Phosphodiesterase 6 (PDE6) is the effector enzyme in the phototransduction cascade and is critical for the health of both rod and cone photoreceptors. Its dysfunction, caused by mutations in either the enzyme itself or AIPL1 (aryl hydrocarbon receptor-interacting protein-like 1), leads to retinal diseases culminating in blindness. Progress in research on PDE6 and AIPL1 has been severely hampered by failure to express functional PDE6 in a heterologous expression system. Here, we demonstrated that AIPL1 is an obligate chaperone of PDE6 and that it enables low yield functional folding of cone PDE6C in cultured cells. We further show that the AIPL1-mediated production of folded PDE6C is markedly elevated in the presence of the inhibitory P␥-subunit of PDE6. As illustrated in this study, a simple and sensitive system in which AIPL1 and P␥ are co-expressed with PDE6 represents an effective tool for probing structure-function relationships of AIPL1 and reliably establishing the pathogenicity of its variants.Cyclic nucleotide phosphodiesterases of the sixth family (PDE6) 2 are the key effectors in the visual transduction cascade in rod and cone photoreceptors. In the dark, activity of the PDE6 catalytic dimers is restrained by two tightly bound inhibitory ␥-subunits (P␥). This allows cGMP to maintain depolarizing "dark" current through a cGMP-gated channel in the photoreceptor plasma membrane. Photoexcitation leads to G-protein-mediated activation of PDE6 followed by a drop in cytoplasmic cGMP, channel closure, and propagation of an electrical signal to downstream retinal neurons (1, 2). In addition to being essential to photoreceptor physiology, PDE6 is critical to the health and survival of rods and cones. Malfunctions caused by mutations in genes that encode either PDE6 or its putative chaperone, aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1), lead to severe blinding retinal diseases.Mutations in the PDE6A, PDE6B, and PDE6G genes, which encode the catalytic PDE6AB subunits and P␥ of rod PDE6, respectively, are responsible for a significant proportion of cases of recessive retinitis pigmentosa (3-5) and can also lead to autosomal dominant congenital stationary night blindness (6). Mutations in their cone counterparts, PDE6C and PDE6H, cause autosomal recessive achromatopsia (7-10).PDE6 deficiency also appears to underlie one of the most severe forms of Leber congenital amaurosis (LCA type 4), a condition caused by mutations in the AIPL1 gene (11,12). LCA is an early onset inherited retinopathy and one of the main causes of blindness in children (13). The link between PDE6 and AIPL1 was discovered in studies of AIPL1 knock-out and knockdown mouse models, which revealed rapid severe retinal degeneration and a marked reduction in PDE6 protein levels and activity prior to the loss of photoreceptor cells (14, 15). Thus, the animal models recapitulated the hallmarks of LCA and suggested that AIPL1 is a potential chaperone of PDE6. This notion is consistent with the fact that AIPL1 contains an FK506-binding pro...

Inherited retinal degenerations are a common cause of untreatable blindness worldwide, with retinitis pigmentosa and cone dystrophy affecting approximately 1 in 3500 and 1 in 10,000 individuals, respectively. A major limitation to the development of effective therapies is the lack of availability of animal models that fully replicate the human condition. Particularly for cone disorders, rodent, canine, and feline models with no true macula have substantive limitations. By contrast, the cone-rich macula of a nonhuman primate (NHP) closely mirrors that of the human retina. Consequently, well-defined NHP models of heritable retinal diseases, particularly cone disorders that are predictive of human conditions, are necessary to more efficiently advance new therapies for patients. We have identified 4 related NHPs at the California National Primate Research Center with visual impairment and findings from clinical ophthalmic examination, advanced retinal imaging, and electrophysiology consistent with achromatopsia. Genetic sequencing confirmed a homozygous R565Q missense mutation in the catalytic domain of PDE6C, a cone-specific phototransduction enzyme associated with achromatopsia in humans. Biochemical studies demonstrate that the mutant mRNA is translated into a stable protein that displays normal cellular localization but is unable to hydrolyze cyclic GMP (cGMP). This NHP model of a cone disorder will not only serve as a therapeutic testing ground for achromatopsia gene replacement, but also for optimization of gene editing in the macula and of cone cell replacement in general.

