Hakim Muradov

Intrinsically disordered gamma-subunit of cGMP phosphodiesterase encodes functionally relevant transient secondary and tertiary structure.

The retinal phosphodiesterase (PDE6) inhibitory γ-subunit (PDEγ) plays a central role in vertebrate phototransduction through alternate interactions with the catalytic αβ-subunits of PDE6 and the α-subunit of transducin (αt). Detailed structural analysis of PDEγ has been hampered by its intrinsic disorder. We present here the NMR solution structure of PDEγ, which reveals a loose fold with transient structural features resembling those seen previously in the x-ray structure of PDEγ46–87 when bound to αt in the transition-state complex. NMR mapping of the interaction between PDEγ46–87 and the chimeric PDE5/6 catalytic domain confirmed that C-terminal residues 74–87 of PDEγ are involved in the association and demonstrated that its W70 indole group, which is critical for subsequent binding to αt, is left free at this stage. These results indicate that the interaction between PDEγ and αt during the phototransduction cascade involves the selection of preconfigured transient conformations.

The Inhibitory γ Subunit of the Rod cGMP Phosphodiesterase Binds the Catalytic Subunits in an Extended Linear Structure

The unique feature of rod photoreceptor cGMP phosphodiesterase (PDE6) is the presence of inhibitory subunits (Pγ), which interact with the catalytic heterodimer (Pαβ) to regulate its activity. This uniqueness results in an extremely high sensitivity and sophisticated modulations of rod visual signaling where the Pγ/Pαβ interactions play a critical role. The quaternary organization of the αβγγ heterotetramer is poorly understood and contradictory patterns of interaction have been previously suggested. Here we provide evidence that supports a specific interaction, by systematically and differentially analyzing the Pγ-binding regions on Pα and Pβ through photolabel transfer from various Pγ positions throughout the entire molecule. The Pγ N-terminal Val16–Phe30 region was found to interact with the Pαβ GAFa domain, whereas its C terminus (Phe73–Ile87) interacted with the Pαβ catalytic domain. The interactions of Pγ with these two domains were bridged by its central Ser40–Phe50 region through interactions with GAFb and the linker between GAFb and the catalytic domain, indicating a linear and extended interaction between Pγ and Pαβ. Furthermore, a photocross-linked product αβγ(γ) was specifically generated by the double derivatized Pγ, in which one photoprobe was located in the polycationic region and the other in the C terminus. Taken together the evidence supports the conclusion that each Pγ molecule binds Pαβ in an extended linear interaction and may even interact with both Pα and Pβ simultaneously.

Asymmetric Interaction between Rod Cyclic GMP Phosphodiesterase γ Subunits and αβ Subunits

Rod phosphodiesterase (PDE6) is the central effector enzyme in vertebrate visual transduction. Holo-PDE6 consists of two similar catalytic subunits (Pαβ) and two identical inhibitory subunits (Pγ). Pαβ is the only heterodimer in the PDE superfamily, yet its significance for the function of PDE6 is poorly understood. An unequal interaction of Pγ with Pβ as compared with Pα in the PDE6 complex has not been reported. We investigated the interaction interface between full-length Pγ and Pαβ, by differentiating Pγ interaction with each individual Pαβ subunit through radiolabel transfer from various positions throughout the entire Pγ molecule. The efficiency of radiolabel transfer indicates that the close vicinity of serine 40 on Pγ makes a major contribution to the interaction with Pαβ. In addition, a striking asymmetry of interaction between the Pγ polycationic region and the Pαβ subunits was observed when the stoichiometry of Pγ versus the Pαβ dimer was below 2. Preferential photolabeling on Pβ from Pγ position 40 and on Pα from position 30 increased while lowering the Pγ/Pαβ ratio, but diminished when the Pγ/Pαβ ratio was over 2. Our finding leads to the conclusion that two classes of Pγ binding sites exist on Pαβ, each composed of GAF domains in both Pα and Pβ, differing from the conventional models suggesting that each Pγ binds only one of the Pαβ catalytic subunits. This new model leads to insight into how the unique Pαβ heterodimer contributes to the sophisticated regulation in visual transduction through interaction with Pγ.

