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Outer retinal and renal glomerular functions rely on specialized vasculature maintained by VEGF that is produced by neighboring epithelial cells, the retinal pigment epithelium (RPE) and podocytes, respectively. Dysregulation of RPE- and podocyte-derived VEGF is associated with neovascularization in wet age-related macular degeneration (ARMD), choriocapillaris degeneration, and glomerular thrombotic microangiopathy (TMA). Since complement activation and genetic variants in inhibitory complement factor H (CFH) are also features of both ARMD and TMA, we hypothesized that VEGF and CFH interact. Here, we demonstrated that VEGF inhibition decreases local CFH and other complement regulators in the eye and kidney through reduced VEGFR2/PKC-α/CREB signaling. Patient podocytes and RPE cells carrying disease-associated CFH genetic variants had more alternative complement pathway deposits than controls. These deposits were increased by VEGF antagonism, a common wet ARMD treatment, suggesting that VEGF inhibition could reduce cellular complement regulatory capacity. VEGF antagonism also increased markers of endothelial cell activation, which was partially reduced by genetic complement inhibition. Together, these results suggest that VEGF protects the retinal and glomerular microvasculature, not only through VEGFR2-mediated vasculotrophism, but also through modulation of local complement proteins that could protect against complement-mediated damage. Though further study is warranted, these findings could be relevant for patients receiving VEGF antagonists.
A number of genetic diseases are a result of missense mutations in protein structure. These mutations can lead to severe protein destabilization and misfolding. The unfolding mutation screen (UMS) is a computational method that calculates unfolding propensities for every possible missense mutation in a protein structure. The UMS validation demonstrated a good agreement with experimental and phenotypical data. 15 protein structures (a combination of homology models and crystal structures) were analyzed using UMS. The standard and clustered heat maps, and patterned protein structure from the analysis were stored in a UMS library. The library is currently composed of 15 protein structures from 14 inherited eye diseases including retina degenerations, glaucoma, and cataracts, and contains data for 181,110 mutations. The UMS protein library introduces 13 new human models of eye disease related proteins and is the first collection of the consistently calculated unfolding propensities, which could be used as a tool for the express analysis of novel mutations in clinical practice, next generation sequencing, and genotype-to-phenotype relationships in inherited eye disease.
Exosomes contain proteins, lipids, RNA, and DNA that mediate intercellular signaling. Exosomes can contribute to the pathological processes of various diseases, although their roles in ocular diseases are unclear. We aimed to isolate exosomes from tear fluids (TF) of patients with Thyroid eye disease (TED) and analyze the exosomal proteins. TFs were collected from eight patients with TED and eight control subjects. The number of TF exosomes were measured using nanoparticle-tracking analysis. The expression of specific proteins in the purified exosome pellets were analyzed using a Proteome Profiler Array Kit. Cultured normal orbital fibroblasts were incubated with TF exosomes from patients with TED and control subjects, and changes in inflammatory cytokine levels were compared. TF exosomes from TED patients showed more exosomes than the control subjects. The expression levels of exosomal proteins vitamin D-binding (VDB) protein, C-reactive protein (CRP), chitinase 3-like 1 (CHI3L1), matrix metalloproteinase-9 (MMP-9), and vascular adhesion molecule-1 (VCAM-1) were significantly increased in patients with TED, compared to those of controls. Orbital fibroblasts exposed to TF exosomes from patients with TED showed significantly higher levels of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) production than those treated with control TF exosomes. Specific proteins showed higher expression in exosomes from TED patients, implying that they may play keys roles in TED pathogenesis.
Infection by Leptospira interrogans has been causally associated with human and equine uveitis. Studies in our laboratories have demonstrated that leptospiral lipoprotein LruA and LruB are expressed in the eyes of uveitic horses, and that antibodies directed against LruA and LruB react with equine lenticular and retinal extracts, respectively. These reactivities were investigated further by performing immunofluorescent assays on lenticular and retinal tissue sections. Incubation of lens tissue sections with LruA-antiserum and retinal sections with LruB-antiserum resulted in positive fluorescence. By employing two-dimensional gel analyses followed by immunoblotting and mass spectrometry, lens proteins cross-reacting with LruA antiserum were identified to be alpha-crystallin B and vimentin. Similarly, mass spectrometric analyses identified beta-crystallin B2 as the retinal protein cross-reacting with LruB-antiserum. Purified recombinant human alpha-crystallin B and vimentin were recognized by LruA-directed antiserum, but not by control pre-immune serum. Recombinant beta-crystallin B2 was likewise recognized by LruB-directed antiserum, but not by pre-immune serum. Moreover, uveitic eye fluids contained significantly higher levels of antiibodies that recognized alpha-crystallin B, beta-crystallin B2 and vimentin than did normal eye fluids. Our results indicate that LruA and LruB share immuno-relevant epitopes with eye proteins, suggesting that cross-reactive antibody interactions with eye antigens may contribute to immunopathogenesis of Leptospira-associated recurrent uveitis.
