstrate that at least five of the 29 GGDEF/EAL genes in Escherichia coli are not only stationary phase-induced under the control of the general stress response master regulator sigma(S) (RpoS), but also exhibit differential control by additional environmental and temporal signals. Two of the corresponding proteins, YdaM (GGDEF only) and YciR (GGDEF + EAL), which in vitro show DGC and PDE activity, respectively, play an antagonistic role in the expression of the biofilm-associated curli fimbriae. This control occurs at the level of transcription of the curli and cellulose regulator CsgD. Moreover, we show that H-NS positively affects curli expression by inversely controlling the expression of ydaM and yciR. Furthermore, we demonstrate a temporally fine-tuned GGDEF cascade in which YdaM controls the expression of another GGDEF protein, YaiC. By genome-wide microarray analysis, evidence is provided that YdaM and YciR strongly and nearly exclusively control CsgD-regulated genes. We conclude that specific GGDEF/EAL proteins have very distinct expression patterns, and when present in physiological amounts, can act in a highly precise, non-global and perhaps microcompartmented manner on a few or even a single specific target(s).
Mol Microbiol 2006 Nov
PMID:Cyclic-di-GMP-mediated signalling within the sigma network of Escherichia coli. 1701 Jan 56

The adaptor protein 1 (AP1) complex is a heterotetramer that participates in cargo sorting into clathrin-coated vesicles at the trans-Golgi network (TGN) and endosomes. The gamma subunit of AP1 possesses a C-terminal "ear" domain that recruits a cohort of accessory proteins through recognition of a shared canonical motif, PsiG[PDE][PsiLM] (where Psi is an aromatic residue). The physiological relevance of these ear-motif interactions, however, remains to be demonstrated. Here we report that the cyclin G-associated kinase (GAK) has two sequences fitting this motif, FGPL and FGEF, which mediate binding to the AP1-gamma-ear domain in vitro. Mutation of both gamma-ear-binding sequences or depletion of AP1-gamma by RNA interference (RNAi) decreases the association of GAK with the TGN in vivo. Depletion of GAK by RNAi impairs the sorting of the acid hydrolase, cathepsin D, to lysosomes. Importantly, expression of RNAi-resistant GAK restores the lysosomal sorting of cathepsin D in cells depleted of endogenous GAK, whereas expression of a similar construct bearing mutations in both gamma-ear-binding sequences fails to correct the sorting defect. Thus, interactions between the PsiG[PDE][PsiLM]-motif sequences in GAK and the AP1-gamma-ear domain are critical for the recruitment of GAK to the TGN and the function of GAK in lysosomal enzyme sorting.
Mol Biol Cell 2007 Aug
PMID:Canonical interaction of cyclin G associated kinase with adaptor protein 1 regulates lysosomal enzyme sorting. 1753 18

Human leishmaniasis is a major public health problem in many countries, but chemotherapy is in an unsatisfactory state. Leishmania major phosphodiesterases (LmjPDEs) have been shown to play important roles in cell proliferation and apoptosis of the parasite. Thus LmjPDE inhibitors may potentially represent a novel class of drugs for the treatment of leishmaniasis. Reported here are the kinetic characterization of the LmjPDEB1 catalytic domain and its crystal structure as a complex with 3-isobutyl-1-methylxanthine (IBMX) at 1.55 A resolution. The structure of LmjPDEB1 is similar to that of human PDEs. IBMX stacks against the conserved phenylalanine and forms a hydrogen bond with the invariant glutamine, in a pattern common to most inhibitors bound to human PDEs. However, an extensive structural comparison reveals subtle, but significant differences between the active sites of LmjPDEB1 and human PDEs. In addition, a pocket next to the inhibitor binding site is found to be unique to LmjPDEB1. This pocket is isolated by two gating residues in human PDE families, but constitutes a natural expansion of the inhibitor binding pocket in LmjPDEB1. The structure particularity might be useful for the development of parasite-selective inhibitors for the treatment of leishmaniasis.
Mol Microbiol 2007 Nov
PMID:Crystal structure of the Leishmania major phosphodiesterase LmjPDEB1 and insight into the design of the parasite-selective inhibitors. 1794 32

