Cloning and Characterization of the Human and Mouse PDE7B, a Novel cAMP-Specific Cyclic Nucleotide Phosphodiesterase (original) (raw)
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Biochemical Journal, 2004
PDE4A7 is an isoform encoded by the human PDE4A cAMP-specific phosphodiesterase gene that fails to hydrolyse cAMP and whose transcripts are widely expressed. Removal of either the N- or C-terminal unique portions of PDE4A7 did not reconstitute catalytic activity, showing that they did not exert a chronic inhibitory effect. A chimera (Hyb2), formed by swapping the unique N-terminal portion of PDE4A7 with that of the active PDE4A4C form, was not catalytically active. However, one formed (Hyb1) by swapping the unique C-terminal portion of PDE4A7 with that common to all active PDE4 isoforms was catalytically active. Compared with the active PDE4A4B isoform, Hyb1 exhibited a similar Km value for cAMP and IC50 value for rolipram inhibition, but was less sensitive to inhibition by Ro-20-1724 and denbufylline, and considerably more sensitive to thermal denaturation. The unique C-terminal region of PDE4A7 was unable to support an active catalytic unit, whereas its unique N-terminal region ca...
Biochemical Journal, 2003
We have isolated cDNAs encoding PDE4B4, a new cAMPspecific phosphodiesterase (PDE4) isoform with novel properties. The amino acid sequence of PDE4B4 demonstrates that it is encoded by the PDE4B gene, but that it differs from the previously isolated PDE4B1, PDE4B2 and PDE4B3 isoforms by the presence of a novel N-terminal region of 17 amino acids. PDE4B4 contains both of the upstream conserved region 1 (UCR1) and UCR2 regulatory units that are characteristic of ' long ' PDE4 isoforms. RNase protection demonstrated that PDE4B4 mRNA is expressed preferentially in liver, skeletal muscle and various regions of the brain, which differs from the pattern of tissue distribution of the other known PDE4B long forms, PDE4B1 and PDE4B3. Expression of PDE4B4 cDNA in COS7 cells produced a protein of 85 kDa under denaturing conditions. Subcellular fractionation of recombinant, COS7-cell expressed PDE4B4 showed that the protein was localized within
Molecular cloning and characterization of a distinct human phosphodiesterase gene family: PDE11A
Proceedings of the National Academy of Sciences, 2000
We report here the cloning, expression, and characterization of human PDE11A1, a member of a distinct cyclic nucleotide phosphodiesterase (PDE) family. PDE11A exhibits <50% amino acid identity with the catalytic domains of all other PDEs, being most similar to PDE5, and has distinct biochemical properties. The human PDE11A1 cDNA isolated contains a complete open reading frame encoding a 490-amino acid enzyme with a predicted molecular mass of 55,786 Da. At the N terminus PDE11A1 has a single GAF domain homologous to that found in other signaling molecules, including PDE2, PDE5, PDE6, and PDE10, which constitutes a potential allosteric binding site for cGMP or another small ligand. Tissue distribution studies indicate that PDE11A mRNA occurs at highest levels in skeletal muscle, prostate, kidney, liver, pituitary, and salivary glands and testis. PDE11A is expressed as at least three major transcripts of Ϸ10.5, Ϸ8.5, and Ϸ6.0 kb, thus suggesting the existence of multiple subtypes. This possibility is further supported by the detection of three distinct proteins of Ϸ78, Ϸ65, and Ϸ56 kDa by Western blotting of human tissues for PDE11A isoforms. Recombinant human PDE11A1 hydrolyzes both cGMP and cAMP with K m values of 0.52 M and 1.04 M, respectively, and similar V max values. Therefore, PDE11A represents a dual-substrate PDE that may regulate both cGMP and cAMP under physiological conditions. PDE11A is sensitive to the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) as well as zaprinast and dipyridamole, inhibitors that are generally considered relatively specific for the cGMP-selective PDEs, with IC50 values of 49.8 M, 12.0 M, and 0.37 M, respectively.
