| Abstract No.: | A-B1034 |
| Country: | Canada |
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| Title: | VISININ-LIKE PROTEIN-3 MODULATES THE INTERACTION BETWEEN CYTOCHROME B5 AND NADH-CYTOCHROME B5 REDUCTASE IN A CA2+-DEPENDENT MANNER |
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| Authors/Affiliations: | 1 Kensuke Oikawa*; 1 Solmaz Nafez; 1 Gary Odero; 1 Ning Ge; 1 Dali Zhang; 1 Benedict Albensi;
1 Division of Neurodegenerative Disorders, St. Boniface General Hospital Research Centre, Winnipeg, MB, Canada
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| Content: | Objective: Visinin-like protein-3 (VILIP-3) belongs to the neuronal calcium sensor (NCS) protein family expressed primarily in retinal photoreceptors or neurons and neuroendocrine cells. VILIP-3 expression was predominantly shown in cerebellar Purkinje cells. We discovered that VILIP-3 interacts with microsomal cytochrome b5 (Cyb5) in a Ca2+-dependent manner (J Biol Chem. 2004; 279(15):15142-52). These findings suggested that VILIP-3 may link Ca2+-signaling to the machinery of the microsomal monooxygenase (MMO) complex in the endoplasmic reticulum. However the physiological relevance of the interaction between VILIP-3 and Cyb5 remains to be determined. To answer the question, we examined if VILIP-3 can interact with NADH-cytochrome b5 reductase (b5R), which is one of the components of MMO and the physiological electron donor for Cyb5. We also tried to identify cytochrome P450 isoforms in the cerebellum to assign a terminal enzyme of MMO system.
Materials and Methods: For the co-immunoprecipitation experiments, 293T cells were transfected with 3xFLAG-tagged protein expression vectors and c-myc epitope-tagged protein expression vectors. The protein extractions of transfected cells were immunoprecipitated with anti-FLAG M2-agarose beads (Sigma). The immunocomplexes were eluted with 3xFLAG peptide (Sigma). The eluted samples were resolved by SDS-PAGE gels, and were subjected to Western Blotting analysis with anti c-Myc antibody. The total RNAs were extracted from the C57BL/6 mouse brain tissues using RNeasy Plus Mini (Qiagen). Expression of transcripts for cytochrome P450s were examined by reverse transcription-PCR. [Results] We obtained evidence that VILIP-3 interacts with Cyb5-Reductase (b5R) in a Ca2+-" independent" manner. Furthermore, Ca2+- bound VILIP-3 enhanced the interaction between Cyb5 and b5R. Inversely, Ca2+-unbound VILIP-3 attenuated the affinity between Cyb5 and b5R. Three out of five isoforms of Cyp 4f family, Cyp 4f13, Cyp 4f14, and Cyp 4f16, as well as Cyp 4x1, Cyp 2j9, and Cyp 2u1, were present in all of the regions of brain including cerebellum.
Conclusion: The current evidence strongly supports that the ternary complex of VILIP-3, Cyb5, and b5R might function as a Ca2+-dependent switching apparatus for MMO. In summary, when intracellular Ca2+ concentrations reach a high level, Ca2+-bound form of VILIP-3 enhances the coupling between Cyb5 and b5R, and facilitates production of bioactive molecules, such as eicosanoids, through activities of some isoforms of cytochrome P450s in cerebellar Purkinje cells. |
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