Vardenafil has higher affinity to phosphodiesterase-5 (PDE5) than sildenafil and lower administered dosage for the treatment of erectile dysfunction. However, the molecular basis for these differences is puzzling because two drugs have similar chemical structures. Reported here is a crystal structure of the fully active and nonmutated PDE5A1 catalytic domain in complex with vardenafil. The structure shows that the conformation of the H-loop in the PDE5A1-vardenafil complex is different from those of any known structures of the unliganded PDE5 and its complexes with the inhibitors. In addition, the molecular configuration of vardenafil differs from that of sildenafil when bound to PDE5. It is noteworthy that the binding of vardenafil causes loss of the divalent metal ions that have been observed in all the previously published PDE structures. The conformational variation of both PDE5 and the inhibitors provides structural insight into the different potencies of the drugs.
Mol Pharmacol 2008 Jan
PMID:Conformational variations of both phosphodiesterase-5 and inhibitors provide the structural basis for the physiological effects of vardenafil and sildenafil. 1795 9

We report here the generation and pharmacological characterization of a phosphodiesterase 2A (PDE2A) reporter cell line. Human PDE2A was stably transfected in a parental cell line expressing the atrial natriuretic peptide (ANP) receptor and the cyclic nucleotide-gated (CNG) cation channel CNGA2, acting as the biosensor for intracellular cGMP. In this reporter cell line, cGMP levels can be monitored in real-time via aequorin luminescence stimulated by calcium influx through the CNG channel. By using different PDE inhibitors, we could show that our PDE2A reporter assay specifically monitors PDE2A inhibition with high sensitivity. In the absence of ANP stimulation, the PDE2A selective inhibitors EHNA, BAY 60-7550 and PDP did not increase basal luminescence levels in this experimental setting. However, in combination with ANP, these inhibitors stimulated luminescence signals and induced leftward shifts of ANP concentration-response curves. Similar results were obtained when using IBMX, trequinsin and dipyridamole, which inhibit PDE2A nonselectively with lower potency. PDP, the most potent PDE2A inhibitor known to date, was found to exhibit much lower cellular activity as anticipated from its biochemical PDE2A inhibitory activity. By cellular uptake and transport studies we could show that PDP's cell permeability is low and that the compound is a substrate for an efflux transporter. Other PDE inhibitors including vinpocetine, milrinone, rolipram, sildenafil, zaprinast, BRL 50481 and BAY 73-6691 did not stimulate luminescence signals on our PDE2A reporter cell line. The results imply that this novel PDE2A reporter assay provides an efficient, high throughput means for the identification and characterization of PDE2A inhibitors.
Mol Pharm
PMID:A novel PDE2A reporter cell line: characterization of the cellular activity of PDE inhibitors. 1904 45

