Fluorescence Micro-spectroscopy Assessment of the in vitro Dimerization of BACE1-GFP Fusion Protein in Cultured Cells
2015-11
Loading...
View/Download File
Persistent link to this item
Statistics
View StatisticsJournal Title
Journal ISSN
Volume Title
Title
Fluorescence Micro-spectroscopy Assessment of the in vitro Dimerization of BACE1-GFP Fusion Protein in Cultured Cells
Authors
Published Date
2015-11
Publisher
Type
Thesis or Dissertation
Abstract
Alzheimer’s Disease (AD) is a neurodegenerative disorder that results from the formation of beta-amyloid (Aβ) plaques in the brain, which according to the amyloid cascade hypothesis triggers known symptoms, such as memory loss, in AD patients. Aβ plaques are formed by the proteolytic cleavage of the amyloid precursor protein (APP) by the proteases BACE1 and γ secretase. These enzyme-facilitated cleavages lead to the production of Aβ fragments that then aggregate to form plaques, which ultimately lead to neuronal cell death. Recent detergent protein extraction studies suggest that the untreated BACE1 protein forms a dimer that has significantly higher catalytic activity than its monomeric counterpart. Currently, however, there are no studies that support the dimerization hypothesis of BACE1 in living cells. In this contribution, we describe our effort to examine the dimerization hypothesis of BACE1 in cultured HEK293 cells using complementary fluorescence spectroscopy and microscopy methods. Cells were transfected with a BACE1-EGFP fusion protein construct and imaged using confocal and differential interference contrast (DIC) microscopy to monitor labeled BACE1 localization and distribution within the cell. Subsequently, single molecule fluctuation analysis allowed us to test the dimerization hypothesis of the labeled BACE1 using fluorescence measurements of the diffusion coefficient (size-dependent observable) and the molecular brightness as a function of BACE1 substrate analog inhibitor binding. Complementary two-photon fluorescence lifetime and anisotropy imaging enabled us to monitor BACE1 conformational changes and its local environment as a function of substrate binding. These studies provide evidence that BACE1 substrate-mediated dimerization occurs in cells and may be dependent on cellular location.
Keywords
Description
University of Minnesota M.S. thesis. November 2015. Major: Chemistry. Advisors: Joseph Johnson, Ahmed Heikal. 1 computer file (PDF); xv, 124 pages.
Related to
Replaces
License
Series/Report Number
Funding information
Isbn identifier
Doi identifier
Previously Published Citation
Other identifiers
Suggested citation
Gardeen, Spencer. (2015). Fluorescence Micro-spectroscopy Assessment of the in vitro Dimerization of BACE1-GFP Fusion Protein in Cultured Cells. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/193406.
Content distributed via the University Digital Conservancy may be subject to additional license and use restrictions applied by the depositor. By using these files, users agree to the Terms of Use. Materials in the UDC may contain content that is disturbing and/or harmful. For more information, please see our statement on harmful content in digital repositories.