RESUMEN
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.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6 , Hidrolasas Diéster Fosfóricas , Enfermedades de la Retina , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Humanos , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Células Fotorreceptoras Retinianas Conos , Enfermedades de la Retina/tratamiento farmacológico , Enfermedades de la Retina/genética , Transducina/química , Transducina/metabolismoRESUMEN
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.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Células Fotorreceptoras Retinianas Conos/enzimología , Células Fotorreceptoras Retinianas Bastones/enzimología , Visión Ocular/fisiología , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/química , Humanos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Células Fotorreceptoras Retinianas Conos/química , Células Fotorreceptoras Retinianas Bastones/químicaRESUMEN
Phosphodiesterase-6 (PDE6) plays a central role in both rod and cone phototransduction pathways. In the dark, PDE6 activity is suppressed by its inhibitory γ-subunit (Pγ). Rhodopsin-catalyzed activation of the G protein transducin relieves this inhibition and enhances PDE6 catalysis. We hypothesized that amino acid sequence differences between rod- and cone-specific Pγs underlie transducin's ability to more effectively activate cone-specific PDE6 than rod PDE6. To test this, we analyzed rod and cone Pγ sequences from all major vertebrate and cyclostome lineages and found that rod Pγ loci are far more conserved than cone Pγ sequences and that most of the sequence differences are located in the N-terminal region. Next we reconstituted rod PDE6 catalytic dimer (Pαß) with various rod or cone Pγ variants and analyzed PDE6 activation upon addition of the activated transducin α-subunit (Gtα*-GTPγS). This analysis revealed a rod-specific Pγ motif (amino acids 9-18) that reduces the ability of Gtα*-GTPγS to activate the reconstituted PDE6. In cone Pγ, Asn-13 and Gln-14 significantly enhanced Gtα*-GTPγS activation of cone Pγ truncation variants. Moreover, we observed that the first four amino acids of either rod or cone Pγ contribute to Gtα*-GTPγS-mediated activation of PDE6. We conclude that physiological differences between rod and cone photoreceptor light responsiveness can be partially ascribed to ancient, highly conserved amino acid differences in the N-terminal regions of Pγ isoforms, demonstrating for the first time a functional role for this region of Pγ in the differential activation of rod and cone PDE6 by transducin.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/química , Guanosina 5'-O-(3-Tiotrifosfato)/química , Células Fotorreceptoras Retinianas Conos/enzimología , Células Fotorreceptoras Retinianas Bastones/enzimología , Animales , Catálisis , Bovinos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismoRESUMEN
Photoreceptor phosphodiesterase 6 (PDE6) is the central effector of the visual excitation pathway in both rod and cone photoreceptors, and PDE6 mutations that alter PDE6 structure or regulation can result in several human retinal diseases. The rod PDE6 holoenzyme consists of two catalytic subunits (Pαß) whose activity is suppressed in the dark by binding of two inhibitory γ-subunits (Pγ). Upon photoactivation of rhodopsin, the heterotrimeric G protein (transducin) is activated, resulting in binding of the activated transducin α-subunit (Gtα) to PDE6, displacement of Pγ from the PDE6 active site, and enzyme activation. Although the biochemistry of this pathway is understood, a lack of detailed structural information about the PDE6 activation mechanism hampers efforts to develop therapeutic interventions for managing PDE6-associated retinal diseases. To address this gap, here we used a cross-linking MS-based approach to create a model of the entire interaction surface of Pγ with the regulatory and catalytic domains of Pαß in its nonactivated state. Following reconstitution of PDE6 and activated Gtα with liposomes and identification of cross-links between Gtα and PDE6 subunits, we determined that the PDE6-Gtα protein complex consists of two Gtα-binding sites per holoenzyme. Each Gtα interacts with the catalytic domains of both catalytic subunits and induces major changes in the interaction sites of the Pγ subunit with the catalytic subunits. These results provide the first structural model for the activated state of the transducin-PDE6 complex during visual excitation, enhancing our understanding of the molecular etiology of inherited retinal diseases.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/química , Proteínas de Unión al GTP/química , Visión Ocular , Animales , Sitios de Unión , Dominio Catalítico , Bovinos , Reactivos de Enlaces Cruzados , Microscopía por Crioelectrón , Holoenzimas/química , Espectrometría de Masas , Mutación , Unión Proteica , Retina/enzimología , Rodopsina/química , Transducina/químicaRESUMEN
