Quantitative phosphoproteomics reveals involvement of multiple signaling pathways in early phagocytosis by the retinal pigmented epithelium.

One of the major biological functions of the retinal pigmented epithelium (RPE) is the clearance of shed photoreceptor outer segments (POS) through a multistep process resembling phagocytosis. RPE phagocytosis helps maintain the viability of photoreceptors that otherwise could succumb to the high metabolic flux and photo-oxidative stress associated with visual processing. The regulatory mechanisms underlying phagocytosis in the RPE are not fully understood, although dysfunction of this process contributes to the pathogenesis of multiple human retinal degenerative disorders, including age-related macular degeneration. Here, we present an integrated transcriptomic, proteomic, and phosphoproteomic analysis of phagocytosing RPE cells, utilizing three different experimental models: the human-derived RPE-like cell line ARPE-19, cultured murine primary RPE cells, and RPE samples from live mice. Our combined results indicated that early stages of phagocytosis in the RPE are mainly characterized by pronounced changes in the protein phosphorylation level. Global phosphoprotein enrichment analysis revealed involvement of PI3K/Akt, mechanistic target of rapamycin (mTOR), and MEK/ERK pathways in the regulation of RPE phagocytosis, confirmed by immunoblot analyses and in vitro phagocytosis assays. Most strikingly, phagocytosis of POS by cultured RPE cells was almost completely blocked by pharmacological inhibition of phosphorylation of Akt. Our findings, along with those of previous studies, indicate that these phosphorylation events allow the RPE to integrate multiple signals instigated by shed POS at different stages of the phagocytic process.

Receptor MER Tyrosine Kinase Proto-oncogene (MERTK) Is Not Required for Transfer of Bis-retinoids to the Retinal Pigmented Epithelium.

Accumulation of bis-retinoids in the retinal pigmented epithelium (RPE) is a hallmark of aging and retinal disorders such as Stargardt disease and age-related macular degeneration. These aberrant fluorescent condensation products, including di-retinoid-pyridinium-ethanolamine (A2E), are thought to be transferred to RPE cells primarily through phagocytosis of the photoreceptor outer segments. However, we observed by two-photon microscopy that mouse retinas incapable of phagocytosis due to a deficiency of the c-Mer proto-oncogene tyrosine kinase (Mertk) nonetheless contained fluorescent retinoid condensation material in their RPE. Primary RPE cells from Mertk-/- mice also accumulated fluorescent products in vitro Finally, quantification of A2E demonstrated the acquisition of retinal condensation products in Mertk-/- mouse RPE prior to retinal degeneration. In these mice, we identified activated microglial cells that likely were recruited to transport A2E-like condensation products to the RPE and dispose of the dying photoreceptor cells. These observations demonstrate a novel transport mechanism between photoreceptor cells and RPE that does not involve canonical Mertk-dependent phagocytosis.

Transcriptome analysis reveals rod/cone photoreceptor specific signatures across mammalian retinas.

A defined set of genetic instructions encodes functionality in complex organisms. Delineating these unique genetic signatures is essential to understanding the formation and functionality of specialized tissues. Vision, one of the five central senses of perception, is initiated by the retina and has evolved over time to produce rod and cone photoreceptors that vary in a species-specific manner, and in some cases by geographical region resulting in higher order visual acuity in humans. RNA-sequencing and use of existing and de novo transcriptome assemblies allowed ocular transcriptome mapping from a diverse set of rodent and primate species. Global genomic refinements along with systems-based comparative and co-expression analyses of these transcriptome maps identified gene modules that correlated with specific features of rod versus cone retinal cellular composition. Organization of the ocular transcriptome demonstrated herein defines the molecular basis of photoreceptor architecture and functionality, providing a new paradigm for neurogenetic analyses of the mammalian retina in health and disease.

Animals deficient in C2Orf71, an autosomal recessive retinitis pigmentosa-associated locus, develop severe early-onset retinal degeneration.

