May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Aquaporin-4 Is a Target for Ubiquitin-Proteasomal Degradation
Author Affiliations & Notes
  • A. Dibas
    Pharmacology & Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas
  • M.-H. Yang
    Texas Christian University, Fort Worth, Texas
  • J. Bobich
    Texas Christian University, Fort Worth, Texas
  • T. Yorio
    Pharmacology & Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas
  • Footnotes
    Commercial Relationships  A. Dibas, None; M. Yang, None; J. Bobich, None; T. Yorio, None.
  • Footnotes
    Support  None.
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 3169. doi:https://doi.org/
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      A. Dibas, M.-H. Yang, J. Bobich, T. Yorio; Aquaporin-4 Is a Target for Ubiquitin-Proteasomal Degradation. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3169. doi: https://doi.org/.

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

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Abstract

Purpose: : To understand mechanisms involved in regulating the fate of Aquaporin-4, a key channel regulating the formation of aqueous humor formation in ciliary epithelium and astrogliosis in optic nerve head astrocytes.

Methods: : Changes in cell volume was assessed following excitation of fura-2-labeled cells at 360 nm. Western blotting using anti-aquaporin-4 antibodies was used to follow changes in AQP4 in human primary brain astrocytes and U373MG astrocytoma cell-line. Real-time PCR was used for measuring changes in AQP4 and beta-actin following hypotonic shock and inhibition of the ubiquitin proteasomal system. Immunoprecipitation was performed using monoclonal anti-AQP4. Affinity purification of ubiquitinated proteins was performed using S5a column.

Results: : Mg132, a proteasomal inhibitor, increased AQP4 protein levels in primary human brain astrocytes and U373MG an astrocytoma cell-line without significantly affecting mRNA as judged by Q-PCR. Immunoblotting using rabbit anti-AQP4 antibodies showed the presence of several 30-37 KDa proteins and higher molecular weight proteins (87 and 100 KDa). Affinity purified ubiquitinated proteins on a S5a column revealed the presence of an 87 and 100 KDa AQP4 isoforms. In addition, two subunits of the 19S regulatory complex of the ubiquitin proteasome system (UPS): S6’ (Rpt5), and S5a (Rpn10), whose recognize polyubiquitinated substrates of the proteasome and, ubiquitin carboxy-terminal hydrolase L1 (UCH-L1, deubiquitinating enzyme for recycling of ubiquitin) are detected in AQP4 immunoprecipitated fractions. Mg132 treatment enhanced the rate of cellular swelling after a hypotonic shock due in part to increased presence of AQP4 proteins.

Conclusions: : This is the first report showing that AQP4 is a target for degradation by the proteasomal system. The regulation of AQP4 levels by the proteasome may provide insight into novel therapeutic options for reducing aqueous humor formation and astrogliosis.

Keywords: astrocyte • astrocytes: optic nerve head • aqueous 
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