ArticleLiterature Review

Characteristics of photoreceptor PDE (PDE6): Similarities and differences to PDE5

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Abstract

Phosphodiesterase 6 (PDE6) is highly concentrated in the retina. It is most abundant in the internal membranes of retinal photoreceptors, where it reduces cytoplasmic levels of cyclic guanosine monophosphate (cGMP) in rod and cone outer segments in response to light. The rod PDE6 holoenzyme comprises alpha and beta catalytic subunits and two identical inhibitory gamma subunits. Each catalytic subunit contains three distinct globular domains corresponding to the catalytic domain and two GAF domains (responsible for allosteric cGMP binding). The PDE6 catalytic subunits resemble PDE5 in amino-acid sequence as well as in three-dimensional structure of the catalytic dimer; preference for cGMP over cyclic adenosine monophosphate (cAMP) as a substrate; and the ability to bind cGMP at the regulatory GAF domains. Most PDE5 inhibitors inhibit PDE6 with similar potency, and electroretinogram studies show modest effects of PDE5 inhibitors on visual function-an observation potentially important in designing PDE5-specific therapeutic agents.

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... Due to the amino acid sequence and the secondary structural similarity of PDE6 and PDE11 with the catalytic domain of PDE5, first-generation PDE5 inhibitors (i.e. sildenafil and vardenafil) are also competitive inhibitors of PDE6 and PDE11 [9]. Thus, inhibition of PDE6 by PDE5 inhibitors can disrupt phototransduction processes, leading to transient visual changes, including altered color perception and sensitivity to light [9]. ...
... sildenafil and vardenafil) are also competitive inhibitors of PDE6 and PDE11 [9]. Thus, inhibition of PDE6 by PDE5 inhibitors can disrupt phototransduction processes, leading to transient visual changes, including altered color perception and sensitivity to light [9]. Understanding the distinct roles of PDE5 and PDE6 in cellular signaling pathways is essential for elucidating the Disclaimer/Publisher's Note: The statements, opinions, and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). ...
... PDE6, a crucial enzyme involved in phototransduction, is responsible for the hydrolysis of cyclic guanosine monophosphate (cGMP) in photoreceptor cells. By inhibiting PDE6 activity, PDE5 inhibitors disrupt the delicate balance of cGMP levels in the retina, leading to alterations in visual perception [9]. The mechanism underlying visual disturbances involves perturbations in the phototransduction cascade, affecting processes such as light sensitivity and color discrimination. ...
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Phosphodiesterase type 5 (PDE5) inhibitors are widely used in the treatment of various conditions, including erectile dysfunction and pulmonary hypertension. Despite their clinical efficacy, these drugs quite often cause visual disturbances due to off-target effects on Phosphodiesterase type 5 (PDE6) in the retina. This review explores the application of a general intermolecular binding affinity calculator (GIBAC)-based binding selectivity strategy in the design of PDE5 inhibitors, aiming to enhance binding selectivity and minimize visual side effects. This GIBAC strategy integrates computational structural biological approaches to iteratively refine drug-target binding affinities, thereby improving target specificity and the structural biophysical limit for the efficacy-safety balance of PDE5 inhibitors. Through detailed analysis of PDE5’s and PDE6’s biological role and the molecular mechanisms underlying visual disturbances, this article underscores the necessity of target binding selectivity in PDE5 inhibitor design in future. Additionally, this article discusses the practical applications of GIBAC in computational drug discovery and design, along with the future directions and the potential for GIBAC to transform PDE5 inhibitor development, ultimately enhancing therapeutic outcomes and patient safety. Finally, this article calls for a GIBAC-based paradigm shift in computational drug discovery and design towards the continued development of the pharmaceutical industry.
... The family of phosphodiesterase 6 (PDE6) is almost exclusively expressed in photoreceptor cells of the retina where it is highly abundant in the internal membrane discs of the outer segment (289). Depending on the photoreceptor type, the tetrameric PDE6 enzymes with a total molecular mass of around 220 kDa differ in their composition. ...
... Both PDE6 isozymes contain two inhibitory γ-subunits which is unique compared to other PDE families. Also here, rod PDE6γ and cone PDE6γ`, encoded by the PDE6G and PDE6H gene, slightly differ in their size (9.4 versus 9.7 kDa) and amino-acid composition (289,291,292). Concerning their catalytic properties and interaction with the respective PDE6γ subunit, the catalytic PDE6α, PDE6β and PDE6C subunits seem to be similar (293). ...
... In particular, PDE5, which is expressed in a variety of tissues, seems to be a candidate as it is sequentially and structurally closely related to PDE6. They share cGMP as substrate and most PDE5 inhibitors are also potential PDE6 inihbitors (289). Of course it would also be interesting to test whether other retinal proteins such as rhodopsin or RPE65 ...
... The PDE6 family is one of five families whose N-terminal regulatory region consists of two tandem GAF domains (socalled due to their occurrence in mammalian cGMP-binding PDEs, Anabaena adenylyl cyclases, and E. coli FhlA) that bind cyclic nucleotides and are generally believed to allosterically communicate with the catalytic domain [60,149]. While structurally and pharmacologically most closely related to PDE5 [32], PDE6 has several features that distinguish it from the other PDE families: (1) whereas cone PDE6 catalytic subunits (gene name PDE6C, common name α'-subunit) form a homodimer as is the case for the other ten PDE families, most vertebrates have two homologous rod PDE6 catalytic subunit genes (PDE6A (α-subunit; Pα) and PDE6B (β-subunit; Pβ) that form a catalytic heterodimer (Pαβ); (2) the catalytic activity of rod and cone PDE6 operates at the diffusion-controlled limit for enzyme catalyzed reactions, more than 100-fold greater than the catalytic turnover number for any other PDE family; (3) unlike other PDE families, PDE6 is directly activated upon binding of a G-protein α-subunit; (4) rod and cone PDE6 catalytic activity is controlled by the binding of an inhibitory γ-subunit (Pγ; gene names: PDE6G (rod) and PDE6H (cone; abbreviated as Pγ')) at the entrance of the enzyme active site; (5) proper protein folding of PDE6 catalytic subunits requires the participation of a photoreceptor-specific chaperone protein, aryl-hydrocarbon receptor interacting protein-like 1 (AIPL1); and (6) PDE6 catalytic subunits are post-translationally modified at their C-termini in a sequential manner by prenylation (farnesyl moiety for Pα, geranylgeranyl group for Pβ and for the cone α'-subunit), proteolysis, and carboxymethylation (previously reviewed in [7,33,54,76,136,139]. ...
... This therapeutic strategy has seen remarkable success with the introduction of numerous FDAapproved PDE inhibitors that selectively target individual PDE families, and, in a few instances, specific isoforms of a particular PDE family [9]. Since there are no obvious clinical applications for inhibitor compounds targeting rod and cone PDE6, most pharmacological research on PDE6 has focused on designing inhibitor compounds for other PDE families that have low affinity for PDE6 [32]. ...
... PDE6 is most closely related to the PDE5 enzyme family, based on primary sequence, three-dimensional structure, substrate specificity, as well as known susceptibility to inhibition by compounds designed to target the PDE5 active site [32]. Whereas most so-called PDE5-selective inhibitors are more accurately characterized as PDE5/6-selective inhibitors due to lack of selectivity for PDE5 over rod or cone PDE6 (e.g., sildenafil, vardenafil [145]), a few compounds have been identified that inhibit PDE5 with 100-fold or greater potency than PDE6 (e.g., tadalafil [39,145]). ...
Article
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Rod and cone photoreceptors of the vertebrate retina utilize cGMP as the primary intracellular messenger for the visual signaling pathway that converts a light stimulus into an electrical response. cGMP metabolism in the signal-transducing photoreceptor outer segment reflects the balance of cGMP synthesis (catalyzed by guanylyl cyclase) and degradation (catalyzed by the photoreceptor phosphodiesterase, PDE6). Upon light stimulation, rapid activation of PDE6 by the heterotrimeric G-protein (transducin) triggers a dramatic drop in cGMP levels that lead to cell hyperpolarization. Following cessation of the light stimulus, the lifetime of activated PDE6 is also precisely regulated by additional processes. This review summarizes recent advances in the structural characterization of the rod and cone PDE6 catalytic and regulatory subunits in the context of previous biochemical studies of the enzymological properties and allosteric regulation of PDE6. Emphasis is given to recent advances in understanding the structural and conformational changes underlying the mechanism by which the activated transducin α-subunit binds to—and relieves inhibition of—PDE6 catalysis that is controlled by its intrinsically disordered, inhibitory γ-subunit. The role of the regulator of G-protein signaling 9–1 (RGS9-1) in regulating the lifetime of the transducin-PDE6 is also briefly covered. The therapeutic potential of pharmacological compounds acting as inhibitors or activators targeting PDE6 is discussed in the context of inherited retinal diseases resulting from mutations in rod and cone PDE6 genes as well as other inherited defects that arise from excessive cGMP accumulation in retinal photoreceptor cells.
... In the rod photoreceptor cells, PDE6B forms the PDE6 complex with PDE6α and PDE6γ subunits [13][14][15]. The inactive PDE6 in the dark allows cGMP to bind to cGMP gated ion channels, which keeps these cGMP bound channels open. ...
... Light absorption by rhodopsin triggers the phototransduction cascade. The PDE6 is activated by the release of the PDE6γ subunit from the PDE6αβ, causing the hydrolysis of cGMP; the ion channels close without the cGMP binding, leading to hyperpolarization of the cell membrane [13,16]. PDE6B-Y347X results in a nonfunctional PDE in the rd1 mice, leading to increased cGMP and calcium levels in the photoreceptors [17,18] and, ultimately, the death of rod photoreceptor cells [10,11]. ...
Article
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The cGMP-phosphodiesterase 6 beta subunit (PDE6B) is an essential component in the phototransduction pathway for light responses in photoreceptor cells. PDE6B gene mutations cause the death of rod photoreceptors, named as hereditary retinitis pigmentosa (RP) in humans and retinal degeneration (RD) in rodents. Here, we report a new RD model, identified from a phenotypic screen of N-ethyl-N-nitrosourea (ENU)-induced mutant mice, which displays retinal degeneration caused by a point mutation in the Pde6b gene that results in PDE6B-T592I mutant protein. The homozygous mutant mice show an extensive loss of rod photoreceptors at the age of 3 weeks; unexpectedly, the loss of rod photoreceptors can be partly rescued by dark rearing. Thus, this RD mutant model displays a light-dependent loss of rod photoreceptors. Both western blot and immunostaining results show very low level of mutant PDE6B-T592I protein in the retina. Structure modeling suggests that the T592I mutation probably affects the function and stability of PDE6B protein by changing intramolecular interactions. We further demonstrate that the expression of wild-type PDE6B delivered by subretinally injected adeno-associated virus (rAAV) prevents photoreceptor cell death in this RD model in vivo. The PDE6B-T592I mutant is, therefore, a valuable RD model for evaluating rAAV-mediated treatment and for investigating the molecular mechanism of light-dependent rod photoreceptor cell death that is related to impaired PDE6B function.
... Cyclic-nucleotide phosphodiesterases of the sixth family (PDE6) have a key role in phototransduction cascade [9]. In rods, this enzyme is a heterodimer of PDE6A and PDE6B subunits and in cones it is a homodimer of PDE6C subunits [10]. ...
... The PDE6C gene encodes the cone α subunit of cyclic guanosine monophosphate (cGMP) phosphodiesterase [10,11]. During light exposure the photopigment is excited and, via guanosine diphosphate (GDP)/guanosine-5′-triphosphate (GTP) exchange and release of the Ga-subunit, activates the G-protein transducing. ...
Article
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We report the clinical phenotype and genetic findings of two variants in PDE6C underlying achromatopsia (ACHM). Four patients with the variant c.1670G>A in exon 13 of the PDE6C gene were identified. Additionally, one had compound heterozygous genotype, with two variants in the PDE6C gene, a variant of c.2192G>A in exon 18 and c.1670G>A in exon 13. All patients presented the symptomatic triad of decreased visual acuity, severe photophobia, and colour vision disturbances. SD-OCT showed an absence of the ellipsoid zone, creating an optically empty cavity at the fovea in three patients. The patient with the compound heterozygous genotype presented a more severe subfoveal outer retina atrophy. ERG recordings showed extinguished responses under photopic and 30-Hz flicker stimulation, with a normal rod response. We identified two new variants in the PDE6C gene that leads to ACHM.