Background: Mutations in AIPL1, a chaperone of the lipidated visual effector phosphodiesterase-6, cause severe childhood blindness. Results: AIPL1 binds the farnesyl lipid moiety. The unique insert region of AIPL1 is critical for this interaction. Conclusion: The AIPL1-farnesyl interaction suggests its role in the interaction with phosphodiesterase-6 and normal function of AIPL1. Significance: This study describes a novel mechanism of AIPL1 in retina disease.

Mutations in PDE6 genes encoding the effector enzymes in rods and cones underlie severe retinal diseases including retinitis pigmentosa (RP), autosomal dominant congenital stationary night blindness (adCSNB), and achromatopsia (ACHM). Here we examined a spectrum of pathogenic missense mutations in PDE6 using the system based on co-expression of cone PDE6C with its specialized chaperone AIPL1 and the regulatory Pγ subunit as a potent co-chaperone. We uncovered two mechanisms of PDE6C mutations underlying ACHM: (a) folding defects leading to expression of catalytically inactive proteins and (b) markedly diminished ability of Pγ to co-chaperone mutant PDE6C proteins thereby dramatically reducing the levels of functional enzyme. The mechanism of the Rambusch adCSNB associated with the H258N substitution in PDE6B was probed through the analysis of the model mutant PDE6C-H262N. We identified two interrelated deficits of PDE6C-H262N: disruption of the inhibitory interaction of Pγ with mutant PDE6C that markedly reduced the ability of Pγ to augment the enzyme folding. Thus, we conclude that the Rambusch adCSNB is triggered by low levels of the constitutively active PDE6. Finally, we examined PDE6C-L858V, which models PDE6B-L854V, an RP-linked mutation that alters the protein isoprenyl modification. This analysis suggests that the type of prenyl modifications does not impact the folding of PDE6, but it modulates the enzyme affinity for its trafficking partner PDE6D. Hence, the pathogenicity of PDE6B-L854V likely arises from its trafficking deficiency. Taken together, our results demonstrate the effectiveness of the PDE6C expression system to evaluate pathogenicity and elucidate the mechanisms of PDE6 mutations in retinal diseases.

A recently discovered enzyme in the mandelate pathway of Pseudomonas putida, mandelamide hydrolase (MAH), catalyzes the hydrolysis of mandelamide to mandelic acid and ammonia. Sequence analysis suggests that MAH is a member of the amidase signature family, which is widespread in nature and contains a novel Ser-cis-Ser-Lys catalytic triad. Here we report the expression in Escherichia coli, purification, and characterization of both wild-type and His(6)-tagged MAH. The recombinant enzyme was stable, exhibited a pH optimum of 7.8, and was able to hydrolyze both enantiomers of mandelamide with little enantiospecificity. The His-tagged variant showed no significant change in kinetic constants. Phenylacetamide was found to be the best substrate, with changes in chain length or replacement of the phenyl group producing greatly decreased values of k(cat)/K(m). As with another member of this family, fatty acid amide hydrolase, MAH has the uncommon ability to hydrolyze esters and amides at similar rates. MAH is even more unusual in that it will only hydrolyze esters and amides with little steric bulk. Ethyl and larger esters and N-ethyl and larger amides are not substrates, suggesting that the MAH active site is very sterically hindered. Mutation of each residue in the putative catalytic triad to alanine resulted in total loss of activity for S204A and K100A, while S180A exhibited a 1500-fold decrease in k(cat) and significant increases in K(m) values. Overall, the MAH data are similar to those of fatty acid amide hydrolase and support the suggestion that there are two distinct subgroups within the amidase signature family.

