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Results: 4

1.
Fig. 4.

Fig. 4. From: The initial substrate-binding site of ?-secretase is located on presenilin near the active site.

Proposed models for inhibitors (a) and substrate (b) interactions with γ-secretase. (a)(Top) Substrate-based d-10 helical peptide interacts with PS1 at the initial binding site. (Middle) Transition-state analogue III-31-C binds to PS1 NTF/CTF heterodimer at the active site, located internally and containing two aspartates (denoted as “D”). (Bottom) d-13 peptide interacts with both active and initial binding sites. (b) Upon docking to the initial binding site, a substrate passes through PS1 subunits fully (path A) or partially (path B) to access the nearby active site.

Anna Y. Kornilova, et al. Proc Natl Acad Sci U S A. 2005 March 1;102(9):3230-3235.
2.
Fig. 2.

Fig. 2. From: The initial substrate-binding site of ?-secretase is located on presenilin near the active site.

Photoactivatable 13-residue helical d-peptide inhibitor of γ-secretase covalently labels PS1-NTF and -CTF, but differently from 10-residue helical photoprobe. (a) d-13-Bpa-Bt (50 nM) was incubated with γ-30 lysates in the absence or presence of d-13 (250 nM), and the samples were treated and analyzed as in Fig. 1a. (b) d-13-Bpa-Bt (50 nM) was incubated with HeLa lysates in the absence or presence of III-31-C (250 nM). (c) d-13-Bpa-Bt (50 nM) was incubated with HeLa lysates in the absence or presence of d-13 (250 nM). Immunoblotting was performed with antibodies directed against the PS2 CTF. (d) d-13-Bpa-Bt (100 nM) was incubated with HeLa lysates in the absence or presence of Compound E (2 μM) and DAPT (2 μM). (e) III-63 (50 nM) was incubated with HeLa lysates in the absence or presence of d-13 (250 nM) and III-31-C (250 nM). (f)(Left) d-13-Bpa-Bt (100 nM) was incubated with HeLa lysates in the absence or presence of d-10 (2 μM). (Right) d-10-Bpa-Bt (500 nM) was incubated with HeLa lysates in the absence or presence of d-13 (5 μM) and d-10 (10 μM).

Anna Y. Kornilova, et al. Proc Natl Acad Sci U S A. 2005 March 1;102(9):3230-3235.
3.
Fig. 3.

Fig. 3. From: The initial substrate-binding site of ?-secretase is located on presenilin near the active site.

FAD-PS1 mutation L166P affects the photolabeling by the active-site-directed III-63, with little or no effect on both helical peptide photoprobes. (a) CHAPSO-solubilized membranes derived from HEK cells stably expressing PS1-L166P, PS1-G384A, and PS1-wt were normalized by the amount of PS1. (b–d) Membranes were incubated with d-10-Bpa-Bt (500 nM) (b), d-13-Bpa-Bt (500 nM) (c), and III-63 (500 nM) (d). Samples were treated and analyzed as in Fig. 1a. (e) γ-Secretase activity measured in the solubilized membranes. C100-Flag was incubated with membranes for 4 h, and APP intracellular domain (AICD)-Flag (shown), the product of the proteolysis, was detected with anti-Flag antibody.

Anna Y. Kornilova, et al. Proc Natl Acad Sci U S A. 2005 March 1;102(9):3230-3235.
4.
Fig. 1.

Fig. 1. From: The initial substrate-binding site of ?-secretase is located on presenilin near the active site.

Photoactivatable 10-residue helical d-peptide inhibitor of γ-secretase covalently labels PS1 heterodimer at a site distinct from the active site. (a) d-10-Bpa-Bt (500 nM) was incubated with γ-30 lysates in the absence or presence of d-10 (10 μM), and the samples were irradiated at 350 nm. Biotinylated proteins were precipitated with immobilized streptavidin and blotted for NCT, PS1-NTF, PS1-CTF, the Flag-tag of Pen-2, and the HA-tag of Aph-1. Western blotting reveals that PS1-NTF is specifically tagged, but not NCT, APH-1, or Pen-2. Small traces of the labeled PS1-CTF also were observed that were only visible upon longer exposure. (b) d-10-Bpa-Bt (500 nM) was incubated with HeLa lysates in the absence or presence of d-10 (10 μM), L-10 (10 μM), and III-31-C (5 μM). (c) d-10-Bpa-Bt (500 μM) was incubated with HeLa lysates in the absence or presence of Compound E (5 μM) and DAPT (5 μM).

Anna Y. Kornilova, et al. Proc Natl Acad Sci U S A. 2005 March 1;102(9):3230-3235.

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