Original Article

Acta Pharmacologica Sinica advance online publication, 17 Apr 2017; doi: 10.1038/aps.2017.35

Defining the minimum substrate and charge recognition model of gamma-secretase
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Yan Yan1,2,3,#, Ting-Hai Xu1,2,3,#, Karsten Melcher2 and H Eric Xu1,2

  1. 1Key Laboratory of Receptor Research, VARI-SIMM Center, Center for Structure and Function of Drug Targets, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
  2. 2Laboratory of Structural Sciences and Laboratory of Structural Biology and Biochemistry, Van Andel Research Institute, Grand Rapids, MI 49503, USA
  3. 3University of Chinese Academy of Sciences, Beijing 100049, China

Correspondence: Karsten Melcher, E-mail Karsten.melcher@vai.org; H Eric Xu, eric.xu@vai.org

#These authors contributed equally to this work.

Received 5 March 2017; Accepted 13 March 2017
Advance online publication 17 April 2017

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Abstract

γ-Secretase is an intramembrane aspartyl protease that cleaves the C99 fragment of amyloid precursor protein to generate extracellular Aβ peptides. These peptides can oligomerize and aggregate to form amyloid plaques, processes that are widely believed to be causal for Alzheimer's disease. In spite of this critical function, it remains unknown how γ-secretase recognizes C99 and its other substrates, including Notch. In this study we determined E22-K55 as the minimal C99 fragment that was sufficient and required for initial cleavage. Within this fragment, we identified four determinants: (i) a transferable extracellular determinant that differed between C99 and Notch, and which included negative charge in the case of C99, (ii) the amino acid sequence of the C-terminal half of the transmembrane helix, (iii) an invariant lysine or arginine at the intracellular membrane border, and (iv) a positive charge cluster that included the invariant lysine/arginine. We demonstrated that the charge clusters of C99 and Notch receptors could directly bind phosphatidylinositol 4,5-bisphosphate (PIP2). The PIP2-binding cluster was required for γ-secretase cleavage, and modulation of membrane PIP2 levels strongly affected γ-secretase cleavage levels and the Aβ40/Aβ42 ratio, providing support for the importance of the PIP2 interaction in cells. Together, these studies provide critically needed insight into substrate recognition by γ-secretase.

Keywords:

Alzheimer's disease; amyloid beta (Aβ); amyloid precursor protein (APP); C99; γ-secretase; minimum substrate; Notch protein; recognition model