As the central effector of visual transduction, the regulation of photoreceptor phosphodiesterase (PDE6) is controlled by both allosteric mechanisms and extrinsic binding partners. However, the conformational changes and interactions of PDE6 with known interacting proteins are poorly understood. Using a fluorescence detection system for the analytical ultracentrifuge, we examined allosteric changes in PDE6 structure and protein-protein interactions with its inhibitory γ-subunit, the prenyl-binding protein (PrBP/δ), and activated transducin. In solution, the PDE6 catalytic dimer (Pαß) exhibits a more asymmetric shape (axial ratio of 6.6) than reported previously. The inhibitory Pγ subunit behaves as an intrinsically disordered protein in solution but binds with high affinity to the catalytic dimer to reconstitute the holoenzyme without a detectable change in shape. Whereas the closely related PDE5 homodimer undergoes a significant change in its sedimentation properties upon cGMP binding to its regulatory cGMP binding site, no such change was detected upon ligand binding to the PDE6 catalytic dimer. However, when Pαß was reconstituted with Pγ truncation mutants lacking the C-terminal inhibitory region, cGMP-dependent allosteric changes were observed. PrBP/δ bound to the PDE6 holoenzyme with high affinity (K(D) = 6.2 nm) and induced elongation of the protein complex. Binding of activated transducin to PDE6 holoenzyme resulted in a concentration-dependent increase in the sedimentation coefficient, reflecting a dynamic equilibrium between transducin and PDE6. We conclude that allosteric regulation of PDE6 is more complex than for PDE5 and is dependent on interactions of regions of Pγ with the catalytic dimer.
Asunto(s)
GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Proteínas del Ojo/metabolismo , Células Fotorreceptoras Retinianas Bastones/enzimología , Transducina/metabolismo , Regulación Alostérica/fisiología , Animales , Bovinos , GMP Cíclico/química , GMP Cíclico/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/química , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/química , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Proteínas del Ojo/química , Proteínas del Ojo/genética , Humanos , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Células Fotorreceptoras Retinianas Bastones/citología , Transducina/química , Transducina/genéticaRESUMEN
The 11 families of the Class I cyclic nucleotide phosphodiesterases (PDEs) are critical for regulation of cyclic nucleotide signaling. PDE5 (important in regulating vascular smooth muscle contraction) and PDE6 (responsible for regulating visual transduction in vertebrate photoreceptors) are structurally similar but have several functional differences whose structural basis is poorly understood. Using evolutionary trace analysis and structural homology modeling in conjunction with site-directed mutagenesis, we have tested the hypothesis that class-specific differences between PDE5 and PDE6 account for the biochemical and pharmacological differences in the two enzyme families. Replacing human PDE5 residues in the M-loop region of the binding site for the PDE5-selective inhibitor tadalafil (Cialis®) with the corresponding class-specific cone PDE6 residues (P773E, I778V, E780L, F787W, E796V, D803P, L804M, N806D, I813L, S815K) reduces tadalafil binding affinity to levels characteristic of PDE6. These mutations fail to alter vardenafil (Levitra®) affinity for the active site. Class-specific differences in PDE5 versus cone PDE6 that contribute to the accelerated catalytic efficiency of PDE6 were identified but required heterologous expression of full-length PDE5 constructs. Introduction of PDE6 residues into the background of the PDE5 protein sequence often led to loss of catalytic activity and reduced protein solubility, supporting the idea that multiple structural elements of PDE6 are highly susceptible to misfolding during heterologous expression. This work validates the use of PDE5 as a template to identify functional differences between PDE5 and PDE6 that will accelerate efforts to develop the next generation of PDE5-selective inhibitors with fewer adverse side effects resulting from PDE6 inhibition.
