title: Proteomic profiling of human retinal pigment epithelium exposed to an advanced glycation-modified substrate.
abstract: OBJECTIVE
  The retinal pigment epithelium (RPE) and underlying Bruch's membrane undergo significant modulation during ageing. Progressive, age-related modifications of lipids and proteins by advanced glycation end products (AGEs) at this cell-substrate interface have been implicated in RPE dysfunction and the progression to age-related macular degeneration (AMD). The pathogenic nature of these adducts in Bruch's membrane and their influence on the overlying RPE remains unclear. This study aimed to identify alterations in RPE protein expression in cells exposed to AGE-modified basement membrane (AGE-BM), to determine how this "aged" substrate impacts RPE function and to map the localisation of identified proteins in ageing retina.
  
  
  METHODS
  Confluent ARPE-19 monolayers were cultured on AGE-BM and native, non-modified BM (BM). Following 28-day incubation, the proteome was profiled using 2-dimensional gel electrophoresis (2D), densitometry and image analysis was employed to map proteins of interest that were identified by electrospray ionisation mass spectrometry (ESI MS/MS). Immunocytochemistry was employed to localise identified proteins in ARPE-19 monolayers cultured on unmodified and AGE-BM and to analyze aged human retina.
  
  
  RESULTS
  Image analysis detected altered protein spot densities between treatment groups, and proteins of interest were identified by LC ESI MS/MS which included heat-shock proteins, cytoskeletal and metabolic regulators. Immunocytochemistry revealed deubiquitinating enzyme ubiquitin carboxyterminal hydrolase-1 (UCH-L1), which was upregulated in AGE-exposed RPE and was also localised to RPE in human retinal sections.
  
  
  CONCLUSIONS
  This study has demonstrated that AGE-modification of basement membrane alters the RPE proteome. Many proteins are changed in this ageing model, including UCHL-1, which could impact upon RPE degradative capacity. Accumulation of AGEs at Bruch"s membrane could play a significant role in age-related dysfunction of the RPE.
authors.affiliation: School of Medicine, Dentistry and Biomedical Science, Queen's University Belfast, Northern Ireland, UK.
authors.forename: J V
authors.initials: JV
authors.lastname: Glenn
authors.forename: H
authors.initials: H
authors.lastname: Mahaffy
authors.forename: S
authors.initials: S
authors.lastname: Dasari
authors.forename: M
authors.initials: M
authors.lastname: Oliver
authors.forename: M
authors.initials: M
authors.lastname: Chen
authors.forename: M E
authors.initials: ME
authors.lastname: Boulton
authors.forename: H
authors.initials: H
authors.lastname: Xu
authors.forename: W J
authors.initials: WJ
authors.lastname: Curry
authors.forename: Alan W
authors.initials: AW
authors.lastname: Stitt
mesh_terms: D016570:Bruch Membrane / Q000187:drug effects; D002478:Cells, Cultured; D003720:Densitometry; D015180:Electrophoresis, Gel, Two-Dimensional; D005136:Eye Proteins / Q000378:metabolism*; D017127:Glycation End Products, Advanced / Q000494:pharmacology*; D006801:Humans; D007150:Immunohistochemistry; D040081:Protein Array Analysis; D040901:Proteomics; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D043222:Ubiquitin Thiolesterase / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D005136:Eye Proteins; D017127:Glycation End Products, Advanced; C482860:UCHL1 protein, human; D043222:Ubiquitin Thiolesterase
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