Endothelial nitric oxide synthase (NOS3) regulates the functional response to beta-adrenergic (beta-AR) stimulation via modulation of the L-type Ca(2+) current (I(Ca)). However, the NOS3 signaling pathway modulating I(Ca) is unknown. This study investigated the contribution of soluble guanylate cyclase (sGC) and phosphodiesterase type 5 (PDE5), a cGMP-specific PDE, in the NOS3-mediated regulation of I(Ca). Myocytes were isolated from NOS3 knockout (NOS3(-/-)) and wildtype (WT) mice. We measured I(Ca) (whole-cell voltage-clamp), and simultaneously measured Ca(2+) transients (Fluo-4 AM) and cell shortening (edge detection). Zaprinast (selective inhibitor of PDE5), decreased beta-AR stimulated (isoproterenol, ISO)-I(Ca), and Ca(2+) transient and cell shortening amplitudes in WT myocytes. However, YC-1 (NO-independent activator of sGC) only reduced ISO-stimulated I(Ca), but not cardiac contraction. We further investigated the NOS3/sGC/PDE5 pathway in NOS3(-/-) myocytes. PDE5 is mislocalized in these myocytes and we observed dissimilar effects of PDE5 inhibition and sGC activation compared to WT. That is, zaprinast had no effect on ISO-stimulated I(Ca), or Ca(2+) transient and cell shortening amplitudes. Conversely, YC-1 significantly decreased both ISO-stimulated I(Ca), and cardiac contraction. Further confirming that PDE5 localizes NOS3/cGMP signaling to I(Ca); YC-1, in the presence of zaprinast, now significantly decreased ISO-stimulated Ca(2+) transient and cell shortening amplitudes in WT myocytes. The effects of YC-1 on I(Ca) and cardiac contraction were blocked by KT5823 (a selective inhibitor of the cGMP-dependent protein kinase, PKG). Our data suggests a novel physiological role for PDE5 in restricting the effects of NOS3/sGC/PKG signaling pathway to modulating beta-AR stimulated I(Ca), while limiting effects on cardiac contraction.
J Mol Cell Cardiol 2009 Aug
PMID:Phosphodiesterase 5 restricts NOS3/Soluble guanylate cyclase signaling to L-type Ca2+ current in cardiac myocytes. 1934 27

The traditional Chinese medicines (TCM), Epimedium sagittatum (ESs), Cnidium monnieri (CMs), and Semen cuscutae (SCs), were used for treating erectile dysfunction since the ancient Han dynasty (202 BC-AD 220). Phosphodiesterase-5 (PDE-5) is deemed the target protein for inhibition to treat erectile dysfunction. In this study, a reliable multiple linear regression (MLR) model (r value=0.8484) was used to predict the activities of new candidates which were designed from ES, CM, and SC. From docking and pharmacophore analysis, the potent candidates among ES, CM, and SC were screened. SC01, SC03, and ES03b were predicted to have high potencies based on MLR analysis and high docking scores. Additionally, from our analysis, we make the follow conclusion (1) Hydrophobic compounds tend to be more potent PDE-5 inhibitors; (2) Because of the big binding site, inhibitors with molecular weights over 500 remain potent; (3) From the pharmacophore analysis, the features of hydrogen bond acceptors are the basis for designing novel inhibitors of PDE-5 and (4) According to MLR analysis, the number of ring groups could be up to 6, but the number of aromatic rings was limited to 4 to be potent. This study offers an alternative way to screen PDE-5 inhibitors from TCM and provides a scientific basis for confirming pharmacological actions of TCM.
J Mol Graph Model 2009 Oct
PMID:Computational screening and design of traditional Chinese medicine (TCM) to block phosphodiesterase-5. 1974 66

Regulatory T cells (Treg) are crucial for the suppression of antigen-specific immune responses by activated conventional T cells (Tcon). It has been recently reported that this suppression is mediated by cyclic adenosine monophosphate (cAMP) transported from Treg to Tcon via gap junctions. However, the underlying biochemical mechanisms of cAMP accumulation in activated Treg are still unclear. Here we reported that although non-activated murine Treg and Tcon displayed similar intracellular cAMP amounts, both subpopulations showed distinct expression of enzymes regulating cAMP metabolism. Thus, in Treg, activities of both anabolic (adenylyl cyclase, AC) and catabolic (phosphodiesterase, PDE) enzymes were lower than in Tcon. Furthermore, we demonstrated for the first time the expression of the PDE11 protein in murine Treg and Tcon. Treg activation by IL-2 induced a strong AC7 activation and cAMP accumulation in Treg. In contrast, Tcon showed a significant decrease in the AC7 activity and cAMP amounts under these conditions. Our data suggest that the mechanism of cAMP accumulation in stimulated Treg involves the AC7 activation and provide new insight into the modulation of Treg activities via AC inhibition or stimulation in various pathological processes like tumor and autoimmune diseases.
Mol Immunol 2010 Jan
PMID:Distinct metabolism of cyclic adenosine monophosphate in regulatory and helper CD4+ T cells. 1993 55


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