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 (FT) 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.

Clinically-identified C-terminal mutations in fibulin-3 are prone to misfolding and destabilization

“…This approximate region of F3 (amino acids 259-493) has also been shown to bind to tissue inhibitor of matrix metalloproteinase 3 (TIMP3) 44 , a critical extracellular matrix regulatory component that is also associated with Sorsby's Fundus Dystrophy 45 . Failure of F3 to bind to TIMP3 or other known F3 interacting partners 46,47 due to partial misfolding in its C-terminus may ultimately influence its fate in the cell and at the organismal level. Additionally, recent studies have indicated that the C-terminus of F3 may be the source of amyloid fibrils found in the veins of aged individuals 48 , suggesting that this portion of the protein has a propensity for β-sheet formation and aggregation.…”

The Pathophysiological Significance of Fibulin-3

“…Moreover, its many intramolecular disulphide bonds could form abnormally and propagate a detrimental cycle of bond reduction, glutathione consumption, and reactive oxygen species production, which also drives ER stress and apoptosis [ 71 ]. Substantiating a role for mutant fibulin-3-induced ER stress in the pathogenesis of ML is more recent evidence that fibulin-3 has numerous intracellular interacting partners, the majority of which are involved in protein folding or protein degradation in the ER [ 72 ], thus confirming the feasibility that part of the pathological impact of mutant fibulin-3 could be the perturbation of normal cellular proteostasis.…”

Prospective Application of Activity-Based Proteomic Profiling in Vision Research-Potential Unique Insights into Ocular Protease Biology and Pathology

“…Serine protease activity is not only dependent on the abundance of the protease, but also on the absence or presence of endogenous serine protease inhibitors, or serpins, which have been associated with a variety of angiogenesis-related eye diseases (e.g., pigment epithelium derived factor, PEDF) [70], detected in ocular-derived cell lines (e.g., serine protease inhibitor E1, SERPINE1 [a.k.a. heat-shock protein 47, HSP47]) [71], as well as in human tear fluid (e.g., secretory leukocyte protease inhibitor) [72]. Therefore, how the activities of these serine proteases are modulated and play a role in AMD and other eye diseases is still unknown.…”