Asunto(s)
Aminoácidos/química , Carbolinas/química , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/química , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/química , Proteínas del Ojo/química , Secuencia de Aminoácidos , Dominio Catalítico , Clonación Molecular , GMP Cíclico/metabolismo , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Evolución Molecular , Humanos , Imidazoles/química , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Inhibidores de Fosfodiesterasa 5/farmacología , Piperazinas/química , Unión Proteica , Retina/metabolismo , Homología de Secuencia de Aminoácido , Sulfonas/química , Tadalafilo , Triazinas/química , Diclorhidrato de VardenafilRESUMEN
The cGMP phosphodiesterase (PDE6) involved in visual transduction in photoreceptor cells contains two inhibitory γ-subunits (Pγ) which bind to the catalytic core (Pαß) to inhibit catalysis and stimulate cGMP binding to the GAF domains of Pαß. During visual excitation, interaction of activated transducin with Pγ relieves inhibition. Pγ also participates in a complex with RGS9-1 and other proteins to accelerate the GTPase activity of activated transducin. We studied the structural determinants for these important functions of Pγ. First, we identified two important sites in the middle region of Pγ (amino acids 27-38 and 52-54) that significantly stabilize the overall binding affinity of Pγ with Pαß. The ability of Pγ to stimulate noncatalytic cGMP binding to the GAF domains of PDE6 has been localized to amino acids 27-30 of Pγ. Transducin activation of PDE6 catalysis critically depends on the presence of Ile54 in the glycine-rich region of Pγ in order to relieve inhibition of catalysis. The central glycine-rich region of Pγ is also required for transducin to increase cGMP exchange at the GAF domains. Finally, Thr-65 and/or Val-66 of Pγ are critical residues for Pγ to stimulate GTPase activity of transducin in a complex with RGS9-1. We propose that the glycine-rich region of Pγ is a primary docking site for PDE6-interacting proteins involved in the activation/inactivation pathways of visual transduction. This functional mapping of Pγ with its binding partners demonstrates the remarkable versatility of this multifunctional protein and its central role in regulating the activation and lifetime of visual transduction.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/química , Proteínas RGS/química , Transducina/química , Animales , Sitios de Unión , Bovinos , GMP Cíclico/química , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Cistina/química , Proteínas del Ojo/química , Proteínas del Ojo/metabolismo , Isoleucina/química , Fragmentos de Péptidos/química , Mapeo Peptídico , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Subunidades de Proteína/química , Proteínas RGS/metabolismo , Segmento Externo de las Células Fotorreceptoras Retinianas/enzimología , Segmento Externo de las Células Fotorreceptoras Retinianas/metabolismo , Transducina/metabolismoRESUMEN
Phosphodiesterase-5 (PDE5) is responsible for regulating the concentration of the second messenger molecule cGMP by hydrolyzing it into 5'-GMP. PDE5 is implicated in erectile dysfunction and cardiovascular diseases. The substrate binding site in the catalytic domain of PDE5 is surrounded by several dynamic structural motifs (including the α14 helix, M-loop, and H-loop) that are known to switch between inactive and active conformational states via currently unresolved structural intermediates. We evaluated the conformational dynamics of these structural motifs in the apo state and upon binding of an allosteric inhibitor (evodiamine) or avanafil, a competitive inhibitor. We employed enhanced sampling-based replica exchange solute scaling (REST2) method, principal component analysis (PCA), time-lagged independent component analysis (tICA), molecular dynamics (MD) simulations, and well-tempered metadynamics simulations to probe the conformational changes in these structural motifs. Our results support a regulatory mechanism for PDE5, where the α14 helix alternates between an inward (lower activity) conformation and an outward (higher activity) conformation that is accompanied by the folding/unfolding of the α8' and α8â³ helices of the H-loop. When the allosteric inhibitor evodiamine is bound to PDE5, the inward (inactive) state of the α14 helix is preferred, thus preventing substrate access to the catalytic site. In contrast, competitive inhibitors of PDE5 block catalysis by occupying the active site accompanied by stabilization of the outward conformation of the α14 helix. Defining the conformational dynamics underlying regulation of PDE5 activation will be helpful in rational design of next-generation small molecules modulators of PDE5 activity.
