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H.Z. Malina, B.E. Frueh; Xanthurenic Acid Changes Signaling of 14–3–3 Proteins and Calmodulin: Involvement in the Diseases Development and Drug Targeting . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5291. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Xanthurenic acid accumulation in the cells leads to unfolded protein formation, translocation of pro–apoptotic proteins into mitochondria and cytochrome c release. The pathological apoptosis is characterized by cell death associated with accumulation of unfolded proteins leading to plaque formation. The signaling proteins like calmodulin and 14–3–3 proteins keep the correct protein network in the cells. Here, we show that xanthurenic acid changes interaction between calmodulin and 14–3–3 proteins leading to cell pathology, and the interaction of these proteins are different in the knockout cells. Methods: Primary retinal pigment epithelial (RPE) cells were isolated from post–mortem eye (37 years old women) harvested for cornea transplantation and murine embryonic fibroblast (MEF) (wide type, Bid–/–, Bax/Bak–/–) were obtained from Dr. S. Korsmeyer, Harvard University, U.S.A. The cell extract from control cells and the cells growing the in the presence of xanthurenic acid were used. Immunoprecipitation, Western blot analysis were applied to study protein–protein interaction. The immunofluorescent staining and confocal microscopy were used to show protein localization. Results: Calmodulin uniformly stained cytoplasm of the primary RPE cells and wide type MEF cells. Antibody against calmodulin binding site of MARCKS protein was prepared and used for immunoprecipitation of RPE and MEF cell extracts. Western blots of the immunoprecipited proteins were probed with calmodulin. In cells growing in the presence of 10 µM xanthurenic acid calmodulin binding site of MARCKS binds strongly and covalently to calmodulin. This was not observed in control cells and the interaction at 20 µM xanthurenic acid was weak. In Bid–/– this covalent binding was not observed at all, and in Bax /Bak –/– this binding was observed in the control cells, but decreased with xanthurenic acid. Xanthurenic acid changes also the interaction of 14–3–3 protein with calmodulin. Xanthurenic acid led to wrong signaling of the proteins and the cell pathology. The rescue of the cell pathology was observed by antibody against the xanthurenic acid–modified protein. Conclusions: Folding of signaling proteins in the knockout cells is different then in the normal cells. A knockout animal is not appropriated as a model to study the aging–associated diseases. Xanthurenic acid with aging binds covalently to proteins and changes the protein–protein interactions leading to cell pathology. The pathology could be rescued by respective antibodies, which can be developed as drug against degenerative diseases associated with aging.
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