Identification of Inhibitor Specificity Determinants in a Mammalian Phosphodiesterase (original) (raw)
Related papers
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 and Biophysical Research Communications, 2000
We have identified and characterised a novel member of the PDE7 family of cyclic nucleotide phosphodiesterases (PDE), which we have designated PDE7B. Mouse and human full-length cDNAs were isolated encoding a protein of 446 and 450 amino acids, respectively. The predicted protein sequence of PDE7B showed highest homology (70% identity) to that of PDE7A. Northern blot analysis identified a single 5.5-kb transcript with highest levels detected in brain, heart, and liver. Kinetic analysis of the mouse and human purified recombinant enzymes show them to specifically hydrolyse cAMP with a K m of 0.1 and 0.2 M respectively. Inhibitor studies show sensitivity to dipyridamole, IC 50 of 0.51 and 1.94 M, and IBMX, IC 50 of 3.81 and 7.37 M, for the mouse and human enzymes, respectively. This shows that dipyridamole is not selective for cGMP over cAMP PDEs as previously believed. Other standard PDE inhibitors including zaprinast, rolipram, and milrinone do not significantly inhibit PDE7B.
The Molecular Basis for Different Recognition of Substrates by Phosphodiesterase Families 4 and 10
Journal of Molecular Biology, 2007
Phosphodiesterases (PDEs) are key enzymes that control the cellular concentrations of the second messengers cAMP and cGMP. The mechanism for selective recognition of substrates cAMP and cGMP by individual PDE families remains a puzzle. To understand the mechanism for substrate recognition by PDE enzymes, the crystal structure of the catalytic domain of an inactive D201N mutant of PDE4D2 in complex with substrate cAMP has been determined at 1.56 Å resolution. The structure shows that Gln369 forms only one hydrogen bond with the adenine of cAMP. This finding provides experimental evidence against the hypothesis of two hydrogen bonds between the invariant glutamine and the substrate cAMP in PDE4, and thus suggests that the widely circulated "glutamine switch" model is unlikely the mechanism for substrate recognition by PDEs. A structure comparison between PDE4D2-cAMP and PDE10A2-cAMP reveals an anti configuration of cAMP in PDE4D2 but syn in PDE10A2, in addition to different contact patterns of cAMP in these two structures. These observations imply that individual PDE families have their characteristic mechanisms for substrate recognition.
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...
Intracellular Compartmentalization of PDE4 Cyclic AMP-Specific Phosphodiesterases
Methods, 1998
on chromosome 19p13.2. The catalytic unit of the The PDE4 cyclic AMP-specific phosphodiesterase family various PDE4 family members is encoded by Ç7 excomprises a large number of different isoforms encoded by ons, the sequences of which show high homology for four distinct genes, with additional complexity arising the four different PDE4 families (4A, 4B, 4C, 4D). through alternate mRNA splicing. This generates a number However, the extreme 3 exons, which encode the Cof distinct PDE4 isoforms with unique N-terminal regions. terminal tail of the various PDE4 enzymes, are very The range of such splice variants emanating from the four different and encode amino acid sequences that show PDE4 genes appears to be highly conserved across species. no similarity between the C-terminal regions of One key role for such regions appears to be their potential members of any of the four PDE4 gene families. Nevto target isoforms to specific intracellular sites. Evidence ertheless, within a particular PDE4 gene family, all for such a targeting role for these N-terminal regions can active isoforms appear to have identical C-terminal be gleaned by a variety of techniques. These include subcelregions. This observation has been usefully exploited lular fractionation, confocal microscopy, binding assays to (4-9) to generate antibodies/antisera that are able show association with proteins having src homology 3 (SH3) to detect all active PDE4 isoforms of a particular domains, and generation of chimeric constructs of these N-PDE4 gene family. Similarly, generic reverse tranterminal regions with proteins that are normally expressed scription polymerase chain reaction (RT-PCR) primin the cytosol. ᭧ 1998 Academic Press ers can also be designed to identify the presence of transcripts for all active members of a particular PDE4 gene family without a priori knowledge of the range of splice variants present (10, 11). This is because alternative mRNA splicing appears, for active The PDE4 cyclic AMP specific phosphodiesterase species, to be uniquely associated with 5 domain family comprises a large number of different isoswaps. It should, however, be noted, that regions of forms encoded by four distinct genes with additional sequence within the putative catalytic domain are complexity arising through alternate mRNA splicing PDE4 subfamily specific and have also been usefully (1-3). The genes are complex structures with many exploited to generate generic RT-PCR primers exons. For example, the human PDE4A gene spans aimed, again, at amplifying/detecting transcripts for 50 kb, comprises at least 17 exons, and is located all active members arising from a particular PDE4 gene subfamily. Thus, both molecular and immunological methods can be employed to detect active 1 To whom correspondence and reprint requests should be addressed.