Structural basis of phosphodiesterase 6 inhibition by the C‐terminal region of the γ‐subunit

The inhibitory interaction of phosphodiesterase‐6 (PDE6) with its γ‐subunit (Pγ) is pivotal in vertebrate phototransduction. Here, crystal structures of a chimaeric PDE5/PDE6 catalytic domain (PDE5/6cd) complexed with sildenafil or 3‐isobutyl‐1‐methylxanthine and the Pγ‐inhibitory peptide Pγ70−87 have been determined at 2.9 and 3.0 Å, respectively. These structures show the determinants and the mechanism of the PDE6 inhibition by Pγ and suggest the conformational change of Pγ on transducin activation. Two variable H‐ and M‐loops of PDE5/6cd form a distinct interface that contributes to the Pγ‐binding site. This allows the Pγ C‐terminus to fit into the opening of the catalytic pocket, blocking cGMP access to the active site. Our analysis suggests that disruption of the H–M loop interface and Pγ‐binding site is a molecular cause of retinal degeneration in atrd3 mice. Comparison of the two PDE5/6cd structures shows an overlap between the sildenafil and Pγ70−87‐binding sites, thereby providing critical insights into the side effects of PDE5 inhibitors on vision.

Rod phosphodiesterase-6 PDE6A and PDE6B Subunits Are Enzymatically Equivalent

Phosphodiesterase-6 (PDE6) is the key effector enzyme of the phototransduction cascade in rods and cones. The catalytic core of rod PDE6 is a unique heterodimer of PDE6A and PDE6B catalytic subunits. The functional significance of rod PDE6 heterodimerization and conserved differences between PDE6AB and cone PDE6C and the individual properties of PDE6A and PDE6B are unknown. To address these outstanding questions, we expressed chimeric homodimeric enzymes, enhanced GFP (EGFP)-PDE6C-A and EGFP-PDE6C-B, containing the PDE6A and PDE6B catalytic domains, respectively, in transgenic Xenopus laevis. Similar to EGFP-PDE6C, EGFP-PDE6C-A and EGFP-PDE6C-B were targeted to the rod outer segments and concentrated at the disc rims. PDE6C, PDE6C-A, and PDE6C-B were isolated following selective immunoprecipitation of the EGFP fusion proteins. All three enzymes, PDE6C, PDE6C-A, and PDE6C-B, hydrolyzed cGMP with similar Km (20–23 μm) and kcat (4200–5100 s−1) values. Likewise, the Ki values for PDE6C, PDE6C-A, and PDE6C-B inhibition by the cone- and rod-specific PDE6 γ-subunits (Pγ) were comparable. Recombinant cone transducin-α (Gαt2) and native rod Gαt1 fully and potently activated PDE6C, PDE6C-A, and PDE6C-B. In contrast, the half-maximal activation of bovine rod PDE6 required markedly higher concentrations of Gαt2 or Gαt1. Our results suggest that PDE6A and PDE6B are enzymatically equivalent. Furthermore, PDE6A and PDE6B are similar to PDE6C with respect to catalytic properties and the interaction with Pγ but differ in the interaction with transducin. This study significantly limits the range of mechanisms by which conserved differences between PDE6A, PDE6B, and PDE6C may contribute to remarkable differences in rod and cone physiology.

Unique transducins expressed in long and short photoreceptors of lamprey Petromyzon marinus

Lampreys represent the most primitive vertebrate class of jawless fish and serve as an evolutionary model of the vertebrate visual system. Transducin-alpha (G alpha(t)) subunits were investigated in lamprey Petromyzon marinus in order to understand the molecular origins of rod and cone photoreceptor G proteins. Two G alpha(t) subunits, G alpha(tL) and G alpha(tS), were identified in the P. marinus retina. G alpha(tL) is equally distant from cone and rod G proteins and is expressed in the lampreys long photoreceptors. The short photoreceptor G alpha(tS) is a rod-like transducin-alpha that retains several unique features of cone transducins. Thus, the duplication of the ancestral transducin gene giving rise to rod transducins has already occurred in the last common ancestor of the jawed and jawless vertebrates.

Characterization of human cone phosphodiesterase-6 ectopically expressed in Xenopus laevis rods.

PDE6 (phosphodiesterase-6) is the effector molecule in the vertebrate phototransduction cascade. Progress in understanding the structure and function of PDE6 has been hindered by lack of an expression system of the enzyme. Here we report ectopic expression and analysis of compartmentalization and membrane dynamics of the enhanced green fluorescent protein (EGFP) fusion protein of human cone PDE6C in rods of transgenic Xenopus laevis. EGFP-PDE6C is correctly targeted to the rod outer segments in transgenic Xenopus, where it displayed a characteristic striated pattern of EGFP fluorescence. Immunofluorescence labeling indicated significant and light-independent co-localization of EGFP-PDE6C with the disc rim marker peripherin-2 and endogenous frog PDE6. The diffusion of EGFP-PDE6C on disc membranes investigated with fluorescence recovery after photobleaching was markedly slower than theoretically predicted. The enzymatic characteristics of immunoprecipitated recombinant PDE6C were similar to known properties of the native bovine PDE6C. PDE6C was potently inhibited by the cone- and rod-specific PDE6 γ-subunits. Thus, transgenic Xenopus laevis is a unique expression system for PDE6 well suited for analysis of the mechanisms of visual diseases linked to PDE6 mutations.

