Visualizing Active Enzyme Complexes Using a Photoreactive Inhibitor for Proximity Ligation – Application on γ-Secretase

<div><p>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.</p></div

PLA with GTB and PS1-CTF antibody.

<p>PLA was done as schematically depicted in A in the absence(B, C and D) or, as depicted in E, in the presence(F, G and H) of competitor L-685,458. The entire neuron is shown (B and F), a close-up of the cell body from the corresponding neuron (C and G) and a selected part of the neurites (D and H). The dots were quantified with Duolink Image Tool and expressed as fold increase to the sum of the negative controls (lacking the PS1 antibody or GTB) and as % inhibition in the presence of L-685,458, calculated by two different ways, as described in the results section (I and J). Mean values ± SE from at least three cells in four different experiments are shown. The p-value was calculated using one-tailed, paired t-test.</p

PLA with GTB and PS1-NTF antibody.

<p>PLA was done as schematically depicted in A in the absence (B, C and D) or as depicted in E in the presence(F, G and H) of competitor L-685,458. A neuron is shown (B and F), a close-up of the cell body from the corresponding neuron (C and G) and a selected part of the neurites (D and H). The number of dots were quantified with Duolink Image Tool and expressed as fold increase compared to the sum of the negative controls (lacking the PS1 antibody or GTB) and to the % inhibition in the presence of L-685,458, calculated in two different ways, as described in the results section (I and J). Mean values ± SE from at least three cells in three different experiments are shown. The p-value was calculated using one-tailed, paired t-test.</p

Structure and characterization of GTB.

<p>(A) Structure of the γ-secretase inhibitor with a transferable biotin group, denoted GTB, designed with a PEG linker, a disulfide bond, a photoreactive group and a biotin group. The distance between the inhibitor part and the biotin moiety was determined by 3D ChemDraw (CambridgeSoft). (B) Solubilized γ-secretase prepared from rat brain was incubated with different concentrations of GTB and isolated with streptavidin beads. The captured γ-secretase complex was eluted by SDS sample buffer and subjected to western blot for the indicated γ-secretase subunit. (C) Solubilized γ-secretase prepared from rat brain was incubated with 100 nM GTB in the presence (+) or the absence (-) of 10 µM L-685,458 and isolated with streptavidin beads and analyzed as described above.</p

PLA with GTB and nicastrin antibody.

<p>PLA was done as schematically depicted in Fig. A and E. Mouse nicastrin-CTF antibody was used with GTB in the absence(B, C and D) or presence(F, G and H) of the competitor L-685,458. The entire neuron is shown (B and F), a close-up of the cell body from the corresponding neuron (C and G) and a selected part of the neurites (D and H). The PLA signals (red dots) were quantified with Duolink Image Tool and expressed as the ratio between the sample and the sum of the negative controls (lacking the nicastrin antibody or GTB) and as % inhibition in the presence of L-685,458, calculated by two different ways, as described in the results section (I and J). Mean values ± SE from at least three cells in three different experiments are shown. The p-value was calculated using one-tailed, paired t-test.</p

Antibody validation.

<p>The applicability of antibodies for PLA was validated by a single recognition PLA experiment, as described in Experimental Procedures and schematically outlined in Fig. 2A–C. In addition to the PLA signals (red dots), the cells (embryonic mouse primary hippocampal neurons) were stained with DAPI and phalloidin for nuclei (blue) and actin (green), respectively. DAPI and PLA staining are shown for the PS1 antibody recognizing the N-terminus, the PS1 antibody recognizing the cytosolic loop and the nicastrin antibody recognizing a cytosolic epitope in D, E and F, respectively. DAPI, PLA and phalloidin staining for the same antibodies are shown in G, H and I, respectively.</p