SUMMARY UNC119 is widely expressed among vertebrates and invertebrates. Here we report that UNC119 recognized the acylated N-terminus of the rod photoreceptor transducin α-subunit (Tα) as well as C. elegans G proteins Odr-3 and Gpa-13. The crystal structure of human UNC119 at 1.95 Å resolution revealed an immunoglobulin-like β-sandwich fold. Pulldowns and isothermal titration calorimetry revealed a tight interaction between UNC119 and acylated Gα peptides. Co-crystallization of UNC119 with an acylated Tα N-terminal peptide at 2.0 Å revealed that the lipid chain is buried deeply into UNC119's hydrophobic cavity. UNC119 bound TαGTP inhibiting its GTPase activity, thereby providing a stable UNC119-TαGTP complex that is capable of diffusing from the inner segment back to the outer segment following light-induced translocation. UNC119 deletion in both mouse and C. elegans lead to G protein mislocalization. These results establish UNC119 as a novel Gα-subunit cofactor that is essential for G-protein trafficking in sensory cilia.

BackgroundAge-related macular degeneration (AMD) is a leading cause of blindness among the elderly characterized by retinal pigment epithelium (RPE) degeneration with accumulation of abnormal intracellular deposits (lipofuscin) and photoreceptor death. RPE is vital for the retina and integrity of photoreceptors through its phagocytic function which is closely linked to formation of lipofuscin through daily phagocytosis of discarded photoreceptor outer segments (POS). Although phagocytosis has been implicated in AMD, it has not been directly shown to be altered in AMD. RPE phagocytic defect was previously shown to be rescued by subretinal injection of human umbilical tissue derived cells (hUTC) in a rodent model of retinal degeneration (RCS rat) through receptor tyrosine kinase (RTK) ligands and bridge molecules. Here, we examined RPE phagocytic function directly in the RPE from AMD patients and the ability and mechanisms of hUTC to affect phagocytosis in the human RPE.MethodsHuman RPE was isolated from the post-mortem eyes of normal and AMD-affected subjects and cultured. RPE phagocytic function was measured in vitro using isolated POS. The effects of hUTC conditioned media, recombinant RTK ligands brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF), and glial cell-derived neurotrophic factor (GDNF), as well as bridge molecules milk-fat-globule-EGF-factor 8 (MFG-E8), thrombospondin (TSP)-1, and TSP-2 on phagocytosis were also examined in phagocytosis assays using isolated POS. RNA was isolated from normal and AMD RPE treated with hUTC conditioned media and subjected to transcriptome profiling by RNA-Seq and computational analyses.ResultsRPE phagocytosis, while showing a moderate decline with age, was significantly reduced in AMD RPE, more than expected for age. hUTC conditioned media stimulated phagocytosis in the normal human RPE and significantly rescued the phagocytic dysfunction in the AMD RPE. RTK ligands and bridge molecules duplicated the rescue effect. Moreover, multiple molecular pathways involving phagocytosis, apoptosis, oxidative stress, inflammation, immune activation, and cholesterol transport were affected by hUTC in the RPE.ConclusionsWe demonstrated for the first time RPE phagocytic dysfunction in AMD, highlighting its likely importance in AMD, and the ability of hUTC to correct this dysfunction, providing insights into the therapeutic potential of hUTC for AMD.Electronic supplementary materialThe online version of this article (10.1186/s12967-018-1434-6) contains supplementary material, which is available to authorized users.