Asunto(s)
GMP Cíclico , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/química , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Sitios de Unión , Dominio Catalítico , GMP Cíclico/químicaRESUMEN
G-protein coupled signaling pathways are organized into multi-protein complexes called signalosomes that are located within and on cellular membranes. We describe the use of silica nanoparticles coated with a unilamellar phospholipid bilayer (lipobeads) to reconstitute the activated photoreceptor G-protein α-subunit (Gtα*) with its cognate effector (phosphodiesterase-6; PDE6) for biochemical and structural studies of the activation mechanism regulating this GPCR signaling pathway. Lipobeads are prepared by resuspending dried-down phospholipid mixtures with monodisperse 70 nm silica particles, followed by extrusion through a 100 nm membrane filter. This uniform and supported liposomal preparation is easily sedimented, permitting the separation of soluble from membrane-associated proteins. Upon loading lipobeads with Gtα* and PDE6, we find that activation of PDE6 catalysis by Gtα* occurs much more efficiently than in the absence of membranes. Chemical cross-linking of membrane-confined proteins allows detection of changes in protein-protein interactions, resulting from G-protein activation of PDE6. The advantages of using lipobeads over partially purified membrane preparations or traditional liposomal preparations are generally applicable to the study of other membrane-confined signal transduction pathways.
RESUMEN
The central enzyme of the visual transduction cascade, cGMP phosphodiesterase (PDE6), is regulated by its gamma-subunit (Pgamma), whose inhibitory constraint is released upon binding of activated transducin. It is generally believed that the last four or five C-terminal amino acid residues of Pgamma are responsible for blocking catalysis. In this paper, we showed that the last 10 C-terminal residues (Pgamma78-87) are the minimum required to completely block catalysis. The kinetic mechanism of inhibition by the Pgamma C terminus depends on which substrate is undergoing catalysis. We also discovered a second mechanism of Pgamma inhibition that does not require this C-terminal region and that is capable of inhibiting up to 80% of the maximal cGMP hydrolytic rate. Furthermore, amino acids 63-70 and/or the intact alpha2 helix of Pgamma stabilize binding of C-terminal Pgamma peptides by 100-fold. When PDE6 catalytic subunits were reconstituted with portions of the Pgamma molecule and tested for activation by transducin, we found that the C-terminal region (Pgamma63-87) by itself could not be displaced but that transducin could relieve inhibition of certain Pgamma truncation mutants. Our results are consistent with two distinct mechanisms of Pgamma inhibition of PDE6. One involves direct interaction of the C-terminal residues with the catalytic site. A second regulatory mechanism may involve binding of other regions of Pgamma to the catalytic domain, thereby allosterically reducing the catalytic rate. Transducin activation of PDE6 appears to require interaction with both the C terminus and other regions of Pgamma to effectively relieve its inhibitory constraint.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/química , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Holoenzimas/química , Holoenzimas/metabolismo , Transducina/metabolismo , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/fisiología , Animales , Bovinos , Mutación , Conformación Proteica , Relación Estructura-ActividadRESUMEN
Retinal photoreceptor phosphodiesterase (PDE6) is unique among the phosphodiesterase enzyme family not only for its catalytic heterodimer but also for its regulatory gamma-subunits (Pgamma) whose inhibitory action is released upon binding to the G-protein transducin. It is generally assumed that during visual excitation both catalytic sites are relieved of Pgamma inhibition upon binding of two activated transducin molecules. Because PDE6 shares structural and pharmacological similarities with PDE5, we utilized radiolabeled PDE5 inhibitors to probe the catalytic sites of PDE6. The membrane filtration assay we used to quantify [(3)H]vardenafil binding to PDE6 required histone II-AS to stabilize drug binding to the active site. Under these conditions, [(3)H]vardenafil binds stoichiometrically to both the alpha- and beta-subunits of the activated PDE6 heterodimer. [(3)H]vardenafil fails to bind to either the PDE6 holoenzyme or the PDE6 catalytic dimer reconstituted with Pgamma, consistent with Pgamma blocking access to the drug-binding sites. Following transducin activation of membrane-associated PDE6 holoenzyme, [(3)H]vardenafil binding increases in proportion to the extent of PDE6 activation. Both [(3)H]vardenafil binding and hydrolytic activity of transducin-activated PDE6 fail to exceed 50% of the value for the PDE6 catalytic dimer. However, adding a 1000-fold excess of activated transducin can stimulate the hydrolytic activity of PDE6 to its maximum extent. These results demonstrate that both subunits of the PDE6 heterodimer are able to bind ligands to the enzyme active site. Furthermore, transducin relieves Pgamma inhibition of PDE6 in a biphasic manner, with only one-half of the maximum PDE6 activity efficiently attained during visual excitation.