The transparent and refractive properties of the ocular lens are dependent on its precise cellular structure, supported by the regulation of lens cellular processes of proliferation and differentiation that are essential throughout life. The ERK/MAPK-signalling pathway plays a crucial role in regulating lens cell proliferation and differentiation, and in turn is regulated by inhibitory molecules including the Spred family of proteins to modulate and attenuate the impact of growth factor stimulation. Given Spreds are strongly and distinctly expressed in lens, along with their established inhibitory role in a range of different tissues, we investigated the role these antagonists play in regulating lens cell proliferation and differentiation, and their contribution to lens structure and growth. Using established mice lines deficient for either or both Spred 1 and Spred 2, we demonstrate their role in regulating lens development by negatively regulating ERK1/2 activity. Mice deficient for both Spred 1 and Spred 2 have impaired lens and eye development, displaying irregular lens epithelial and fibre cell activity as a result of increased levels of phosphorylated ERK1/2. While Spred 1 and Spred 2 do not appear to be necessary for induction and early stages of lens morphogenesis (prior to E11.5), nor for the formation of the primary fibre cells, they are required for the continuous embryonic growth and differentiation of the lens.
Sleep has a pivotal role in learning-memory and sleep deprivation (SD) negatively affects synaptic functioning. Cytidine-5-diphosphocholine (Citicoline) has been known to improve learning and memory functions. Our objective was to explore the effects of Citicoline on hippocampal and cortical synaptic proteins in rapid eye movement (REM) sleep-deprived rats.
Ranibizumab is a recombinant VEGF-A antibody used to treat the wet form of age-related macular degeneration. It is intravitreally administered to ocular compartments, and the treatment requires frequent injections, which may cause complications and patient discomfort. To reduce the number of injections, alternative treatment strategies based on relatively non-invasive ranibizumab delivery are desired for more effective and sustained release in the eye vitreous than the current clinical practice. Here, we present self-assembled hydrogels composed of peptide amphiphile molecules for the sustained release of ranibizumab, enabling local high-dose treatment. Peptide amphiphile molecules self-assemble into biodegradable supramolecular filaments in the presence of electrolytes without the need for a curing agent and enable ease of use due to their injectable nature-a feature provided by shear thinning properties. In this study, the release profile of ranibizumab was evaluated by using different peptide-based hydrogels at varying concentrations for improved treatment of the wet form of age-related macular degeneration. We observed that the slow release of ranibizumab from the hydrogel system follows extended- and sustainable release patterns without any dose dumping. Moreover, the released drug was biologically functional and effective in blocking the angiogenesis of human endothelial cells in a dose-dependent manner. In addition, an in vivo study shows that the drug released from the hydrogel nanofiber system can stay in the rabbit eye's posterior chamber for longer than a control group that received only a drug injection. The tunable physiochemical characteristics, injectable nature, and biodegradable and biocompatible features of the peptide-based hydrogel nanofiber show that this delivery system has promising potential for intravitreal anti-VEGF drug delivery in clinics to treat the wet form age-related macular degeneration.
The SIX family of homeodomain-containing DNA-binding proteins play crucial roles in both Drosophila and vertebrate retinal specification. In flies, three such family members exist, but only two, Sine oculis (So) and Optix, are expressed and function within the eye. In vertebrates, the homologs of Optix (Six3 and Six6) and probably So (Six1 and Six2) are also required for proper eye formation. Depending upon the individual SIX protein and the specific developmental context, transcription of target genes can either be activated or repressed. These activities are thought to occur through physical interactions with the Eyes absent (Eya) co-activator and the Groucho (Gro) co-repressor, but the relative contribution that each complex makes to overall eye development is not well understood. Here, we attempt to address this issue by investigating the role that each complex plays in the induction of ectopic eyes in Drosophila. We fused the VP16 activation and Engrailed repressor domains to both So and Optix, and attempted to generate ectopic eyes with these chimeric proteins. Surprisingly, we find that So and Optix must initially function as transcriptional repressors to trigger the formation of ectopic eyes. Both factors appear to be required to repress the expression of non-retinal selector genes. We propose that during early phases of eye development, SIX proteins function, in part, to repress the transcription of non-retinal selector genes, thereby allowing induction of the retina to proceed. This model of repression-mediated induction of developmental programs could have implications beyond the eye and might be applicable to other systems.
