September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Mutant Bestrophin-1 (BEST1) is degraded via the endo-lysosomal pathway
Author Affiliations & Notes
  • Andrea Milenkovic
    Institute of Humangenetics, University of Regensburg, Regensburg, Germany
  • Bernhard HF Weber
    Institute of Humangenetics, University of Regensburg, Regensburg, Germany
  • Footnotes
    Commercial Relationships   Andrea Milenkovic, None; Bernhard Weber, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 6591. doi:
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    • Get Citation

      Andrea Milenkovic, Bernhard HF Weber; Mutant Bestrophin-1 (BEST1) is degraded via the endo-lysosomal pathway. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6591.

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

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Purpose : Bestrophin-1 (BEST1), an integral membrane protein in the basolateral aspect of the RPE, is thought to act as a volume-regulated anion channel. Mutations in BEST1 cause Best vitelliforme macular dystrophy (BVMD) and are assumed to give rise to trafficking problems prompting the protein to remain within so far undefined cellular compartments. The precise molecular pathomechanisms of the disease-associated mutations are still unclear.

Methods : Protein half-life was determined in polarized MDCKII cell lines, constitutively expressing wildtype BEST1 and seven disease-associated mutants. Cultivated cells were treated with cycloheximide (CHX, 20µg/ml) and harvested at various time points. Protein lysates were analyzed by densitometry of Western blot signals. To examine degradation pathways, cells were treated with CHX and a series of inhibitors for the proteasomal, lysosomal and autophagy pathway. BEST1 was localized by immunocytochemistry.

Results : Normal BEST1 and BEST1-R218C revealed localization to the plasma membrane (PM) in contrast to intracellular localization of BEST1 mutants T6P, L21V, W93C, L224M, Y227N and F305S. Protein level of wildtype BEST1 remains stable even after 24h CHX treatment and arrest of protein synthesis, whereas six out of seven mutants degraded within 3 hours and mutant R218C within 12h. Similar to the majority of integral PM proteins, wildtype BEST1 is degraded via the endo-lysosomal pathway, inhibited by ammonium chloride (20mM) and chloroquine (10µM). Unexpectedly, all mutants tested do not reveal degradation via the proteasomal but instead via the endo-lysosomal degradation pathway. Consequently, low temperature (26°C) for 36h failed to foster trafficking of mutant BEST1 protein to the cell surface as described for many endoplasmic reticulum (ER)-retained mutants.

Conclusions : Our data support the hypothesis that loss of BEST1 function is caused by a decrease in mutant protein stability with instable, temperature-insensitive mutant BEST1 degraded via the endo-lysosomal pathway. As a consequence, a chaperon-mediated rescue as shown for many mutants in disorders like CFTR, Parkinson Disease and Alzheimer disease may not lead to success in BVMD.

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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