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L. Subramanian, P.R. van Ginkel, T.M. Walker, A.S. Polans; Apoptosis-linked Gene-2 (ALG-2)- A Potential Site for Therapeutic Intervention in Retinal Degeneration and Uveal Melanoma . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2844.
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Purpose: To characterize calcium-induced conformational changes leading to the interaction of ALG-2 with AIP1, a putative effector molecule. ALG-2 was identified as a pro-apoptotic calcium-binding protein. In the eye, ALG-2 is expressed predominantly in photoreceptor cells. ALG-2 could play a role in the calcium-mediated apoptosis associated with retinal degeneration. Further, ALG-2 is down-regulated in uveal melanoma cell lines endowing a selective advantage to these tumor cells. Methods: TNS fluorescence to study conformational changes, cross-linking and analytical centrifugation to study dimerization, covalent modification, site-directed mutagenesis and co-immunoprecipitation to study regions of interaction. Results: Cross-linking experiments demonstrate that ALG-2 is present as a dimer in both normal melanocytes and uveal melanoma cells independent of intracellular calcium concentration. This is consistent with in vitro cross-linking results using recombinant ALG-2. Analytical centrifugation in the absence of calcium shows that ALG-2 exists predominantly as a dimer. In the presence of calcium, ALG-2 forms aggregates. Deletion of the N-terminal region results in improved solubility of the protein but does not affect dimerization. TNS fluorescence studies show similar calcium-induced conformational changes with or without the N-terminal region. Covalent modification experiments to identify residues affected by the conformational change are in progress. The N-terminal region, which appears to be involved in the calcium-induced aggregation of ALG-2 could potentially be important for target recognition and interaction. Co-immunoprecipitation experiments combined with site-directed mutagenesis will help delineate the mechanism of interaction at the molecular level. Conclusions: ALG-2 could potentially be involved in two major blinding diseases: retinal degeneration by mediating aberrant levels of calcium and uveal melanoma by down-regulation. Understanding the mechanisms of calcium-induced ALG-2 activation could lead to opportunities for therapeutic intervention.
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