In Alzheimer’s disease (AD), β-site amyloid precursor protein cleaving enzyme 1 (BACE1) proteolysis is the rate limiting step that leads to amyloid beta (Aβ) formation, which is thought to be the main cause of Alzheimer’s disease. The process begins with BACE1 cleaving the amyloid precursor protein (APP), an integral Type I membrane protein, with subsequent cleavage by γ-secretase creating the 40 or 42 amino acid Aβ peptides. BACE1 and BACE2 form a small family of Type I membrane aspartyl proteases with 75% homology, which are both expressed in the brain and able to cleave APP at the β-secretase cleavage site. In addition, both BACE1 and BACE2 have alternatively spliced isoforms. Published studies indicated that the shorter BACE1 isoforms did not contribute to Aβ production; however, the purpose of these isoforms remains unclear. No information was available on the BACE2 isoforms until a recently published report indicated BACE2C protein concentration and activity were associated with the progression of neurodegenerative disease. Since alternative splicing is a common event that typically results in changes of activity and function, we hypothesize that this is also the case for the BACE isoforms. BACE1 is a prime therapeutic target for the treatment of AD but its inhibition could yield unexpected results if the shorter BACE1 or BACE2 isoforms are also inhibited, especially if they are active and have specific physiological functions. Our goal was to express and purify recombinant BACE1 and BACE2 isoforms and characterize their activity utilizing in vitro FRET assays. Recombinant BACE2 isoforms were synthesized and cloned into the pSec insect expression vector and subsequently expressed in Drosophila S2 cells along with previously synthesized deglycosylated BACE1 isoforms. Isoforms were partially purified using ion exchange chromatography and characterized using two FRET peptide substrate assays. All isoforms showed activity and Km values for most isoforms were determined. Using a BACE1 inhibitor, IC50 values were also determined for the BACE isoforms. These data show that the isoforms are active and have distinct binding affinities, suggesting that the BACE isoforms likely have alternative functions when compared to the full length proteins. New BACE clones have also been constructed to aid in the purification of the isoforms which will allow for better kinetic analyses.
University of Minnesota M.S. thesis. July 2012. Major: Chemistry. Advisor: Dr. Joseph Johnson. 1 computer file (PDF); viii, 60 pages.
Bicknese, Christopher Lynn.
Cloning, expression,and characterization of recombinant bace isoforms..
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