Abstract
Extracellular deposition of β-amyloid (Aβ) peptides in the brain is a hallmark of Alzheimer's disease (AD). Upon β-secretase-mediated cleavage of the β C-terminal fragment (β-CTF) from the Aβ precursor protein, the γ-secretase complex produces the Aβ peptides associated with AD. The familial T43I mutation within the transmembrane domain of the β-CTF (also referred to as C99) increases the ratio between the Aβ42 and Aβ40 peptides largely due to a decrease in Aβ40 formation. Aβ42 is the principal component of amyloid deposits within the brain parenchyma, and an increase in the Aβ42/Aβ40 ratio is correlated with early-onset AD. Using NMR and FTIR spectroscopy, here we addressed how the T43I substitution influences the structure of C55, the minimal sequence containing the entire extracellular and transmembrane (TM) domains of C99 needed for γ-secretase processing. 13C NMR chemical shifts indicated that the T43I substitution increases helical structure within the TM domain of C55. These structural changes were associated with a shift of the C55 dimer to the monomer and an increase in the tilt of the TM helix relative to the membrane normal in the T43I mutant compared with that of WT C55. The A21G (Flemish) mutation was previously found to increase secreted Aβ40 levels; here, we combined this mutation in the extracellular domain of C99 with T43I and observed that the T43I/A21G double mutant decreases Aβ40 formation. We discuss how the observed structural changes in the T43I mutant may decrease Aβ40 formation and increase the Aβ42/Aβ40 ratio.
Highlights
The C99 substrate is in a monomer– dimer equilibrium in membrane bilayers, where the structure, dynamics, and membrane interactions of the substrate influence its initial binding to the enzyme complex away from the catalytic site (Fig. 8B)
T43I C99 has been shown to have a weaker interaction with ␥-secretase than WT C99 (37)
Polarized Attenuated total reflection (ATR) FTIR spectra were obtained on a Bruker IFS 66V/S spectrometer
Summary
A, -amyloid; APP, amyloid precursor protein; ATR, attenuated total reflection; -CTF,  C-terminal fragment; DARR, dipolar assisted rotational resonance; DMPC, dimyristoyl-phosphatidylcholine; DMPG dimyristoyl-phosphatidylglycerol; DPC, dodecylphosphocholine; AD, Alzheimer’s disease; FAD, familial Alzheimer’s disease; MAS, magic angle spinning; TM, transmembrane; POPC, 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3phosphoserine; CHO, Chinese hamster ovary; LB, Luria–Bertani broth; TROSY, transverse relaxation– optimized spectroscopy; 2D and 3D, twodimensional and three-dimensional, respectively. A second cluster of amino acids in the -CTF that are mutated in familial AD occurs between the cleavage site (Ala42) that releases the A42 peptide and the cytoplasmic end of the TM domain (Leu). A second cluster of amino acids in the -CTF that are mutated in familial AD occurs between the cleavage site (Ala42) that releases the A42 peptide and the cytoplasmic end of the TM domain (Leu52) These include the T43A Iranian (13), T43I. We have previously shown that the A21G mutation lengthens the TM helix, stabilizes the TM dimer, and destabilizes -sheet structure in the extracellular region of the -CTF (8, 12) These structural changes are associated with an increase in the total amount of A peptide released upon ␥-secretase cleavage as well as with an increase in the A42/ A40 ratio (8, 9). FTIR measurements provide information on the global secondary structure of these mutants in membrane bilayers
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