... Unfortunately, they generally cause dosedependent side effects, most of which are due to cross-reactivity with other PDEs (Huang and Lie, 2013). PDE6 enzyme is a key effector enzyme in the cascade of light transduction in mammalian rod and cone photoreceptor cells (Marmor and Kessler, 1999;Cote, 2004). It plays a role in visual signal transduction and responds to light through a mechanism of transition from an inactive to an active state, which is regulated by its unique "γ-subunit (Granovsky and Artemyev, 2001;Zhang et al., 2005;Zhang and Artemyev, 2010). ...
Article
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Introduction: In clinical practice, phosphodiesterase 5 (PDE5) inhibitors are commonly used to treat erectile dysfunction and pulmonary arterial hypertension. However, due to the high structural similarity between PDE5 and Phosphodiesterase 6 (PDE6), there is a risk that existing drugs will cause off-target effects on PDE6 resulting in visual disorders such as low visual acuity and color blindness. Previous research on the selectivity of PDE5 inhibitors focused on marketed drugs such as sildenafil and tadalafil. Methods: In this study, a highly selective PDE5 inhibitor, ligand3, was used as the subject, and molecular docking, molecular dynamics simulations, MM-GBSA, alanine scanning, and independent gradient model analysis were employed to investigate the biological mechanism underlying the selectivity of PDE5 inhibitors. Results and Discussion: The present work revealed that the binding mode of ligand3 to the PDE5A and PDE6C targets was distinctly different. Ligand3 exhibited stronger coulombic forces when binding to PDE5A, while showing stronger van der waals forces when binding to PDE6C. Ligand3 binds more deeply at the active site of PDE5A than at PDE6C, allowing its side chains to effectively bind to the critical TYR612, whereas in the case of the shallow binding to PDE6C, ligand3 lacks a similar effect. Mechanism investigations of highly selective inhibitors through computational simulation might provide an insight into potent treatment of drugs.
... (ii) the Q pocket, a central region of the catalytic site including Tyr612, His613, Ala767, Ile768, Val782, Gln817, and Phe820; (iii) the H pocket, a hydrophobic pocket including Ala783, Phe786, Phe787, Leu804, and Ile813; and (iv) the L region, a subdomain associated with catalytic site opening, also named the lid region including Phe664, Met816, Gly819, and Ala823 [21]. In case of the PDE6, the catalytic domain of PDE6 is found to be similar to that of PDE5 [22,23]. As compared to the PDE5 catalytic domain, the important residues are also shown in Supplementary Fig. S1. ...
Article
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Phosphodiesterase type 5 (PDE5) inhibitors play a crucial role in blocking PDE5 to improve erectile dysfunction (ED). However, most PDE5 drugs revealed side effects including the loss of vision due to the PDE6 inhibition. Phenanthrene derivatives isolated from E. macrobulbon were previously reported as PDE5 inhibitors. Two phenanthrene derivatives (cpds 1–2) revealed better inhibition to PDE5 than PDE6 and cpd 1 is more selective to PDE5 than cpd 2. To elucidate why the phenanthrene derivatives could inhibit PDE5 and PDE6, their binding modes were investigated using molecular dynamics simulations and quantum chemical calculations, as compared to the PDE5 drugs. From the results, all four drugs and phenanthrene derivatives revealed similar π–π interactions to Phe820 in PDE5. Additional H-bond interaction to Gln817 in PDE5 resulted in better PDE5 inhibition of vardenafil and tadalafil. Moreover, cpds 1–2 were able to form the H-bond interaction with Asp764 in PDE5. In the case of the PDE6, the loss of π–π interaction to Phe776 and H-bond interaction to Gln773 indicated the important points for losing the PDE6 inhibition. In conclusion, to develop the new potent PDE5 inhibitors, not only the important interaction with PDE5 but also the interaction with PDE6 should be considered. In phenanthrene derivatives, the middle ring was significant to form π–π interactions to Phe820 in PDE5 and hydroxyl substituent was also the key part to form the H-bond interaction with Asp764 in PDE5. Principal component analysis (PCA) and free energy landscape (FEL) analysis indicated the stability of the system. The bioavailability, drug-likeness, and pharmacokinetics of phenanthrene derivatives were also predicted. These derivatives revealed good drug-likeness and GI absorption. The obtained results showed that phenanthrene derivatives could be interesting for the development of PDE5 inhibitors in the future.
... Long-term application of vardenafil successfully prevented the development of heart failure with preserved ejection fraction in both type 1 and type 2 diabetic rat models (Radovits et al., 2009;Mátyás et al., 2017). Existing PDE5 inhibitors, however, are known to be non-specific, with measurable inhibitory effects on the retinal PDE6 enzyme as well (Zhang et al., 2005;Cote, 2004). Side effects, such as vision deterioration, color vision problems (red desaturation), and perception of increased brightness of light, have been regularly reported in a dose-dependent manner (Marmor and Kessler, 1999) when these drugs were administered for erectile dysfunctionwith a cumulative dose of just a fraction of what is lately being proposed against diabetes. ...
... Dysregulation of PDE6α leads to cGMP accumulation in photoreceptor cells, which can be cytotoxic to the retina [174]. Under normal conditions, PDE6α promotes the hydrolysis of cGMP in response to light, resulting in membrane hyperpolarization [175]. On the other hand, PDE6β expression is directly proportional to the rod population, demonstrating the differentiation efficiency of the protocol used [37]. ...
Article
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The human eye plays a critical role in vision perception, but various retinal degenerative diseases such as retinitis pigmentosa (RP), glaucoma, and age-related macular degeneration (AMD) can lead to vision loss or blindness. Although progress has been made in understanding retinal development and in clinical research, current treatments remain inadequate for curing or reversing these degenerative conditions. Animal models have limited relevance to humans, and obtaining human eye tissue samples is challenging due to ethical and legal considerations. Consequently, researchers have turned to stem cell-based approaches, specifically induced pluripotent stem cells (iPSCs), to generate distinct retinal cell populations and develop cell replacement therapies. iPSCs offer a novel platform for studying the key stages of human retinogenesis and disease-specific mechanisms. Stem cell technology has facilitated the production of diverse retinal cell types, including retinal ganglion cells (RGCs) and photoreceptors, and the development of retinal organoids has emerged as a valuable in vitro tool for investigating retinal neuron differentiation and modeling retinal diseases. This review focuses on the protocols, culture conditions, and techniques employed in differentiating retinal neurons from iPSCs. Furthermore, it emphasizes the significance of molecular and functional validation of the differentiated cells.
... PDE6 plays an important role in converting light to electrical signals within the neural retina 14 . When the PDE6 enzyme is activated, intracellular cGMP is hydrolyzed, leading to propagation of the visual cycle 15 . While rods have a PDE6 catalytic core composed of a heterodimer of PDE6A and PDE6B subunits which are inhibited by PDE6G, cones have a catalytic homodimer of PDE6C, which is inhibited by PDE6H subunits 16 . ...
Article
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To report the association of autoimmune polyglandular syndrome type 1 (APS1) with cone dystrophy in a large Saudi family. This is a Retrospective chart review and prospective genetic testing and ophthalmic examination of a large multiplex consanguineous family. Genetic testing was performed on 14 family members, seven of whom had detailed ophthalmic examinations. Medical history, ocular history and evaluation, visual field testing, full-field electroretinogram (ERG), and Whole Exome Sequencing (WES) results were analyzed. Three family members were homozygous for c.205_208dupCAGG;p.(Asp70Alafs*148) in AIRE and homozygous for c.481-1G>A in PDE6C. One additional family member was homozygous for only the AIRE variant and another additional family member was homozygous for only the PDE6C variant. All patients with homozygosity for the PDE6C variant had cone dystrophy, and all patients with homozygosity for the AIRE variant had APS1. In addition, two of the family members who were homozygous for the PDE6C and AIRE variants had reduced rod function on ERG. We report the co-inheritance for APS1 and PDE6C-related cone dystrophy, an unusual example of two seemingly independent recessive conditions coinciding within a family. Dual molecular diagnosis must be taken into account by ophthalmologists facing unusual constellations of findings, especially in consanguineous families.
... PDE enzymes are a superfamily containing 11 members (PDE1-11) in humans, which are structurally related but consist of multiple genes and function distinctly [36]. PDE6 belongs to 3 ,5 -cyclic nucleotide PDEs that catalyze the hydrolysis of the phosphodiester bond of cyclic nucleotides [principally, cGMP and cyclic adenosine monophosphate (cAMP)] [37,38]. There are rod PDE6 complexes and cone PDE6 complexes. ...
Article
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Photoreceptors in the retina are highly specialized neurons with photosensitive molecules in the outer segment that transform light into chemical and electrical signals, and these signals are ultimately relayed to the visual cortex in the brain to form vision. Photoreceptors are composed of rods and cones. Rods are responsible for dim light vision, whereas cones are responsible for bright light, color vision, and visual acuity. Photoreceptors undergo progressive degeneration over time in many hereditary and age-related retinal diseases. Despite the remarkable heterogeneity of disease-causing genes, environmental factors, and pathogenesis, the progressive death of rod and cone photoreceptors ultimately leads to loss of vision/blindness. There are currently no treatments available for retinal degeneration. Cyclic guanosine 3′, 5′-monophosphate (cGMP) plays a pivotal role in phototransduction. cGMP governs the cyclic nucleotide-gated (CNG) channels on the plasma membrane of the photoreceptor outer segments, thereby regulating membrane potential and signal transmission. By gating the CNG channels, cGMP regulates cellular Ca2+ homeostasis and signal transduction. As a second messenger, cGMP activates the cGMP-dependent protein kinase G (PKG), which regulates numerous targets/cellular events. The dysregulation of cGMP signaling is observed in varieties of photoreceptor/retinal degenerative diseases. Abnormally elevated cGMP signaling interferes with various cellular events, which ultimately leads to photoreceptor degeneration. In line with this, strategies to reduce cellular cGMP signaling result in photoreceptor protection in mouse models of retinal degeneration. The potential mechanisms underlying cGMP signaling-induced photoreceptor degeneration involve the activation of PKG and impaired Ca2+ homeostasis/Ca2+ overload, resulting from overactivation of the CNG channels, as well as the subsequent activation of the downstream cellular stress/death pathways. Thus, targeting the cellular cGMP/PKG signaling and the Ca2+-regulating pathways represents a significant strategy for photoreceptor protection in retinal degenerative diseases.
... cGMP signalling is critical in photoreceptor cell death caused by disease-causing mutations. Studies suggest the hypothesis that high intracellular cGMP levels trigger photoreceptors' degeneration [18]; examples of such a situation are genes that encode for photoreceptor phosphodiesterase-6 (PDE6) [19]. Among all PDE6 family genes, Pde6a, Pde6b, and Pde6g can cause RP [20][21][22]. ...
Article
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Usher syndrome (USH) is characterised by degenerative vision loss known as retinitis pigmentosa (RP), sensorineural hearing loss, and vestibular dysfunction. RP can cause degeneration and the loss of rod and cone photoreceptors, leading to structural and functional changes in the retina. Cep250 is a candidate gene for atypical Usher syndrome, and this study describes the development of a Cep250 KO mouse model to investigate its pathogenesis. OCT and ERG were applied in Cep250 and WT mice at P90 and P180 to access the general structure and function of the retina. After recording the ERG responses and OCT images at P90 and P180, the cone and rod photoreceptors were visualised using an immunofluorescent stain. TUNEL assays were applied to observe the apoptosis in Cep250 and WT mice retinas. The total RNA was extracted from the retinas and executed for RNA sequencing at P90. Compared with WT mice, the thickness of the ONL, IS/OS, and whole retina of Cep250 mice was significantly reduced. The a-wave and b-wave amplitude of Cep250 mice in scotopic and photopic ERG were lower, especially the a-wave. According to the immunostaining and TUNEL stain results, the photoreceptors in the Cep250 retinas were also reduced. An RNA-seq analysis showed that 149 genes were upregulated and another 149 genes were downregulated in Cep250 KO retinas compared with WT mice retinas. A KEGG enrichment analysis indicated that cGMP-PKG signalling pathways, MAPK signalling pathways, edn2-fgf2 axis pathways, and thyroid hormone synthesis were upregulated, whereas protein processing in the endoplasmic reticulum was downregulated in Cep250 KO eyes. Cep250 KO mice experience a late-stage retinal degeneration that manifests as the atypical USH phenotype. The dysregulation of the cGMP-PKG-MAPK pathways may contribute to the pathogenesis of cilia-related retinal degeneration.
