Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Here we identify an 11-residue helical module in the unique N-terminal region of the cyclic AMP-specific phosphodiesterase PDE4A1 that determines association with phospholipid bilayers and shows a profound selectivity for interaction with phosphatidic acid (PA). This module contains a core bilayer insertion unit that is formed by two tryptophan residues, Trp(19) and Trp(20), whose orientation is optimized for bilayer insertion by the Leu(16):Val(17) pairing. Ca(2+), at submicromolar levels, interacts with Asp(21) in this module and serves to gate bilayer insertion, which is completed within 10 ms. Selectivity for interaction with PA is suggested to be achieved primarily through the formation of a charge network of the form (Asp(21-):Ca(2+):PA(2-):Lys(24+)) with overall neutrality at the bilayer surface. This novel phospholipid-binding domain, which we call TAPAS-1 (tryptophan anchoring phosphatidic acid selective-binding domain 1), is here identified as being responsible for membrane association of the PDE4A1 cAMP-specific phosphodiesterase. TAPAS-1 may not only serve as a paradigm for other PA-binding domains but also aid in detecting related phospholipid-binding domains and in generating simple chimeras for conferring membrane association and intracellular targeting on defined proteins.
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PMID:TAPAS-1, a novel microdomain within the unique N-terminal region of the PDE4A1 cAMP-specific phosphodiesterase that allows rapid, Ca2+-triggered membrane association with selectivity for interaction with phosphatidic acid. 1199 73

The phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis can be activated by nonsubstrate interfaces such as phosphatidylcholine micelles or bilayers. This activation corresponds with partial insertion into the interface of two tryptophans, Trp-47 in helix B and Trp-242 in a loop, in the rim of the alphabeta-barrel. Both W47A and W242A have much weaker binding to interfaces and considerably lower kinetic interfacial activation. Tryptophan rescue mutagenesis, reinsertion of a tryptophan at a different place in helix B in the W47A mutant or in the loop (residues 232-244) of the W242A mutant, has been used to determine the importance and orientation of a tryptophan in these two structural features. Phosphotransferase and phosphodiesterase assays, and binding to phosphatidylcholine vesicles were used to assess both orientation and position of tryptophans needed for interfacial activity. Of the helix B double mutants, only one mutant, I43W/W47A, has tryptophan in the same orientation as Trp-47. I43W/W47A shows recovery of phosphatidylinositol-specific phospholipase C (PC) activation of d-myo-inositol 1,2-cyclic phosphate hydrolysis. However, the specific activity toward phosphatidylinositol is still lower than wild type enzyme and high activity with phosphatidylinositol solubilized in 30% isopropyl alcohol (a hallmark of the native enzyme) is lost. Reinserting a tryptophan at several positions in the loop composed of residues 232-244 partially recovers PC activation and affinity of the enzyme for lipid interfaces as well as activation by isopropyl alcohol. G238W/W242A shows an enhanced activation and affinity for PC interfaces above that of wild type. These results provide constraints on how this bacterial phosphatidylinositol-specific phospholipase C binds to activating PC interfaces.
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PMID:Optimizing the interfacial binding and activity of a bacterial phosphatidylinositol-specific phospholipase C. 1271 98