Genetic mapping was recently used to identify the underlying cause for a previously uncharacterized cohort of autosomal recessive retinitis pigmentosa cases. Genetic mapping of affected individuals resulted in the identification of an uncharacterized gene, C2Orf71, as the causative locus. However, initial homology searches failed to reveal similarities to any previously characterized protein or domain. To address this issue, we characterized the mouse homolog, BC027072. Immunohistochemistry with a custom polyclonal antibody showed staining localized to the inner segments (IS) of photoreceptor cells, as well as the outer segments (OS) of cone cells. A knockout mouse line (BC(-/-)) was generated and demonstrated that loss of this gene results in a severe, early-onset retinal degeneration. Histology and electron microscopy (EM) revealed disorganized OS as early as 3 weeks with complete loss by 24 weeks of age. EM micrographs displayed packets of cellular material containing OS discs or IS organelles in the OS region and abnormal retinal pigmented epithelium cells. Analyses of retinoids and rhodopsin levels showed <20% in BC(-/-) versus wild-type mice early in development. Electroretinograms demonstrated that affected mice were virtually non-responsive to light by 8 weeks of age. Lastly, RNAseq analysis of ocular gene expression in BC(-/-) mice revealed clues to the causes of the progressive retinal degenerations. Although its function remains unknown, this protein appears essential for normal OS development/maintenance and vision in humans and mice. RNAseq data are available in the GEO database under accession: GSE63810.

A polyketide synthase acyltransferase domain structure suggests a recognition mechanism for its hydroxymalonyl-acyl carrier protein substrate.

We have previously shown that the acyl transferase domain of ZmaA (ZmaA-AT) is involved in the biosynthesis of the aminopolyol polyketide/nonribosomal peptide hybrid molecule zwittermicin A from cereus UW85, and that it specifically recognizes the precursor hydroxymalonyl-acyl carrier protein (ACP) and transfers the hydroxymalonyl extender unit to a downstream second ACP via a transacylated AT domain intermediate. We now present the X-ray crystal structure of ZmaA-AT at a resolution of 1.7 Å. The structure shows a patch of solvent-exposed hydrophobic residues in the area where the AT is proposed to interact with the precursor ACP. We addressed the significance of the AT/ACP interaction in precursor specificity of the AT by testing whether malonyl- or methylmalonyl-ACP can be recognized by ZmaA-AT. We found that the ACP itself biases extender unit selection. Until now, structural information for ATs has been limited to ATs specific for the CoA-linked precursors malonyl-CoA and (2S)-methylmalonyl-CoA. This work contributes to polyketide synthase engineering efforts by expanding our knowledge of AT/substrate interactions with the structure of an AT domain that recognizes an ACP-linked substrate, the rare hydroxymalonate. Our structure suggests a model in which ACP interaction with a hydrophobic motif promotes secondary structure formation at the binding site, and opening of the adjacent substrate pocket lid to allow extender unit binding in the AT active site.

Structural and functional analysis of the native peripherin-ROM1 complex isolated from photoreceptor cells.

Peripherin and its homologue ROM1 are retina-specific members of the tetraspanin family of integral membrane proteins required for morphogenesis and maintenance of photoreceptor outer segments, regions that collect light stimuli. Over 100 pathogenic mutations in peripherin cause inherited rod- and cone-related dystrophies in humans. Peripherin and ROM1 interact in vivo and are predicted to form a core heterotetrameric complex capable of creating higher order oligomers. However, structural analysis of tetraspanin proteins has been hampered by their resistance to crystallization. Here we present a simplified methodology for high yield purification of peripherin-ROM1 from bovine retinas that permitted its biochemical and biophysical characterization. Using size exclusion chromatography and blue native gel electrophoresis, we confirmed that the core native peripherin-ROM1 complex exists as a tetramer. Peripherin, but not ROM1, is glycosylated and we examined the glycosylation site and glycan composition of ROM1 by liquid chromatographic tandem mass spectrometry. Mass spectrometry was used to analyze the native complex in detergent micelles, demonstrating its tetrameric state. Our electron microscopy-generated structure solved to 18 Å displayed the tetramer as an elongated structure with an apparent 2-fold symmetry. Finally, we demonstrated that peripherin-ROM1 tetramers induce membrane curvature when reconstituted in lipid vesicles. These results provide critical insights into this key retinal component with a poorly defined function.