... Photoreceptor cell-specific PDE6 protein complex comprises three genes: PDE6A, PDE6B and PDE6C encoding the catalytic subunits; PDE6G and PDE6H genes encoding the inhibitory subunits and PDE6D responsible for their solubilization ( Figure 6) [113]. ...
Article
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Phosphodiesterases are key regulators that fine tune the intracellular levels of cyclic nucleotides, given their ability to hydrolyze cAMP and cGMP. They are critical regulators of cAMP/cGMP-mediated signaling pathways, modulating their downstream biological effects such as gene expression, cell proliferation, cell-cycle regulation but also inflammation and metabolic function. Recently, mutations in PDE genes have been identified and linked to human genetic diseases and PDEs have been demonstrated to play a potential role in predisposition to several tumors, especially in cAMP-sensitive tissues. This review summarizes the current knowledge and most relevant find- ings regarding the expression and regulation of PDE families in the testis focusing on PDEs role in testicular cancer development.
... The predominant form of PDE in the retina is PDE6, which is found in the internal membranes of rod and cone photoreceptors and regulates cGMP levels in response to light [46]. There have been some studies that suggest PDE5 inhibitors cause side effects in the eye, for example, non-arteritic ischemic neuropathy from sildenafil use; however, more studies are needed to determine if there is a cause-and-effect relationship [47]. ...
Article
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Bestrophin 1 (Best1) is a chloride channel that localises to the plasma membrane of retinal pigment epithelium (RPE) cells. Mutations in the BEST1 gene are associated with a group of untreatable inherited retinal dystrophies (IRDs) called bestrophinopathies, caused by protein instability and loss-of-function of the Best1 protein. 4PBA and 2-NOAA have been shown to rescue the function, expression, and localisation of Best1 mutants; however, it is of interest to find more potent analogues as the concentration of the drugs required is too high (2.5 mM) to be given therapeutically. A virtual docking model of the COPII Sec24a site, where 4PBA has been shown to bind, was generated and a library of 1416 FDA-approved compounds was screened at the site. The top binding compounds were tested in vitro in whole-cell patch-clamp experiments of HEK293T cells expressing mutant Best1. The application of 25 μM tadalafil resulted in full rescue of Cl− conductance, comparable to wild type Best1 levels, for p.M325T mutant Best1 but not for p.R141H or p.L234V mutants.
... The PDE enzymatic superfamily consists of 11 gene families (PDE1 to PDE11), the majority of which contain multiple PDE genes [12]. Thus, some PDE families are cGMP-specific (PDE5, 6, and 9), while others are cAMP-specific (PDE4, 7 and 8), and others hydrolyze both cAMP and cGMP (PDE1, 2, 3, 10, and 11) [13,14]. Resulting low cAMP concentrations promote inflammation by increasing the production of interleukins (IL)-8, -12, -17, -22, -23, tumor necrosis factor (TNF)α, interferon, and chemokines, while high cAMP concentrations produce an anti-inflammatory response by inducing the synthesis of IL-6 and IL-10 [15]. ...
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Acute respiratory distress syndrome (ARDS) is a common complication of critical illness characterized by lung inflammation, epithelial and endothelial dysfunction, alveolar-capillary leakage, and worsening respiratory failure. The present study aimed to investigate the anti-inflammatory effects of non-selective phosphodiesterase (PDE) inhibitor aminophylline. New Zealand white rabbits were randomly divided into 3 groups: animals with respiratory failure defined as PaO2/FiO2 ratio (P/F) below < 26.7 kPa, and induced by saline lung lavage (ARDS), animals with ARDS treated with intravenous aminophylline (1 mg/kg; ARDS/AMINO), and healthy ventilated controls (Control). All animals were oxygen ventilated for an additional 4 h and respiratory parameters were recorded regularly. Post mortem, the lung tissue was evaluated for oedema formation, markers of inflammation (tumor necrosis factor, TNFα, interleukin (IL)-1β, -6, -8, -10, -13, -18), markers of epithelial damage (receptor for advanced glycation end products, RAGE) and endothelial injury (sphingosine 1-phosphate, S1P), oxidative damage (thiobarbituric acid reactive substances, TBARS, 3-nitrotyrosine, 3NT, total antioxidant capacity, TAC). Aminophylline therapy decreased the levels of pro-inflammatory cytokines, markers of epithelial and endothelial injury, oxidative modifications in lung tissue, reduced lung oedema, and improved lung function parameters compared to untreated ARDS animals. In conclusion, non-selective PDE inhibitor aminophylline showed a significant anti-inflammatory activity suggesting a potential of this drug to be a valuable component of ARDS therapy.
... As a consequence, interest in the pharmacology of PDE6 has focused on assessing possible adverse side effects arising from administration of PDE inhibitors targeting other PDE families. This is particularly relevant to inhibitors targeting PDE5 (e.g., sildenafil, vardenafil, tadalafil), since PDE6 is most closely related (structurally and biochemically) to PDE5 [122] and several marketed PDE5 inhibitors are known to have moderate, reversible adverse effects on vision-either via direct inhibition of PDE6 activity in photoreceptor cells or indirectly by inhibition of PDE5 present in ocular blood vessels [123][124][125]. ...
... Several medications have also been available to overcome erectile dysfunction (ED), such as sildenafil, vardenafil, tadalafil, and avanafil [8]. However, these drugs have the side effect of the retinal defect [8,9]. ...
Conference Paper
The root bark of yellow ginseng (Rennellia elliptica Korth.) is traditionally used as tonic and aphrodisiac by local people in West Kalimantan, Indonesia. This study determined the aphrodisiac activity of the ethanol extract of yellow ginseng root in male DDY mice. A Completely Randomized Design was used in this study, with 5 treatments, i.e., negative control (1% Na-CMC), positive control (sildenafil citrate) (5 mg/kg BW), yellow ginseng ethanol extract (50 mg/kg), yellow ginseng ethanol extract (100 mg/kg BW), yellow ginseng ethanol extract (250 mg/kg BW). The treatment was administered orally once a day by oral gavage. The duration of extract administration was 3, 5, and 10 days. The results showed that the administration of yellow ginseng ethanol extract at a dose of 250 mg/kg BW for 3 days increased libido as an indication of aphrodisiac activity. The administration of yellow ginseng extract for 10 days indicated prolonged ejaculation. However, this result was not statistically significant (p>0.05) compared to negative and positive control treatments.
... This effect has not been linked to any retinal structural or functional alterations. 66,67 Back pain and myalgias are caused by a high concentration of the PDE11 enzyme in skeletal muscle, which has a strong cross-reactivity with tadalafil. The FDA issued a warning on the possibility of hearing loss linked with PDE5I usage due to the pathophysiology that causes sensorineural hearing loss, which has been described in a few case studies. ...
Article
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Phosphodiesterase type 5 (PDE5) is a cyclic GMP (cGMP) specific protein. It hydrolyzes the phosphodiesterase linkage and catalyzes the conversion of cGMP to 5’ GMP, which controls different physiological activities of the body. PDE5 is associated with biomedical conditions like neurological disorders, pulmonary arterial hypertension, cardiomyopathy, cancer, erectile dysfunction, and lower urinary tract syndrome. Inhibition of PDE5 has now been proven pharmaceutically effective in a variety of therapeutic conditions. Avanafil, tadalafil, sildenafil, and vardenafil are the most commonly used PDE5 inhibitors (PDE5i) today which are often used for the management of erectile dysfunction, lower urinary tract syndromes, malignancy, and pulmonary arterial hypertension. However, these synthetic PDE5i come with a slew of negative effects. Some of the most common side effects include mild headaches, flushing, dyspepsia, altered color vision, back discomfort, priapism, melanoma, hypotension and dizziness, non-arteritic anterior ischemic optic neuropathy (NAION), and hearing loss. In light of the potential negative effects of this class of medications, there is a lot of room for new, selective PDE5 inhibitors to be discovered. We have found 25 plant botanical compounds effectively inhibiting PDE5 which might be useful in treating a variety of disorders with minimal or no adverse effects.
... Antibodies directed against PDE6 and ARL13B were used to assay for OS elaboration in central versus peripheral retina. PDE6 is a key phototransduction enzyme [27] and ARL13B is a small GTPase functioning as a guanine nucleotide exchange factor (GEF) of ARL3 [28]. In cryosections of Cep164 +/F ;Egfp-Cetn2 control retina, PDE6 (MOE antibody) and ARL13B localize in the photoreceptor OS as expected (Fig 2A and 2D). ...
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Centrosomal protein of 164 kDa (CEP164) is located at distal appendages of primary cilia and is necessary for basal body (BB) docking to the apical membrane. To investigate the function of photoreceptor CEP164 before and after BB docking, we deleted CEP164 during retina embryonic development (Six3Cre), in postnatal rod photoreceptors (iCre75) and in mature retina using tamoxifen induction (Prom1-ETCre). BBs dock to the cell cortex during postnatal day 6 (P6) to extend a connecting cilium (CC) and an axoneme. P6 retina-specific knockouts (retCep164-/-) are unable to dock BBs, thereby preventing formation of CC or outer segments (OSs). In rod-specific knockouts (rodCep164-/-), Cre expression starts after P7 and CC/OS form. P16 rodCep164-/- rods have nearly normal OS lengths, and maintain OS attachment through P21 despite loss of CEP164. Intraflagellar transport components (IFT88, IFT57 and IFT140) were reduced at P16 rodCep164-/- BBs and CC tips and nearly absent at P21, indicating impaired intraflagellar transport. Nascent OS discs, labeled with a fluorescent dye on P14 and P18 and harvested on P19, showed continued rodCep164-/- disc morphogenesis but absence of P14 discs mid-distally, indicating OS instability. Tamoxifen induction with PROM1ETCre;Cep164F/F (tamCep164-/-) adult mice affected maintenance of both rod and cone OSs. The results suggest that CEP164 is key towards recruitment and stabilization of IFT-B particles at the BB/CC. IFT impairment may be the main driver of ciliary malfunction observed with hypomorphic CEP164 mutations.
... The contemporary drug therapies are solely focused on Phosphodiesterase type 5 antagonists such as sildenafil citrate, which raises the amount of cyclic Guanosine monophosphate (cGMP) in the cavernosal vasculature, contributing to the facilitation and longer duration of penile erection and hold a stable penile rigidity [6]. Upon further emphasis on the pharmacological activity of such molecules shows a systematic idea about enhancement of sexual stimulation by crossing the blood brain barrier and trying to mimic or stimulating some area of sexual excitement in the central nervous system. ...
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Background: Aphrodisiac drugs usage is increasing as a sexual enhancement resources among both men and women without a medical indication and efficacy. Recreational usage of aphrodisiacs has been correlated with elevated sexual risk activity, an increased risk for STIs, including HIV infection, and high levels of concomitant illegal substance usage. The aim of the research was to do the demographic study and various regulation associated with the class of drug.
... The PDE enzymatic superfamily consists of 11 gene families (PDE1 to PDE11), the majority of which contain multiple PDE genes [13]. Thus, some PDE families are cGMP-speci c (PDE5, 6, and 9), while others are cAMP-speci c (PDE4, 7 and 8), and others hydrolyze both cAMP and cGMP (PDE1, 2, 3, 10, and 11) [14,15]. Resulting low cAMP concentrations promote in ammation by increasing the production of interleukins (IL)-8, -12, -17, -22, -23, tumor necrosis factor (TNF)α, interferon, and chemokines, while high cAMP concentrations produce an anti-in ammatory response by inducing the synthesis of IL-6 and IL- 10 [16]. ...
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Acute respiratory distress syndrome (ARDS) is a common complication of critical illness characterized by lung inflammation, epithelial and endothelial dysfunction, alveolar-capillary leakage, and worsening respiratory failure. The present study aimed to investigate the anti-inflammatory effects of non-selective phosphodiesterase (PDE) inhibitor aminophylline. New Zealand white rabbits were randomly divided into 3 groups: animals with respiratory failure defined as PaO 2 /FiO 2 ratio (P/F) below < 26.7 kPa, and induced by saline lung lavage (ARDS), animals with ARDS treated with intravenous aminophylline (1 mg/kg; ARDS/AMINO), and healthy ventilated controls (Control). All animals were oxygen ventilated for an additional 4 h and respiratory parameters were recorded regularly. Post mortem , the lung tissue was evaluated for oedema formation, markers of inflammation (tumor necrosis factor, TNFα, interleukin (IL)-1β, -6, -8, -10, -13, -18), markers of epithelial damage (receptor for advanced glycation end products, RAGE) and endothelial injury (sphingosine 1-phosphate, S1P), oxidative damage (thiobarbituric acid reactive substances, TBARS, 3-nitrotyrosine, 3NT, total antioxidant capacity, TAC). Aminophylline therapy decreased the levels of pro-inflammatory cytokines, markers of epithelial and endothelial injury, oxidative modifications in lung tissue, reduced lung oedema, and improved lung function parameters compared to untreated ARDS animals. In conclusion, non-selective PDE inhibitor aminophylline showed a significant anti-inflammatory activity suggesting a potential of this drug to be a valuable component of ARDS therapy.
