gamma-Secretase substrate selectivity can be modulated directly via interaction with a nucleotide-binding site - PubMed (original) (raw)

gamma-Secretase substrate selectivity can be modulated directly via interaction with a nucleotide-binding site

Patrick C Fraering et al. J Biol Chem. 2005.

Abstract

gamma-Secretase is an unusual protease with an intramembrane catalytic site that cleaves many type I membrane proteins, including the amyloid beta-protein (Abeta) precursor (APP) and the Notch receptor. Genetic and biochemical studies have identified four membrane proteins as components of gamma-secretase: heterodimeric presenilin composed of its N- and C-terminal fragments, nicastrin, Aph-1, and Pen-2. Here we demonstrated that certain compounds, including protein kinase inhibitors and their derivatives, act directly on purified gamma-secretase to selectively block cleavage of APP- but not Notch-based substrates. Moreover, ATP activated the generation of the APP intracellular domain and Abeta, but not the generation of the Notch intracellular domain by the purified protease complex, and was a direct competitor of the APP-selective inhibitors, as were other nucleotides. In accord, purified gamma-secretase bound specifically to an ATP-linked resin. Finally, a photoactivable ATP analog specifically labeled presenilin 1-C-terminal fragments in purified gamma-secretase preparations; the labeling was blocked by ATP itself and APP-selective gamma-secretase inhibitors. We concluded that a nucleotide-binding site exists within gamma-secretase, and certain compounds that bind to this site can specifically modulate the generation of Abeta while sparing Notch. Drugs targeting the gamma-secretase nucleotide-binding site represent an attractive strategy for safely treating Alzheimer disease.

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Figures

FIGURE 1

FIGURE 1. Nonhydrolyzed ATP can activate the in vitro generation of Aβ40 and Aβ42

A, effect of ATP on purified γ-secretase activity. γ-Secretase diluted in 0.2% CHAPSO/HEPES, pH 7.5, was incubated at 37 °C for 4 h in the presence of 0.1% PC, 0.025% PE, the indicated concentrations of ATP, and 1 μ

m

C100FLAG (an APP-based substrate) or 1 μ

m

N100FLAG (a Notch-based substrate); both substrates were adjusted to 0.5% SDS prior to addition to the reactions (6). The reactions were Western-blotted for AICD-FLAG (with M2 anti-FLAG antibody) and for NICD-FLAG (with Notch Ab1744 antibody). Levels of Aph1-HA serve as equal loading controls. Aβ40 and Aβ42 were measured by ELISA (B, means ± S.D.; n = 3). Levels of AICD-FLAG and NICD-FLAG were estimated by densitometry (values are single determinations from the blot shown). Asterisks indicate significant differences in Aβ40 (*, p < 0.01) and Aβ42 (**, p < 0.01) production compared with samples without ATP. C, ATPase assays on purified γ-secretase. [α-32P]ATP was incubated at 37 °C for the indicated times in the reaction buffer (0.2% CHAPSO/HEPES, pH 7.5, 150 m

m

NaCl, 5 m

m

MgCl2, 5 m

m

CaCl2, 0.025% PE, and 0.10% PC) alone (lanes 11–15) or in the presence of purified γ-secretase (lanes 6 –10), C100FLAG substrate (lanes 16 –20), or both purified γ-secretase and C100FLAG substrate (lanes 21–25). Two μl of each reaction were then analyzed by TLC to separate ATP from ADP. As a positive control to show ATP hydrolysis products, [α-32P]ATP was incubated at the indicated times in the presence of canine kidney phosphatase (lanes 1–5).

FIGURE 2

FIGURE 2. Gleevec itself is not a direct γ-secretase inhibitor, but a Gleevec extract inhibits the generation of Aβ by purified γ-secretase without affecting the cleavage of a Notch-based substrate

A, effect of III-31C and DAPT on the cleavage by purified γ-secretase of C100FLAG and N100FLAG. γ-Secretase diluted in 0.2% CHAPSO/HEPES, pH 7.5, was incubated at 37 °C for 4 h with 1 μ

m

C100FLAG or N100FLAG substrate, 0.1% PC, 0.025% PE, and the indicated concentrations of III-31C and DAPT. The reactions were Western-blotted for AICD-FLAG (M2 antibody) and NICD-FLAG (Ab1744 antibody). Levels of Aph1-HA serve as equal loading controls. B, effect of Gleevec extract on Aβ40 and Aβ42 generation by purified γ-secretase. γ-Secretase diluted in 0.2% CHAPSO/HEPES, pH 7.5, was incubated at 37 °C for 4 h with 1 μ

