June 2015
Volume 56, Issue 7
ARVO Annual Meeting Abstract  |   June 2015
missense mutations induce the endoplasmic reticulum unfolded protein response (ER-UPR) stress complex.
Author Affiliations & Notes
  • Glenn Lobo
    Ophthalmic Research, Cleveland Clinic, Cleveland, OH
  • Adrian Au
    Ophthalmic Research, Cleveland Clinic, Cleveland, OH
  • Lindsey A Ebke
    Ophthalmic Research, Cleveland Clinic, Cleveland, OH
  • Stephanie A Hagstrom
    Ophthalmic Research, Cleveland Clinic, Cleveland, OH
    Ophthalmology, Case Western Reserve University, Cleveland, OH
  • Footnotes
    Commercial Relationships Glenn Lobo, None; Adrian Au, None; Lindsey Ebke, None; Stephanie Hagstrom, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 5394. doi:
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      Glenn Lobo, Adrian Au, Lindsey A Ebke, Stephanie A Hagstrom; missense mutations induce the endoplasmic reticulum unfolded protein response (ER-UPR) stress complex.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5394.

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

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Purpose: Mutations in the TULP1 gene are associated with early-onset retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). However, the pathologic mechanism causing photoreceptor cell death remains unknown. We hypothesize that TULP1 mutations produce misfolded protein products that accumulate in the endoplasmic reticulum (ER) and induce cellular apoptosis via the unfolded protein response (UPR) pathway.

Methods: Protein stability of missense TULP1 mutations were evaluated using the programs PolyPhen, SIFT, I-Mutant and RaptorX. Full-length TULP1 was amplified from human retinal RNA and cloned into the mammalian expression vector pEGFP-N1. The pTULP1-wt-GFP plasmid was used as a template to engineer each of the TULP1 mutations (R420P, I459K, F491L) by site directed mutagenesis. hTERT-RPE-1 cells were transiently transfected with 3 µg of individual purified DNA plasmids. Six days after transfection, subcellular localization patterns of the GFP-tagged TULP1 proteins were achieved by confocal imaging. Total protein from transfected hTERT-RPE-1 cells was isolated for Western blot analysis.

Results: In-silico analyses of known TULP1 missense mutations predict the mutant proteins will be unstable and misfolded under physiological conditions. Confocal microscopy revealed that GFP-tagged wt TULP1 localized predominantly to the cytoplasm and plasma membrane. In contrast, all three mutant TULP1 proteins showed cytoplasmic punctate staining which co-localized with the ER using ER tracker. Western blot analysis of cells expressing mutant TULP1 proteins revealed an induction of the three key ER stress response proteins, BiP, CHOP and phospho-PERK.

Conclusions: Our in-silico and in-vitro analyses suggest that mutant TULP1 proteins accumulate within the ER leading to induction of the UPR stress response complex as the pathologic mechanism causing photoreceptor degeneration. However, in vivo models are required to further validate this mechanistic pathway and allow for investigation of therapeutics capable of modulating the UPR to aid in the attenuation of TULP1-induced photoreceptor cell death.


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