title: Substrate sequence influences γ-secretase modulator activity, role of the transmembrane domain of the amyloid precursor protein.
abstract: A subset of non-steroidal anti-inflammatory drugs modulates the γ cleavage site in the amyloid precursor protein (APP) to selectively reduce production of Aβ42. It is unclear precisely how these γ-secretase modulators (GSMs) act to preferentially spare Aβ40 production as well as Notch processing and signaling. In an effort to determine the substrate requirements in NSAID/GSM activity, we determined the effects of sulindac sulfide and flurbiprofen on γ-cleavage of artificial constructs containing several γ-secretase substrates. Using FLAG-tagged constructs that expressed extracellularly truncated APP, Notch-1, or CD44, we found that these substrates have different sensitivities to sulindac sulfide. γ-Secretase cleavage of APP was altered by sulindac sulfide, but CD44 and Notch-1 were either insensitive or only minimally altered by this compound. Using chimeric APP constructs, we observed that the transmembrane domain (TMD) of APP played a pivotal role in determining drug sensitivity. Substituting the APP TMD with that of APLP2 retained the sensitivity to γ-cleavage modulation, but replacing TMDs from Notch-1 or ErbB4 rendered the resultant molecules insensitive to drug treatment. Specifically, the GXXXG motif within APP appeared to be critical to GSM activity. Consequently, the modulatory effects on γ-cleavage appears to be substrate-dependent. We hypothesize that the substrate present in the γ-secretase complex influences the conformation of the complex so that the binding site of GSMs is either stabilized or less favorable to influence the cleavage of the respective substrates.
authors.affiliation: Department of Neurosciences, University of California San Diego, La Jolla, California 92093, USA.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Sagi
authors.forename: Christian B
authors.initials: CB
authors.lastname: Lessard
authors.forename: Kellen D
authors.initials: KD
authors.lastname: Winden
authors.forename: Hiroko
authors.initials: H
authors.lastname: Maruyama
authors.forename: Jeremy C
authors.initials: JC
authors.lastname: Koo
authors.forename: Sascha
authors.initials: S
authors.lastname: Weggen
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Kukar
authors.forename: Todd E
authors.initials: TE
authors.lastname: Golde
authors.forename: Edward H
authors.initials: EH
authors.lastname: Koo
mesh_terms: D020816:Amino Acid Motifs; D053829:Amyloid Precursor Protein Secretases / Q000235:genetics / Q000378:metabolism*; D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000378:metabolism*; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013467:Sulindac / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: C545487:APP protein, human; D016564:Amyloid beta-Protein Precursor; D000894:Anti-Inflammatory Agents, Non-Steroidal; C497483:CD44 protein, human; D018960:Hyaluronan Receptors; C497761:NOTCH1 protein, human; D051881:Receptor, Notch1; D011993:Recombinant Fusion Proteins; D013467:Sulindac; C025462:sulindac sulfide; D053829:Amyloid Precursor Protein Secretases