Parkinson´s disease is characterized by the accumulation of proteinaceous aggregates in Lewy bodies and Lewy Neurites. The main component found in such aggregates is α-synuclein. Here, we investigate how bovine eye lens crystallin proteins influence the aggregation kinetics of α-synuclein at mildly acidic pH (5.5) where the underlying aggregation mechanism of this protein is dominated by secondary nucleation of monomers on fibril surface providing an autocatalytic amyloid amplification process. Bovine α-, βH- and γB-crystallins were found to display chaperone-like activity inhibiting α-synuclein aggregation. This effect was shown to be time-dependent, with early additions of α-crystallin capable of retarding and even inhibiting aggregation during the time frame of the experiment. The inhibitory nature of crystallins was further investigated using trap and seed kinetic experiments. We propose crystallins interact with mature α-synuclein fibrils, possibly binding along the surfaces and at fibril free ends, inhibiting both elongation and monomer-dependent secondary nucleation processes in a mechanism that may be generic to some chaperones that prevent the onset of protein misfolding related pathologies.
Post-translational modification (PTM) of lens proteins is believed to play various roles in age-related lens function and development. Among the different types of PTM, phosphorylation is most noteworthy to play a major role in the regulation of various biosignaling pathways in relation to metabolic processes and cellular functions. The present study reported the quantitative analysis of the in vivo phosphoproteomics profiles of human normal and cataractous lenses with the aim of identifying specific phosphorylation sites which may provide insights into the physiologic significance of phosphorylation in relation to cataract formation.
Fragile X syndrome (FXS) is the most common form of hereditary mental retardation. FXS patients have a deficit for the fragile X mental retardation protein (FMRP) that results in abnormal neuronal dendritic spine morphology and behavioral phenotypes, including sleep abnormalities. In a Drosophila model of FXS, flies lacking the dfmr1 protein (dFMRP) have abnormal circadian rhythms apparently as a result of altered clock output. In this study, we present biochemical and genetic evidence that dFMRP interacts with a known clock output component, the LARK RNA-binding protein. Our studies demonstrate physical interactions between dFMRP and LARK, that the two proteins are present in a complex in vivo, and that LARK promotes the stability of dFMRP. Furthermore, we show genetic interactions between the corresponding genes indicating that dFMRP and LARK function together to regulate eye development and circadian behavior.
The chemical nature of the non-tryptophan (non-Trp) fluorescence of porcine and human eye lens proteins was identified by Mass Spectrometry (MS) and Fluorescence Steady-State and Lifetime spectroscopy as post-translational modifications (PTM) of Trp and Arg amino acid residues. Fluorescence intensity profiles measured along the optical axis of human eye lenses with age-related nuclear cataract showed increasing concentration of fluorescent PTM towards the lens centre in accord with the increased optical density in the lens nucleolus. Significant differences between fluorescence lifetimes of "free" Trp derivatives hydroxytryptophan (OH-Trp), N-formylkynurenine (NFK), kynurenine (Kyn), hydroxykynurenine (OH-Kyn) and their residues were observed. Notably, the lifetime constants of these residues in a model peptide were considerably greater than those of their "free" counterparts. Fluorescence of Trp, its derivatives and argpyrimidine (ArgP) can be excited at the red edge of the Trp absorption band which allows normalisation of the emission spectra of these PTMs to the fluorescence intensity of Trp, to determine semi-quantitatively their concentration. We show that the cumulative fraction of OH-Trp, NFK and ArgP emission dominates the total fluorescence spectrum in both emulsified post-surgical human cataract protein samples, as well as in whole lenses and that this correlates strongly with cataract grade and age.
