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W.-H. Lee, A. Junk, B. Govindarajan, J. Laird, R. G. Salomon, S. K. Bhattacharya; Calpain-1 Processing Differences in Normal and Diseased Ocular Tissues. Invest. Ophthalmol. Vis. Sci. 2008;49(13):675.
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To determine whether in vitro modification of cysteine proteases and calpains in particular with lipid adduct isolevuglandin E2 (IsoLGE2 ) affects its ubiquitination and degradation by cellular proteasome machinery in the glaucomatous trabecular meshwork (TM) in contrast to controls. Whether there exists expression differences for calpain-1 in TM and the optic nerve.
Enzymatic assays for cysteine proteases and calpains were made using standard substrates and inhibitors and TM tissue extracts. Western analyses were performed on human TM extracts (from 35 donors) with antibodies to IsoLGE2, ubiquitin and proteasome. Calpain-1 was modified in vitro by IsoLGE2. Plugged gel electrophoresis and dynamic light scattering experiments were performed to analyze protein aggregates. The translational differences were probed using Northern Blot and other published protocols.
Lipid peroxidation product modification of calpain-1 was observed in the human glaucomatous TM (but not in the optic nerve). Modified calpain-1 was conjugated and loaded onto the 26S proteosomal machinery resulting in the impairment of the later. In contrast to the TM, elevated levels of calpain-1occur in the glaucomatous optic nerve and is due to translational control.
Calpains are modified by lipid peroxidation products in vivo. In vitro IsoLGE2modification of calpain-1 makes it resistant to protease digestion and impairs proteasome. Calpain-1 overexpression occurs in the optic nerve (and controlled at the level of translation) but not in the TM.
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