title: Visualizing active enzyme complexes using a photoreactive inhibitor for proximity ligation--application on γ-secretase.
abstract: Here, we present a highly sensitive method to study protein-protein interactions and subcellular location selectively for active multicomponent enzymes. We apply the method on γ-secretase, the enzyme complex that catalyzes the cleavage of the amyloid precursor protein (APP) to generate amyloid β-peptide (Aβ), the major causative agent in Alzheimer disease (AD). The novel assay is based on proximity ligation, which can be used to study protein interactions in situ with very high sensitivity. In traditional proximity ligation assay (PLA), primary antibody recognition is typically accompanied by oligonucleotide-conjugated secondary antibodies as detection probes. Here, we first performed PLA experiments using antibodies against the γ-secretase components presenilin 1 (PS1), containing the catalytic site residues, and nicastrin, suggested to be involved in substrate recognition. To selectively study the interactions of active γ-secretase, we replaced one of the primary antibodies with a photoreactive γ-secretase inhibitor containing a PEG linker and a biotin group (GTB), and used oligonucleotide-conjugated streptavidin as a probe. Interestingly, significantly fewer interactions were detected with the latter, novel, assay, which is a reasonable finding considering that a substantial portion of PS1 is inactive. In addition, the PLA signals were located more peripherally when GTB was used instead of a PS1 antibody, suggesting that γ-secretase matures distal from the perinuclear ER region. This novel technique thus enables highly sensitive protein interaction studies, determines the subcellular location of the interactions, and differentiates between active and inactive γ-secretase in intact cells. We suggest that similar PLA assays using enzyme inhibitors could be useful also for other enzyme interaction studies.
authors.affiliation: KI-Alzheimer Disease Research Center-KI-ADRC, Karolinska Institutet, Department of Neurobiology, Care Sciences and Society-NVS, Novum Level 5, Stockholm, Sweden. sophia.schedin.weiss@ki.se
authors.forename: Sophia
authors.initials: S
authors.lastname: Schedin-Weiss
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Inoue
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Teranishi
authors.forename: Natsuko Goto
authors.initials: NG
authors.lastname: Yamamoto
authors.forename: Helena
authors.initials: H
authors.lastname: Karlström
authors.forename: Bengt
authors.initials: B
authors.lastname: Winblad
authors.forename: Lars O
authors.initials: LO
authors.lastname: Tjernberg
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D053595:Embryonic Stem Cells; D057075:Enzyme Assays; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D051379:Mice; D009097:Multienzyme Complexes; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects
chemical_list: D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D053829:Amyloid Precursor Protein Secretases