Photoreceptor phagocytosis is mediated by phosphoinositide signaling.

Circadian oscillations in peripheral tissues, such as the retinal compartment of the eye, are critical to anticipating changing metabolic demands. Circadian shedding of retinal photoreceptor cell discs with subsequent phagocytosis by the neighboring retinal pigmented epithelium (RPE) is essential for removal of toxic metabolites and lifelong survival of these postmitotic neurons. Defects in photoreceptor phagocytosis can lead to severe retinal pathology, but the biochemical mechanisms remain poorly defined. By first documenting a 2.8-fold burst of photoreceptor phagocytosis events in the mouse eye in the morning compared with the afternoon by serial block face imaging, we established time points to assess transcriptional readouts by RNA sequencing (RNA-Seq). We identified 365 oscillating protein-coding transcripts that implicated the phosphoinositide lipid signaling network mediating the discrete steps of photoreceptor phagocytosis. Moreover, examination of overlapping cistromic sites by core clock transcription factors and promoter elements of these effector genes provided a functional basis for the circadian cycling of these transcripts. RNA-Seq also revealed oscillating expression of 16 long intergenic noncoding RNAs and key histone modifying enzymes critical for circadian gene expression. Our phenotypic and genotypic characterization reveals a complex global landscape of overlapping and temporally controlled networks driving the essential circadian process in the eye.

Evolutionarily conserved long intergenic non-coding RNAs in the eye.

The discovery that the mammalian transcriptome encodes thousands of long intergenic non-coding (linc) RNA transcripts, together with recent evidence that lincRNAs can regulate protein-coding genes, has added a new level of complexity to cellular transcriptional/translational regulation. Indeed several reports now link mutations in lincRNAs to heritable human disorders. Here, we identified a subset of lincRNAs in terminally differentiated adult human retinal neurons based on their sequence conservation across species. RNA sequencing of eye tissue from several mammalian species with varied rod/cone photoreceptor content identified 18 lincRNAs that were highly conserved across these species. Sixteen of the 18 were conserved in human retinal tissue with 14 of these also conserved in the macular region. A subset of lincRNAs exhibited restricted tissue expression profiles in mice, with preferential expression in the retina. Mouse models with different populations of retinal cells as well as in situ hybridization provided evidence that these lincRNAs localized to specific retinal compartments, most notably to the photoreceptor neuronal layer. Computational genomic loci and promoter region analyses provided a basis for regulated expression of these conserved lincRNAs in retinal post-mitotic neurons. This combined approach identified several lincRNAs that could be critical for retinal and visual maintenance in adults.

Photoreceptor proteins initiate microglial activation via Toll-like receptor 4 in retinal degeneration mediated by all-trans-retinal.

Although several genetic and biochemical factors are associated with the pathogenesis of retinal degeneration, it has yet to be determined how these different impairments can cause similar degenerative phenotypes. Here, we report microglial/macrophage activation in both a Stargardt disease and age-related macular degeneration mouse model caused by delayed clearance of all-trans-retinal from the retina, and in a retinitis pigmentosa mouse model with impaired retinal pigment epithelium (RPE) phagocytosis. Mouse microglia displayed RPE cytotoxicity and increased production of inflammatory chemokines/cytokines, Ccl2, Il1b, and Tnf, after coincubation with ligands that activate innate immunity. Notably, phagocytosis of photoreceptor proteins increased the activation of microglia/macrophages and RPE cells isolated from model mice as well as wild-type mice. The mRNA levels of Tlr2 and Tlr4, which can recognize proteins as their ligands, were elevated in mice with retinal degeneration. Bone marrow-derived macrophages from Tlr4-deficient mice did not increase Ccl2 after coincubation with photoreceptor proteins. Tlr4(-/-)Abca4(-/-)Rdh8(-/-) mice displayed milder retinal degenerative phenotypes than Abca4(-/-)Rdh8(-/-) mice. Additionally, inactivation of microglia/macrophages by pharmacological approaches attenuated mouse retinal degeneration. This study demonstrates an important contribution of TLR4-mediated microglial activation by endogenous photoreceptor proteins in retinal inflammation that aggravates retinal cell death. This pathway is likely to represent an underlying common pathology in degenerative retinal disorders.

