September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Identification and validation of wild-type and mutant fibulin-3 interacting partners in RPE cells
Author Affiliations & Notes
  • John Hulleman
    Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • Annie Nguyen
    Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • Joseph Genereux
    Chemistry, University of California at Riverside, Riverside, California, United States
  • Footnotes
    Commercial Relationships   John Hulleman, None; Annie Nguyen, None; Joseph Genereux, None
  • Footnotes
    Support  Research to Prevent Blindness Career Development Award
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 5814. doi:
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      John Hulleman, Annie Nguyen, Joseph Genereux; Identification and validation of wild-type and mutant fibulin-3 interacting partners in RPE cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5814.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Fibulin-3 (F3) is a secreted protein which is expressed in the retinal pigment epithelium (RPE). An autosomal dominant R345W mutation in F3 has been associated with a rare retinal dystrophy called Malattia Leventinese, or dominant drusen. This disease is characterized by early-onset, extensive drusen formation. Additional evidence suggests that the wild-type (WT) form of F3 may also impact pathogenic drusen formation in age-related macular degeneration patients. The purpose of this study was to identify and manipulate F3 interacting partners in the hope of gaining insight into how RPE cells fold and secrete WT vs. mutant F3.

Methods : To unbiasedly identify F3 interacting partners, we used a quantitative proteomics approach known as stable isotope labeling by amino acids in cell culture (SILAC) followed by mass spectrometry (MS). ARPE-19 cells were labeled in media with either 'heavy' or 'light' arginine for >8 passages to achieve a fully labeled proteome. Confluent, isotope labeled ARPE-19 cells were infected with low levels of adenovirus encoding for FLAG-tagged WT or R345W F3. Cellular proteins were crosslinked and F3 binding partners were extracted by immunoprecipitation followed by MS. In separate experiments, MS-identified binding partners were confirmed by conventional western blotting. The influence of the identified binding partners on F3 secretion and intracellular steady-state levels were subsequently evaluated by using RNA interference followed by western blotting.

Results : Interestingly, WT and R345W F3 share a number of common intracellular binding partners, yet the stoichiometryof their binding to these partners differs. We confirmed 4 previously identifed F3 interacting partners (i.e., GRP78, GRP94, PDIA3, CALR) and found 18 new intracellular F3 binding partners. A number of the newly identified F3 interacting partners (e.g., P4HB, UGGT1, etc.) belonged to a multi-protein endoplasmic reticulum (ER) chaperone complex. Using siRNA, we were able to confirm that knockdown of the majority of the newly identified F3 interacting proteins resulted in increased secreted and intracellular F3, indicating that they likely play a role in facilitating ER-associated degradation of WT and R345W F3.

Conclusions : Identification of these novel interacting proteins provides a basis for genetically manipulating the folding and secretion of F3 in an attempt to treat certain retinal diseases.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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