Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Summary

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

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 cells⁴. 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 apoptosis⁸, to modulate signaling pathways⁹, and to regulate calcium signaling¹⁰. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology¹¹, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques¹². 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.

Protocol

1. Expression of Recombinant APP Intracellular Domain (AICD)

1. Transform E. coli strain BL21 with human AICD (residues 649-695 of APP, neuronal isoform numbering) cloned into vector pGEX-4T-1 (GE Healthcare). This vector will express AICD as the C-terminal moiety of a fusion protein of glutathione-S-transferase (GST). This vector also encodes a thrombin cleavage sequence to facilitate removal of the GST moiety. Details of cloning AICD into pGEX-4T-1 can be found in our previous publication⁴.
2. From a single colony, inoculate 10 ml of LB broth with ampicillin (100 μg/ml), and incubate overnight at 37 °C with vigorous shaking.
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Discussion

In this protocol we have outlined a procedure for obtaining highly pure AICD for structural, biophysical, and biochemical analyses. This procedure does not require sophisticated chromatography equipment and is therefore accessible to most laboratories. Other groups have purified AICD^5-7,16, including GST-AICD^17-19, for biochemical/structural analyses. Disadvantages to previous protocols include poor solubility of AICD¹⁶, less than ideal purity¹⁷, and the requirement for size ex.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
pGEX-4T-1	GE Healthcare	28-9545-49
Thrombin	GE Healthcare	27-0846-01
Ampicillin	Fisher Scientific	BP1760
Bradford protein assay reagent	Bio-Rad	500-0002
Coomassie blue	Bio-Rad	161-0786
IPTG ( isopropyl-beta-D thiogalactopyranoside)	Sigma-Aldrich	I6758
Glutathione-agarose	Sigma-Aldrich	G4510
p-aminobenzamidine-agarose	Sigma-Aldrich	A7155
Complete protease inhibitor cocktail	Roche	11836170001
Slide-A-Lyzer dialysis cassettes	Thermo Scientific	66380
Chromatography columns	Evergreen Scientific	208-3367-050
Emulsifier	Avestin, Inc	EmulsiFlex-C3	Highly recommended
Eppendorf Thermomixer	Eppendorf	022670107
Mica Disks	Ted Pella	50-12
AFM cantilevers	Bruker	MSNL-10
WSxM software	Nanotec	N/A	Free download