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/antagonistas & inhibidores , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Imidazoles/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Células Fotorreceptoras de Vertebrados/enzimología , Piperazinas/farmacología , Transducina/metabolismo , Animales , Sitios de Unión , Catálisis , Dominio Catalítico , Bovinos , Dimerización , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Histonas/metabolismo , Holoenzimas , Modelos Moleculares , Conformación Proteica , Subunidades de Proteína , Ensayo de Unión Radioligante , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Sulfonas/farmacología , Triazinas/farmacología , Diclorhidrato de VardenafilRESUMEN
Regulation of photoreceptor phosphodiesterase (PDE6) activity is responsible for the speed, sensitivity, and recovery of the photoresponse during visual signaling in vertebrate photoreceptor cells. It is hypothesized that physiological differences in the light responsiveness of rods and cones may result in part from differences in the structure and regulation of the distinct isoforms of rod and cone PDE6. Although rod and cone PDE6 catalytic subunits share a similar domain organization consisting of tandem GAF domains (GAFa and GAFb) and a catalytic domain, cone PDE6 is a homodimer whereas rod PDE6 consists of two homologous catalytic subunits. Here we provide the x-ray crystal structure of cone GAFab regulatory domain solved at 3.3â¯Å resolution, in conjunction with chemical cross-linking and mass spectrometric analysis of conformational changes to GAFab induced upon binding of cGMP and the PDE6 inhibitory γ-subunit (Pγ). Ligand-induced changes in cross-linked residues implicate multiple conformational changes in the GAFa and GAFb domains in forming an allosteric communication network. Molecular dynamics simulations of cone GAFab revealed differences in conformational dynamics of the two subunits forming the homodimer and allosteric perturbations on cGMP binding. Cross-linking of Pγ to GAFab in conjunction with solution NMR spectroscopy of isotopically labeled Pγ identified the central polycationic region of Pγ interacting with the GAFb domain. These results provide a mechanistic basis for developing allosteric activators of PDE6 with therapeutic implications for halting the progression of several retinal degenerative diseases.
Asunto(s)
Proteínas Aviares/metabolismo , Pollos/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Regulación Alostérica , Animales , Proteínas Aviares/química , Dominio Catalítico , Cristalografía por Rayos X , GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/química , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Dominios Proteicos , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismoRESUMEN
Photoreceptor phosphodiesterase (PDE6) is the central effector enzyme in the visual excitation pathway in rod and cone photoreceptors. Its tight regulation is essential for the speed, sensitivity, recovery, and adaptation of visual signaling. The rod PDE6 holoenzyme (Pαßγ2) is composed of a catalytic heterodimer (Pαß) that binds two inhibitory γ subunits. Each of the two catalytic subunits (Pα and Pß) contains a catalytic domain responsible for cGMP hydrolysis and two tandem GAF domains, one of which binds cGMP noncatalytically. Unlike related GAF-containing PDEs where cGMP binding allosterically activates catalysis, the physiological significance of cGMP binding to the GAF domains of PDE6 is unknown. To elucidate the structural determinants of PDE6 allosteric regulators, we biochemically characterized PDE6 complexes in various allosteric states (Pαß, Pαß-cGMP, Pαßγ2, and Pαßγ2-cGMP) with a quantitative cross-linking/mass spectrometry approach. We employed a normalization strategy to dissect the cross-linking reactivity of individual residues in order to assess the spatial cross-linking propensity of detected pairs. In addition to identifying cross-linked pairs that undergo conformational changes upon ligand binding, we observed an asymmetric binding of the inhibitory γ-subunit and the noncatalytic cGMP to the GAFa domains of rod PDE6, as well as a stable open conformation of Pαß catalytic dimer in different allosteric states. These results advance our understanding of the exquisite regulatory control of the lifetime of rod PDE6 activation/deactivation during visual signaling, as well as providing a structural basis for interpreting how mutations in rod PDE6 subunits can lead to retinal diseases.