The diuretic helicokinins YFSPWG-amide (Hez KI), VRFSPWG-amide (Hez KII) and KVKFSAWG-amide (Hez KIII) are potent contractants of the isolated gut of the caterpillar Spodoptera frugiperda at doses ranging from 0.1 to 10nM. In comparison, the pentapeptide FSPWG-amide was a full agonist with greatly reduced potency while SPWG-amide and PWG-amide were weak partial agonists. Substitution of individual amino acids in Hez KI with alanine revealed that replacement of the [phenylalanine(2)] residue caused a large fall in potency while replacement of [tryptophan(5)] residue caused complete loss of myogenic activity. The striking fall in potency of YASPWG-amide and the lack of activity of YFSPAG-amide confirm the requirement for aromatic groups in positions 2 and 3 of the core pentapeptide as well as supporting the ideas that the active core of these peptides adopts a beta-turn when interacting with receptors, bringing together the [Phe] and [Trp] residues that are critical for activity. Neither the pentapeptide proctolin nor the potent mammalian gut contractant Substance P were able to cause contraction when applied to caterpillar gut tissue. Incubation of isolated gut tissue in the phosphodiesterase inhibitor theophylline (10-100&mgr;M) caused significant potentiation of the response to applied Hez KI. Conversely, in the presence of the L-type Ca(2+) channel blocker verapamil (10&mgr;M-1mM) or Co(2+) (1-50mM) the contractile effects of Hez KI were attentuated significantly. These data suggest that the gut of S. frugiperda contains G-protein-linked kinin receptors that utilise cyclic AMP as their second messenger system and cause contraction by promoting the entry of extracellular Ca(2+).
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PMID:Structure-activity relationship of contractile effects induced by helicokinins in the isolated gut of the lepidopteran caterpillar Spodoptera frugiperda. 1277 Jan 34

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis catalyzes the hydrolysis of phosphatidylinositol (PI) in a Ca(2+)-independent two-step mechanism: (i) an intramolecular phosphotransferase reaction to form inositol 1,2-(cyclic)-phosphate (cIP), followed by (ii) a cyclic phosphodiesterase activity that converts cIP to inositol 1-phosphate (I-1-P). Moderate amounts of water-miscible organic solvents have previously been shown to dramatically enhance the cyclic phosphodiesterase activity, that is, hydrolysis of cIP. Cosolvents [isopropanol (iPrOH), dimethylsufoxide (DMSO), and dimethylformamide (DMF)] also enhance the phosphotransferase activity of PI-PLC toward PI initially presented in vesicles, monomers, or micelles. Although these water-miscible organic cosolvents caused large changes in PI particle size and distribution (monitored with pyrene-labeled PI fluorescence, 31P NMR spectroscopy, gel filtration, and electron microscopy) that differed with the activating solvent, the change in PI substrate structure in different cosolvents was not correlated with the enhanced catalytic efficiency of PI-PLC toward its substrates. PI-PLC stability was decreased in water/organic cosolvent mixtures (e.g., the T(m) for PI-PLC thermal denaturation decreased linearly with added iPrOH). However, the addition of myo-inositol, a water-soluble inhibitor of PI-PLC, helped stabilize the protein. At 30% iPrOH and 4 degrees C (well below the T(m) for PI-PLC in the presence of iPrOH), cosolvent-induced changes in protein secondary structure were minimal. iPrOH and diheptanoylphosphatidylcholine, each of which activates PI-PLC for cIP hydrolysis, exhibited a synergistic effect for cIP hydrolysis that was not observed with PI as substrate. This behavior is consistent with a mechanism for cosolvent activation that involves changes in active site polarity along with small conformational changes involving the barrel rim tryptophan side chains that have little effect on protein secondary structure.
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PMID:Water-miscible organic cosolvents enhance phosphatidylinositol-specific phospholipase C phosphotransferase as well as phosphodiesterase activity. 1283 83

Indolicidin, ILPWKWPWWPWRR-NH(2), a short 13-residue antimicrobial and cytolytic peptide characterized from bovine neutrophils, has the calmodulin-recognition 1-5-10 hydrophobic pattern (indicated by amino acids in bold), is cationic, and thereby fulfills the requirements to interact with calmodulin. Hence, we have investigated the calmodulin-binding properties of indolicidin. Indolicidin interacted with calmodulin with fairly high affinity in a Ca(2+)-dependent manner. However, when bound, the peptide did not adopt helical conformation. Indolicidin also inhibited calmodulin-stimulated phosphodiesterase activity with IC(50) values in the nanomolar range. Replacement of either the proline residues of indolicidin with alanines or tryptophan residues with phenylalanines did not affect binding to calmodulin. However, these replacements had distinctive effects on the conformations of the bound peptides. While the alanine analog of indolicidin adopted predominantly alpha-helical conformation, the phenylalanine analog remained largely unordered. Differences in the ability of these analogs to inhibit the calmodulin-stimulated phosphodiesterase activity were observed. While the alanine analog was capable of inhibiting the activity with IC(50) values comparable to that of indolicidin, the phenylalanine analog did not inhibit the activity. Our results indicate that ability to adopt amphiphilic alpha-helical structure is not a prerequisite for binding to calmodulin and also binding does not necessarily result in inhibition of calmodulin-stimulated enzyme activities.
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PMID:Indolicidin, a 13-residue basic antimicrobial peptide rich in tryptophan and proline, interacts with Ca(2+)-calmodulin. 1367 55