... The phototransduction pathway contains a lot more DEGs than the number expected by chance (highest enrichment ratio); besides, this pathway is one of the most highly deregulated (ranking 22). This fact is interesting, in particular since genes of the PDE6 family previously associated with the action of photoreceptors as signal transducers of luminal stimuli [102,103] have also recently been argued to be significantly over-expressed in human cancers, in particular in breast carcinomas [36]. PDE6A and PDE6G are present among the DEGs of this pathway. ...
Article
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Retinoblastoma (Rb) is a rare intraocular tumour in early childhood, with an approximate incidence of 1 in 18 000 live births. Experimental studies for Rb are complex due to the challenges associated with obtaining a normal retina to contrast with diseased tissue. In this work, we reanalyse a dataset that contains normal retina samples. We identified the individual genes whose expression is different in Rb in contrast with normal tissue, determined the pathways whose global expression pattern is more distant from the global expression observed in normal tissue, and finally, we identified which transcription factors regulate the highest number of differentially expressed genes (DEGs) and proposed as transcriptional master regulators (TMRs). The enrichment of DEGs in the phototransduction and retrograde endocannabinoid signalling pathways could be associated with abnormal behaviour of the processes leading to cellular differentiation and cellular proliferation. On the other hand, the TMRs nuclear receptor subfamily 5 group A member 2 and hepatocyte nuclear factor 4 gamma are involved in hepatocyte differentiation. Therefore, the enrichment of aberrant expression in these transcription factors could suggest an abnormal retina development that could be involved in Rb origin and progression.
... The catalytic activity of PDE6 is regulated by the inhibitory subunits encoded by pde6g and pde6h. The pde6a and pde6b genes, which give rise to a catalytic heterodimer, and the inhibitory subunit gene pde6g were expressed in rod cells, whereas pde6c, which results in a catalytic homodimer, and the inhibitory subunit gene pde6h were expressed in cone cells [44,45]. The expression of all the tested PDE6 genes decreased in seleniumstressed embryos at 96 hpf. ...
Article
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Selenium is an essential trace element for humans and other vertebrates, playing an important role in antioxidant defense, neurobiology and reproduction. However, the toxicity of excessive selenium has not been thoroughly evaluated, especially for the visual system of vertebrates. In this study, fertilized zebrafish embryos were treated with 0.5 µM L-selenomethionine to investigate how excessive selenium alters zebrafish eye development. Selenium-stressed zebrafish embryos showed microphthalmia and altered expression of genes required for retinal neurogenesis. Moreover, ectopic proliferation, disrupted mitochondrial morphology, elevated ROS-induced oxidative stress, apoptosis and ferroptosis were observed in selenium-stressed embryos. Two antioxidants-reduced glutathione (GSH) and N-acetylcysteine (NAC)-and the ferroptosis inhibitor ferrostatin (Fer-1) were unable to rescue selenium-induced eye defects, but the ferroptosis and apoptosis activator cisplatin (CDDP) was able to improve microphthalmia and the expression of retina-specific genes in selenium-stressed embryos. In summary, our results reveal that ferroptosis and apoptosis might play a key role in selenium-induced defects of embryonic eye development. The findings not only provide new insights into selenium-induced cellular damage and death, but also important implications for studying the association between excessive selenium and ocular diseases in the future.
... 94 More importantly, sildenafil also affects PDE6, which is expressed in rods and cones, differing slightly in its catalytic subunit composition. 95 It plays a major part in the phototransduction cascade where its activation is triggered by a G protein, transducin, upon photon absorption. A high intracellular cGMP concentration prevalent in the dark is reduced by PDE6, hydrolyzing cGMP to GMP. ...
Article
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Retinal drug toxicity screening is essential for the development of safe treatment strategies for a large number of diseases. To this end, retinal organoids derived from human pluripotent stem cells (hPSCs) provide a suitable screening platform due to their similarity to the human retina and the ease of generation in large-scale formats. In this study, two hPSC cell lines were differentiated to retinal organoids, which comprised all key retinal cell types in multiple nuclear and synaptic layers. Single-cell RNA-Seq of retinal organoids indicated the maintenance of retinal ganglion cells and development of bipolar cells: both cell types segregated into several subtypes. Ketorolac, digoxin, thioridazine, sildenafil, ethanol, and methanol were selected as key compounds to screen on retinal organoids because of their well-known retinal toxicity profile described in the literature. Exposure of the hPSC-derived retinal organoids to digoxin, thioridazine, and sildenafil resulted in photoreceptor cell death, while digoxin and thioridazine additionally affected all other cell types, including Müller glia cells. All drug treatments caused activation of astrocytes, indicated by dendrites sprouting into neuroepithelium. The ability to respond to light was preserved in organoids although the number of responsive retinal ganglion cells decreased after drug exposure. These data indicate similar drug effects in organoids to those reported in in vivo models and/or in humans, thus providing the first robust experimental evidence of their suitability for toxicological studies.
... AIPL1 acts as a specialized molecular co-chaperone, together with HSP90, enabling the correct folding and assembly of the guanosine-3',5'-cyclic monophosphate (cGMP)-specific phosphodiesterase 6 (PDE6), a critical holoenzyme in the phototransduction cascade that hydrolyses cGMP in rods and cones upon light stimulation. 8,9 It has been shown that the AIPL1 FKBP-like domain binds the isoprenylated PDE6 catalytic subunits, [10][11][12] while the AIPL1 TPR domain interacts with the regulatory PDE6g subunit 13 and the EEVD motif located at the Cterminus of HSP90. 14,15 Several studies in mouse retina revealed that, with the reduction or absence of AIPL1, cone and rod PDE6 levels decrease [16][17][18] and rod PDE6 subunits are misassembled and targeted to proteasomes for degradation. ...
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Leber Congenital Amaurosis type 4 (LCA4), caused by AIPL1 mutations, is characterised by severe sight impairment in infancy and rapidly progressive degeneration of photoreceptor cells. We generated retinal organoids using induced pluripotent stem cells (iPSCs) from renal epithelial cells obtained from four children with AIPL1 nonsense mutations. iPSC-derived photoreceptors exhibited the molecular hallmarks of LCA4, including undetectable AIPL1 and rod cGMP phosphodiesterase (PDE6) compared to control or CRISPR corrected organoids. Moreover, increased levels of cGMP were detected. The translational readthrough inducing drug (TRID) PTC124 was investigated as a potential therapeutic. LCA4 retinal organoids exhibited rescue of AIPL1 and PDE6; however, the level of full-length, functional AIPL1 induced through PTC124 treatment was insufficient to reduce cGMP levels and fully rescue the LCA4 phenotype. LCA4 retinal organoids are a valuable platform for the in vitro investigation of the molecular mechanisms that drive photoreceptor loss and for the evaluation of novel therapeutics.
... As a consequence, interest in the pharmacology of PDE6 has focused on assessing possible adverse side effects arising from administration of PDE inhibitors targeting other PDE families. This is particularly relevant to inhibitors targeting PDE5 (e.g., sildenafil, vardenafil, tadalafil), since PDE6 is most closely related (structurally and biochemically) to PDE5 [122] and several marketed PDE5 inhibitors are known to have moderate, reversible adverse effects on vision-either via direct inhibition of PDE6 activity in photoreceptor cells or indirectly by inhibition of PDE5 present in ocular blood vessels [123][124][125]. ...
Article
The photoreceptor phosphodiesterase (PDE6) is a member of large family of Class I phosphodiesterases responsible for hydrolyzing the second messengers cAMP and cGMP. PDE6 consists of two catalytic subunits and two inhibitory subunits that form a tetrameric protein. PDE6 is a peripheral membrane protein that is localized to the signal-transducing compartment of rod and cone photoreceptors. As the central effector enzyme of the G-protein coupled visual transduction pathway, activation of PDE6 catalysis causes a rapid decrease in cGMP levels that results in closure of cGMP-gated ion channels in the photoreceptor plasma membrane. Because of its importance in the phototransduction pathway, mutations in PDE6 genes result in various retinal diseases that currently lack therapeutic treatment strategies due to inadequate knowledge of the structure, function, and regulation of this enzyme. This review focuses on recent progress in understanding the structure of the regulatory and catalytic domains of the PDE6 holoenzyme, the central role of the multi-functional inhibitory γ-subunit, the mechanism of activation by the heterotrimeric G protein, transducin, and future directions for pharmacological interventions to treat retinal degenerative diseases arising from mutations in the PDE6 genes.
... There are PDE5 located especially in cardiovascular system, PDE6 found in the retina and PDE9 present in heart muscle and brain. In this condition, modulation of PDE5 and PDE9 function is interesting as a cardiovascular therapeutic option [73][74][75]. ...
Article
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For years, guanylate cyclase seemed to be homogenic and tissue nonspecific enzyme; however, in the last few years, in light of preclinical and clinical trials, it became an interesting target for pharmacological intervention. There are several possible options leading to an increase in cyclic guanosine monophosphate concentrations. The first one is related to the uses of analogues of natriuretic peptides. The second is related to increasing levels of natriuretic peptides by the inhibition of degradation. The third leads to an increase in cyclic guanosine monophosphate concentration by the inhibition of its degradation by the inhibition of phosphodiesterase type 5. The last option involves increasing the concentration of cyclic guanosine monophosphate by the additional direct activation of soluble guanylate cyclase. Treatment based on the modulation of guanylate cyclase function is one of the most promising technologies in pharmacology. Pharmacological intervention is stable, effective and safe. Especially interesting is the role of stimulators and activators of soluble guanylate cyclase, which are able to increase the enzymatic activity to generate cyclic guanosine monophosphate independently of nitric oxide. Moreover, most of these agents are effective in chronic treatment in heart failure patients and pulmonary hypertension, and have potential to be a first line option.
... Nonetheless, despite this high selectivity, each of these drugs inhibits other PDE isoenzymes to some extent. Sildenafil and vardenafil, for example, have shown only 10 and 15 times lower specificity for PDE6 than for PDE5, respectively (reviewed in [14]), which could be explained by the fact that the kinetic and catalytic properties of PDE6 are very similar to those of PDE5 [15][16][17]. ...
Article
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Phosphodiesterase type 5 (PDE5) inhibitors such as Viagra® (sildenafil citrate) have demonstrated efficacy in the treatment of erectile dysfunction (ED) by inducing cyclic guanosine monophosphate (cGMP) elevation followed by vasodilation and increased blood flow. It also exerts minor inhibitory action against PDE6, which is present exclusively in rod and cone photoreceptors. The effects of sildenafil on the visual system have been investigated in a wide variety of clinical and preclinical studies due to the fact that a high dose of sildenafil may cause mild and transient visual symptoms in some patients. A literature review was performed using PubMed, Cochrane Library and Clinical Trials databases from 1990 up to 2020, focusing on the pathophysiology of visual disorders induced by sildenafil. The aim of this review was not only to gather and summarize the information available on sildenafil clinical trials (CTs), but also to spot subpopulations with increased risk of developing undesirable visual side effects. This PDE inhibitor has been associated with transient and reversible ocular side effects, including changes in color vision and light perception, blurred vision, photophobia, conjunctival hyperemia and keratitis, and alterations in the electroretinogram (ERG). Sildenafil may induce a reversible increase in intraocular pressure (IOP) and a few case reports suggest it is involved in the development of nonarteritic ischemic optic neuropathy (NAION). Reversible idiopathic serous macular detachment, central serous retinopathy and ERG disturbances have been related to the significant impact of sildenafil on retinal perfusion. So far, sildenafil does not seem to cause permanent toxic effects on chorioretinal tissue and photoreceptors as long as the therapeutic dose is not exceeded and is taken under a physician’s direction to treat a medical condition. However, the recreational use of sildenafil can lead to harmful side effects, including vision changes.
