Purification and aggregation of the amyloid precursor protein intracellular domain

Amina El Ayadi, Emily S. Stieren, José M. Barral, Andres Oberhauser, Darren Boehning

Research output: Contribution to journalArticle

Abstract

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed α, β, and γ-secretases1. Sequential proteolytic cleavage of APP with β and γ-secretases leads to the production of a small proteolytic peptide, termed Aβ, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes 2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro 5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells 4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis 8, to modulate signaling pathways9, and to regulate calcium signaling 10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology 11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques 12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

Original languageEnglish (US)
JournalJournal of Visualized Experiments
Issue number66
StatePublished - Aug 28 2012

Fingerprint

Amyloid beta-Protein Precursor
Amyloid
Purification
Agglomeration
Proteins
Amyloid Precursor Protein Secretases
Alzheimer Disease
Molecular Chaperones
Calcium Signaling
Atomic Force Microscopy
Secretory Pathway
Amyloid Plaques
Protein C
Transgenic Mice
Peptide Hydrolases
Hot Temperature
Western Blotting
Pathology
Transcription
Apoptosis

Keywords

  • Aggregation
  • AICD
  • Alzheimer's disease
  • Amyloid precursor protein
  • APP
  • Atomic force microscopy
  • Cellular biology
  • Issue 66
  • Medicine
  • Molecular biology
  • Molecular chaperone
  • Neuroscience
  • Ubiquilin-1

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)

Cite this

Purification and aggregation of the amyloid precursor protein intracellular domain. / El Ayadi, Amina; Stieren, Emily S.; Barral, José M.; Oberhauser, Andres; Boehning, Darren.

In: Journal of Visualized Experiments, No. 66, 28.08.2012.

Research output: Contribution to journalArticle

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