A novel γ-secretase assay based on detection of the putative C-terminal fragment-γ of amyloid β protein precursor

Inga Pinnix, Usha Musunuru, Han Tun, Arati Sridharan, Todd Golde, Christopher Eckman, Chewki Ziani-Cherif, Luisa Onstead, Kumar Sambamurti

Research output: Contribution to journalArticle

127 Scopus citations

Abstract

Alzheimer's disease is characterized by the deposits of the 4-kDa amyloid β peptide (Aβ). The Aβ protein precursor (APP) is cleaved by β-secretase to generate a C-terminal fragment, CTFβ, which in turn is cleaved by γ-secretase to generate Aβ. Alternative cleavage of the APP by α-secretase at Aβ16/17 generates the C-terminal fragment, CTFα. In addition to Aβ, endoproteolytic cleavage of CTFα and CTFβ by γ-secretase should yield a C-terminal fragment of 57-59 residues (CTFγ). However, CTFγ has not yet been reported in either brain or cell lysates, presumably due to its instability in vivo. We detected the in vitro generation of Aβ as well as an ∼6-kDa fragment from guinea pig brain membranes. We have provided biochemical and pharmacological evidence that this 6-kDa fragment is the elusive CTFγ, and we describe an in vitro assay for γ-secretase activity. The fragment migrates with a synthetic peptide corresponding to the 57-residue CTFγ fragment. Three compounds previously identified as γ-secretase inhibitors, pepstatin-A, MG132, and a substrate-based difluoroketone (t-butoxycarbonyl-Val-Ile-(S)-4-amino-3-oxo-2,2-difluoropentanoyl-Val-Ile-OMe ), reduced the yield of CTFγ, providing additional evidence that the fragment arises from γ-secretase cleavage. Consistent with reports that presenilins are the elusive γ-secretases, subcellular fractionation studies showed that presenilin-1, CTFα, and CTFβ are enriched in the CTFγ-generating fractions. The in vitro γ-secretase assay described here will be useful for the detailed characterization of the enzyme and to screen for γ-secretase inhibitors.

Original languageEnglish (US)
Pages (from-to)481-487
Number of pages7
JournalJournal of Biological Chemistry
Volume276
Issue number1
DOIs
StatePublished - Jan 5 2001

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'A novel γ-secretase assay based on detection of the putative C-terminal fragment-γ of amyloid β protein precursor'. Together they form a unique fingerprint.

  • Cite this

    Pinnix, I., Musunuru, U., Tun, H., Sridharan, A., Golde, T., Eckman, C., Ziani-Cherif, C., Onstead, L., & Sambamurti, K. (2001). A novel γ-secretase assay based on detection of the putative C-terminal fragment-γ of amyloid β protein precursor. Journal of Biological Chemistry, 276(1), 481-487. https://doi.org/10.1074/jbc.M005968200