There are no reliable biomarkers to predict thyroid eye disease (TED) in patients with autoimmune thyroid disease (AITD) currently. Several evidences support the involvement of the lacrimal gland in TED. The aim of our study was to quantitatively correlate the changes in tear protein profile with increasing severity of TED. Tear samples were collected from four groups of patients; AITD without TED (AITD), AITD with mild TED (mild TED), AITD with severe TED (severe TED) and normal controls. A total of 72 patients were recruited for the study. In discovery phase, isobaric tags for relative and absolute quantification (iTRAQ) 4-plex was used for quantitative proteomics analysis. For verification of results from discovery phase, sequential window acquisition of all theoretical fragment ion spectra (SWATH) was used to analyze an independent cohort from normal controls, AITD, mild TED and severe TED. Two proteins, S100A4 and PIP showed consistent dysregulation trends in the discovery and validation phase experiments. Our study demonstrated the differences in tear proteome across the spectrum of different severity and activity of TED in patients with AITD. Two tear proteins, S100A4 and PIP may serve as potential biomarkers to predict progression to severe TED in patients with AITD.
Phosphorylation is an important post-translational modification for the cellular regulation of various biosignaling pathways. We have identified in vivo phosphorylation sites of various lens proteins including especially the major structural proteins of the crystallin family from porcine eye lenses by means of two-dimensional gel electrophoresis (2-DE) or immobilized metal affinity chromatography (IMAC) followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).
Most studies about dry eye disease (DED) chose unilateral eye for investigation and drew conclusions based on monocular results, whereas most studies involving tear proteomics were based on the results of pooling tears from a group of DED patients. Patients with DED were consecutively enrolled for binocular clinical tests, tear biochemical markers of DED, and tear proteome. We found that bilateral eyes of DED patients may have similar but different ocular surface performance and tear proteome. Most ocular surface homeostatic markers and tear biomarkers were not significantly different in the bilateral eyes of DED subjects, and most clinical parameters and tear biomarkers were correlated significantly between bilateral eyes. However, discrepant binocular presentation in the markers of ocular surface homeostasis and the associations with tear proteins suggested that one eye's performance cannot represent that of the other eye or both eyes. Therefore, in studies for elucidating tear film homeostasis of DED, we may lose some important messages hidden in the fellow eye if we collected clinical and proteomic data only from a unilateral eye. For mechanistic studies, it is recommended that researchers collect tear samples from the eye with more severe DED under sensitive criteria for identifying the more severe eye and evaluating the tear biochemical and proteomic markers with binocular concordance drawn in prior binocular studies.
Genes that code for proteins involved in organelle biogenesis and intracellular trafficking produce products that are critical in normal cell function . Conserved orthologs of these are present in most or all eukaryotes, including Drosophila melanogaster Some of these genes were originally identified as eye color mutants with decreases in both types of pigments found in the fly eye. These criteria were used for identification of such genes, four eye color mutations that are not annotated in the genome sequence: chocolate, maroon, mahogany, and red Malpighian tubules were molecularly mapped and their genome sequences have been evaluated. Mapping was performed using deletion analysis and complementation tests. chocolate is an allele of the VhaAC39-1 gene, which is an ortholog of the Vacuolar H+ ATPase AC39 subunit 1. maroon corresponds to the Vps16A gene and its product is part of the HOPS complex, which participates in transport and organelle fusion. red Malpighian tubule is the CG12207 gene, which encodes a protein of unknown function that includes a LysM domain. mahogany is the CG13646 gene, which is predicted to be an amino acid transporter. The strategy of identifying eye color genes based on perturbations in quantities of both types of eye color pigments has proven useful in identifying proteins involved in trafficking and biogenesis of lysosome-related organelles. Mutants of these genes can form the basis of valuable in vivo models to understand these processes.
Age-related macular degeneration (AMD), in which choroidal neovascularization (CNV) affects the center of the retina (macula), leads to the irreversible visual loss. The intravitreal injection of anti-angiogenesis antibodies improved the prognosis of AMD, but relatively less invasive therapies should be explored. In the present study, we show that a high-density lipoprotein (HDL) mutant is a therapeutically active drug carrier capable of treating a posterior eye disease in mice via instillation. Various HDL mutants were prepared with apoA-I proteins fused with different cell-penetrating peptides (CPPs) and phospholipids with different alkyl chain lengths; their sizes were further controlled in the range of 10-25nm. They were screened based on the efficiency of fluorescent dye delivery to the inner retinal layer in mice. The best mutant was found to have penetratin (PEN) as a CPP, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and a size of 15nm. In preclinical studies on a laser-induced CNV murine model, 1week of instillation of the best mutant carrying the anti-angiogenesis drug pazopanib had dramatic therapeutic effects in reducing the CNV size. Importantly, the HDL mutant by itself contributed to the therapeutic effects. Future clinical trials for treating AMD with instillation of the HDL mutant are expected.
