Focus on molecules: rod cGMP phosphodiesterase type 6.
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ABSTRACT:
SUBMITTER: Ionita MA
PROVIDER: S-EPMC10546788 | biostudies-literature | 2007 Jan
REPOSITORIES: biostudies-literature
Publications
Focus on molecules: rod cGMP phosphodiesterase type 6.
Ionita Marcel A MA Pittler Steven J SJ
Experimental eye research 20060323 1
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Project description: Not available
| S-EPMC3132568 | biostudies-literature
Project description:Rod cGMP phosphodiesterase 6 (PDE6) is a key enzyme of the phototransduction cascade, consisting of PDE6alpha, PDE6beta, and two regulatory PDE6gamma subunits. PDE6 is membrane associated through isoprenyl membrane anchors attached to the C-termini of PDE6alpha and PDE6beta and can form a complex with prenyl-binding protein delta (PrBP/delta), an isoprenyl-binding protein that is highly expressed in photoreceptors. The stoichiometry of PDE6-PrBP/delta binding and the mechanism by which the PDE6-PrBP/delta complex assembles have not been fully characterized, and the location of regulatory PDE6gamma subunits within the protein assembly has not been elucidated. To clarify these questions, we have developed a rapid purification method for PDE6-PrBP/delta from bovine rod outer segments utilizing recombinant PrBP/delta. Transmission electron microscopy of negatively stained samples revealed the location of PrBP/delta and, thus, where the carboxyl-termini of PDE6alpha and PDE6beta must be located. The three-dimensional structure of the PDE6alphabetagamma complex was determined up to 18 A resolution from single-particle projections and was interpreted by model building to identify the probable location of isoprenylation, PDE6gamma subunits, and catalytic sites.
| S-EPMC2917047 | biostudies-literature
Project description:Emerging evidence reveals crucial roles of wild type RAS in liver cancer. The delta subunit of rod-specific photoreceptor cGMP phosphodiesterase (PDE6D) regulates the trafficking of RAS proteins to the plasma membrane and thereby contributes to RAS activation. However, the expression and specific function of PDE6D in hepatocellular carcinoma (HCC) were completely unknown. In this study, PDE6D was newly found to be markedly upregulated in HCC tissues and cell lines. Overexpression of PDE6D in HCC correlated with enhanced tumor stages, tumor grading, and ERK activation. PDE6D depletion significantly reduced proliferation, clonogenicity, and migration of HCC cells. Moreover, PDE6D was induced by TGF-β1, the mediator of stemness, epithelial-mesenchymal transition (EMT), and chemoresistance. In non-resistant cells, overexpression of PDE6D conferred resistance to sorafenib-induced toxicity. Further, PDE6D was overexpressed in sorafenib resistance, and inhibition of PDE6D reduced proliferation and migration in sorafenib-resistant HCC cells. Together, PDE6D was found to be overexpressed in liver cancer and correlated with tumor stages, grading, and ERK activation. Moreover, PDE6D contributed to migration, proliferation, and sorafenib resistance in HCC cells, therefore representing a potential novel therapeutic target.
| S-EPMC6468542 | biostudies-literature
Project description:We report the chromosomal localization, mutant gene identification, ophthalmic appearance, histology, and functional analysis of two new hereditary mouse models of retinal degeneration not having the Pde6brd1("r", "rd", or "rodless") mutation. One strain harbors an autosomal recessive mutation that maps to mouse chromosome 5. Sequence analysis showed that the retinal degeneration is caused by a missense point mutation in exon 13 of the beta-subunit of the rod cGMP phosphodiesterase (beta-PDE) gene (Pde6b). The gene symbol for this strain was set as Pde6brd10, abbreviated rd10 hereafter. Mice homozygous for the rd10 mutation showed histological changes at postnatal day 16 (P16) of age and sclerotic retinal vessels at four weeks of age, consistent with retinal degeneration. Retinal sections were highly positive for TUNEL and activated caspase-3 immunoreactivity, specifically in the outer nuclear layer (ONL). ERGs were never normal, but rod and cone ERG a- and b-waves were easily measured at P18 and steadily declined over 90% by two months of age. Protein extracts from rd10 retinas were positive for beta-PDE immunoreactivity starting at about the same time as wild-type (P10), though signal averaged less than 40% of wild-type. Interestingly, rearing rd10 mice in total darkness delayed degeneration for at least a week, after which morphological and functional loss progressed irregularly. With the second strain, a complementation test with rd1 mice revealed that the retinal degeneration phenotype observed represents a possible new allele of Pde6b. Sequencing demonstrated a missense point mutation in exon 16 of the beta-subunit of rod phosphodiesterase gene, different from the point mutations in rd1 and rd10. The gene symbol for this strain was set as Pde6bnmf137, abbreviated nmf137 hereafter. Mice homozygous for this mutation showed retinal degeneration with a mottled retina and white retinal vessels at three weeks of age. The exon 13 missense mutation (rd10) is the first known occurrence of a second mutant allele spontaneously arising in the Pde6b gene in mice and may provide a model for studying the pathogenesis of autosomal recessive retinitis pigmentosa (arRP) in humans. It may also provide a better model for experimental pharmaceutical-based therapy for RP because of its later onset and milder retinal degeneration than rd1 and nmf137.
| S-EPMC2562796 | biostudies-literature
Project description:Network Map States Transitions Functions Protein Classes Sequence Interactions Pathways Domains & Motifs Protein Structure Orthologs Sequence Interactions Pathways Domains & Motifs Protein Structure Orthologs Blast Data.