... The above side effects in retina are commonly thought to arise from inhibition of PDE 5 located in cells of the inner retina and in the retinal pigment epithelium, as well as from inhibition of PDE 6 found in photoreceptors [14,15]. One hypothesized consequence of such inhibition is a greater-than-normal cGMP content causing cyclic nucleotide-gated channels to remain open even in the light, resulting in widespread oxidative stress via increased intracellular calcium levels and ion pumping [16][17][18][19]. ...
Article
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Purpose The phosphodiesterase inhibitor sildenafil is a promising treatment for neurodegenerative disease, but it can cause oxidative stress in photoreceptors ex vivo and degrade visual performance in humans. Here, we test the hypotheses that in wildtype mice sildenafil causes i) wide-spread photoreceptor oxidative stress in vivo that is linked with ii) impaired vision. Methods In dark or light-adapted C57BL/6 mice ± sildenafil treatment, the presence of oxidative stress was evaluated in retina laminae in vivo by QUEnch-assiSTed (QUEST) magnetic resonance imaging, in the subretinal space in vivo by QUEST optical coherence tomography, and in freshly excised retina by a dichlorofluorescein assay. Visual performance indices were also evaluated by QUEST optokinetic tracking. Results In light-adapted mice, 1 hr post-sildenafil administration, oxidative stress was most evident in the superior peripheral outer retina on both in vivo and ex vivo examinations; little evidence was noted for central retina oxidative stress in vivo and ex vivo. In dark-adapted mice 1 hr after sildenafil, no evidence for outer retina oxidative stress was found in vivo. Evidence for sildenafil-induced central retina rod cGMP accumulation was suggested as a panretinally thinner, dark-like subretinal space thickness in light-adapted mice at 1 hr but not 5 hr post-sildenafil. Cone-based visual performance was impaired by 5 hr post-sildenafil and not corrected with anti-oxidants; vision was normal at 1 hr and 24 hr post-sildenafil. Conclusions The sildenafil-induced spatiotemporal pattern of oxidative stress in photoreceptors dominated by rods was unrelated to impairment of cone-based visual performance in wildtype mice.
... Eye disorder is another ADE worth noticing. Sildenafil is a highly selective PDE-5 inhibitor, but it also affects PDE-6 receptors, which regulate rod and cone photoreceptors [97]. Thus, sildenafil may disturb the visual transduction cascade and lead to retinopathy in premature neonates treated with sildenafil [98]. ...
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Introduction:Persistent Pulmonary Hypertension of the Newborn (PPHN) is a life-threatening neonatal condition, mostly treated with inhaled nitric oxide (iNO), intravenous prostaglandins, oral bosentan, sildenafil and tadalafil. However, the utility of non-oral agents is limited in PPHN for their side effects and inconvenient deliveries. Therefore, oral agents such as bosentan, sildenafil and tadalafil are becoming appealing for their satisfactory efficacy, easy mode of administration and acceptable side effects. Areas covered: We conducted a comprehensive search on Pubmed, Scopus, Web of Sciences concerning the use of bosentan, sildenafil and tadalafil to treat PPHN and summarized their efficacy, safety and pharmacokinetics. Expert opinion: Current randomized controlled trials (RCTs) have demonstrated the favorable responses and tolerable side effects of bosentan and sildenafil. Nevertheless, those RCTs are small and only one study has described the pharmacokinetics of sildenafil in neonates. Accordingly, bosentan, sildenafil and tadalafil remain off-label in clinical use. More well-designed RCTs with large samples and long-term follow-up and pharmacometrics studies are needed to demonstrate the efficacy, safety and pharmacokinetics of bosentan, sildenafil and tadalafil in PPHN.
Article
Background: The safety of neonatal sildenafil use remains uncertain. This study aimed to investigate adverse events (AEs) associated with sildenafil use in neonates. Research design and methods: We collected data on AEs associated with sildenafil use in neonates from the US Food and Drug Administration Adverse Event Reporting System database, spanning from its inception of the database in 2004 to 2023. Disproportionality measures were employed to analyze the correlation between AEs and sildenafil. Results: Sildenafil was identified as the primary suspect drug in 75 AE reports, involving 214 AEs. Three system organ classes, namely, eye disorders, hepatobiliary disorders, and vascular disorders were associated with sildenafil use. Six preferred terms, namely, flushing, retinopathy of prematurity, hyperbilirubinemia, pulmonary hemorrhage, hypotension, and diarrhea were associated with sildenafil use. Notably, hyperbilirubinemia and pulmonary hemorrhage were previously unreported AEs associated with sildenafil use. Conclusion: The results highlight the ongoing uncertainty surrounding the safety of neonatal sildenafil use and provide vital support for risk monitoring and identification in neonates receiving sildenafil. Additionally, the study underscores the need for continuous safety surveillance in neonates treated with sildenafil and suggests further exploration of the precise causal relationships between AEs and sildenafil.
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There is increasing concern regarding pollutants disrupting the vertebrate thyroid hormone (TH) system, which is crucial for development. Thus, identification of TH system–disrupting chemicals (THSDCs) is an important requirement in the Organisation for Economic Co‐operation and Development (OECD) testing framework. The current OECD approach uses different model organisms for different endocrine modalities, leading to a high number of animal tests. Alternative models compatible with the 3Rs (replacement, reduction, refinement) principle are required. Zebrafish embryos, not protected by current European Union animal welfare legislation, represent a promising model. Studies show that zebrafish swim bladder inflation and eye development are affected by THSDCs, and the respective adverse outcome pathways (AOPs) have been established. The present study compared effects of four THSDCs with distinct molecular modes of action: Propylthiouracil (PTU), potassium perchlorate, iopanoic acid, and the TH triiodothyronine (T3) were tested with a protocol based on the OECD fish embryo toxicity test (FET). Effects were analyzed according to the AOP concept from molecular over morphological to behavioral levels: Analysis of thyroid‐ and eye‐related gene expression revealed significant effects after PTU and T3 exposure. All substances caused changes in thyroid follicle morphology of a transgenic zebrafish line expressing fluorescence in thyrocytes. Impaired eye development and swimming activity were observed in all treatments, supporting the hypothesis that THSDCs cause adverse population‐relevant changes. Findings thus confirm that the FET can be amended by TH system–related endpoints into an integrated protocol comprising molecular, morphological, and behavioral endpoints for environmental risk assessment of potential endocrine disruptors, which is compatible with the 3Rs principle. Environ Toxicol Chem 2024;00:1–18. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.
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Phosphodiesterases (PDEs) function to hydrolyze intracellular cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), regulating a variety of intracellular signal transduction and physiological activities. PDEs can be divided into 11 families (PDE1~11) and the diversity and complex expression of PDE family genes suggest that different subtypes may have different mechanisms. PDEs are involved in various disease pathologies such as inflammation, asthma, depression, and erectile dysfunction and are thus targets of interest for several drug discovery campaigns. Natural products have always been an important source of bioactive compounds for drug discovery, over the years several natural compounds have shown potential as inhibitors of PDEs. In this article, phosphodiesterase inhibitors of natural origin have been reviewed with emphasis on their chemistry and biological activities.
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Since the dawn of time, people have been using various plant-based preparations as medicines, such as tinctures, teas, powders, and other herbal concoctions. This practise dates back thousands of years. Eulophia is used in Ayurvedic medicine as an aphrodisiac, a sperm count enhancer, a cardiac and general tonic, a blood purifier, a remedy on impotency and gynaecological problems, an expectorant, anabolic, diuretic, astringent, digestive, and soft purgative. It is also used as a remedy on impotence and gynaecological problems. Androgens are male steroid sex hormones that work directly to accelerate the development of male sexual organs and male secondary sexual traits. They do this by activating at receptors on target tissues in the body. Corticotrophin, a hormone produced by the pituitary gland, is in charge of regulating its production. The apparent presence of androgenic as well as estrogenic properties in Eulophia species, in particular E. ochreata, has been an important finding of the current study. These properties are associated with MEEH and MEEO's antidepressant, antimicrobial, analgesic, anthelmintic, antiinflammatory, antinociceptive, antioxidant, antipyretic, and neutralising oxidative stress qualities. An erg from this work is being used as a support for the efficacy of the plants for arthritic, adaptogenic, aphrodisiac, and implantation activity; wound healing; diabetes; diarrhoea; and antiscrofulous activity; wound healing; diabetes; diarrhoea; and antiscrofulous activity.
Research
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Since the dawn of time, people have been using various plant-based preparations as medicines, such as tinctures, teas, powders, and other herbal concoctions. This practise dates back thousands of years. Eulophia is used in Ayurvedic medicine as an aphrodisiac, a sperm count enhancer, a cardiac and general tonic, a blood purifier, a remedy on impotency and gynaecological problems, an expectorant, anabolic, diuretic, astringent, digestive, and soft purgative. It is also used as a remedy on impotence and gynaecological problems. Androgens are male steroid sex hormones that work directly to accelerate the development of male sexual organs and male secondary sexual traits. They do this by activating at receptors on target tissues in the body. Corticotrophin, a hormone produced by the pituitary gland, is in charge of regulating its production. The apparent presence of androgenic as well as estrogenic properties in Eulophia species, in particular E. ochreata, has been an important finding of the current study. These properties are associated with MEEH and MEEO's antidepressant, antimicrobial, analgesic, anthelmintic, antiinflammatory, antinociceptive, antioxidant, antipyretic, and neutralising oxidative stress qualities. An erg from this work is being used as a support for the efficacy of the plants for arthritic, adaptogenic, aphrodisiac, and implantation activity; wound healing; diabetes; diarrhoea; and antiscrofulous activity; wound healing; diabetes; diarrhoea; and antiscrofulous activity.
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Photoreceptor cells generate neuronal signals in response to capturing light. This process, called phototransduction, takes place in a highly specialized outer segment organelle. There are significant discrepancies in the reported amounts of many proteins supporting this process, particularly those of low abundance, which limits our understanding of their molecular organization and function. In this study, we used quantitative mass spectrometry to simultaneously determine the abundances of 20 key structural and functional proteins residing in mouse rod outer segments. We computed the absolute number of molecules of each protein residing within an individual outer segment and the molar ratio among all 20 proteins. The molar ratios of proteins comprising three well-characterized constitutive complexes in outer segments differed from the established subunit stoichiometries of these complexes by less than 7%, highlighting the exceptional precision of our quantification. Overall, this study resolves multiple existing discrepancies regarding the outer segment abundances of these proteins, thereby advancing our understanding of how the phototransduction pathway functions as a single, well-coordinated molecular ensemble.
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Retinoblastoma (Rb) is a pediatric intraocular malignancy that is proposed to originate from maturing cone cell precursors in the developing retina. The molecular mechanisms underlying the biological and clinical behaviors are important to understand in order to improve the management of advanced-stage tumors. While the genetic causes of Rb are known, an integrated understanding of the gene expression and metabolic processes in tumors of human eyes is deficient. By integrating transcriptomic profiling from tumor tissues and metabolomics from tumorous eye vitreous humor samples (with healthy, age-matched pediatric retinae and vitreous samples as controls), we uncover unique functional associations between genes and metabolites. We found distinct gene expression patterns between clinically advanced and non-advanced Rb. Global metabolomic analysis of the vitreous humor of the same Rb eyes revealed distinctly altered metabolites, indicating how tumor metabolism has diverged from healthy pediatric retina. Several key enzymes that are related to cellular energy production, such as hexokinase 1, were found to be reduced in a manner corresponding to altered metabolites; notably, a reduction in pyruvate levels. Similarly, E2F2 was the most significantly elevated E2F family member in our cohort that is part of the cell cycle regulatory circuit. Ectopic expression of the wild-type RB1 gene in the Rb-null Y79 and WERI-Rb1 cells rescued hexokinase 1 expression, while E2F2 levels were repressed. In an additional set of Rb tumor samples and pediatric healthy controls, we further validated differences in the expression of HK1 and E2F2. Through an integrated omics analysis of the transcriptomics and metabolomics of Rb, we uncovered a significantly altered tumor-specific metabolic circuit that reduces its dependence on glycolytic pathways and is governed by Rb1 and HK1.
