Investigating γ-secretase protein interactions in live cells using active site-directed clickable dual-photoaffinity probes

被引：7

作者：

Ballard, T. Eric ^{[1
,2
]}

Murrey, Heather E. ^{[1
]}

Geoghegan, Kieran F. ^{[3
]}

Ende, Christopher W. Am ^{[2
]}

Johnson, Douglas S. ^{[1
]}

机构：

[1] Pfizer Worldwide Res & Dev, Neurosci Med Chem & Chem Biol, Cambridge, MA 02139 USA

[2] Pfizer Worldwide Res & Dev, Neurosci Med Chem, Groton, CT 06340 USA

[3] Pfizer Worldwide Res & Dev, Struct Biol & Biophys, Groton, CT 06340 USA

来源：

MEDCHEMCOMM | 2014年 / 5卷 / 03期

关键词：

PHOTO-CROSS-LINKING; ALZHEIMERS-DISEASE; MASS-SPECTROMETRY; ESCHERICHIA-COLI; LIVING CELLS; AMINO-ACID; PRESENILIN-1; COMPLEXES; DISTINCT; IDENTIFICATION;

D O I：

10.1039/c3md00283g

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

We have developed a suite of clickable gamma-secretase active site-directed dual-photoaffinity probes possessing photoactivatable benzophenones that are located within the inhibitor scaffold as well as spaced away from the core by a linker. Through photoactivation of these dual photoprobes and subsequent click chemistry mediated conjugation of a reporter group, we have been able to specifically label PS1-N-terminal fragment (PS1-NTF), PS1-C-terminal fragment (PS1-CTF) and nicastrin and form a pseudo-full length PS1 through crosslinks between PS1-NTF and PS1-CTF. Probe-mediated protein-protein crosslinks were confirmed by in-gel fluorescence, western blotting and LC-MS/MS detection of tryptic peptides of the electrophoretically separated proteins.

引用

页码：321 / 327

页数：7

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