Visualizing active enzyme complexes using a photoreactive inhibitor for proximity ligation--application on...

Area of Science:

• Biochemistry
• Cell Biology
• Neuroscience

Background:

• Protein-protein interactions are vital for enzyme function.
• γ-secretase (GCase) cleaves amyloid precursor protein (APP), producing amyloid β-peptide (Aβ), a key factor in Alzheimer disease (AD).
• Existing methods struggle to selectively study active enzyme complexes in situ.

Purpose of the Study:

• To develop a highly sensitive method for studying active multicomponent enzyme interactions and subcellular localization.
• To apply this novel assay to γ-secretase to understand its active forms and localization.
• To differentiate between active and inactive γ-secretase complexes within intact cells.

Main Methods:

• Utilized a modified proximity ligation assay (PLA) based on enzyme-inhibitor recognition.
• Employed a photoreactive γ-secretase inhibitor (GTB) conjugated to a PEG linker and biotin as a probe.
• Used oligonucleotide-conjugated streptavidin for detection, enabling selective interaction studies of active γ-secretase.

Main Results:

• The novel assay detected significantly fewer interactions compared to traditional antibody-based PLA, reflecting the proportion of inactive γ-secretase.
• PLA signals localized more peripherally when the inhibitor probe was used, suggesting γ-secretase maturation occurs away from the ER.
• The method successfully determined the subcellular location of active γ-secretase interactions.

Conclusions:

• The developed inhibitor-based PLA is a sensitive tool for studying active enzyme interactions and localization in situ.
• This technique provides new insights into γ-secretase activity, localization, and maturation in Alzheimer disease research.
• The approach is adaptable for studying interactions of other enzyme complexes.