NMR structural studies of FAD mutations in APPTM and amyloid-β peptide

Clemente, Nicolina Marie
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Wang, Chunyu
Barquera, Blanca L.
BBystroff, Christopher, 1960-
Colón, Wilfredo
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Biochemistry and biophysics
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Attribution-NonCommercial-NoDerivs 3.0 United States
This electronic version is a licensed copy owned by Rensselaer Polytechnic Institute, Troy, NY. Copyright of original work retained by author.
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Aβ protein is the main aggregate component found in senile plaque in the AD brain. Familial Alzheimer’s disease (FAD) point mutations found within the Aβ sequence are known to cause early onset AD that occurs prior to the age of 65. Although FAD constitutes a small number of AD cases its clinical behavior is similar to sporadic AD. Aβ FAD mutants usually aggregate faster than Aβ Wild Type and in some cases form unique aggregates. We hypothesize that FAD mutations change Aβ monomer conformation and affect their aggregation. Due to Aβ’s intrinsically disordered nature, it cannot be studied using basic structure determination techniques such as x-ray crystallography or cryo-EM. The following study used combined solution NMR and high-pressure techniques for the characterization of conformational differences between Wild Type, protective and FAD Aβ monomers. Basic 2D experiments at atmospheric pressure revealed variation in backbone amide chemical shifts at and around the site of mutation, as expected, while high-pressure NMR studies showed differences in increased pressure coefficients at residues 12-17 in FAD mutants. This region is close to the central hydrophobic cluster of Aβ, which is known to play an important role in Aβ aggregation. These conformational changes could contribute to the distinct properties of FAD mutant Aβ species. The experimental outcome from these studies demonstrates the utility of high-pressure NMR as a tool for characterizing regions of residual structure in less ordered protein systems.
Amyloid precursor protein (APP) is best known for its role in Alzheimer’s disease (AD) as the protein from which Amyloid-β (Aβ) is cleaved and released. The enzyme that ultimately liberates Aβ from APP is an aspartyl protease called γ-secretase. The two main forms of Aβ produced by amyloidgenic APP processing are Aβ40 and Aβ42, the latter of the two aggregating into a toxic higher order species that make up the major component of senile plaques in the AD brain. Early onset AD point mutations within the transmembrane region of APP (APPTM) have been proposed to increase the Aβ42/Aβ40 ratio by disrupting APPTM structure and dynamics. In the following study, we determined how point mutations in APPTM affect the initial binding interaction between the catalytic subunit (Presenilin) of γ-secretase using recently discovered presenilin homolog enzymes (PSH). NMR chemical shift perturbation identified the juxtamembrane regions of APPTM as the binding interface, including the ε-cleavage site. Additional findings indicate binding differences between Wild Type and V44M-APPTM substrates with the mutant showing an increase in chemical shift perturbation (CSP) at the ε-cleavage site in V44M. Furthermore, when comparing PSHs, the MAMRE50 enzyme causes larger CSP in APPTM than MCMJR1 implying increased binding affinity. Higher substrate perturbation correlates well with increased substrate cleavage rate by MAMRE50 shown in both NMR and gel based assays. This study demonstrates the importance of enzyme substrate interplay for the determination of catalytic efficiency and specificity in intramembrane proteolysis involved in AD pathogenesis.
December 2017
School of Science
Biochemistry and Biophysics Program
Rensselaer Polytechnic Institute, Troy, NY
Rensselaer Theses and Dissertations Online Collection
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