Defective photoreceptor phagocytosis in a mouse model of enhanced S-cone syndrome causes progressive retinal degeneration.

Enhanced S-cone syndrome (ESCS), featuring an excess number of S cones, manifests as a progressive retinal degeneration that leads to blindness. Here, through optical imaging, we identified an abnormal interface between photoreceptors and the retinal pigment epithelium (RPE) in 9 patients with ESCS. The neural retina leucine zipper transcription factor-knockout (Nrl(-/-)) mouse model demonstrates many phenotypic features of human ESCS, including unstable S-cone-positive photoreceptors. Using massively parallel RNA sequencing, we identified 6203 differentially expressed transcripts between wild-type (Wt) and Nrl(-/-) mouse retinas, with 6 highly significant differentially expressed genes of the Pax, Notch, and Wnt canonical pathways. Changes were also obvious in expression of 30 genes involved in the visual cycle and 3 key genes in photoreceptor phagocytosis. Novel high-resolution (100 nm) imaging and reconstruction of Nrl(-/-) retinas revealed an abnormal packing of photoreceptors that contributed to buildup of photoreceptor deposits. Furthermore, lack of phagosomes in the RPE layer of Nrl(-/-) retina revealed impairment in phagocytosis. Cultured RPE cells from Wt and Nrl(-/-) mice illustrated that the phagocytotic defect was attributable to the aberrant interface between ESCS photoreceptors and the RPE. Overcoming the retinal phagocytosis defect could arrest the progressive degenerative component of this disease.

Phagocytosis of retinal rod and cone photoreceptors.

Photoreceptor cells maintain a roughly constant length by continuously generating new outer segments from their base while simultaneously releasing mature outer segments engulfed by the retinal pigment epithelium (RPE). Thus postmitotic RPE cells phagocytose an immense amount of material over a lifetime, disposing of photoreceptor cell waste while retaining useful content. This review focuses on current knowledge of outer segment phagocytosis, discussing the steps involved along with their critical participants as well as how various perturbations in outer segment (OS) disposal can lead to retinopathies.

Identification of Solanum habrochaites loci that quantitatively influence tomato fruit ripening-associated ethylene emissions.

The phytohormone ethylene is essential for ripening of climacteric fruits such as tomato. While many of the genes responsible for ethylene synthesis and perception have been identified, the regulatory network controlling autocatalytic climacteric ethylene synthesis is not well understood. In order to better understand the regulation of ripening-associated ethylene, we have exploited the genetic variation within Solanum Sect. Lycopersicon. In particular, we have used a near-isogenic population of S. habrochaites introgression lines to identify chromosome segments affecting ethylene emissions during ripening. S. habrochaites fruits produce much larger quantities of ethylene during ripening than do cultivated S. lycopersicum tomatoes. A total of 17 segments were identified; 3 had emissions more than twice the level of the tomato parent, 11 had less than a twofold increase and 3 had significantly reduced emissions at one or more ripening stages. While several of these segments co-segregate with known ethylene-related genes, many do not correspond to known genes. Thus, they may identify novel modes of regulation. These results illustrate the utility of wild relatives and their introgression lines to understand regulation of fruit ripening-related processes.

Biosynthesis of polyketide synthase extender units.

This review covers the biosynthesis of extender units that are utilized for the assembly of polyketides by polyketide synthases. The metabolic origins of each of the currently known polyketide synthase extender units are covered.

Investigations into viomycin biosynthesis by using heterologous production in Streptomyces lividans.