| S-EPMC4618378 | biostudies-literature
Project description:Irish setter dogs affected with a rod/cone dysplasia (locus designation, rcd1) display markedly elevated levels of retinal cGMP during postnatal development. The photoreceptor degeneration commences approximately 25 days after birth and culminates at about 1 year when the population of rods and cones is depleted. A histone-sensitive retinal cGMP phosphodiesterase (PDE; EC 3.1.4.35) activity, a marker for photoreceptor PDEs, was shown previously to be present in retinal homogenates of immature, affected Irish setters. Here we report that, as judged by HPLC separation, this activity originates exclusively from cone photoreceptors, whereas rod PDE activity is absent. An immunoreactive product the size of the PDE alpha subunit, but none the size of the beta subunit, can be detected on immunoblots of retinal extracts of affected dogs, suggesting a null mutation in the PDE beta-subunit gene. Using PCR amplification of Irish setter retinal cDNA, we determined the complete coding sequence of the PDE beta subunit in heterozygous and affected animals. The affected PDE beta-subunit mRNA contained a nonsense amber mutation at codon 807 (a G-->A transition converting TGG to TAG), which was confirmed to be present in putative exon 21 of the affected beta-subunit gene. The premature stop codon truncates the beta subunit by 49 residues, thus removing the C-terminal domain that is required for posttranslational processing and membrane association. These results suggest that the rcd1 gene encodes the rod photoreceptor PDE beta subunit and that a nonsense mutation in this gene is responsible for the production of a nonfunctional rod PDE and the photoreceptor degeneration in the rcd1/rcd1 Irish setter dogs.
| S-EPMC46427 | biostudies-other
Project description:Retinitis pigmentosa (RP) is the most common form of hereditary retinal degeneration, with a worldwide prevalence of 1 in 4000. Over 30 genes and loci have been implicated in nonsyndromic autosomal-recessive (ar) RP. Genome-wide homozygosity mapping was conducted in two sibships from an extended consanguineous Muslim Arab Israeli family segregating ar severe early-onset RP. A shared homozygous region on chromosome 17q25.3 was identified in both sibships, with an overlap of 4.7 Mb. One of the genes located in this interval is PDE6G, encoding for the inhibitory gamma subunit of rod photoreceptor cyclic GMP-phosphodiesterase. Mutations in the genes encoding for the catalytic subunits of this holoenzyme, PDE6A and PDE6B, cause arRP. Sequencing of all coding exons, including exon-intron boundaries, revealed a homozygous single base change (c.187+1G>T) located in the conserved intron 3 donor splice site of PDE6G. This mutation cosegregated with the disease in the extended family. We used an in vitro splicing assay to demonstrate that this mutation leads to incorrect splicing. Affected individuals had markedly constricted visual fields. Both scotopic and photopic electroretinograms were severely reduced or completely extinct. Funduscopy showed typical bone spicule-type pigment deposits spread mainly at the midperiphery, as well as pallor of the optic disk. Macular involvement was indicated by the lack of foveal reflex and typical cystoid macular edema, proved by optical coherence tomography. These findings demonstrate the positive role of the gamma subunit in maintaining phosphodiesterase activity and confirm the contribution of PDE6G to the etiology of RP in humans.
| S-EPMC2917712 | biostudies-literature
Project description: Not available
| S-EPMC2821900 | biostudies-literature
Project description:StmF mutants are chemotactic mutants that are defective in a cGMP phosphodiesterase (PDE) activity. We identified a novel gene, PdeD, that harbors two cyclic nucleotide-binding domains and a metallo-beta-lactamase homology domain. Similar to stmF mutants, pdeD-null mutants displayed extensively streaming aggregates, prolonged elevation of cGMP levels after chemotactic stimulation, and reduced cGMP-PDE activity. PdeD transcripts were lacking in stmF mutant NP377, indicating that this mutant carries a PdeD lesion. Expression of a PdeD-YFP fusion protein in pdeD-null cells restored the normal cGMP response and showed that PdeD resides in the cytosol. When purified by immunoprecipitation, the PdeD-YFP fusion protein displayed cGMP-PDE activity, which was retained in a truncated construct that contained only the metallo-beta-lactamase domain.
| S-EPMC133599 | biostudies-literature
Project description:Retinal degeneration in the mouse mutant, rd, was previously shown to be a disorder of cyclic nucleotide metabolism involving a deficiency in the activity of the rod photoreceptor cGMP phosphodiesterase (PDE). We have characterized the normal and rd PDE beta-subunit gene, and their respective transcripts, by PCR and direct sequence analysis. We show that the gene consists of at least 22 exons ranging in size from 48 base pairs to several hundred base pairs, covering greater than 25 kilobases. Within a 67-base-pair exon of the rd PDE beta-subunit gene, we identified a nonsense ochre mutation (a C----A transversion in codon 347) that truncates the normal gene product, eliminating more than one-half of the peptide chain, including the putative catalytic domain. The consequences of the truncation are consistent with the observed phenotypes in rd mice heterozygous and homozygous for the disorder. The nonsense mutation was also found in another related and in six unrelated strains displaying the rd phenotype, indicating that the rd allele arose from a single genetic event. The results strongly argue for the nonsense mutation being responsible for retinal degeneration in the rd mouse.
| S-EPMC52500 | biostudies-other