(TM6-7 loop). For this, a GFP-PS1-RFP (G-PS1-R) construct was generated by tagging PS1 NT with green fluorescent protein (GFP) and TM6-7 loop with red fluorescent protein (mRFP). We demonstrate that various Ab42 raising manipulations, including direct manipulations of the c-secretase components (Pen2, Aph1 and PS1) or pharmacological targeting of APP (fenofibrate) lead to a common conformational change of PS1: closer proximity of the PS1 NT and TM6-7 loop. Conversely, treatment with the Ab42– lowering agent (ibuprofen) moves PS1 NT and loop farther apart. Thus, we found that a broad range of Ab42-altering manipu- lations, regardless of their precise molecular mechanism or their target, lead to a consistent change in PS1 conformation. This suggests that PS1 conformation could be a reliable predictor of Ab production, and presents an allosteric modulation of the ‘‘pathogenic’’ PS1 conformation as an attractive therapeutic strategy. Results GFP-PS1-RFP Probe Reconstitutes c-Secretase Activity GFP-PS1-RFP (G-PS1-R) construct was generated to monitor PS1 conformation (PS1 NT-loop proximity) in live cells. For this, green fluorescent protein (GFP) was fused to the PS1 N-terminus and red fluorescent protein (mRFP) was inserted into the TM6-7 loop domain (Figure 1A). The resulting G-PS1-R fusion protein represents an ideal molecular probe for FRET-based analysis with a donor and an acceptor fluorophore in a 1:1 ratio. When expressed in cells, the G-PS1-R probe shows perinuclear distribution of GFP and RFP, a pattern similar to that of endogenous wt PS1 (Supplementary Figure S1, and Figure 1B). To examine whether the G-PS1-R probe can traffic though the secretory pathway to the plasma membrane, we assessed the expression pattern of the G-PS1-R protein in cells by using total internal reflection fluorescence microscopy (TIRFM), which detects fluorescent signal within 100 nm of the cell surface. A dotty pattern of the TIRFM signal was detected at the cell surface in G-PS1-R expressing cells, indicating that a few percent of the G-PS1-R molecules reach the cell surface (Figure 1C). Next, we asked whether the G-PS1-R probe could be incorporated into the c-secretase complex to exert its enzymatic function. To test this, we monitored the ability of G-PS1-R to undergo endoproteolysis, and to cleave APP and Notch c-secretase substrates. First, CHO cells were transiently transfected with G- PS1-R or infected with lenti-G-PS1-R, lysed, and analyzed by Western blot. As shown in Figure 1D, G-PS1-R undergoes endoproteolysis to generate ,60 kDa PS1 NTF-GFP and ,40 kDa CTF-RFP. Noticeably, the endogenous PS1 was reduced in cells transfected with G-PS1-R, suggesting that other components of the c-secretase are forming a complex with exogenous G-PS1-R, destabilizing endogenous wild-type (wt) PS1 (the ‘replacement phenomena’) (Figure 1D, bottom panel). To determine if G-PS1-R is able to reconstitute c-secretase activity, PS1/2 dKO MEF cells were transfected with G-PS1-R, and APP processing was assessed by monitoring APP C-terminal fragment (CTF) levels in cell lysate. Using an APP C-terminal specific antibody, we observed the reduction in the level of APP CTFs in PS1/2 dKO cells transfected with G-PS1-R compared to that in the ‘‘no PS’’ lane (Figure 1 Etop panel ). This indicates that APP CTFs, immediate substrates of the c-secretase, are cleaved by G-PS1-R containing c-secretase complex. Further- more, when PS1/2 dKO MEF cells were transfected with another immediate substrate of c-secretase, the constitutively active NEC construct, the cleavage product Notch intracellular domain PS1 Conformation and Ab Figure 1. Expression, processing, and activity of the G-PS1-R protein. (A)- Schematic representation of the G-PS1-R construct. The GFP is fused to the N-terminus of PS1, and serves as a donor fluorophore in the FRET assays, whereas RFP (an acceptor fluorophore) is introduced by PCR into the TM6-7 loop region of PS1 between amino acids 351 and 352. (B–C) The expression of the G-PS1-R in CHO cells. (B) -Epi-fluorescent signal shows strongest fluorescence intensity in the perinuclear region of the cell by confocal microscopy. (C) - Cell surface expression of the G-PS1-R probe shown by TIRFM. A speckled pattern of the TIRF signal indicates that G-PS1-R protein reaches the cell surface. (D) CHO cells were transfected with G-PS1-R construct and cell lysates were subjected to the western blot analysis. (D, Top) G-PS1-R undergoes endoproteolysis to yield PS1 NTF and CTF. A ,100 kDa full-length G-PS1-R, ,50 kDa PS1 NTF-GFP and ,40 kDa CTF PS1-RFP were observed by blotting with the anti-GFP antibody. Asterisk: non- specific bands. (D, Bottom) Blotting by anti-PS1 CTF is shown. Full- length G-PS1-R as well as ,40 kDa PS1-RFP CTF were detected. Note, that endogenous PS1 CTF is reduced in the G-PS1-R expressing CHO cells (replacement phenomena). (E) G-PS1-R reconstitutes c-secretase activity in PS1/2 dKO MEF cells. (E, Top) APP CTF is reduced in PS1/ 2 dKO MEF cells expressing G-PS1-R; probed with anti-APP CTF antibody. (E, Bottom) PS1/2 dKO cells were transfected with NEC with or without G-PS1-R construct, lysed, and analyzed by western blotting using NICD-specific (Val1744) antibody. Note that NICD can only be observed in cells expressing G-PS1-R. doi:10.1371/journal.pone.0007893.g001 (NICD) was detected only in G-PS1-R co-transfected cells (Figure 1E bottom panel ). Thus, we conclude that G-PS1-R can assemble into the c-secretase complex and is able to reconstitute c-secretase activity in PS1/2 dKO cells. Detection of Inter- versus Intra-Molecular FRET Using G- PS1-R Probe To test whether GFP on the N-terminus and RFP in the loop domain of PS1 are close enough to support FRET, CHO cells were transfected with GFP-PS1 (negative control) or G-PS1-R constructs, and analyzed by Fluorescence Lifetime Imaging PLoS ONE | www.plosone.org 2 November 2009 | Volume 4 | Issue 11 | e7893