Identification of BACE1 Cleavage Sites in Human Voltage-Gated Sodium Channel Beta 2 Subunit

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Identification of BACE1 Cleavage Sites in Human Voltage-Gated Sodium Channel Beta 2 Subunit

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Title: Identification of BACE1 Cleavage Sites in Human Voltage-Gated Sodium Channel Beta 2 Subunit
Author: Gersbacher, Manuel T; Kim, Doo Yeon; Bhattacharyya, Raja; Kovacs, Dora M.

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Citation: Gersbacher, Manuel T., Doo Yeon Kim, Raja Bhattacharyya, and Dora M. Kovacs. 2010. Identification of BACE1 cleavage sites in human voltage-gated sodium channel beta 2 subunit. Molecular Neurodegeneration 5: 61.
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Abstract: Background: The voltage-gated sodium channel β2 subunit (Navβ2) is a physiological substrate of BACE1 (β-site APP cleaving enzyme) and γ-secretase, two proteolytic enzymes central to Alzheimer's disease pathogenesis. Previously, we have found that the processing of Navβ2 by BACE1 and γ-secretase regulates sodium channel metabolism in neuronal cells. In the current study we identified the BACE1 cleavage sites in human Navβ2. Results: We found a major (147-148 L↓M, where ↓ indicates the cleavage site) and a minor (144145 L↓Q) BACE1 cleavage site in the extracellular domain of human Navβ2 using a cell-free BACE1 cleavage assay followed by mass spectrometry. Next, we introduced two different double mutations into the identified major BACE1 cleavage site in human Navβ2: 147LM/VI and 147LM/AA. Both mutations dramatically decreased the cleavage of human Navβ2 by endogenous BACE1 in cell-free BACE1 cleavage assays. Neither of the two mutations affected subcellular localization of Navβ2 as confirmed by confocal fluorescence microscopy and subcellular fractionation of cholesterol-rich domains. Finally, wildtype and mutated Navβ2 were expressed along BACE1 in B104 rat neuroblastoma cells. In spite of α-secretase still actively cleaving the mutant proteins, Navβ2 cleavage products decreased by ~50% in cells expressing Navβ2 (147LM/VI) and ~75% in cells expressing Navβ2 (147LM/AA) as compared to cells expressing wildtype Navβ2. Conclusion: We identified a major (147-148 L↓M) and a minor (144-145 L↓Q) BACE1 cleavage site in human Navβ2. Our in vitro and cell-based results clearly show that the 147-148 L↓M is the major BACE1 cleavage site in human Navβ2. These findings expand our understanding of the role of BACE1 in voltage-gated sodium channel metabolism.
Published Version: doi:10.1186/1750-1326-5-61
Other Sources: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3022600/pdf/
Terms of Use: This article is made available under the terms and conditions applicable to Other Posted Material, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA
Citable link to this page: http://nrs.harvard.edu/urn-3:HUL.InstRepos:4878063

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