Bovine PDEdelta was originally copurified with rod cGMP phosphodiesterase (PDE) and shown to interact with prenylated, carboxymethylated C-terminal Cys residues. Other studies showed that PDEdelta can interact with several small GTPases including Rab13, Ras, Rap, and Rho6, all of which are prenylated, as well as the N-terminal portion of retinitis pigmentosa GTPase regulator and Arl2/Arl3, which are not prenylated. We show by immunocytochemistry with a PDEdelta-specific antibody that PDEdelta is present in rods and cones. We find by yeast two-hybrid screening with a PDEdelta bait that it can interact with farnesylated rhodopsin kinase (GRK1) and that prenylation is essential for this interaction. In vitro binding assays indicate that both recombinant farnesylated GRK1 and geranylgeranylated GRK7 co-precipitate with a glutathione S-transferase-PDEdelta fusion protein. Using fluorescence resonance energy transfer techniques exploiting the intrinsic tryptophan fluorescence of PDEdelta and dansylated prenyl cysteines as fluorescent ligands, we show that PDEdelta specifically binds geranylgeranyl and farnesyl moieties with a Kd of 19.06 and 0.70 microm, respectively. Our experiments establish that PDEdelta functions as a prenyl-binding protein interacting with multiple prenylated proteins.
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PMID:Photoreceptor cGMP phosphodiesterase delta subunit (PDEdelta) functions as a prenyl-binding protein. 1456 60

The GTP-binding protein (G protein), transducin, serves as a key molecular switch in vertebrate vision through the tight regulation of its GTP-binding (activation)/GTP hydrolytic (deactivation) cycle by the photoreceptor rhodopsin. To better understand the structure-function characteristics of transducin activation, we have set out to identify spectroscopic probes that bind to the guanine nucleotide-binding site of this G protein and maintain its ability to interact with its specific cellular target/effector, the cyclic GMP phosphodiesterase (PDE). In this study, we describe the characterization of a fluorescently labeled GTP analogue, BODIPY-FL GTPgammaS (BOD-GTPgammaS), that binds to the alpha subunit of transducin (alpha(T)) in a rhodopsin- and Gbetagamma-dependent manner, similar to the binding of GTP or GTPgammaS, with an apparent dissociation constant of 100 nM. The rhodopsin-dependent binding of BOD-GTPgammaS to alpha(T) is slow, relative to the rate of binding of GTPgammaS, particularly under conditions where rhodopsin must act catalytically to stimulate the exchange of BOD-GTPgammaS for GDP on multiple alpha(T) subunits. This reflects a slower rate of dissociation of rhodopsin and Gbetagamma from alpha(T)-BOD-GTPgammaS complexes, relative to their rates of dissociation from alpha(T)-GTPgammaS. The binding of BOD-GTPgammaS occurs without a change in the intrinsic tryptophan fluorescence of alpha(T), indicating that only a subtle movement of the Switch 2 domain on alpha(T) accompanies the binding of this GTPgammaS analogue. Nevertheless, the BOD-GTPgammaS-bound alpha(T) subunit is able to bind with high affinity to the recombinant, purified gamma subunit of PDE (gamma(PDE)) labeled with 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS (K(d) approximately 13 nM)), as well as bind to and stimulate the activity of PDE, albeit less efficiently compared to alpha(T)-GTPgammaS. Taken together, these findings suggest that the binding of BOD-GTPgammaS to transducin causes it to adopt a distinct conformation that appears to be intermediate between the inactive and fully active states of alpha(T), and this fluorescent nucleotide analogue can be used as a reporter group to characterize the interactions of alpha(T) in this conformational state with its biological target/effector.
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PMID:Stabilization of an intermediate activation state for transducin by a fluorescent GTP analogue. 1523 86