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Alzheimer's disease (AD), one of the greatest threats to human health, is characterized by declined cognition and changed behavior. Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) that play an important role in learning and memory are hydrolyzed by phosphodiesterases (PDEs). Most PDE isoforms are highly expressed in the brain, and the inhibition of PDEs is beneficial to counteract AD. Thus, targeting PDEs represents a therapeutic potential for this disease. So far, a variety of PDE inhibitors have been discovered with significant cognitive enhancement effects in animal models and more than ten agents have entered into clinical trials. In this review, we summarize PDE mediated cyclic nucleotide signaling pathways, PDE family members involved in AD and recent advance of PDE inhibitors in preclinical and clinical studies, trying to provide an outlook of PDE inhibitors for the treatment of AD in future.
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Purpose: Cytoplasmic dynein-1 (henceforth dynein) moves cargo in conjunction with dynactin toward the minus end of microtubules. The dynein heavy chain, DYNC1H1, comprises the backbone of dynein, a retrograde motor. Deletion of Dync1h1 abrogates dynein function. The purpose of this communication is to demonstrate effects of photoreceptor dynein inactivation during late postnatal development and in adult retina. Methods: We mated Dync1h1F/F mice with iCre75 and Prom1-CreERT2 mice to generate conditional rod and tamoxifen-induced knockout in rods and cones, respectively. We documented retina degeneration with confocal microscopy at postnatal day (P) 10 to P30 for the iCre75 line and 1 to 4 weeks post tamoxifen induction (wPTI) for the Prom1-CreERT2 line. We performed scotopic and photopic electroretinography (ERG) at P16 to P30 in the iCre75 line and at 1-week increments in the Prom1-CreERT2 line. Results were evaluated statistically using Student's t-test, two-factor ANOVA, and Welch's ANOVA. Results: Cre-induced homologous recombination of Dync1h1F/F mice truncated DYNC1H1 after exon 23. rodDync1h1-/- photoreceptors degenerated after P14, reducing outer nuclear layer (ONL) thickness and combined inner segment/outer segment (IS/OS) length significantly by P18. Scotopic ERG a-wave amplitudes decreased by P16 and were extinguished at P30. Cones were stable under rod-knockout conditions until P21 but inactive at P30. In tamDync1h1-/- photoreceptors, the IS/OS began shortening by 3wPTI and were nearly eliminated by 4wPTI. The ONL shrank significantly over this interval, indicating rapid photoreceptor degeneration following the loss of dynein. Conclusions: Our results demonstrate dynein is essential for the secretory pathway, formation of outer segments, and photoreceptor maintenance.
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Proteolysis targeting chimeras (PROTACs), which hijack proteins of interest (POIs) and recruit E3 ligases for target degradation via the ubiquitin-proteasome pathway, are a novel drug discovery paradigm that has been widely used as biological tools and medicinal molecules with the potential of clinical application value. To date, a wide variety of small molecule PROTACs have been developed. Importantly, VHL-based PROTACs have emerged to be a promising approach for proteins, including those non-druggable ones, such as transcriptional factors and scaffold proteins. VHL-based PRTOACs have been developed for the treatment of diseases that are difficult to be dealt with by conventional methods, such as radiotherapy, chemotherapy, and small molecule inhibitors. In this review, the recent advances of VHL-based PRTOACs were summarized, and the chances and challenges associated with this area were also highlighted.
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Protease-targeted chimeras (PROTACs) are a new technology that is receiving much attention in the treatment of diseases. The mechanism is to inhibit protein function by hijacking the ubiquitin E3 ligase for protein degradation. Heterogeneous bifunctional PROTACs contain a ligand for recruiting E3 ligase, a linker, and another ligand to bind to the target protein for degradation. A variety of small-molecule PROTACs (CRBN, VHL, IAPs, MDM2, DCAF15, DCAF16, and RNF114-based PROTACs) have been identified so far. In particular, CRBN-based PROTACs (e.g., ARV-110 and ARV-471) have received more attention for their promising therapeutic intervention. To date, CRBN-based PRTOACs have been extensively explored worldwide and have excelled not only in cancer diseases but also in cardiovascular diseases, immune diseases, neurodegenerative diseases, and viral infections. In this review, we will provide a comprehensive update on the latest research progress in CRBN-based PRTOACs area. Following the criteria, such as disease area and drug target class, we will present the degradants in alphabetical order by target. We also provide our own perspective on the future prospects and potential challenges facing PROTACs.
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All vertebrates share a canonical retina with light-sensitive photoreceptors in the outer retina. These photoreceptors are of two kinds: rods and cones, adapted to low and bright light conditions, respectively. They both show a peculiar morphology, with long outer segments, comprised of ordered stacks of disc-shaped membranes. These discs host numerous proteins, many of which contribute to the visual transduction cascade. This pathway converts the light stimulus into a biological signal, ultimately modulating synaptic transmission. Recently, the zebrafish (Danio rerio) has gained popularity for studying the function of vertebrate photoreceptors. In this review, we introduce this model system and its contribution to our understanding of photoreception with a focus on the cone visual transduction cascade.
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Accounts in Drug Discovery describes recent case studies in medicinal chemistry with a particular emphasis on how the inevitable problems that arise during any project can be surmounted or overcome. The Editors cover a wide range of therapeutic areas and medicinal chemistry strategies, including lead optimization starting from high throughput screening "hits" as well as rational, structure-based design. The chapters include "follow-ons" and "next generation" compounds that aim to improve upon first generation agents. This volume surveys the range of challenges commonly faced by medicinal chemistry researchers, including the optimization of metabolism and pharmacokinetics, toxicology, pharmaceutics and pharmacology, including proof of concept in the clinic for novel biological targets. The case studies include medicinal chemistry stories on recently approved and marketed drugs, but also chronicle "near-misses", i.e., exemplary compounds that may have proceeded well into the clinic but for various reasons did not result in a successful registration. As the vast majority of projects fail prior to registration, much can be learned from such narratives. By sharing a wide range of drug discovery experiences and information across the community of medicinal chemists in both industry and academia, we believe that these accounts will provide insights into the art of medicinal chemistry as it is currently practiced and will help to serve the needs of active medicinal chemists.
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The long-held principle that functionally important proteins evolve slowly has recently been challenged by studies in mice and yeast showing that the severity of a protein knockout only weakly predicts that protein's rate of evolution. However, the relevance of these studies to evolutionary changes within proteins is unknown, because amino acid substitutions, unlike knockouts, often only slightly perturb protein activity. To quantify the phenotypic effect of small biochemical perturbations, we developed an approach to use computational systems biology models to measure the influence of individual reaction rate constants on network dynamics. We show that this dynamical influence is predictive of protein domain evolutionary rate in vertebrates and yeast, even after controlling for expression level and breadth, network topology, and knockout effect. Thus, our results not only demonstrate the importance of protein domain function in determining evolutionary rate, but also the power of systems biology modeling to uncover unanticipated evolutionary forces.
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We investigated the specificity of CAAX box-related isoprenylation of rod photoreceptor cGMP phosphodiesterase (PDE) subunits expressed in bacteria and the consequences of this modification on rod disk membrane association. Full-length cDNA sequences of the alpha and beta-subunits of mouse PDE, inserted into bacterial pET expression vectors, were overexpressed as fusion proteins containing 28 (bMP-alpha) and 26 (bMP-beta) additional amino acid residues at their N termini. Both fusion proteins were overexpressed and stored in inclusion bodies. Purified bMP-alpha and bMP-beta were recognized by bovine PDE-specific polyclonal antibodies, but did not associate with depleted rod disk membranes and were catalytically inactive. Using bovine brain or retina extracts as sources of protein prenyltransferases and tritiated farnesyl- or geranylgeranylpyrophosphate as donors, bMP-alpha (CAAX sequence CCIQ) was exclusively farnesylated, and bMP-beta (CAAX sequence CCIL) was exclusively geranylgeranylated. After isoprenylation, bMP-alpha and bMP-beta each associated with rod photoreceptor outer segment disk membranes under isotonic, but not under hypotonic, conditions. The results indicate that isoprenylated bMP-alpha and bMP-beta each interact independently with membranes and that isoprenylation is the key modification that facilitates membrane association.
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Photoreceptor cGMP phosphodiesterase (PDE6) is the effector enzyme in the G protein-mediated visual transduction cascade. In the dark, the activity of PDE6 is shut off by the inhibitory γ subunit (Pγ). Chimeric proteins between cone PDE6α′ and cGMP-binding and cGMP-specific PDE (PDE5) have been constructed and expressed in Sf9 cells to study the mechanism of inhibition of PDE6 catalytic activity by Pγ. Substitution of the segment PDE5-(773–820) by the corresponding PDE6α′-(737–784) sequence in the wild-type PDE5 or in a PDE5/PDE6α′ chimera containing the catalytic domain of PDE5 results in chimeric enzymes capable of inhibitory interaction with Pγ. The catalytic properties of the chimeric PDEs remained similar to those of PDE5. Ala-scanning mutational analysis of the Pγ-binding region, PDE6α′-(750–760), revealed PDE6α′ residues essential for the interaction. The M758A mutation markedly impaired and the Q752A mutation moderately impaired the inhibition of chimeric PDE by Pγ. The analysis of the catalytic properties of mutant PDEs and a model of the PDE6 catalytic domain suggest that residues Met758 and Gln752directly bind Pγ. A model of the PDE6 catalytic site shows that PDE6α′-(750–760) forms a loop at the entrance to the cGMP-binding pocket. Binding of Pγ to Met758 would effectively block access of cGMP to the catalytic cavity, providing a structural basis for the mechanism of PDE6 inhibition.
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A simple purification method has been developed for isolation of bovine cGMP phosphodiesterase from photoreceptor rod outer segments. The enzyme is peripherally membrane bound in its native state and is present in relatively high concentrations. In the bovine photoreceptors its molar ratio to rhodopsin can be estimated to be as great as 1:40 and not lower than 1: 170. The isolated enzyme is purified to homogeneity as demonstrated by gel electrophoresis under native and denaturing conditions and analytical ultracentrifuga- Con. The core enzyme has a molecular weight of approximately 170,000, as demonstrated by sucrose gradient centrifugation and analytical ultracentrifugation, and is composed of two major subunits, 88,000 (CY) and 84,000 (p), augmented by a small subunit of 13,000 (y). In its purified state, the enzyme is activated neither by light nor GTP in contrast to the native membranebound enzyme. As isolated, the molar activity of the enzyme is 45 mol of cGMP hydrolized s-’ mol-’ with a Km = 150 PM (cGMP) and Km > 4 XnM (CAMP). Protamine activates the enzyme to 360 mol of cGMP see-’ mol-’ @-fold) and limited digestion by trypsin activates it to as great as 2100 mol of cGMP s-l mol-’ @O-fold). Journal Article
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A number of phototransducing proteins in vertebrate photoreceptors contain a carboxyl terminal -CXXX motif (where C = cysteine and X = any amino acid), known to be a signal sequence for their post-translational prenylation and carboxyl methylation. To study the roles of these modifications in the visual excitation process, we have utilized an intravitreal injection method to radiolabel the prenylated proteins of rat retinas in vivo. We showed that two of the major prenylated polypeptides in the rod outer segments are the PDE alpha and PDE beta subunits of cyclic GMP phosphodiesterase PDE alpha and PDE beta subunits of cyclic GMP phosphodiesterase (PDE). By chromatographic analyses of the amino acid constituents generated by exhaustive proteolysis of PDE alpha and PDE beta, we further demonstrated that they are differentially prenylated by farnesylation and geranylgeranylation, respectively. While a number of proteins ending with the -CXXX sequence have already been reported to possess either a farnesyl or a geranylgeranyl group, PDE is the first enzyme shown to be modified by both types of prenyl groups. The prenyl modification of PDE most likely plays a major role in membrane attachment and in correctly positioning the PDE molecule for phototransduction.
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We investigated the specificity of CAAX box-related isoprenylation of rod photoreceptor cGMP phosphodiesterase (PDE) subunits expressed in bacteria and the consequences of this modification on rod disk membrane association. Full-length cDNA sequences of the alpha and beta subunits of mouse PDE, inserted into bacterial pET expression vectors, were overexpressed as fusion proteins containing 28 (bMP-alpha) and 26 (bMP-beta) additional amino acid residues at their N termini. Both fusion proteins were overexpressed and stored in inclusion bodies. Purified bMP-alpha and bMP-beta were recognized by bovine PDE-specific polyclonal antibodies, but did not associate with depleted rod disk membranes and were catalytically inactive. Using bovine brain or retina extracts as sources of protein prenyltransferases and tritiated farnesyl- or geranylgeranylpyrophosphate as donors, bMP-alpha (CAAX sequence CCIQ) was exclusively farnesylated, and bMP-beta (CAAX sequence CCIL) was exclusively geranylgeranylated. After isoprenylation, bMP-alpha and bMP-beta each associated with rod photoreceptor outer segment disk membranes under isotonic, but not under hypotonic, conditions. The results indicate that isoprenylated bMP-alpha and bMP-beta each interact independently with membranes and that isoprenylation is the key modification that facilitates membrane association.