Simultaneous determination of proteins with micrometric resolution is a significant challenge. In this study, laser ablation (LA) inductively coupled plasma - mass spectrometry (ICP-MS) was employed to quantify the distribution of proteins associated to the eye disease age-related macular degeneration (AMD) using antibodies labelled with three different metal nanoclusters (MNCs). PtNCs, AuNCs and AgNCs contain hundreds of metal atoms and were used to detect metallothionein 1/2 (MT1/2), complement factor H (CFH) and amyloid precursor protein (APP) in retina, ciliary body, retinal pigment epithelium (RPE), choroid and sclera from human cadaveric eye sections. First, the labelling of MNCs bioconjugated primary antibodies (Ab) was optimised following an immunolabelling protocol to avoid the non-specific interaction of MNCs with the tissue. Then, the LA and ICP-MS conditions were studied to obtain high-resolution images for the simultaneous detection of the three labels at the same tissue section. A significant signal amplification was found when using AuNCs, AgNCs and PtNCs labelled Ab of 310, 723 and 1194 respectively. After the characterisation of MNCs labelled immunoprobes, the Ab labelling was used for determination of MT1/2, CFH and APP in the RPE-choroid-sclera, where accumulation of extracellular deposits related to AMD was observed. Experimental results suggest that this method is fully suitable for the simultaneous detection of at least three different proteins.
Endocytosis is a multi-step process involving a large number of proteins, both general factors, such as clathrin and adaptor protein complexes, and unique proteins, which modulate specialized endocytic processes, like the EHD proteins. EHDs are a family of Eps15 Homology Domain containing proteins that consists of four mammalian homologs, one C. elegans, one Drosophila melanogaster and two plants orthologs. These membrane-associated proteins are involved in different steps of endocytic trafficking pathways. We have previously shown that the Drosophila EHD ortholog, PAST1, associates predominantly with the plasma membrane. Mutations in Past1 result in defects in endocytosis, male sterility, temperature sensitivity and premature death of the flies. Also, Past1 genetically interacts with Notch. In the present study, we investigated the role of PAST1 in the developing fly eye. In mutant flies lacking PAST1, abnormal differentiation of photoreceptors R1, R6 and R7 was evident, with partial penetrance. Likewise, five cone cells were present instead of four. Expression of transgenic PAST1 resulted in a dominant negative effect, with a phenotype similar to that of the deletion mutant, and appearance of additional inter-ommatidial pigment cells. Our results strongly suggest a role for PAST1 in differentiation of photoreceptors R1/R6/R7 and cone cells of the fly ommatidia.
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) imaging following in situ enzymatic digestion is a versatile analytical method for the untargeted investigation of protein distributions, which has rarely been used for plants so far. The present study describes a workflow for in situ tryptic digestion of plant seed tissue for MALDI MS imaging. Substantial modifications to the sample preparation procedure for mammalian tissues were necessary to cater to the specific properties of plant materials. For the first time, distributions of tryptic peptides were successfully visualized in plant tissue using MS imaging with accurate mass detection. Sixteen proteins were visualized and identified in chickpea seeds showing different distribution patterns, e.g., in the cotyledons, radicle, or testa. All tryptic peptides were detected with a mass resolution higher than 60,000 as well as a mass accuracy better than 1.5 ppm root-mean-square error and were matched to results from complementary liquid chromatography-MS/MS (LC-MS/MS) data. The developed method was also applied to crab's eye vine seeds for targeted MS imaging of the toxic protein abrin, showing the presence of abrin-a in all compartments. Abrin (59 kDa), as well as the majority of proteins visualized in chickpeas, was larger than 50 kDa and would thus not be readily accessible by top-down MS imaging. Since antibodies for plant proteins are often not readily available, in situ digestion MS imaging provides unique information, as it makes the distribution and identification of larger proteins in plant tissues accessible in an untargeted manner. This opens up new possibilities in the field of plant science as well as to assess the nutritional quality and/or safety of crops.
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