Cell-attached patch-clamp recordings on native striated myofibers from adult dystrophic mdx mice revealed a higher occurrence and open probability compared to non-dystrophic wild-type myofibers of a 30 pS voltage-insensitive Ca2+-permeable channel, inhibited by Gd3+, streptomycin and ruthenium red. Myofibers from in vivo exercised animals had higher channel occurrence and/or open probability. Insulin-like growth factor 1 (3.3 nM) induced and/or enhanced channel activity, via PI3 kinase, in wild-type but not in mdx myofibers. Interestingly, in both genotypes the current was silenced by db-cAMP or pentoxifylline, a phosphodiesterase inhibitor. The channel activity/occurrence in pentoxifylline-treated exercised mdx (50 mg/kg/day i.p. for 4-8 weeks) overlapped that of exercised wild-type mice. Thus, a growth factor-sensitive current, likely due to a TRP channel, is activated in vivo by exercise in native striated fibers; its deregulation in the absence of dystrophin may contribute to Ca2+ homeostasis alteration. The possibility to pharmacologically counteract abnormal channel activity discloses important therapeutic application.
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PMID:Overactivity of exercise-sensitive cation channels and their impaired modulation by IGF-1 in mdx native muscle fibers: beneficial effect of pentoxifylline. 1701 Jun 31

GPR35 is a G protein-coupled receptor recently "de-orphanized" using high-throughput intracellular calcium measurements in clonal cell lines expressing a chimeric G-protein alpha-subunit. From these screens, kynurenic acid, an endogenous metabolite of tryptophan, and zaprinast, a synthetic inhibitor of cyclic guanosine monophosphate-specific phosphodiesterase, emerged as potential agonists for GPR35. To investigate the coupling of GPR35 to natively expressed neuronal signaling pathways and effectors, we heterologously expressed GPR35 in rat sympathetic neurons and examined the modulation of N-type (Ca(V)2.2) calcium channels. In neurons expressing GPR35, calcium channels were inhibited in the absence of overt agonists, indicating a tonic receptor activity. Application of kynurenic acid or zaprinast resulted in robust voltage-dependent calcium current inhibition characteristic of Gbetagamma-mediated modulation. Both agonist-independent and -dependent effects of GPR35 were blocked by Bordetella pertussis toxin pretreatment indicating the involvement of G(i/o) proteins. In neurons expressing GPR35a, a short splice variant of GPR35, zaprinast was more potent (EC(50) = 1 microM) than kynurenic acid (58 microM) but had a similar efficacy (approximately 60% maximal calcium current inhibition). Expression of GPR35b, which has an additional 31 residues at the N terminus, produced similar results but with much greater variability. Both GPR35a and GPR35b appeared to have similar expression patterns when fused to fluorescent proteins. These results suggest a potential role for GPR35 in regulating neuronal excitability and synaptic release.
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PMID:Inhibition of N-type calcium channels by activation of GPR35, an orphan receptor, heterologously expressed in rat sympathetic neurons. 1794 Jan 99

GPR35 is a Gi/o- and G16-coupled receptor abundantly expressed in gastrointestinal tissues and immune cells. Kynurenic acid (a tryptophan metabolite and ionotropic glutamate receptor antagonist) and zaprinast (a phosphodiesterase inhibitor) are GPR35 agonists. Here, we show that the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) is also a GPR35 agonist. NPPB activates the GPR35-Gi/o and GPR35-G16 pathways in human embryonic kidney 293 (HEK293) cells and induces intracellular calcium mobilization in a concentration-dependent manner in HEK293 cells coexpressing human, rat or mouse GPR35 and the chimeric G protein G(qi5). These results suggest a novel pharmacological activity of NPPB and will provide useful information to search for more potent and selective GPR35 agonists.
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PMID:5-Nitro-2-(3-phenylpropylamino)benzoic acid is a GPR35 agonist. 1881 9


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