The carboxyl terminal mutational hotspot of the ciliary disease protein RPGRORF15 (retinitis pigmentosa GTPase regulator) is glutamylated in vivo

Mutations inRPGR(ORF15)(retinitis pigmentosa GTPase regulator) are a major cause of inherited retinal degenerative diseases. RPGR(ORF15)(1152 residues) is a ciliary protein involved in regulating the composition and function of photoreceptor cilia. The mutational hotspot in RPGR(ORF15)is an unusual C-terminal domain encoded by exon ORF15, which is rich in polyglutamates and glycine residues (Glu-Gly domain) followed by a short stretch of basic amino acid residues (RPGR(C2)domain; residues 1072-1152). However, the properties of the ORF15-encoded domain and its involvement in the pathogenesis of the disease are unclear. Here we show that RPGR(ORF15)is glutamylated at the C-terminus, as determined by binding to GT335, which recognizes glutamylated substrates. This reactivity is lost in two mouse mutants ofRpgr, which do not express RPGR(ORF15)due to disease-causing mutations in exon ORF15. Our results indicate that RPGR(ORF15)is posttranslationally glutamylated in the Glu-Gly domain and that the GT335 antibody predominantly recognizes RPGR(ORF15)in photoreceptor cilia

Structural but Not Functional Alterations in Cones in the Absence of the Retinal Disease Protein Retinitis Pigmentosa 2 (RP2) in a Cone-Only Retina

X-linked retinitis pigmentosa 2 (XLRP2) patients and Rp2 (null) mice exhibit severe cone photoreceptor degeneration. However, due to the paucity of cones in mammalian model systems, it is not clear how cones respond to the loss of RP2. Here we have used the Nrl(-/-) mice, which develop a rodless and short wavelength (S) opsin-containing cone-only retina, to generate Rp2 (null)::Nrl(-/-) double knock out (Rp2-DKO) mice. We found that the ciliary axoneme and the outer segments (OSs) of the cones were significantly longer with disorganized membrane infoldings as compared to the Nrl(-/-) mice. Additionally, we found misregulation in the expression of the genes related to ophthalmic disease, cell trafficking, and stress-response in the Rp2-DKO mice prior to the onset of cone degeneration. Surprisingly, the loss of RP2 did not affect progressive photoreceptor dysfunction of the Nrl(-/-) mice and the trafficking of S opsin. Our data suggest that RP2 is a negative regulator of cone OS length but does not affect S-opsin trafficking and S-cone function. Our studies also provide a cone-only platform to design cone-targeted therapeutic strategies for X-linked RP2

Ablation of retinal ciliopathy protein RPGR results in altered photoreceptor ciliary composition

Cilia regulate several developmental and homeostatic pathways that are critical to survival. Sensory cilia of photoreceptors regulate phototransduction cascade for visual processing. Mutations in the ciliary protein RPGR (retinitis pigmentosa GTPase regulator) are a prominent cause of severe blindness disorders due to degeneration of mature photoreceptors. However, precise function of RPGR is still unclear. Here we studied the involvement of RPGR in ciliary trafficking by analyzing the composition of photoreceptor sensory cilia (PSC) in Rpgr(ko) retina. Using tandem mass spectrometry analysis followed by immunoblotting, we detected few alterations in levels of proteins involved in proteasomal function and vesicular trafficking in Rpgr(ko) PSC, prior to onset of degeneration. We also found alterations in the levels of high molecular weight soluble proteins in Rpgr(ko) PSC. Our data indicate RPGR regulates entry or retention of soluble proteins in photoreceptor cilia but spares the trafficking of key structural and phototransduction-associated proteins. Given a frequent occurrence of RPGR mutations in severe photoreceptor degeneration due to ciliary disorders, our results provide insights into pathways resulting in altered mature cilia function in ciliopathies

Prenylated retinal ciliopathy protein RPGR regulates ciliary localization of Joubert Syndrome-associated protein INPP5E in cooperation with PDE6