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A bovine lung cGMP-binding phosphodiesterase (cG-BPDE) was purified to homogeneity and exhibited specific cGMP hydrolytic (Km = 5.6 μM) and cGMP binding (half-maximum ≈ 0.2 μM) activities which comigrated throughout the purification. A chimeric structure was suggested for cG-BPDE since DEAE chromatography of a partial α-chymotryptic digest of cG-BPDE separated cGMP-binding fragments from a cGMP hydrolytic fragment. Native cG-BPDE (178 kDa) appeared to be a homodimer comprised of two 93-kDa subunits. The order of potency of inhibitors of cG-BPDE hydrolysis of cGMP was as follows: zaprinast > dipyridamole > 3-isobutyl-1-methyl-8-methoxymethylxanthine > 3-isobutyl-1-methylxanthine > cilostamide > theophylline > rolipram. Minimum [3H]cGMP binding stoichiometry was 0.93 mol of cGMP bound/mol of monomer, but [3H]cGMP dissociation from cG-BPDE in the presence of excess unlabeled cGMP was curvilinear, suggesting multiple cGMP-binding sites. Two chymotryptic cGMP-binding fragments of 35 and 45 kDa were specifically photoaffinity labeled with [32P] cGMP, exhibited [3H]cGMP association and dissociation behavior indistinguishable from native cG-BPDE, and each had the amino-terminal sequence: Thr-Ser-Pro-Arg-Phe-Asp-Asn-Asp-Glu-Gly-. Cochromatography of the two cGMP-binding fragments suggested that both a dimerization domain and a cGMP-binding domain were located in a 35-kDa segment of cG-BPDE. Increased [3H]cGMP binding to or [32P]cGMP photoaffinity labeling of cG-BPDE binding sites in the presence of hydrolytic site-specific cyclic nucleotide analogs suggested communication between hydrolytic and binding sites. The principle of reciprocity thus predicts that cGMP binding to the binding sites may affect the hydrolytic site. In the presence of cGMP, the binding fragments or native cG-BPDE exhibited an electronegative shift on high performance liquid chromatography-DEAE, consistent with a cGMP-induced change in cG-BPDE conformation.
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A substantial fraction (20-30%) of the bovine rod outer segment phosphodiesterase (PDE) activity is not associated with outer segment membranes prepared with buffers of moderate ionic strength; this PDE activity appears to represent a distinct, soluble isozyme. Although this PDE isozyme can be demonstrated to be present in sealed rod outer segments, it is discarded from most standard rod outer segment preparations. A method was developed that allowed the rapid purification of the soluble rod PDE by 2600-fold, to apparent homogeneity, using a monoclonal antibody column (ROS-1a). The soluble rod PDE isozyme has a novel Mr = 15,000 subunit (delta) in addition to subunits of Mr = 88,000 (alpha sol), 84,000 (beta sol), and 11,000 (gamma sol). The delta subunit comigrates with and may be identical to the cone PDE 15-kDa subunit. The small subunits of the soluble rod PDE and the membrane-associated rod PDE were isolated by reverse-phase chromatography. The gamma sol subunit was a potent inhibitor of trypsin-activated rod PDE, inhibiting 50% of 1 pM PDE activity at a concentration of 11 pM. This concentration was similar to that observed for the gamma subunit of the membrane-associated rod PDE. The purified delta subunit did not appear to affect PDE activity; this subunit was, however, unusually difficult to keep in solution. All of the kinetic and physical properties of the soluble rod PDE tested thus far are similar to those of the membrane-associated form, except for the presence of the delta subunit, suggesting that this unique subunit could mediate the solubility of the soluble rod PDE and the cone PDE in the intact photoreceptor.
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The cGMP phosphodiesterase (PDE) of cattle retinal rod outer segments comprises three types of subunits: the two heavy catalytic ones, PDE alpha and PDE beta, each around 85 kDa, and the light inhibitory one, PDE gamma or I (11 kDa). The relative stoichiometry is usually assumed to be 1:1:1. PDE activation in the visual transduction cascade results from removal of the inhibitor by the alpha subunit of transducin (T alpha). The stoichiometric complex T alpha-I, separated from activated PDE, has been isolated and characterized. Analyzing now the activated PDE, we find that it still contains some inhibitor and is resolvable into two species, one with 50% of the inhibitor content of the native enzyme and the other totally devoid of it. The same two species are observed upon activation of PDE by very short tryptic proteolysis, which specifically degrades the inhibitor. This leads us to conclude that the composition of the native enzyme is PDE alpha beta-I2. The two inhibitory subunits are differentially bound, sequentially removable, and exchangeable between the native complex PDE alpha beta-I2 and the fully active PDE alpha beta. The possibility of this exchange precludes as yet an unambiguous estimate of the actual activity of the intermediate complex PDE alpha beta-I. The differential binding and the exchangeability of the inhibitors raises the possibility of a fast, diffusion controlled, switch-off mechanism of PDE activity after a flash, which would shortcut the inactivation resulting from the slow GTPase rate of transducin.
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The biochemical bases for the differences in cone and rod photoreceptor physiology have not been thoroughly examined because of the difficulty in obtaining cone photoreceptor components. We report here the purification and preliminary characterization of a bovine cyclic GMP phosphodiesterase (PDE) which is enriched in cone photoreceptors. The cone PDE was purified at least 15,000-fold to apparent homogeneity from bovine retinas by DEAE-cellulose and cGMP-Sepharose affinity chromatography. The trypsin-activated cone PDE hydrolyzed cGMP with efficiency similar to that of the rod PDE. However, a number of characteristics distinguished the cone PDE from the rod isozyme including the subunit structure. As previously reported, the apparent molecular weight of the cone PDE large subunit (alpha') was slightly larger than either of the large subunits of the rod PDE (93,500 versus 88,000 and 84,000). Three other smaller polypeptides were associated with the alpha' subunit (Mr = 11,000, 13,000, and 15,000), one of which (11,000) may be identical to the rod PDE gamma subunit. Cone phosphodiesterase binds at least 10-fold more cyclic GMP/mol of PDE than the rod photoreceptor isozyme. Cyclic GMP binds to this noncatalytic site with high affinity (Kd = 11 nM) and dissociates very slowly (t1/2 = 10-20 min at 37 degrees C). Purified rod transducin activated the cone PDE in solution to at least 90% of the trypsin-activated level. The concentration of rod transducin required for half-maximal activation of cone PDE (15 nM) was 50-fold lower than that necessary for half-maximal activation of rod PDE. Thus several properties of the cone phosphodiesterase clearly distinguish it from the rod isozyme and could account for some differences in cone and rod physiology.
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cGMP-binding cGMP-specific phosphodiesterase (cG-BPDE) binds tightly to a Zn(2+)-chelate column (Francis, S. H., and Corbin, J. D. (1988) Methods Enzymol. 159, 722-729). Using three different approaches, Zn2+ is now shown to bind to cG-BPDE, and the Kd is determined to be approximately 0.5 microM, with a binding stoichiometry of approximately 3 mol of Zn2+/mol of monomer. A similar concentration range of Zn2+ (0.05-1 microM Zn2+) also supports phosphodiesterase (PDE) catalytic activity. The Zn2+ binding to cG-BPDE is not diminished by, nor is catalysis supported by, relatively high concentrations of Cu2+, Cd2+, Ca2+, or Fe2+. Neither cGMP nor 3-isobutyl-1-methylxanthine affects Zn2+ binding under the conditions used. Mn2+, Co2+, or Mg2+ supports catalysis, but only at significantly higher concentrations (4-, 15-, and 250-fold, respectively) than that required for Zn2+. Two tandem amino acid sequences, which are conserved in the catalytic domains of all characterized mammalian PDEs, resemble the single sequence motif that has been shown to coordinate Zn2+ in the catalytic sites of Zn2+ hydrolases such as thermolysin.
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Polymerase chain reaction (PCR) methodology and cDNA library screening were used to isolate a cDNA clone encoding a cGMP-binding, cGMP-specific phosphodiesterase (cGB-PDE) from bovine lung. Degenerate oligonucleotides based on cGB-PDE peptide sequences were used as primers for a PCR reaction with bovine lung cDNA as the template. An 824-base pair PCR product was recovered and used as a probe to screen a bovine lung cDNA library. A 4.5-kilobase pair cDNA clone encoding a full-length cGB-PDE was isolated. The open reading frame of this cDNA predicted an 875 amino acid (AA), 99,525-Da polypeptide. By Northern analysis, the cGB-PDE cDNA hybridized to a single lung 6.9-kilobase mRNA. The identity of the cGB-PDE cDNA was verified by comparison of the deduced AA sequence with several peptide sequences obtained from cGB-PDE. COS-7 cells transfected with cGB-PDE cDNA overexpressed cGMP-binding and cGMP-PDE activities characteristic of lung cGB-PDE. The sequence of cGB-PDE contained a segment (AA 578-812) that was homologous to the putative catalytic region conserved among all mammalian PDEs and a segment (AA 142-526) that was homologous to the putative cGMP binding region of the cGMP-stimulated PDE and the photoreceptor PDEs. As noted also for these PDEs, two internally homologous repeats were contained within the putative cGMP binding region of cGB-PDE. The amino-terminal 142 residues of cGB-PDE showed no significant homology to other PDEs and contained the serine (AA 92) which is phosphorylated by cGMP-dependent protein kinase.
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Cyclic GMP phosphodiesterase (PDE) is the effector enzyme in the visual transduction cascade of vertebrate photoreceptor cells. In the dark, the activity of the enzyme catalytic alpha and beta subunits (Palphabeta) is inhibited by two gamma subunits (Pgamma). Previous results have established that approximately 5-7 C-terminal residues of Pgamma comprise the inhibitory domain. To study the interaction between the Pgamma C-terminal region and Palphabeta, the Pgamma mutant (Cys68 --> Ser, and the last 4 C-terminal residues replaced with cysteine, Pgamma-1-83Cys) was labeled with the fluorescent probe 3-(bromoacetyl)-7-diethylaminocoumarin (BC) at the cysteine residue (Pgamma-1-83BC). Pgamma-1-83BC was a more potent inhibitor of PDE activity than the unlabeled mutant, suggesting that the fluorescent probe in part substitutes for the Pgamma C terminus in PDE inhibition. HoloPDE (Palphabetagamma2) had no effect on the Pgamma-1-83BC fluorescence, but the addition of Palphabeta to Pgamma-1-83BC resulted in an approximately 8-fold maximal fluorescence increase. A Kd for the Pgamma-1-83BC-Palphabeta interaction was 4.0 +/- 0.5 nM. Zaprinast, a specific competitive inhibitor of PDE, effectively displaced the Pgamma-1-83BC C terminus from its binding site on Palphabeta (IC50 = 0.9 microM). cGMP and its analogs, 8-Br-cGMP and 2'-butyryl-cGMP, also competed with the Pgamma-1-83BC C terminus for binding to Palphabeta. Our results provide new insight into the mechanism of PDE inhibition by showing that Pgamma blocks the binding of cGMP to the PDE catalytic site.
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The cGMP phosphodiesterase from retinal rods (PDE-6) is an alphabetagamma2 heterotetramer. The alpha and beta subunits contain catalytic sites for cGMP hydrolysis, whereas the gamma subunits serve as a protein inhibitor of the enzyme. Visual excitation of photoreceptors enables the activated GTP-bound form of the G-protein transducin to remove the inhibitory action of the gamma subunit, thereby triggering PDE-6 activation. The type 5 phosphodiesterase (PDE-5) isoform shares a number of similar characteristics with PDE-6, including binding of cGMP to noncatalytic sites, the cyclic nucleotide specificity, and inhibitor sensitivities. Although the functional role of PDE-5 remains unclear, it has been shown to be activated by protein kinase A (PKA) (Burns, F., Rodger, I. W. & Pyne, N. J. (1992) Biochem. J. 283, 487-491). Here we report that both the recombinant gamma subunit and a peptide corresponding to amino acids 24-46 in this protein inhibited the activation of PDE-5 by PKA. Furthermore, immunoblotting airway smooth muscle membranes with a specific antibody against amino acids 24-46 of the PDE-6 gamma subunit identified two major immunoreactive small molecular mass proteins of 14 and 18 kDa (p14 and p18). These appear to form a complex with PDE-5, because PDE activity was immunoprecipitated using antibody against the PDE-6 gamma subunit. p14 and p18 were also substrates for phosphorylation by a unidentified kinase that was stimulated by a pertussis toxin-sensitive G-protein. Phosphorylation of p14/p18 in membranes treated with guanine nucleotides correlated with a concurrent reduction in the activation of PDE-5 by PKA. We suggest that p14 and p18 share an epitope common to PDE-6 gamma and that this region may interact with PDE-5 to prevent its activation by PKA.
