title: The amyloid precursor protein C-terminal fragment C100 occurs in monomeric and dimeric stable conformations and binds γ-secretase modulators.
abstract: The amyloid-β (Aβ) peptide is contained within the C-terminal fragment (β-CTF) of the amyloid precursor protein (APP) and is intimately linked to Alzheimer's disease. In vivo, Aβ is generated by sequential cleavage of β-CTF within the γ-secretase module. To investigate γ-secretase function, in vitro assays are in widespread use which require a recombinant β-CTF substrate expressed in bacteria and purified from inclusion bodies, termed C100. So far, little is known about the conformation of C100 under different conditions of purification and refolding. Since C100 dimerization influences the efficiency and specificity of γ-secretase cleavage, it is also of great interest to determine the secondary structure and the oligomeric state of the synthetic substrate as well as the binding properties of small molecules named γ-secretase modulators (GSMs) which we could previously show to modulate APP transmembrane sequence interactions [Richter et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 14597-14602]. Here, we use circular dichroism and continuous-wave electron spin resonance measurements to show that C100 purified in a buffer containing SDS at micelle-forming concentrations adopts a highly stable α-helical conformation, in which it shows little tendency to aggregate or to form higher oligomers than dimers. By surface plasmon resonance analysis and molecular modeling we show that the GSM sulindac sulfide binds to C100 and has a preference for C100 dimers.
authors.affiliation: Institut für Chemie und Biochemie, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Botev
authors.forename: Lisa-Marie
authors.initials: LM
authors.lastname: Munter
authors.forename: Ringo
authors.initials: R
authors.lastname: Wenzel
authors.forename: Luise
authors.initials: L
authors.lastname: Richter
authors.forename: Veit
authors.initials: V
authors.lastname: Althoff
authors.forename: Jochen
authors.initials: J
authors.lastname: Ismer
authors.forename: Ulla
authors.initials: U
authors.lastname: Gerling
authors.forename: Christoph
authors.initials: C
authors.lastname: Weise
authors.forename: Beate
authors.initials: B
authors.lastname: Koksch
authors.forename: Peter W
authors.initials: PW
authors.lastname: Hildebrand
authors.forename: Robert
authors.initials: R
authors.lastname: Bittl
authors.forename: Gerd
authors.initials: G
authors.lastname: Multhaup
mesh_terms: D000544:Alzheimer Disease / Q000201:enzymology / Q000378:metabolism*; D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases / Q000737:chemistry / Q000378:metabolism*; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D055503:Protein Multimerization; D055550:Protein Stability; D017434:Protein Structure, Tertiary
chemical_list: D016564:Amyloid beta-Protein Precursor; D053829:Amyloid Precursor Protein Secretases