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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.
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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.
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.
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.
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.
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