Asunto(s)
Reactivos de Enlaces Cruzados/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Espectrometría de Masas , Células Fotorreceptoras Retinianas Bastones/enzimología , Regulación Alostérica , Animales , Bovinos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/química , Ligandos , Lisina/metabolismo , Modelos Moleculares , Subunidades de Proteína/metabolismoRESUMEN
Novel chemical controls are needed that selectively target human, animal, and plant parasitic nematodes with reduced adverse effects on the host or the environment. We hypothesize that the phosphodiesterase (PDE) enzyme family represents a potential target for development of novel nematicides and anthelmintics. To test this, we identified six PDE families present in the nematode phylum that are orthologous to six of the eleven human PDE families. We characterized the binding interactions of family-selective PDE inhibitors with human and C. elegans PDE4 in conjunction with molecular dynamics (MD) simulations to evaluate differences in binding interactions of these inhibitors within the PDE4 catalytic domain. We observed that roflumilast (human PDE4-selective inhibitor) and zardaverine (selective for human PDE3 and PDE4) were 159- and 77-fold less potent, respectively, in inhibiting C. elegans PDE4. The pan-specific PDE inhibitor isobutyl methyl xanthine (IBMX) had similar affinity for nematode and human PDE4. Of 32 residues within 5 Å of the ligand binding site, five revealed significant differences in non-bonded interaction energies (van der Waals and electrostatic interaction energies) that could account for the differential binding affinities of roflumilast and zardaverine. One site (Phe506 in the human PDE4D3 amino acid sequence corresponding to Tyr253 in C. elegans PDE4) is predicted to alter the binding conformation of roflumilast and zardaverine (but not IBMX) into a less energetically favorable state for the nematode enzyme. The pharmacological differences in sensitivity to PDE4 inhibitors in conjunction with differences in the amino acids comprising the inhibitor binding sites of human and C. elegans PDE4 catalytic domains together support the feasibility of designing the next generation of anthelmintics/nematicides that could selectively bind to nematode PDEs.
Asunto(s)
Antinematodos/metabolismo , Antinematodos/farmacología , Caenorhabditis elegans/enzimología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Simulación de Dinámica Molecular , Inhibidores de Fosfodiesterasa 4/metabolismo , Inhibidores de Fosfodiesterasa 4/farmacología , Secuencia de Aminoácidos , Animales , Antinematodos/efectos adversos , Caenorhabditis elegans/efectos de los fármacos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/química , Humanos , Inhibidores de Fosfodiesterasa 4/efectos adversos , Unión Proteica , Conformación ProteicaRESUMEN
The visual transduction pathway in vertebrate photoreceptors transforms a light stimulus entering the photoreceptor outer segments into an electrical response at the synapses of rod and cone photoreceptor cells. This process is mediated by complex biochemical pathways that precisely regulate cGMP levels, thereby controlling the extent, duration, and adaptation of the photoreceptor to the light stimulus. This review first summarizes the major mechanisms of regulating cytoplasmic cGMP levels (synthesis, degradation, buffering, and efflux) as well as the primary targets of action of cGMP (cyclic nucleotide-gated ion channels, cGMP-dependent protein kinase, and cGMPregulated phosphodiesterases). This information is applied to our current understanding of how these processes operate in the signal-transducing outer segment of rod and cone photoreceptors to carry out visual excitation, recovery, and adaptation in response to light stimulation.