Medicinal Research Reviews, 2005
The activity of phosphodiesterases (PDEs) is associated with a wide variety of diseases and an intense effort toward the development of specific PDEs inhibitors has been generated for the last years. They are the enzymes responsible for the hydrolysis of intracellular cyclic adenosine and guanosine monophosphate, and their complexity, as well as their different functional role, makes these enzymes a very attractive therapeutic target. This review is focused on the role of PDEs played on immunomodulatory processes and the advance on the development of specific inhibitors, covering PDEs mainly related to the regulation of autoimmune processes, PDE4 and PDE7. The review also highlights the novel structural classes of PDE4 and PDE7 inhibitors, and the therapeutic potential that combined PDE4/PDE7 inhibitors offer as immunomodulatory agents. ß
Classification of Phosphodiesterases and the Therapeutic Effects of their Inhibitors (Review
—Phosphodiesterase (PDE) is an enzyme that catalyses the hydrolysis of phosphodiester bonds. The enzyme is also takes responsibility for the hydrolysis of cyclic 3',5'adenosine monophosphate (cAMP) and 3',5'cyclic guanosine monophosphate (cGMP). The PDE enzymes in mammals are classified into 11 families, namely PDE1-PDE11. The classification is on the basis of amino acid sequences, substrate specificities, regulatory properties, pharmacological properties, tissue distribution. Various PDE of the same family are related with regards to functionality but differs in their specificities for substrates. Some are hydrolases with selective preferences for cAMP (PDE4, 7 and 8), while the selective preference for some others is for cGMP (PDE5, 6 and 9). Some have the ability to hydrolyse both cAMP and cGMP (PDE1, 2, 3, 10 and 11). cAMP, and cGMP both has important roles in the regulation of inotropic mechanisms in the human myocardium. However, cAMP greatly affects other tissues, and different phosphodiesterase isoenzymes are found in many other tissues. Drugs with inhibitory effects on phosphodiesterase (thus reducing the breakdown of cAMP) have a therapeutic action on the heart, lung, and vasculature as well as on platelet function and inflammatory mechanisms. Inhibitors like these are commonly used as "biochemical tools" to study of role which cyclic nucleotides plays in the cell, but they also may be useful to investigate the structural and functional activities of PDE. As therapeutic agents, they can also be utilized in controlling the pathophysiological changes of responses generated by the cyclic nucleotides in the central nervous system (CNS), cardio-vascular, lung, digestive tract and respectively. PDE enzymes are often targets for inhibition by pharmacological processes due to their unique tissue distribution, structural and functional properties and the inflammatory process. The effect of many of these drugs is evident in more than one isoenzyme, and many tissues possess more than one isoenzyme. As a result, phosphodiesterase inhibitors (PDEI) can have a multiplicity of effects. For example, theophylline has effects on the lung, as well as cardiac and vascular effects; amrinone affects cardiac, vascular and platelet functions. The PDE inhibition, change the intracellular response to extra cellular signals by affecting the processes by the the cyclic nucleotides.
FEBS Letters, 1995
We have recently reported increased survival of dopaminergic substantia nigra neurons by inhibition of phosphodiesterase type IV enzymes. As a first step to unravel the involvement of PDE IV subtypes in this process, we isolated phosphodiesterase type IV cDNAs from human substantia nigra. One isolated partial cDNA done was most homologous to the partially cloned rat and human PDE IV-C isogene. Distribution analysis revealed that the enzyme is expressed in various tissues but not in cells of the immune system. Isolation of the full-length human PDE IV-C isogene cDNA and expression in a PDE-deficient yeast strain resulted in functional complementation of the yeast heat shock response. Inhibition of the enzymatic activity by rolipram characterized this enzyme as a typical type IV phosphodiesterase.