Ciliary dysfunction is an underlying cause of severe human disorders (collectively called ciliopathies), such as retinitis pigmentosa (RP), Joubert Syndrome (JBTS), and Bardet-Biedl Syndrome. Ciliary proteins form distinct functional networks for localization to cilia as well as regulation of ciliary function. However, not much is known about the mechanism of ciliary localization and function of RPGR (retinitis pigmentosa GTPase regulator), a ciliary protein frequently associated with RP worldwide. Using tandem mass spectrometry analysis, we show that RPGR interacts with two JBTS-associated proteins: PDE6Π (delta subunit of Phosphodiesterase; a prenyl-binding protein) and INPP5E (inositol polyphosphate-5-phosphatase 5E; a ciliary cargo). Whereas PDE6Π binds in a prenylation-dependent manner to the C-terminus of RPGR, INPP5E associates with the N-terminus of RPGR. Prenylation and interaction of RPGR with PDE6Π are critical for its localization to cilia. We further show that loss of RPGR results in reduced amount of INPP5E in cilia of fibroblasts and in photoreceptor outer segment, a modified sensory cilium. Overall, our results suggest that RPGR, in complex with PDE6D, regulates the trafficking of ciliary cargo INPP5E and implicate reduction in ciliary INPP5E in the pathogenesis of RPGR-ciliopathy

Green synthesis of silver nanoparticles using methanolic root extracts of Diospyros paniculata and their antimicrobial activities

Since the discovery and subsequent widespread use of antibiotics, a variety of bacterial species of human and animal origin have developed numerous mechanisms that render bacteria resistant to some, and in certain cases to nearly all antibiotics, thereby limiting the treatment options and compromising effective therapy. In the present study, the green synthesis of nanoparticles is carried out by the reduction of silver acetate in the presence of crude methanolic root extracts of Diospyros paniculata, a member of family Ebenaceae. The UV-Vis absorption spectrum of the biologically reduced reaction mixture showed the surface plasmon peak at 428 nm, a characteristic peak of silver nanoparticles. X-ray diffraction (XRD) analysis confirmed the face-centered cubic crystalline structure of metallic silver. The average diameter of Ag NPs is about 17 nm from Transmission Electron Microscopy (TEM) which is in good agreement with the average crystallite size (19 nm) calculated from XRD analysis. Further the study has been extended to the antimicrobial activity against test pathogenic Gram (+), Gram (-) bacterial and fungal strains. The biologically synthesized silver nanoparticles showed promising activity against all the tested pathogenic strains and the activity has been enhanced with the increased dose levels. (C) 2016 Elsevier B.V. All rights reserved

A polymorphism in the CYP1B1 promoter is functionally associated with primary congenital glaucoma

Primary congenital glaucoma (PCG) is a childhood autosomal-recessive disorder caused by developmental defects in the trabecular meshwork and anterior chamber angle. These defects cause raised intraocular pressure (IOP) that damages the optic nerve and if left untreated, results in irreversible blindness. Mutations in CYP1B1 gene at the GLC3A locus (2p21) are associated with PCG. However, there has been very limited exploration of its promoter region. We resequenced the CYP1B1 promoter in a large cohort (n = 835) that included patients with PCG (n = 301), other primary glaucomas (primary open-angle glaucoma: n = 115 and primary angle closure glaucoma: n = 100) and unaffected controls (n = 319). We functionally characterized one associated variant by luciferase reporter assay using the trabecular meshwork (TM3) cell line. We found evidence of strong (P = 6.01 × 10-4) association of rs2567206 (T2805C) SNP in PCG and not in other primary glaucomas. Luciferase assay indicated a ˜90% reduction in CYP1B1 promoter activity in the risk-allele (C) compared to the other allele (T). The association of the risk allele was stronger in cases harboring homozygous CYP1B1 mutations (P = 3.42 × 10-12). The risk haplotype 'C-C-G' in the promoter had a strong non-random association to the previously characterized risk haplotype 'C-G-G-T-A' in the coding region. The independent effect of genotype at the promoter T2805C locus (P = 0.001), and the interaction effect of genotypes at the promoter and coding region mutations loci (P = 0.001) were significant for the presenting IOP of the worst affected eye. This is the first study that unequivocally shows the functional involvement of a CYP1B1 promoter variant in PCG