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Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain
Published on: August 28, 2012
APP dimer formation is initiated in the endoplasmic reticulum and differs between APP isoforms
Simone Isbert1, Katja Wagner, Simone Eggert
1Department of Pathobiochemistry, Molecular Neurodegeneration, University Medical Center of the Johannes Gutenberg-University Mainz, Germany.
Cellular and Molecular Life Sciences : CMLS
|November 23, 2011
Summary
Amyloid precursor protein (APP) forms disulfide-bonded dimers in the endoplasmic reticulum (ER), identified using cell models and biochemical analysis. These dimers remain stable during transport to the cell surface.
Area of Science:
- Cell Biology
- Molecular Biology
- Neuroscience
Background:
- Amyloid precursor protein (APP) belongs to a gene family known to form complexes.
- The precise mechanism and origin of APP dimer formation are not fully understood.
Purpose of the Study:
- To investigate the cellular location and mechanism of APP dimerization.
- To determine the role of the endoplasmic reticulum (ER) in APP dimer formation.
Main Methods:
- Generated a CHO-K1 cell model system for stable expression of human APP with organelle-specific retention motifs (ER and Golgi).
- Utilized biochemical approaches, including SDS-polyacrylamide gel electrophoresis under non-reducing and reducing conditions.
- Employed split GFP complementation assays to visualize APP dimerization in specific subcellular compartments.
Main Results:
- APP forms disulfide-bonded dimers within the ER, evidenced by retention of dimers under non-reducing conditions.
- APP dimerization is dependent on intermolecular disulfide bonds, likely involving cysteines in the extracellular E1 domain.
- APP dimers are stable during transport from the ER to the cell surface.
- APP isoforms with the Kunitz-type protease inhibitor (KPI) domain show reduced cis-dimerization in the ER.
- APP/APLP1 heterointeractions are initiated in the ER.
Conclusions:
- The ER is the primary site of APP disulfide-bonded dimer formation.
- APP dimerization is a stable process that can occur even after transport out of the ER.
- The presence of the KPI domain influences APP cis-dimerization within the ER.
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