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Small Rab GTPases are involved in the regulation of membrane trafficking. They cycle between cytosolic and membrane-bound forms. These membrane association/dissociation are tightly controlled by regulatory proteins. To search for proteins interacting with Rab13, a small GTPase associated with vesicles in fibroblasts and predominantly with tight junctions in epithelial cells, we screened a HeLa two-hybrid cDNA library and isolated a clone encoding a protein of 17.4 kDa. This protein, almost identical to the bovine rod cGMP phosphodiesterase delta subunit, was named human delta-PDE. The delta-PDE binds specifically to Rab13. It exhibits two putative C-terminal sequences necessary for the interaction with PDZ (PSD95, Dlg, ZO-1) domains contained in many proteins localized to specific plasma membrane microdomains. Immunofluorescence microscopic studies revealed that the vesicular stomatitis virus (VSV)-tagged delta-PDE is localized in vesicular structures accumulated near the plasma membrane in epithelial cells. Deletion of the PDZ binding motifs impair VSV-delta-PDE subcellular distribution. Purified recombinant delta-PDE had the capacity to dissociate Rab13 from cellular membranes. Our data support the proposal that delta-PDE, but not GDP dissociation inhibitor, may serve to control the dynamic of the association of Rab13 with cellular membranes.
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Chimeric cGMP phosphodiesterases (PDEs) have been constructed using components of the cGMP-binding PDE (PDE5) and cone photoreceptor phosphodiesterase (PDE6α′) in order to study structure and function of the photoreceptor enzyme. A fully functional chimeric PDE6α′/PDE5 enzyme containing the PDE6α′ noncatalytic cGMP-binding sites, and the PDE5 catalytic domain has been efficiently expressed in the baculovirus/High Five cell system. The catalytic properties of this chimera were practically indistinguishable from those of PDE5, whereas the noncatalytic cGMP binding was similar to that of native purified PDE6α′. The inhibitory γ subunit of PDE6 (Pγ) enhanced the affinity of cGMP binding at noncatalytic sites of native PDE6α′ by ∼6-fold. The polycationic region of Pγ, Pγ-24–45, was mainly responsible for this effect, while the inhibitory domain of Pγ, Pγ-63–87, was ineffective. On the contrary, Pγ failed to inhibit catalytic activity of the chimeric PDE6α′/PDE5 or to modulate its noncatalytic cGMP binding. Substitutions of Ala residues for the conserved Asn, Asn¹⁹³ or Asn⁴⁰², in the two N(K/R)XD-like motifs of the chimeric PDE noncatalytic cGMP-binding sites, each led to a loss of the noncatalytic cGMP binding. Our data suggest that both putative noncatalytic sites of PDE6α′ are important for binding of cGMP, and that the two binding sites are coupled. Furthermore, mutation Asn⁴⁰² → Ala resulted in an approximately 10-fold increase of theK m value for cGMP, indicating that occupation of the noncatalytic cGMP- binding sites of PDE6α′ may regulate catalytic properties of the enzyme.
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The photoreceptor 3':5'-cyclic nucleotide phosphodiesterase (PDE) is the central enzyme of visual excitation in rod photoreceptors. The hydrolytic activity of PDE is precisely regulated by its inhibitory gamma subunit (Pgamma), which binds directly to the catalytic site. We examined the inhibition of frog rod outer segment PDE by endogenous Pgamma, as well as by synthetic peptides corresponding to its central and C-terminal domains, to determine whether the non-catalytic cGMP-binding sites on the catalytic alphabeta dimer of PDE allosterically regulate PDE activity. We found that the apparent binding affinity of Pgamma for PDE was 28 pM when cGMP occupied the non-catalytic sites, whereas Pgamma had an apparent affinity only 1/16 of this when the sites were empty. The elevated basal activity of PDE with empty non-catalytic sites can be decreased by the addition of nanomolar levels of cGMP, demonstrating that the high-affinity non-catalytic sites on the PDE catalytic dimer mediate this effect. No evidence for a direct allosteric effect of the non-catalytic sites on catalysis could be detected for the activated enzyme lacking bound Pgamma. The intrinsic affinity of a synthetic C-terminal (residues 63-87) Pgamma peptide to bind and to inhibit the hydrolytic activity of activated PDE was enhanced 300-fold in the presence of cGMP compared with cAMP. We conclude that the binding of cGMP to the non-catalytic sites of PDE induces an allosteric change in the structure of the catalytic domain that greatly enhances the interaction of the C-terminus of Pgamma with the catalytic domain.
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The binding of cGMP to the noncatalytic sites on two isoforms of the phosphodiesterase (PDE) from mammalian rod outer segments has been characterized to evaluate their role in regulating PDE during phototransduction. Nonactivated, membrane-associated PDE (PDE-M, alpha beta gamma2) has one exchangeable site for cGMP binding; endogenous cGMP remains nonexchangeable at the second site. Non-activated, soluble PDE (PDE-S, alpha beta gamma2 delta) can release and bind cGMP at both noncatalytic sites; the delta subunit is likely responsible for this difference in cGMP exchange rates. Removal of the delta and/or gamma subunits yields a catalytic alphabeta dimer with identical catalytic and binding properties for both PDE-M and PDE-S as follows: high affinity cGMP binding is abolished at one site (KD >1 microM); cGMP binding affinity at the second site (KD approximately 60 nM) is reduced 3-4-fold compared with the nonactivated enzyme; the kinetics of cGMP exchange to activated PDE-M and PDE-S are accelerated to similar extents. The properties of nonactivated PDE can be restored upon addition of gamma subunit. Occupancy of the noncatalytic sites by cGMP may modulate the interaction of the gamma subunit with the alphabeta dimer and thereby regulate cytoplasmic cGMP concentration and the lifetime of activated PDE during visual transduction in photoreceptor cells.
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The photoreceptor cGMP phosphodiesterase (PDE6) plays a key role in vertebrate vision, but its enzymatic mechanism and the roles of metal ion co-factors have yet to be determined. We have determined the amount of endogenous Zn(2+) in rod PDE6 and established a requirement for tightly bound Zn(2+) in catalysis. Purified PDE6 contained 3-4-g atoms of zinc/mole, consistent with an initial content of two tightly bound Zn(2+)/catalytic subunit. PDE with only tightly bound Zn(2+) and no free metal ions was inactive, but activity was fully restored by Mg(2+), Mn(2+), Co(2+), or Zn(2+). Mn(2+), Co(2+), and Zn(2+) also induced aggregation and inactivation at higher concentrations and longer times. Removal of 93% of the tightly bound Zn(2+) by treatment with dipicolinic acid and EDTA at pH 6.0 resulted in almost complete loss of activity in the presence of Mg(2+). This activity loss was blocked almost completely by Zn(2+), less potently by Co(2+) and almost not at all by Mg(2+), Mn(2+), or Cu(2+). The lost activity was restored by the addition of Zn(2+), but Co(2+) restored only 13% as much activity, and other metals even less. Thus tightly bound Zn(2+) is required for catalysis but could also play a role in stabilizing the structure of PDE6, whereas distinct sites where Zn(2+) is rapidly exchanged are likely occupied by Mg(2+) under physiological conditions.
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The rod photoreceptor phosphodiesterase (PDE) is unique among all known vertebrate PDE families for several reasons. It is a catalytic heterodimer (alphabeta); it is directly activated by a G-protein, transducin; and its active sites are regulated by inhibitory gamma subunits. Rod PDE binds cGMP at two noncatalytic sites on the alphabeta dimer, but their function is unclear. We show that transducin activation of frog rod PDE introduces functional heterogeneity to both the noncatalytic and catalytic sites. Upon PDE activation, one noncatalytic site is converted from a high affinity to low affinity state, whereas the second binding site undergoes modest decreases in binding. Addition of gamma to transducin-activated PDE can restore high affinity binding as well as reducing cGMP exchange kinetics at both sites. A strong correlation exists between cGMP binding and gamma binding to activated PDE; dissociation of bound cGMP accompanies gamma dissociation from PDE, whereas addition of either cGMP or gamma to alphabeta dimers can restore high affinity binding of the other molecule. At the active site, transducin can activate PDE to about one-half the turnover number for catalytic alphabeta dimers completely lacking bound gamma subunit. These results suggest a mechanism in which transducin interacts primarily with one PDE catalytic subunit, releasing its full catalytic activity as well as inducing rapid cGMP dissociation from one noncatalytic site. The state of occupancy of the noncatalytic sites on PDE determines whether gamma remains bound to activated PDE or dissociates from the holoenzyme, and may be relevant to light adaptation in photoreceptor cells.
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Cyclic nucleotides are second messengers that are essential in vision, muscle contraction, neurotransmission, exocytosis, cell growth, and differentiation. These molecules are degraded by a family of enzymes known as phosphodiesterases, which serve a critical function by regulating the intracellular concentration of cyclic nucleotides. We have determined the three-dimensional structure of the catalytic domain of phosphodiesterase 4B2B to 1.77 angstrom resolution. The active site has been identified and contains a cluster of two metal atoms. The structure suggests the mechanism of action and basis for specificity and will provide a framework for structure-assisted drug design for members of the phosphodiesterase family.
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This chapter discusses the content of the book “Molecular mechanism in visual transduction.” It highlights the topic and gives a brief description of the chapters in the book. Visual transduction is presently one of the most intensely studied areas in the field of signal transduction research in biological cells. The sense of vision plays a primary role in animal biology and has been subject to long evolutionary development: therefore, the molecular and cellular mechanisms underlying vision have a high degree of sensitivity and versatility. The aims of visual transduction research are first to determine which molecules participate, and then to understand how they act in concert to produce the exquisite electrical responses of the photoreceptor cells. The various molecular mechanisms participating in inactivation are discussed. Molecular biology is now an indispensable tool in signal transduction studies. Numerous vertebrate and invertebrate visual pigments have been characterized and cloned.
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In addition to its cGMP-selective catalytic site, cGMP-binding cGMP-specific phosphodiesterase (PDE5) contains two allosteric cGMP-binding sites and at least one phosphorylation site (Ser92) on each subunit [Thomas, M.K., Francis, S.H. & Corbin, J.D. (1990) J. Biol. Chem.265, 14971–14978]. In the present study, prior incubation of recombinant bovine PDE5 with a phosphorylation reaction mixture [cGMP-dependent protein kinase (PKG) or catalytic subunit of cAMP-dependent protein kinase (PKA), MgATP, cGMP, 3-isobutyl-1-methylxanthine], shown earlier to produce Ser92 phosphorylation, caused a 50–70% increase in enzyme activity and also increased the affinity of cGMP binding to the allosteric cGMP-binding sites. Both effects were associated with increases in its phosphate content up to 0.6 mol per PDE5 subunit. Omission of any one of the preincubation components caused loss of stimulation of catalytic activity. Addition of the phosphorylation reaction mixture to a crude bovine lung extract, which contains PDE5, also produced a significant increase in cGMP PDE catalytic activity. The increase in recombinant PDE5 catalytic activity brought about by phosphorylation was time-dependent and was obtained with 0.2–0.5 μm PKG subunit, which is approximately the cellular level of this enzyme in vascular smooth muscle. Significantly greater stimulation was observed using cGMP substrate concentrations below the Km value for PDE5, although stimulation was also seen at high cGMP concentrations. Considerably higher concentration of the catalytic subunit of PKA than of PKG was required for activation. There was no detectable difference between phosphorylated and unphosphorylated PDE5 in median inhibitory concentration for the PDE5 inhibitors, sildenafil, or zaprinast 3-isobutyl-1-methylxanthine. Phosphorylation reduced the cGMP concentration required for half-maximum binding to the allosteric cGMP-binding sites from 0.13 to 0.03 μm. The mechanism by which phosphorylation of PDE5 by PKG could be involved in physiological negative-feedback regulation of cGMP levels is discussed.