1Department of Surgery, University of Texas Medical Branch, 2Department of Neuroscience and Cell Biology, University of Texas Medical Branch
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El Ayadi, A., Stieren, E. S., Barral, J. M., Oberhauser, A. F., Boehning, D. Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain. J. Vis. Exp. (66), e4204, doi:10.3791/4204 (2012).
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 genes2,3. 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 vitro5,6, 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.
1. Expression of Recombinant APP Intracellular Domain (AICD)
At this point it may be desirable to take out a small sample (~10 μl) to follow the purification by SDS-PAGE (see Figure 1A,B). Each subsequent purification step should also have a small aliquot removed for analysis by SDS-PAGE.
2. Purification of GST-AICD
At this point, it is critical that all steps are performed at 4 °C to limit proteolysis. All tubes, buffers, and other reagents should be pre-chilled. If using a French press or emulsifier for lysis, these should be pre-chilled as well.
The main advantage of these two instruments is that they minimize heating of the sample. The commonly used technique of sonication generates significant heat at the tip of the sonication probe and can result in aggregation of recombinant proteins.
The only step of the protocol which is performed at room temperature is the elution step. Thus, the elution buffer is kept at room temperature. Elution buffer should be prepared fresh.
3. Thrombin Cleavage of GST-AICD and Purification of AICD
The quality and purity of thrombin varies considerably among manufacturers, and can be a significant source of contamination. We use thrombin from GE Healthcare (see table of reagents). After overnight incubation, >95% of the fusion protein should be cleaved (Figure 1B).
Purified AICD should be prepared fresh for biochemical/biophysical characterization. Unused material should be discarded. It is also possible to concentrate the AICD by lyophilizing the sample and resuspending in a smaller volume of buffer6. If detecting AICD requires blotting (such as after a filter trap or dot blot assay), antigen retrieval on the membrane should be performed as described by Pimplikar and Suryanarayana12.
4. AICD Aggregation for Atomic Force Microscopy (AFM)
It is also possible to aggregate larger reaction volumes for biochemical experiments such as filter trap assays and light scattering4. We have had success performing filter trap assays on reaction mixtures as large as 400 μl with AICD diluted to concentrations as low as 1 μM.
Thermal aggregation is a commonly used method to induce the structural transition of a protein from a native to a non-native structure (which is usually beta sheet rich) which results in the formation of intermolecular aggregates. Thermal aggregation is also used to examine the function of molecular chaperones, which shield hydrophobic segments from forming inappropriate intermolecular interactions (and thus retard or prevent thermal aggregation).
We found that image quality and contrast of different protein aggregates depended on the applied force and the duration of the experiment. To minimize sample perturbation by the tip, we maintained the applied force relatively low by keeping a set-point ratio above 95% and limiting the scanning of each sample to 5-10 min. Image processing was performed with WSxM software (Nanotec). Standard image processing consisted of plane subtraction and flattening. Our laboratories use a "home-built" AFM15 interfaced to a commercial scanning probe microscope control system (Nanotec).
5. Representative Results
The expression and relative enrichment of GST-AICD is shown in Figure 1A. After lysis, the majority of the fusion protein is present in the soluble fraction, and therefore extraction from inclusion bodies is not required. The material eluted from the glutathione agarose column is >95% pure. In the preparation shown in Figure 1, the concentration of protein eluted from the column was 14.5 mg/ml, and the yield was 22 mg (from a starting culture of 400 ml). Cleavage of 200 μl of this preparation overnight with 20 U of thrombin resulted in almost 100% cleavage of the fusion protein (Figure 1B). The amount of thrombin used is low enough as to not be detectable by Coomassie blue staining (see "Cleaved" lane, Figure 1B). Removal of the thrombin and GST resulted in some loss of material, however it was >90% pure with a yield in this prep of 70 μg of AICD at a concentration of 0.35 mg/ml. Figure 1C shows the flow chart for aggregation of AICD and subsequent analysis. AICD aggregates imaged by AFM are typically spheroid/amorphous, and range in size from 50 to 100 nm (Figure 1D,F). Aggregated material is not detectable when an equimolar amount of the chaperone ubiquilin-1 is added to the aggregation reaction (Figure 1E)4.
Figure 1. Purification and aggregation of AICD. A) Coomassie blue staining of samples collected at each step of the purification process. For the uninduced and induced samples, 20 μl of total bacteria were run on the gel. For the supernatant fraction, 5 μl of lysate (from a total volume of 20 ml) were run on the gel. For the pellet fraction, the pellet was resuspended in 20 ml of lysis buffer, and 5 μl of this material were sonicated in a water bath sonicator for 30 min at 4 °C prior to loading onto the gel. For the flowthrough, 5 μl were run on the gel. The amounts of eluate loaded onto the gel are indicated. B) Coomassie blue stained gel of uncleaved and cleaved GST-AICD (2 μl) and 1 and 5 μl purified AICD. C) Flowchart of AICD purification, aggregation, and analysis. D,E) Representative AFM image of thermally aggregated AICD in the absence (D) and presence (E) of the molecular chaperone ubiquilin-1. F) Histogram of the size distribution of AICD aggregates.
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 AICD5-7,16, including GST-AICD17-19, for biochemical/structural analyses. Disadvantages to previous protocols include poor solubility of AICD16, less than ideal purity17, and the requirement for size exclusion16 or reversed phase chromatography6,20. Thus, the protocol described here should greatly facilitate in vitro studies of AICD. We have also described a method for thermal aggregation of AICD and detection by AFM. Although increasingly used in the amyloid field21, AFM is a highly specialized technique. More approachable assays such as light scattering, sedimentation, and filter trap assays can also be used to examine the thermal aggregation of AICD4. Finally, the ability to examine directly in vitro the ability of purified molecular chaperones, such as ubiquilin-14, to prevent the thermal aggregation of AICD opens the door for elucidating the functional role of other cytosolic chaperones in modulating APP biology.
No conflicts of interest declared.
The authors would like to thank Dr. Hui Zheng (Baylor College of Medicine) for the APP cDNA. This work was funded by NIH grants R21AG031948 (D.B., J.M.B.), F30AG030878 (E.S.S.), R01DK073394 (AFO), the John Sealy Memorial Endowment Fund for Biomedical Research (AFO), and the Jean C. and William D. Willis Neuroscience Research Endowment (E.S.S.). J.M.B. is a scholar in the Translational Research Scholar Program and a member of the University Of Texas Medical Branch Claude E. Pepper Older Americans Independence Center (supported by NIH Grants UL1RR029876 and P30-AG-024832, respectively).
|Bradford protein assay reagent||Bio-Rad||500-0002|
|IPTG ( isopropyl-beta-D thiogalactopyranoside)||Sigma-Aldrich||I6758|
|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|
|Mica Disks||Ted Pella||50-12|
|WSxM software||Nanotec||N/A||Free download|