Asunto(s)
GMP Cíclico/metabolismo , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Animales , GMP Cíclico/fisiología , Humanos , Células Fotorreceptoras de Vertebrados/metabolismo , Transducción de Señal/fisiologíaRESUMEN
A 17-kDa prenyl-binding protein, PrBP(PDEdelta), is highly conserved among various species from human to Caenorhabditis elegans. First identified as a putative regulatory delta subunit of the cyclic nucleotide phosphodiesterase (PDE6) purified from mammalian photoreceptor cells, PrBP(PDEdelta) has been hypothesized to reduce activation of PDE6 by the heterotrimeric G-protein, transducin, thereby desensitizing the photoresponse. However, recent work shows that PrBP(PDEdelta) interacts with numerous prenylated proteins at their farnesylated or geranylgeranylated C-termini, as well as with non-prenylated proteins. These polypeptides include small GTPases such as Rab13, Ras, Rap, and Rho6, as well as components involved in phototransduction (e.g., rod and cone PDE6, rod and cone opsin kinases). Expression of PrBP(PDEdelta) in tissues and organisms not expressing PDE6, the demonstration of multiple interacting partners with PrBP(PDEdelta), and its low abundance in rod outer segments all argue against it being a regulatory PDE6 subunit. This raises intriguing questions as to its physiological functions. In this chapter, we review the current status of PrBP(PDEdelta) and describe some of the assays used to determine these interactions in detail. In mammalian photoreceptors, the results are consistent with a role of PrBP(PDEdelta) in the transport of prenylated proteins from their site of synthesis in the inner segment to the outer segment where phototransduction occurs.
Asunto(s)
Hidrolasas Diéster Fosfóricas/metabolismo , Anfibios , Animales , Western Blotting , Bovinos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6 , ADN Complementario , Humanos , Ratones , Unión Proteica , Especies Reactivas de Oxígeno , Retina/enzimología , Segmento Externo de la Célula en Bastón/enzimología , Técnicas del Sistema de Dos HíbridosRESUMEN
PURPOSE: Phosphodiesterase (PDE) inhibitors are important therapeutic agents, but their effects on photoreceptor PDE (PDE6) and photoreceptor cells are poorly understood. The potency and selectivity of various classes of PDE inhibitors on purified rod and cone PDE6 and on intact rod outer segments (ROS) were characterized. METHODS: The inhibition constant (K(i)) of isozyme-selective PDE inhibitors was determined for purified rod and cone PDE6. Perturbations of cGMP levels in isolated ROS suspensions by PDE inhibitors were quantitated by a cGMP enzyme-linked immunoassay. RESULTS: Most PDE5-selective inhibitors were excellent PDE6 inhibitors. Vardenafil, a potent PDE5 inhibitor (K(i) = 0.2 nM), was the most potent PDE6 inhibitor tested (K(i) = 0.7 nM). Zaprinast was the only drug that inhibited PDE6 more potently than did PDE5. PDE1-selective inhibitors were equally effective in inhibiting PDE6. In intact ROS, PDE inhibitors elevated cGMP levels, but none fully inhibited PDE6. Their potency for elevating cGMP levels in ROS was much lower than their ability to inhibit the purified enzyme. Competition between PDE5/6-selective drugs and the inhibitory gamma-subunit for the active site of PDE6 is proposed to reduce the effectiveness of drugs at the enzyme-active site. CONCLUSIONS: Several classes of PDE inhibitors inhibit PDE6 equally as well as the PDE family to which they are targeted. In intact ROS, high PDE6 concentrations, binding of the gamma-subunit to the active site, and calcium feedback mechanisms attenuate the effectiveness of PDE inhibitors to inhibit PDE6 and disrupt the cGMP signaling pathway during visual transduction.