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The key visual G protein, transducin undergoes bi-directional translocations between the outer segment (OS) and inner compartments of rod photoreceptors in a light-dependent manner thereby contributing to adaptation and neuroprotection of rods. A mammalian uncoordinated 119 protein (UNC119), also known as Retina Gene 4 protein (RG4), has been recently implicated in transducin transport to the OS in the dark through its interaction with the N-acylated GTP-bound transducin-␣ subunit (G␣ t1 ). Here, we demonstrate that the interaction of human UNC119 (HRG4) with transducin is dependent on the N-acylation, but does not require the GTP-bound form of G␣ t1 . The lipid specificity of UNC119 is unique: UNC119 bound the myristoylated N terminus of G␣ t1 with much higher affinity than a prenylated substrate, whereas the homologous prenyl-binding protein PrBP/␦ did not interact with the myristoylated peptide. UNC119 was capable of interacting with G␣ t1 GDP as well as with heterotrimeric transducin (G t ). This interaction of UNC119 with G t led to displacement of G␤ 1 ␥ 1 from the heterotrimer. Furthermore, UNC119 facilitated solubilization of G t from dark-adapted rod OS membranes. Consistent with these observations, UNC119 inhibited rhodopsin-dependent activation of G t , but had no effect on the GTP-hydrolysis by G␣ t1 . A model for the role of UNC119 in the IS3 OS translocation of G t is proposed based on the UNC119 ability to dissociate G t subunits from each other and the membrane. We also found that UNC119 inhibited activation of G o by D2 dopamine receptor in cultured cells. Thus, UNC119 may play conserved inhibitory role in regulation of GPCR-G protein signaling in non-visual tissues.In rod photoreceptors, exposure to bright light causes translocation of the visual G protein, transducin from the photosensitive outer segments (OS) 2 to the inner compartments of the cells (reviewed in Refs. 1-3). The light-dependent translocation of transducin is thought to play an important role in light-adaptation and neuroprotection (4, 5). Significant advances have been made in understanding the mechanism of this phenomenon. The current evidence supports a simple diffusion model, whereby the activation of transducin by photoexcited rhodopsin (R*) causes dissociation of transducin-␣ (G␣ t1 ) and G␤ 1 ␥ 1 subunits allowing them to diffuse into the inner segment (1-9). However, translocated transducin must return to the OS during dark adaptation to restore rod sensitivity. This retrograde translocation occurs on a relatively slow time scale with a halflife of 2.5 h (4). The precise mechanism of transducin return to the OS in the dark is not known. Formation of heterotrimeric G t in the inner segment (IS) appears to be a prerequisite for correct transport of transducin to the OS. Heterotrimeric G t forms in the IS in the absence of R* following hydrolysis of G␣ t1 -bound GTP. GTP and GTP␥S both caused light-dependent transducin redistribution from the OS in permeabilized retinas, but only GTP-translocated G t returned to the OS i...

Hepatitis C is an oncogenic virus although the mechanisms responsible for this behavior are not clear. We studied the effects of hepatitis C virus (HCV) core protein expression on Telomerase, an enzyme closely associated with cellular immortalization and neoplasia. The aim of this study was to investigate the effects of HCV core protein on the regulation of Telomerase activity in human hepatoma cells. Regulation and expression of human Telomerase reverse transcriptase (TERT) was compared in Huh7 cells stably transfected with HCV core protein or cells expressing vector alone. Telomerase activity was measured using Quantitative Telomerase Detection (QTD) and telomere length was measured by fluorescence in situ hybridization (FISH). Transient transfection and luciferase assay were used to evaluate TERT promoter activity. Telomerase activity was increased twofold in Huh7 cells expressing HCV core protein compared to controls (P < 0.01). This was accompanied by a 1.4-fold increase of TERT mRNA and 1.9-fold increase in TERT protein (P < 0.01 in either case). Cellular fractionation and immunocytochemical studies showed increased localization of TERT in the nucleus of core-expressing cells as compared to controls. FISH assay confirmed that telomeres of HCV core-expressing Huh7 cells were relatively longer than those of control cells (0.22 + 0.05 vs. 0.12 + 0.03, P < 0.01). TERT promoter activity was enhanced about 30% in HCV core-expressing Huh7 cells compared to control cells (P < 0.02). HCV core protein is associated with increased Telomerase activity in hepatoma cells. These findings suggest that enhancement of Telomerase activity by HCV core protein may contribute to the oncogenicity of HCV.