Biochemistry, 2008
Cyclic nucleotide phosphodiesterase-8 (PDE8) is a family of cAMP-specific enzymes and plays important roles in many biological processes, including T-cell activation, testosterone production, adrenocortical hyperplasia, and thyroid function. However, no PDE8 selective inhibitors are available for trial treatment of human diseases. Here we report kinetic properties of the highly active PDE8A1 catalytic domain prepared from refolding and its crystal structures in the unliganded and 3-isobutyl-1-methylxanthine (IBMX) bound forms at 1.9 and 2.1 Å resolutions, respectively. The PDE8A1 catalytic domain has K M of 1.8 μM, Vmax of 6.1 μmol/min/mg, k cat of 4.0 s −1 for cAMP, and K M of 1.6 mM, Vmax of 2.5 μmol/min/mg, k cat of 1.6 s −1 for cGMP, thus indicating that the substrate specificity of PDE8 is dominated by K M . The structure of the PDE8A1 catalytic domain has similar topology as those of other PDE families, but contains two extra helices around Asn685-Thr710. Since this fragment is distant from the active site of the enzyme, its impact on the catalysis is unclear. The PDE8A1 catalytic domain is insensitive to the IBMX inhibition (IC 50 = 700 μM). The unfavorable interaction of IBMX in the PDE8A1-IBMX structure suggests an important role of Tyr748 in the inhibitor binding. Indeed, the mutation of Tyr748 to phenylalanine increases the PDE8A1 sensitivity to several non-selective or family-selective PDE inhibitors. Thus, the structural and mutagenesis studies provide not only insight into the enzymatic properties, but also guidelines for design of PDE8 selective inhibitors.
Biochemical Journal, 2008
We have isolated cDNAs encoding PDE4A8 (phosphodiesterase 4 isoform A8), a new human cAMP-specific PDE4 isoform encoded by the PDE4A gene. PDE4A8 has a novel N-terminal region of 85 amino acids that differs from those of the related ‘long’ PDE4A4, PDE4A10 and PDE4A11 isoforms. The human PDE4A8 N-terminal region has diverged substantially from the corresponding isoforms in the rat and other mammals, consistent with rapid evolutionary change in this region of the protein. When expressed in COS-7 cells, PDE4A8 localized predominantly in the cytosol, but approx. 20% of the enzyme was associated with membrane fractions. Cytosolic PDE4A8 was exquisitely sensitive to inhibition by the prototypical PDE4 inhibitor rolipram (IC50 of 11±1 nM compared with 1600 nM for PDE4A4), but was less sensitive to inhibition by cilomilast (IC50 of 101±7 nM compared with 61 nM for PDE4A4). PDE4A8 mRNA was found to be expressed predominantly in skeletal muscle and brain, a pattern that differs from the tissu...
The cDNA of a human lymphocyte cyclic-AMP phosphodiesterase (PDE IV) reveals a multigene family
Gene, 1993
Five protein families are needed to encompass the diversity of cyclic-AMP (CAMP) phosphodiesterases (PDE). Family IV PDEs (PDE IV) specifically hydrolyze CAMP with a low K,, and are selectively inhibited by rolipram (Rp) and related drugs. Cloned cDNAs from rat (r) suggest that the PDE IV family comprises four distinct members, designated A, B, C and D. Using RNA from a human lymphocytic B-cell line (43D-C12), we have isolated a 3.8-kb cDNA by lowstringency screening using a rat PDE IV member B (r-PDE IT/,) probe. Expression of the human (h) cDNA in Escherichia coli results in CAMP-specific PDE activity that is Rp sensitive. A single large open reading frame (ORF) predicts a 564amino-acid protein with 92.9% identity to r-PDE IV,; at the nucleotide level the identity is 86.3%. This h-PDE ZV, clone, HPB106, differs from a related cDNA clone isolated by others from h-monocytes [Livi et al., Mol. Cell. Biol. 10 (1990) 2678-26861. Our analysis identifies the monocyte clone with r-PDE IV,. Southern blots using a 1.2-kb h-PDE ZV, probe at low stringency suggest the presence of additional uncloned human PDE IV family members. Analysis of genomic Southern blots using short specific probes from the h-PDE ZV, and h-PDE IV, cDNAs indicates that distinct genes encode these two PDE IV family members. RNA from fractionated normal human leukocytes shows major specific messages of 3.0 and 4.6 kb for h-PDE IV, and 3.7 kb for h-PDE IV,. Comparison of our cDNA clones, and PCR analysis of cellular mRNA, suggests that alternative 5'-end sequences of h-PDE IV, are utilized. These results demonstrate a phenomenal complexity in the expression of human PDE ZVs which suggests that unique PDE IV isozymes may control CAMP concentrations in cells.