Asunto(s)
Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Animales , GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6 , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Guanilato Ciclasa/metabolismo , Células Fotorreceptoras de Vertebrados/enzimología , Rana catesbeiana , Factores de TiempoRESUMEN
Of the 11 families of mammalian cyclic nucleotide phosphodiesterases (PDEs), 5 contain regulatory domains capable of binding cyclic guanosine 5'-monophosphate (cGMP). The best understood of the GAF-containing PDEs is the family of rod (PDE6R) and cone (PDE6C) photo receptor PDEs. Binding of cGMP to the rod PDE6 catalytic dimer (alphabeta) allosterically regulates the affinity of the inhibitory subunits of PDE6 (gamma) for the enzyme. Two nonidentical, high- affinity cGMP-binding sites exist on the nonactivated mammalian PDE6R holoenzyme (alphabetagammagamma). One of the sites does not readily exchange with free cGMP when the catalytic dimer is complexed with Pgamma. On dissociation of gamma from the catalytic dimer, one of the two cGMP-binding sites undergoes a transition from high to low affinity. This chapter describes techniques to quantify cGMP binding to PDE6 in order to study the regulatory significance of the GAF domains. For high-affinity cGMP binding sites on PDE6, membrane filtration is the method of choice because of its speed, simplicity, and sensitivity. However, lower-affinity cGMP-binding sites require a method that does not perturb the equilibrium between bound and free ligand. The use of ammonium sulfate solutions during filtration extends to lower-binding affinities the useful range of membrane filtration. However, a centrifugal separation technique that minimizes perturbation of the cGMP-binding equilibrium is also presented for measuring lower-affinity cGMP-binding sites. These methods are applicable to understanding the regulatory mechanisms regulating other GAF-containing PDEs as well.
Asunto(s)
3',5'-GMP Cíclico Fosfodiesterasas/química , GMP Cíclico/análisis , Hidrolasas Diéster Fosfóricas/química , Células Fotorreceptoras Retinianas Conos/enzimología , Células Fotorreceptoras Retinianas Bastones/enzimología , 3',5'-GMP Cíclico Fosfodiesterasas/metabolismo , Animales , Dominio Catalítico , GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6 , Dimerización , Proteínas del Ojo , Humanos , Hidrolasas Diéster Fosfóricas/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismoRESUMEN
The photoreceptor phosphodiesterase (PDE6) is the central effector of visual transduction in vertebrate retinal photoreceptors. Distinct isozymes of PDE6 exist in rods and cones. Mammalian retina serves as an abundant source of tissue for PDE6 purification. Methods are described for the isolation and purification of membrane-associated PDE6 from rod outer segment membranes. Purification of cone PDE6 from the soluble fraction of retinal extracts is also described. Several procedures that can purify the rod and cone isozymes to homogeneity, including anion exchange, hydrophobic interaction, gel filtration, hydroxyapatite, and immunoaffinity chromatography, are presented. A method to activate PDE6 by limited proteolysis of its inhibitory gamma-subunit is also provided.
Asunto(s)
3',5'-GMP Cíclico Fosfodiesterasas/química , 3',5'-GMP Cíclico Fosfodiesterasas/aislamiento & purificación , Células Fotorreceptoras Retinianas Conos/química , Células Fotorreceptoras Retinianas Bastones/química , Animales , Bovinos , Cromatografía Liquida/métodos , Activación Enzimática , Isoenzimas/química , Isoenzimas/aislamiento & purificaciónRESUMEN
Photoreceptor phosphodiesterase (PDE6) is the central effector enzyme in visual excitation pathway in rod and cone photoreceptors. Its tight regulation is essential for the speed, sensitivity, recovery and adaptation of visual detection. Although major steps in the PDE6 activation/deactivation pathway have been identified, mechanistic understanding of PDE6 regulation is limited by the lack of knowledge about the molecular organization of the PDE6 holoenzyme (αßγγ). Here, we characterize the PDE6 holoenzyme by integrative structural determination of the PDE6 catalytic dimer (αß), based primarily on chemical cross-linking and mass spectrometric analysis. Our models built from high-density cross-linking data elucidate a parallel organization of the two catalytic subunits, with juxtaposed α-helical segments within the tandem regulatory GAF domains to provide multiple sites for dimerization. The two catalytic domains exist in an open configuration when compared to the structure of PDE2 in the apo state. Detailed structural elements for differential binding of the γ-subunit to the GAFa domains of the α- and ß-subunits are revealed, providing insight into the regulation of the PDE6 activation/deactivation cycle.