Proteome analysis of lipofuscin in human retinal pigment epithelial cells

Schütt, F.; Ueberle, Barbara; Schnölzer, Martina; Holz, Frank G.; Kopitz, Jürgen

doi:10.1016/s0014-5793(02)03312-4

Cited by 90 publications

(76 citation statements)

References 19 publications

(34 reference statements)

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“…Although our results indicate that lipofuscin granules contain only a small amount of protein, multiple reports of protein compositions for human RPE lipofuscin have contributed to a long standing misconception that lipofuscin contains substantial protein (6,7). The resulting confusion highlights the importance of sample preparation for proteomics studies.…”

Section: Fig 4 Phototoxicity Of Lipofuscinmentioning

confidence: 67%

“…Estimates suggest that lipofuscin contains 30 -70% protein depending upon tissue, species, and study (2,6) and that this protein may contribute to pathogenesis. From 2002 to 2007 proteomics investigations have reported protein compositions for RPE lipofuscin (6,7) and RPE melanolipofuscin (8,9). Oxidative protein modifications have also been associated with lipofuscin (6,7,10), but studies to date have not demonstrated that the identified proteins and oxidative modifications are from the granules rather than from the contaminating material that co-purifies with the gran-ules.…”

mentioning

confidence: 99%

“…From 2002 to 2007 proteomics investigations have reported protein compositions for RPE lipofuscin (6,7) and RPE melanolipofuscin (8,9). Oxidative protein modifications have also been associated with lipofuscin (6,7,10), but studies to date have not demonstrated that the identified proteins and oxidative modifications are from the granules rather than from the contaminating material that co-purifies with the gran-ules. Because in vitro bioactivity studies of lipofuscin have also utilized preparations that contain extragranular contaminants, a clear understanding of the source of the bioactivities demonstrated in these studies remains elusive.…”

mentioning

confidence: 99%

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Retinal Pigment Epithelium Lipofuscin Proteomics

Gugiu

Renganathan

et al. 2008

Molecular & Cellular Proteomics

151

133

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Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein (ϳ2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E 2 adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.

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Section: Fig 4 Phototoxicity Of Lipofuscinmentioning

confidence: 67%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Retinal Pigment Epithelium Lipofuscin Proteomics

Gugiu

Renganathan

et al. 2008

Molecular & Cellular Proteomics

151

133

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“…Thus, to fully characterize this fraction, Schut et al performed a proteome analysis of sucrose-puriied RPE granules [30]. They identiied 65 abundant cellular proteins, which included structural, metabolic, mitochondrial, chaperone, transmembrane, and signaling transduction proteins.…”

Section: Rpe Lipofuscin Accumulationmentioning

confidence: 99%

Lipofuscin Accumulation into and Clearance from Retinal Pigment Epithelium Lysosomes: Physiopathology and Emerging Therapeutics

Nociari¹,

Kiss²,

Rodríguez-Boulan³

2017

Lysosomes - Associated Diseases and Methods to Study Their Function

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Photoreceptors undergo a constant renewal of their light sensitive outer segments (POSs). In the renewal process, 10% of the POS mass is daily phagocytized by the adjacent retinal pigment epithelium (RPE). POS contain vast amounts of 11-cis retinal and alltrans-retinal, two highly reactive vitamin A aldehydes that spontaneously dimerize into lipid bisretinoids (LBs) and accumulate into RPE lysosomes during phagocytosis. As LBs are refractory to lysosomal hydrolases and RPE cells do not divide, this accumulation is irreversible and results in the formation of lipofuscin granules. Lipofuscin accumulation is toxic for RPE cells through a variety of light-dependent and light-independent mechanisms. Beyond a threshold, RPE cells die resulting in secondary loss of overlying photoreceptors. Currently, there are no efective treatments for retinal disorders associated with genetic or age-associated LB accumulation, such as Stargardt disease and age-related macular degeneration (AMD). Thus, there is a great need for medical interventions. Here, we discuss the current understanding of lipofuscin's pathogenicity and the status of diferent strategies under development to promote LB elimination from RPE lysosomes.Keywords: lipofuscin, Stargardt, age-related macular degeneration (AMD), bisretinoids, retinal pigment epithelium (RPE), cyclodextrins, cellular clearance, TFEB, lysosome IntroductionTo understand the origin and consequences of the lysosomal accumulation of lipofuscin in the eye, a basic knowledge of retinal function and organization is required.© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The retinal pigment epithelium (RPE) in vertebrate's eyesLight entering the eye gets refracted by the cornea and lens on the neural retina, where photoreceptors (PR) convert photons into a cascade of chemical and electrical events that propagate to second-order (horizontal, bipolar, and amacrine cells) and third-order (ganglion cells) retinal neurons, which distribute this information to various visual centers of the brain through the ibers of the optic nerve. The bodies of PR cells, rods and cones, display three sectors (Figure 1): the outer segment, illed with stacks of disks densely packed with light-sensitive photopigment; the inner segment, illed with genetic, biosynthetic, and metabolic organelles Lysosomes -Associated Diseases and Methods to Study Their Function 4(nucleus, endoplasmic reticulum, Golgi complex, ribosomes, mitochondria); and the synaptic terminal that connects with bipolar neurons of the retina. In vertebrates, the retina is inverted in the sense that light passes through secondary and tertiary neuronal layers in the inner retina before reaching the rods and cones in the outer retina (Figure 1). Photoreceptors are metabolically very active cells that req...

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“…Analysis of the cell surface proteome of cancer cells of a variety of histologies has revealed an abundance of proteins with chaperone function such as GRP78, HSP70 and protein disulfide isomerase. 104 Organellar proteomics Systematic analysis of the proteins expressed by specific intracellular organelles such as the cancercell mitochondria, 105 lymphocyte-derived exosomes, 7 the lipofuscin in human retinal pigment epithelial cells, 106 the phagosome, 107 microsomes from cancer cells, 108 lipid rafts 109 and human nucleolus 110 have provided extensive insights into the organellar proteomes that was previously impossible to obtain. These studies also demonstrate that even intracellular organelles as small as nucleoli are much more complicated in their protein expression profiles, and express hundreds of proteins some of which are not previously associated with their function.…”

Section: Secretomementioning

confidence: 99%