Viomycin and capreomycin are members of the tuberactinomycin family of antituberculosis drugs. As with many antibacterial drugs, resistance to the tuberactinomycins is problematic in treating tuberculosis; this makes the development of new derivatives of these antibiotics to combat this resistance of utmost importance. To take steps towards developing new derivatives of this family of antibiotics, we have focused our efforts on understanding how these antibiotics are biosynthesized by the producing bacteria so that metabolic engineering of these pathways can be used to generate desired derivatives. Here we present the heterologous production of viomycin in Streptomyces lividans 1326 and the use of targeted-gene deletion as a mechanism for investigating viomycin biosynthesis as well as the generation of viomycin derivatives. Deletion of vioQ resulted in nonhydroxylated derivatives of viomycin, while strains lacking vioP failed to acylate the cyclic pentapeptide core of viomycin with beta-lysine. Surprisingly, strains lacking vioL produced derivatives that had the carbamoyl group of viomycin replaced by an acetyl group. Additionally, the acetylated viomycin derivatives were produced at very low levels. These two observations suggested that the carbamoyl group of the cyclic pentapeptide core of viomycin was introduced at an earlier step in the biosynthetic pathway than previously proposed. We present biochemical evidence that the carbamoyl group is added to the beta-amino group of L-2,3-diaminopropionate prior to incorporation of this amino acid by the nonribosomal peptide synthetases that form the cyclic pentapeptide cores of both viomycin and capreomycin.

Characterization of the complete zwittermicin A biosynthesis gene cluster from Bacillus cereus.

Bacillus cereus UW85 produces the linear aminopolyol antibiotic zwittermicin A (ZmA). This antibiotic has diverse biological activities, such as suppression of disease in plants caused by protists, inhibition of fungal and bacterial growth, and amplification of the insecticidal activity of the toxin protein from Bacillus thuringiensis. ZmA has an unusual chemical structure that includes a d amino acid and ethanolamine and glycolyl moieties, as well as having an unusual terminal amide that is generated from the modification of the nonproteinogenic amino acid beta-ureidoalanine. The diverse biological activities and unusual structure of ZmA have stimulated our efforts to understand how this antibiotic is biosynthesized. Here, we present the identification of the complete ZmA biosynthesis gene cluster from B. cereus UW85. A nearly identical gene cluster is identified on a plasmid from B. cereus AH1134, and we show that this strain is also capable of producing ZmA. Bioinformatics and biochemical analyses of the ZmA biosynthesis enzymes strongly suggest that ZmA is initially biosynthesized as part of a larger metabolite that is processed twice, resulting in the formation of ZmA and two additional metabolites. Additionally, we propose that the biosynthesis gene cluster for the production of the amino sugar kanosamine is contained within the ZmA biosynthesis gene cluster in B. cereus UW85.

Fruit-specific suppression of the ethylene receptor LeETR4 results in early-ripening tomato fruit.

Tomato is an economically important crop and a significant dietary source of important phytochemicals, such as carotenoids and flavonoids. Although it has been known for many years that the plant hormone ethylene is essential for the ripening of climacteric fruits, its role in fruit growth and maturation is much less well understood. In this study, data are presented which indicate that fruit-specific suppression of the ethylene receptor LeETR4 causes early ripening, whereas fruit size, yield and flavour-related chemical composition are largely unchanged. Early fruit ripening is a highly desirable and valuable trait, and the approach demonstrated here should be applicable to any fruit species requiring ethylene to ripen. These results demonstrate that ethylene receptors probably act as biological clocks regulating the onset of tomato fruit ripening.

Ethylene receptor degradation controls the timing of ripening in tomato fruit.

Fruit ripening in tomato requires the coordination of both developmental cues and the phytohormone ethylene. The multigene ethylene receptor family has been shown to negatively regulate ethylene signal transduction and suppress ethylene responses. Here we demonstrate that reduction in the levels of either of two family members, LeETR4 or LeETR6, causes an early-ripening phenotype. We provide evidence that the receptors are rapidly degraded in the presence of ethylene, and that degradation probably occurs through the 26S proteasome-dependent pathway. Ethylene exposure of immature fruits causes a reduction in the amount of receptor protein and earlier ripening. The results are consistent with a model in which receptor levels modulate timing of the onset of fruit ripening by measuring cumulative ethylene exposure.