m

C100FLAG substrate, 0.1% PC, 0.025% PE, and the indicated concentrations of Gleevec extract. Aβ40 and Aβ42 were measured by ELISA (means ± S.D., n = 3). C, effects of Gleevec extract on C100FLAG and N100FLAG cleavage by purified γ-secretase. Parallel reaction mixtures in the presence of 1 μ

m

C100FLAG or N100FLAG, 10 μ

m

III-31C, or the indicated concentrations of the Gleevec extract were incubated at 37 °C for 4 h and Western-blotted for AICD-FLAG (M2), Aβ (6E10), and for NICD-FLAG (Ab1744); short exposures (short exp.) are also shown to validate the lack of effect on NICD-FLAG levels. Levels of Aph1-HA serve as equal loading controls. D, effect of Gleevec extract on C100FLAG and N100FLAG cleavage by endogenous γ-secretase solubilized from HeLa membranes. γ-Secretase assays and the generation of AICD-FLAG, Aβ, and NICD-FLAG were performed as in C. Levels of NCT serve as loading controls. E, effect of three different Gleevec samples on C100FLAG cleavage by purified γ-secretase. γ-Secretase was incubated as described in B with C100FLAG and the indicated concentrations of Gleevec extracted from capsules (left panel), Gleevec extracted from tablets (middle panel), or purified Gleevec (right panel). The reactions were Western-blotted for AICD-FLAG and Aβ. Levels of Aph1-HA serve as equal loading controls. Final purity and characterization of the three Gleevec samples was performed by MALDI-TOF mass spectroscopy; the compounds identified specifically in the active Gleevec extract (left panel) are labeled with arrowheads. Note that a very minor peak at 286.6 in the inactive extract is a major peak in the active extract (asterisk).

FIGURE 3

FIGURE 3. Nucleotides prevent the inhibitory effect of Gleevec extract on purified γ-secretase

A, ATP, ADP, and AMP are direct competitors with respect to the Gleevec extract. γ-Secretase diluted in 0.2% CHAPSO/HEPES, pH 7.5, was incubated at 37 °C for 16 h in the absence (lane 1) or presence (lanes 2– 6) of 100 μ

m

Gleevec extract and the indicated concentrations of ATP, ADP, or AMP. The generation of AICD-FLAG was probed by Western blotting with M2 anti-FLAG. Levels of Aph1-HA served as equal loading controls. Note that the lane 1 control without ATP is the same control for the absence of ADP or AMP. B and C, the effects of increasing concentrations of ATP, ADP, and AMP on Aβ40 (B) and Aβ42 (C) generation by purified γ-secretase in the presence of 100 μ

m

Gleevec extract (reactions performed at 37 °C for 4 h) were quantified by ELISA (n = 3).

FIGURE 4

FIGURE 4. ZM39923 (1367), a potent Janus kinase 3 inhibitor, preferentially blocks the generation of Aβ by purified γ-secretase

A, effect of a large number of protein kinase/phosphatase inhibitors or activators on purified γ-secretase activity. γ-Secretase diluted in 0.2% CHAPSO/HEPES, pH 7.5, was incubated at 37 °C for 16 h in the presence of 1 μ

m

C100FLAG, 0.1% PC, 0.025% PE, and 100 μ

m

of the indicated compounds, except for III-31C (10 μ

m

). The generation of AICD-FLAG was probed by Western blotting with M2 anti-FLAG antibody. B, effect of ZM39923 (1367) and its break-down product ZM449829 (1366) (structures shown) on the cleavage by purified γ-secretase of C100FLAG and N100FLAG. Activity assays were performed as described above in the presence of the indicated concentrations of 1367 and 1366, and the generation of AICD-FLAG and NICD-FLAG was probed by Western blotting with M2 and Notch Ab1744 antibody, respectively. In all figures, levels of Aph1-HA are shown as equal loading controls. C, similarly, the effect of SC-9 (0433) (structure shown) on the cleavage by purified γ-secretase of C100FLAG and N100FLAG was probed.

