Allosteric Modulation of PS1/c-Secretase Conformation Correlates with Amyloid b42/40 Ratio Kengo Uemura1, Christina M. Lill1, Xuejing Li1, Jessica A. Peters1, Alexander Ivanov1, Zhanyun Fan1, Bart DeStrooper2, Brian J. Bacskai1, Bradley T. Hyman1, Oksana Berezovska1* 1 Alzheimer Research Unit, MassGeneral Institute for Neurodegenerative Diseases, Massachusetts General Hospital, Charlestown, Massachusetts, United States of America, 2 Laboratory of Neuronal Cell Biology and Gene Transfer, Katholieke Universiteit Leuven, Leuven, Belgium Abstract Background: Presenilin 1(PS1) is the catalytic subunit of c-secretase, the enzyme responsible for the Ab C-terminal cleavage site, which results in the production of Ab peptides of various lengths. Production of longer forms of the Ab peptide occur in patients with autosomal dominant Alzheimer disease (AD) due to mutations in presenilin. Many modulators of c- secretase function have been described. We hypothesize that these modulators act by a common mechanism by allosterically modifying the structure of presenilin. Methodology/Principal Findings: To test this hypothesis we generated a genetically encoded GFP-PS1-RFP (G-PS1-R) FRET probe that allows monitoring of the conformation of the PS1 molecule in its native environment in live cells. We show that G-PS1-R can be incorporated into the c-secretase complex, reconstituting its activity in PS1/2 deficient cells. Using Fo¨ rster resonance energy transfer (FRET)-based approaches we show that various pharmacological and genetic manipulations that target either c-secretase components (PS1, Pen2, Aph1) or c-secretase substrate (amyloid precursor protein, APP) and are known to change Ab42 production are associated with a consistent conformational change in PS1. Conclusions/Significance: These results strongly support the hypothesis that allosteric changes in PS1 conformation underlie changes in the Ab42/40 ratio. Direct measurement of physiological and pathological changes in the conformation of PS1/c-secretase may provide insight into molecular mechanism of Ab42 generation, which could be exploited therapeutically. Citation: Uemura K, Lill CM, Li X, Peters JA, Ivanov A, et al. (2009) Allosteric Modulation of PS1/c-Secretase Conformation Correlates with Amyloid b42/40 Ratio. PLoS ONE 4(11): e7893. doi:10.1371/journal.pone.0007893 Editor: Hitoshi Okazawa, Tokyo Medical and Dental University, Japan Received October 7, 2009; Accepted October 22, 2009; Published November 18, 2009 Copyright: 2009 Uemura et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by NIH grants AG026593 (OB), AG 15379 (BTH, OB), and EB000768 (BJB), and a fellowship from the Deutscher Akademischer Austauschdienst (DAAD) to CML. http://www.nih.gov/ http://www.daad.de/en/index.html The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: oberezovska@partners.org Introduction c-Secretase is an enzymatic complex composed of at least four proteins: presenilin 1 or presenilin 2 (PS1 or PS2), Nicastrin, Pen2 and Aph1 [1–5], with presenilin representing the catalytic core [6– 9]. Change in the precision of the APP substrate cleavage by c- secretase result in change in the Ab42/40 ratio. Ab42 represents a highly fibrillogenic species that is more prone to form neurotoxic oligomers leading to synaptic dysfunction and neuronal death [10,11]. An increase in the Ab42/40 ratio has been implicated in Alzheimer’s disease (AD) pathogenesis. However, it remains unclear what cellular and molecular factors are responsible for the shift in APP c-cleavage site towards toxic Ab42 species. A number of genetic and pharmacological manipulations targeting either c-secretase complex or its APP substrate have been demonstrated to alter Ab42/40 ratio. For example, familial AD-linked mutations in PS1, elongation of Pen2 N-terminus (NT), or expression of Aph1B isoform, increase the Ab42/40 ratio [12– 14]. Targeting the APP substrate directly by introducing mutations near the APP c-secretase cleavage site [15,16] modifies Ab42/40 ratio. In addition, a subset of Ab42-lowering non-steriodal anti-inflammatory drugs [17,18] act as c-secretase modulators (GSMs) indirectly via binding to the APP substrate [19]. These APP-targeting manipulations may alter APP conformation, its positioning within the membrane, APP substrate presentation to c- secretase, and/or stabilize PS1 in a certain conformation, and thus affect the site of APP cleavage by c-secretase. In the present study we test the hypothesis that Ab42/40 ratio correlates with allosteric PS1/c-secretase conformation. We have previously developed an assay to monitor PS1 conformation in fixed and permeabilized cells by labeling different domains of PS1 with fluorophore-tagged antibodies. Based on our findings [20,21] we hypothesize that the mature PS1 molecule may exist in equilibrium of two conformational states: ‘‘open’’ and ‘‘closed’’, corresponding to a predominant cleavage of APP at Ab40 and Ab42 sites, respectively. To test the hypothesis that multiple GSMs act by allosterically influencing PS1 conformation, and to directly monitor PS1 conformation in live cells, we developed a new assay that monitors proximity between the PS1 NT and the major cytosolic loop between PS1 transmembrane domains 6 and 7 PLoS ONE | www.plosone.org 1 November 2009 | Volume 4 | Issue 11 | e7893