Analysis of PDE6 function using chimeric PDE5/6 catalytic domains

cGMP-phosphodiesterases of the PDE6 family are expressed in retinal photoreceptor cells, where they mediate the phototransduction cascade. A system for expression of PDE6 in vitro is lacking, thus straining progress in understanding the structure-function relationships of the photoreceptor enzyme. Here, we report generation and characterization of bacterially expressed chimeric PDE5/6 catalytic domains which are highly soluble, catalytically active, and sensitive to inhibition by the PDE6 Pgamma subunit. Two flexible PDE6 loops, H and M, impart chimeric PDE5/6 catalytic domains with PDE6-like properties. The replacement of the PDE6 H-loop into the PDE5 catalytic domain increases the catalytic rate and the K(m) value for cGMP hydrolysis, whereas the substitution of the M-loop produces catalytic PDE domains responsive to Pgamma. Multiple PDE6 segments preventing functional expression of the catalytic domain are identified, supporting the requirement for specialized photoreceptor chaperones to assist PDE6 folding in vivo.

Exchange of Cone for Rod Phosphodiesterase 6 Catalytic Subunits in Rod Photoreceptors Mimics in Part Features of Light Adaptation.

Despite the expression of homologous phototransduction components, the molecular basis for differences in light-evoked responses between rod and cone photoreceptors remains unclear. We examined the role of cGMP phosphodiesterase (PDE6) in this difference by expressing cone PDE6 (PDE6C) in rd1/rd1 rods lacking rod PDE6 (PDE6AB) using transgenic mice. The expression of PDE6C rescues retinal degeneration observed in rd1/rd1 rods. Double-transgenic rods (PDE6C++) were compared with rd1/+ rods based on similar PDE6 expression. PDE6C increased the basal PDE activity and speeded the rate-limiting step for phototransduction deactivation, causing rod photoresponses to appear light adapted, with reduced dark current and sensitivity and faster response kinetics. When PDE6C++ and rd1/+ rods were exposed to similar background light, rd1/+ rods displayed greater desensitization. These results indicate an increased spontaneous activity and faster deactivation of PDE6C compared with PDE6AB in darkness, but that background light increases steady PDE6C activity to a lesser extent. In addition to accelerating the recovery of the photoresponse, faster PDE6C deactivation may blunt the rise in background-induced steady PDE6C activity. Therefore, higher basal PDE6C activity and faster deactivation together partially account for faster and less sensitive cone photoresponses in darkness, whereas a reduced rise of steady PDE6C activity in background light may allow cones to avoid saturation. Significance Statement Cones are the primary photoreceptors responsible for most of our visual experience. Cone light responses are less sensitive and display speeded responses compared with rods. Despite the fact that rods and cones use a G-protein signaling cascade with similar organization, the mechanistic basis for these differences remains unclear. Here, we examined the role of distinct isoforms of PDE6, the effector enzyme in phototransduction, in these differences. We developed a transgenic mouse model that expresses cone PDE6 in rods and show that the cone PDE6 isoform is partially responsible for the difference in sensitivity and response kinetics between rods and cones.

CAPN5 gene silencing by short hairpin RNA interference.

BackgroundThe purpose of this project was to identify short hairpin RNA (shRNA) sequences that can suppress expression of human CAPN5 in which gain-of-function mutants cause autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV). We created HEK293T cells that stably express an ADNIV disease allele, CAPN5-p.R243L. Transfection protocols were optimized for neuroblastoma SHSY5Y cells. The gene silencing effect of four different shRNA plasmids that target CAPN5 was tested. RNA and protein expression was determined using quantitative RT-PCR and immunoblot analysis.FindingsTwo of four shRNA plasmids reduced mutant CAPN5 RNA in a stable cell line. Similar knockdown was observed in SH-SY5Y cells that natively express CAPN5. Lactose dehydrogenase assays showed that down-regulation of CAPN5 was not cytotoxic.ConclusionsCAPN5 expression can be suppressed by shRNA-based RNA interference. Further testing in ADNIV models will determine the potential of gene silencing as a strategy to treat, delay, or prevent blindness in ADNIV patients.