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
Methods, 1998
zymes [named PDE4 according to a recently pro-With the cloning of cDNAs coding for the different phosphoposed nomenclature (4)] hydrolyze cAMP specifically diesterase 4 (PDE4) isoenzymes present in mammals, homoand are the target of antidepressants such as roligeneous preparations of these forms have become readily pram in the submicromolar range of concentrations. available. This strategy has greatly facilitated the under-Since these initial observations, a large number of standing of the properties of the myriad of isoforms derived studies have focused on these PDEs as they play an from the four PDE4 genes found in mammals, and has opened important role in cAMP homeostasis (5, 6). In many a new avenue to develop inhibitors with a different degree of cells of the body, their activity and expression are selectivity for each isoform. Here we describe the strategies regulated by cAMP through intracellular feedback and methods used to express PDE4 in bacterial, yeast, inloops (5-7). Recently, it has been established that sect, and mammalian cell heterologous systems, and review lymphocytes express mostly PDE4 isoenzymes (8the advantages and disadvantages of each of these expres-11). This observation has renewed interest in these sion strategies. In addition, procedures to purify the recombiproteins, heightening the need for a thorough undernant proteins are described. The recently developed purificastanding of their structure and regulation. Since intion of a PDE4 by immunoaffinity chromatography provides a hibitors of PDE4 are potent anti-inflammatory rapid and efficient method to prepare large quantities of agents (12, 13), a better understanding of the proper-PDE4. This method should be very useful for structural and ties of PDE4 isoenzymes will certainly facilitate the kinetic studies on the PDE4D isoforms. ᭧ 1998 Academic Press development of more potent and more specific drugs. Several obstacles have frustrated the isolation and biochemical characterization of PDE4 proteins. These enzymes are expressed at very low levels in the cell (estimated 1:100,000 of the cell protein) and are remarkably unstable when purification is at-More than 20 years ago a novel phosphodiesterase tempted. Early attempts to purify these PDE forms species that degrades cAMP in the low micromolar often yielded heterogeneous preparations with mulrange of concentrations was separated on ion-extiple peaks on chromatography and polypeptides of change chromatography of rat or bovine brain and different sizes [reviewed in (14)]. dog kidney extracts (1). The development of drugs A better understanding of the properties of these that stimulate the central nervous system (CNS) enzymes has come with the cloning of the genes and function and that specifically inhibit only these cDNAs corresponding to several PDE4s. Using difforms (2, 3) allowed conclusive identification of ferent strategies for the isolation of PDE4 cDNAs, these PDEs as a distinct group of proteins with unique pharmacological properties. These isoen-four genes were first identified in the rat (15-17).
Archives of Biochemistry …, 2001
The human PDE4A catalytic domain (PDE4A 330-723) expressed in Sf9 cells was found to be heavily phosphorylated on both serines of the conserved SPS motif by mass spectrometric analysis. The purified protein exists as a tetramer at a concentration ϳ1 mg/ml from light scattering measurement and has a K m of 2 M in hydrolyzing cAMP. In comparison, a partially purified PDE4A 330-723 expressed in Escherichia coli has an apparent K m of 10 M. The EC 50 values for the Mg 2؉-or Co 2؉-mediated cAMP hydrolysis between the two enzymes differed by less than twofold. In addition, both enzymes exhibit similar sensitivities toward inhibition by a diverse set of inhibitors. Together with the fact that its adjacent peptide was covalently labeled by an electrophilic cAMP analogue, these results support that the SPS motif is not part of but is positioned near the active site. An efficient purification protocol that provides a highly purified PDE4A catalytic domain suitable for crystallization study is described.
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.