Expression and proteolytic activity of BACE1:GFP fusion proteins in CHO-695 cells
Varella, Raphaela A.
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In the United States alone, one in ten people aged 65 and older has Alzheimer’s disease (AD). There is no cure for this devastating disorder, however there are therapeutics that are able to ameliorate some of the symptoms. AD is caused by a buildup of Amyloid Beta (Aβ) plaque which eventually leads to neuronal cell death in the brain. Aβ is secreted by the proteolysis of Amyloid Precursor Protein (APP) by BACE1 and gamma secretase. Even though there are multiple proteases at play, BACE1 is the rate limiting enzyme in the generation of Aβ. There have not been many studies focused on understanding the functional evolution of the BACE1 protease. Therefore, it is still unclear as to when β-secretase proteolytic activity arose. To gain further understanding into the functional evolution of BACE1, BACE1:GFP fusion proteins were transfected into CHO-695 cells and visualized with microscopy. The expression was appropriate; as secretory pathway localization was seen. A functional assay was run on the conditioned media from the CHO-695 cells which unexpectedly found the Aβ levels to be unaffected by the BACE1:GFP fusion protein transfections. The endogenous BACE 1 activity in CHO-695 cells is one factor which may have led to Aβ levels secreted from the BACE1:GFP fusion proteins to have no detectable differences. Another possible cause is the large GFP epitope tag may have inhibited BACE1 proteolytic activity. Future investigations may utilize a cell line with lower to no endogenous BACE1 activity or with a smaller epitope tag in order to determine when BACE1 first acted on Aβ contained APP.