FIGURE 5

FIGURE 5. Purified γ-secretase binds specifically to an ATP resin

Purified γ-secretase (St. indicates starting material) was incubated overnight in the presence or absence of 50 m

m

ATP with two different ATP-immobilized resins as follows: ATP attached to acrylamide through the γ-phosphate (lanes 1–9) or ATP attached to agarose through the ribose hydroxyls (lanes 10 –12). The unbound fractions (Unb.) were recovered, and the resins washed three times in 0.2% CHAPSO/HEPES and resuspended in Laemmli sample buffer to recover the bound proteins (P. indicates precipitate). All samples were electrophoresed on 4 –20% Tris-glycine gels and transferred to polyvinylidene difluoride membranes to detect NCT-GST (αGST), PS1-NTF (Ab14), Aph1-HA (3F10), PS1-CTF (13A1), and FLAG-Pen2 (M2-anti FLAG). Note that purified γ-secretase binds specifically to the resin in which ATP is attached to acrylamide through the γ-phosphate (lanes 1– 6), whereas γ-secretase from a crude lysate (of γ-30 cells) is unable to bind to the same resin (lanes 7–9). Starting material (St.) and unbound (Unb.) lanes were each loaded with the equivalent of 25% of the material that was bound to the resins (P.) so that the unbound and bound protein levels can be compared directly.

FIGURE 6

FIGURE 6. Specific labeling of the γ-secretase component PS1-CTF with 8-azido-[γ-32P]ATP, a photoaffinity ATP analog

A, photolabeling of γ-secretase with 8-azido-[γ-32P]ATP as revealed by nondenaturing BN-PAGE. Purified γ-secretase solubilized in 0.1% digitonin/Tris-buffered saline was incubated with 22.5 μ

m

of 8-azido-[γ-32P]ATP (10 μCi per reaction) in the presence of 10 m

m

ATP (lanes 2 and

) or 1 mm Gleevec extract (Gleevec E., lanes 3 and ), exposed to UV light for 5 min, and subjected to BN-PAGE analysis as described (31). 32P labeling was assessed by autoradiography (lanes 4 – 6) (BioMax MS films used with BioMax Transcreen HE; Kodak). As a control for the migration of γ-secretase, the purified complex solubilized in 0.1% digitonin/Tris-buffered saline (lane 1), in the presence of 10 mm ATP (lane 2) or 1 mm Gleevec extract (lane 3), was exposed to UV light for 5 min, subjected to BN-PAGE analysis on the same gel as above, and probed for the γ-secretase complex using 3F10 antibody to the Aph1-HA component (lanes 1–3). The asterisk denotes nonspecific aggregates containing Aph1-HA (P. C. Fraering, W. Ye, M. J. LaVoie, B. L. Ostaszewski, D. J. Selkoe, and M. S. Wolfe, unpublished data). HMWC, the high molecular weight γ-secretase complex (31). B, photolabeling of γ-secretase with 8-azido-[γ-32P]ATP as revealed by denaturing SDS-PAGE. Purified γ-secretase was incubated with 22.5 μm of 8-azido-[γ-32P]ATP (10 μCi per reaction) in the absence (lane 1) or presence (lane 2) of PC and PE (lipids) or in the presence of 10 mm ATP (lane 3), 1 mm Gleevec extract (lane 4), 1 μm III-31C (lane 5), 1 μm C100FLAG (lane 7), 1 μm N100FLAG (lane 8), or 300 μm 1367 (lane 9). As a control for the specificity of photolabeling, purified γ-secretase was also incubated with [γ-32P]ATP (without the 8-azido group) (lane 6). All samples were exposed to UV light for 5 min, and the reactions were quenched with 1 mm dithiothreitol. The samples were diluted and were incubated overnight at 4 °C with GSH resin for the affinity precipitation of the NCT-GST component and its associated proteins (6). The resins were then washed three times, and all precipitated proteins were electrophoresed on 4 –20% Tris-glycine gels and the photolabeled proteins autoradiographed as in A. As a control for the mobility of the photolabeled proteins, the same sample as shown in lane 1 was electrophoresed on a 4 –20% Tris-glycine gel and Western-blotted simultaneously for PS1-CTF and Aph1-HA with 13A11 and 3F10 antibodies, respectively (lane 10). Molecular weight markers are in lane 11. The levels of the Aph1-HA on the gel in B are shown to demonstrate equal protein loading. C, photolabeling of endogenous γ-secretase with 8-azido-[γ-32P]ATP. Membranes from untransfected HeLa cells were incubated with 8-azido-[γ-32P]ATP (10 μCi per reaction) in the absence (lane 1) or presence (lane 2) of 10 mm ATP and exposed to UV light as described above. The membranes were washed; the proteins were solubilized in 1% CHAPSO/HEPES, and γ-secretase was co-immunoprecipitated with anti-PS1-NTF antibodies. The photolabeled proteins were detected as described above, and levels of PS1-NTF (Western-blotted with mAb1563 antibody) serve as co-immunoprecipitated controls.

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