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H. Z. Malina; Retinal Degeneration Is Associated With a Formation of Calmodulin Covalent Interactions With Signaling Proteins: Drug Targeting. Invest. Ophthalmol. Vis. Sci. 2007;48(13):19.
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Apoptosis is a process associated with a development and a tissue homeostasis. We have published that the apoptosis associated with aging is a different process, which we called the pathological apoptosis (Malina et al. 2001, 2002, 2003, 2004). In the pathological apoptosis the apoptotic degradation of the nucleus is associated with an aggregation of the post-apoptotic proteins. Calmodulin (Cam) is a signaling protein responsible for a regulation of Ca2+, Cam-kinases, and many proteins. An electrostatic binding of Cam is necessary to execute this regulation. We discovered that the covalent binding of the small chemical compounds, like xanthurenic acid, suppresses the possibility of a regulation of these sequences. A covalent binding of calmodulin to the regulating sequences is a major problem leading to aging associated eye diseases.
The cell culture (MEF, RPE, LEC, aortic smooth muscle cells, lymphocytes) were grown in the presence of xanthurenic acid (10, 20 microM). The cell extracts were immunoprecipitated with an antibody against Bax (Santa Cruz Inc.) and analyzed by Western blot. The detection of the proteins was performed with an antibody against calmodulin. The antibody against the misfolded BH3 sequence of Bax was prepared and used for the detection of misfolded Bax in the apoptotic cells. The same experiments were performed with Cam binding sequence of MARCKS and 14-3-3 proteins.
A covalent modification of BH-3 sequence of Bax by xanthurenic acid leads to a covalent binding of Bax to calmodulin, and a translocation of this complex into the mitochondrial membrane. In all primary cell cultures, Bax in the presence of xanthurenic acid was immunoprecipitated with Cam. Bax-Cam was inserted into the mitochondrial membranes and led to release of the cytochrome C. Cam was also covalently bound, in the misfolding conditions, to the signaling proteins MARCKS and 14-3-3. In the SV40 immortalized MEF cell line the misfolded Bax was translocated into cytoskeleton. Apofix, our antibody which blocks the signaling from the misfolded proteins in the lipid rafts, prevents the xanthurenic acid induced cell pathological apoptosis.
Misfolding of the Bax sequence leads in a primary cell culture to the binding of this sequence to calmodulin leading to a translocation of the Bax into mitochondrial membrane and the cytochrome C release. This apoptosis can be prevented by Apofix, which establishes signaling of calmodulin-MARCKS proteins in the lipid rafts. The discovery allows stopping the aging associated wrong network of the proteins involved in e.g. the retinal degeneration.
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