title: Mapping wild-type and R345W fibulin-3 intracellular interactomes.
abstract: Fibulin-3 (F3) is an important, disulfide-rich, extracellular matrix glycoprotein that has been associated with a number of diseases ranging from cancer to retinal degeneration. An Arg345Trp (R345W) mutation in F3 causes the rare, autosomal dominant macular dystrophy, Malattia Leventinese. The purpose of this study was to identify and validate novel intracellular interacting partners of wild-type (WT) and R345W F3 in retinal pigment epithelium cells. We used stable isotope labeling by amino acids in cell culture (SILAC) to generate 'heavy' and 'light' isotopically labeled ARPE-19 cell populations which were subsequently infected with adenovirus encoding for FLAG-tagged WT or R345W F3. After immunoprecipitation, interacting proteins were identified by multidimensional protein identification technology (MudPIT). We identified sixteen new intracellular F3 interacting partners, the vast majority of which are involved in protein folding and/or degradation in the endoplasmic reticulum (ER). Eight of these interactions (ANXA5, ERdj5, PDIA4, P4HB, PDIA6, RCN1, SDF2L1, and TXNDC5) were verified at the western blotting level. These F3 interactome results can serve as the basis for pursuing targeted genetic or pharmacologic approaches in an effort to alter the fate of either WT or mutant F3.
authors: Department of Ophthalmology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9057, USA; Department of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA. Electronic address: John.Hulleman@UTSouthwestern.edu.
authors: John D
authors: JD
authors: Hulleman
authors: Department of Chemistry, University of California Riverside, 900 University Ave, Riverside, CA 92521, USA.
authors: Joseph C
authors: JC
authors: Genereux
authors: Department of Ophthalmology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9057, USA.
authors: Annie
authors: A
authors: Nguyen
mesh_terms: D004247:DNA / Q000235:genetics*; D004252:DNA Mutational Analysis; D004721:Endoplasmic Reticulum / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000235:genetics* / Q000378:metabolism; D005838:Genotype; D006801:Humans; D009154:Mutation; D017510:Protein Folding; D012160:Retina / Q000378:metabolism; D012162:Retinal Degeneration / Q000235:genetics / Q000378:metabolism*
chemical_list: C119630:EFEMP1 protein, human; D016326:Extracellular Matrix Proteins; D004247:DNA
keywords: Endoplasmic reticulum; Fibulin-3; Malattia Leventinese; Mass spectrometry; MudPIT; SILAC