The RPGR (retinitis pigmentosa GTPase regulator) gene for RP3, the most frequent genetic subtype of X-linked retinitis pigmentosa (XLRP), has been shown to be mutated in 10%-15% of European XLRP patients. We have examined the RPGR gene for mutations in a cohort of 80 affected males from apparently unrelated XLRP families, by direct sequencing of the PCR-amplified products from the genomic DNA. Fifteen different putative disease-causing mutations were identified in 17 of the 80 families; these include four nonsense mutations, one missense mutation, six microdeletions, and four intronic-sequence substitutions resulting in splice defects. Most of the mutations were detected in the conserved N-terminal region of the RPGR protein, containing tandem repeats homologous to those present in the RCC-1 protein (a guanine nucleotide-exchange factor for Ran-GTPase). Our results indicate that mutations either in as yet uncharacterized sequences of the RPGR gene or in another gene located in its vicinity may be a more frequent cause of XLRP. The reported studies will be beneficial in establishing genotype-phenotype correlations and should lead to further investigations seeking to understand the mechanism of disease pathogenesis.

We have been using a differential cDNA cloning approach to isolate human retina-specific and retina-enriched genes [1]. A 1,314 bp cDNA was isolated by this approach, representing a highly retina-specific message encoding a 388 amino acid protein showing 58%, 50%, and 49% homology to bovine fl-arrestin, and bovine and human retinal arrestin (S-antigen), respectively. Chromosomal mapping localized this new arrestin gene to the proximal long arm of the X chromosome, hence it was named X-arrestin. In situ hybridization demonstrated its expression in the inner and outer segments and the inner plexiform regions of the retina.

A subtractive cDNA cloning strategy was used to isolate a 1381-base pair human retina-specific cDNA, human retinal gene 4 (HRG4), which hybridized to a 1.4-kilobase message in the retina and encoded a 240-amino acid acidic protein with a calculated molecular mass of 26,964 Da. The proximal 1 ⁄4 of the conceptual protein sequence was rich in glycine (18%) and proline (20%), had a predicted secondary structure of turns, and showed a loose similarity (19 -24%) to various ␣-collagen sequences, while the distal 3 ⁄4 consisted of a mixture of ␣-helices, ␤-sheets, and turns. Genomic Southern analysis with HRG4 showed cross-hybridizing sequences in six different species, and HRG4 was 92% homologous with a 1264-base pair rat cDNA (rat retinal gene 4; RRG4) at the protein level. The region of 100% identity between the two sequences corresponded to the distal 3 ⁄4 of the protein sequence consisting of mixed secondary structures, suggesting a functionally important domain. Isolation and characterization of mammalian genes specifically or abundantly expressed in the retina have contributed greatly toward the understanding of retinal biology and disease. Such genes include rhodopsin, cGMP-phosphodiesterase, transducin, and arrestin that are involved in phototransduction (1-4); peripherin and rom-1 involved in photoreceptor disc structure and function (5, 6); and interphotoreceptor retinol binding protein involved in transport of retinol between the retinal pigment epithelium (RPE) 1 and photoreceptors (7). Candidate gene analyses of these genes in inherited retinal degenerations have identified rhodopsin, peripherin, cGMPphosphodiesterase ␤, and rom-1 (possibly by itself and in combination with peripherin) to be implicated in retinopathies including retinitis pigmentosa and macular pattern dystrophies (8 -16).In order to expand our knowledge of retinal biology and to identify additional retinal genes that may be involved in retinal diseases, we have been isolating new retinal genes by a subtractive cDNA cloning strategy. This strategy has already resulted in the isolation and characterization of the cDNA and gene for human recoverin, a protein previously thought to be involved in the recovery of the depolarized state in phototransduction (17), and X-arrestin, a new retinal arrestin that appears to play a desensitization role in a yet to be defined retina-specific signal transduction (18). We now report another cDNA obtained by this strategy that represents a new retinaspecific gene that is highly conserved in human and rat and that begins to express an acidic, hydrophilic protein with some similarity to collagen specifically in photoreceptors at the time of outer retinal maturation and continues to express this protein throughout adult life. MATERIALS AND METHODS Subtractive cDNA CloningA retina-enriched cDNA library was prepared as described previously (17). Northern Blot AnalysisHuman Tissue Blot-Total RNA was isolated from human retina, cornea, iris, cultured retinal pigment epithelial cells, and fibroblasts using guanidin...