Article pubs.acs.org/biochemistry
Purification and Characterization of the Human γ-Secretase Activating Protein Catherine L. Deatherage, Arina Hadziselimovic, and Charles R. Sanders* Department of Biochemistry, Center for Structural Biology, and Institute of Chemical Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-8725, United States S Supporting Information *
ABSTRACT: Alzheimer’s disease is a fatal neurological disorder that is a leading cause of death, with its prevalence increasing as the average life expectancy increases worldwide. There is an urgent need to develop new therapeutics for this disease. A newly described protein, the γ-secretase activating protein (GSAP), has been proposed to promote elevated levels of amyloid-β production, an activity that seems to be inhibited using the well-establish cancer drug, imatinib (Gleevec). Despite much interest in this protein, there has been little biochemical characterization of GSAP. Here we report protocols for the recombinant bacterial expression and purification of this potentially important protein. GSAP is expressed in inclusion bodies, which can be solubilized using harsh detergents or urea; however, traditional methods of refolding were not successful in generating soluble forms of the protein that contained well-ordered and homogeneous tertiary structure. However, GSAP could be solubilized in detergent micelle solutions, where it was seen to be largely α-helical but to adopt only heterogeneous tertiary structure. Under these same conditions, GSAP did not associate with either imatinib or the 99-residue transmembrane C-terminal domain of the amyloid precursor protein. These results highlight the challenges that will be faced in attempts to manipulate and characterize this protein.
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protein (PION). The domain is proteolytically released from PION under cellular conditions, the resulting protein being termed GSAP. GSAP appears to form a ternary complex with γsecretase and C99, as determined through immunoprecipitation reactions and pull-down assays. Knockdown of GSAP through siRNA in N2a cells selectively lowered Aβ levels and did not reduce the level of cleavage of other γ-secretase substrates. GSAP knockdown also reduced the Aβ plaque burden in a mouse model of AD.8 These data suggest that GSAP may selectively promote Aβ production by promoting γ-secretase cleavage of C99, making GSAP a potential AD drug target. Since the initial discovery of GSAP, one additional research paper has been published; it characterized the immunohistochemical distribution of GSAP in the brains of AD patients.10 GSAP immunoreactivity was observed in four distinct morphological structures present in different regions of the brain in AD patients; one of these structures was largely unique to AD brains as compared to age-matched control brains. GSAP immunoreactivity was also detected in the proximity of presenilin (PS1, a component of γ-secretase) as well as in association with Aβ-containing senile plaques. While recombinant expression and purification of GSAP were briefly mentioned in these reports, methods were not provided. This
lzheimer's disease (AD) is a devastating neurodegenerative disease that impacts millions of people worldwide at enormous personal and economic cost.1 Unfortunately, there is currently no cure or effective treatment, but researchers have made significant progress in characterizing the pathophysiology of AD.2 The most widely accepted hypothesis for the etiology of the disease revolves around the amyloid precursor protein (APP).3 APP is cleaved by β-secretase to generate its 99-residue transmembrane C-terminus (C99), which is then cleaved by γsecretase to produce amyloid-β (Aβ) peptides of different lengths. These peptides form neurotoxic oligomers that are then deposited as neuritic plaques, the pathological markers of the disease. Inhibition of the heterotetrameric γ-secretase to block cleavage of C99 would reduce the level of Aβ production.4−6 Unfortunately, γ-secretase has numerous substrates and has not, so far, been an effective therapeutic target because of the important role that its cleavage of other substrates, particularly Notch, plays in cellular biology.7 As such, there is great interest in exploring how to prevent or modulate C99 cleavage without inhibiting cleavage of other γ-secretase substrates. This imperative resulted in the discovery of the γ-secretase activating protein (GSAP). GSAP was first described by He et al.8 A previous study had shown that the Abl kinase inhibitor imatinib decreases the level of Aβ production, likely by inhibiting γ-secretase activity.9 The search for the imatinib target led to photolabeling of the Cterminal domain of the uncharacterized pigeon homologue © 2012 American Chemical Society
Received: May 8, 2012 Revised: June 7, 2012 Published: June 8, 2012 5153
dx.doi.org/10.1021/bi300605u | Biochemistry 2012, 51, 5153−5159
Biochemistry
Article
confirmed by Western blotting using a monoclonal anti-5X His mouse antibody (Cell Signaling Technology, Danvers, MA). Purification of N- and C-Terminally His6-Tagged GSAP. The harvested cells were weighed and lysed in 20 mL of lysis buffer [75 mM Tris, 300 mM NaCl, and 0.2 mM EDTA (pH 7.8)] per gram of cells. Also added were 5 mM magnesium acetate, 2 mg/mL lysozyme, 0.2 mg/mL DNase and RNase, and 50 μL of protease inhibitor per gram of cells. The suspension was tumbled for 90 min at room temperature. After being tumbled, cells were further disrupted by a 5 min probe sonication with a 50% duty cycle at approximately 57 W using a Misonix (Farmingdale, NY) sonicator. The lysate was centrifuged at 20000 rpm in a Beckman-Coulter (Indianapolis, IN) JA 25.5 rotor (approximately 48000g), and the pellet, which includes inclusion bodies, was retained. The inclusion bodies containing GSAP were solubilized in 20 mL of lysis buffer per original gram of cells using 3% (v/v) Empigen, a harsh detergent. This solution was tumbled at room temperature until a clear mixture was observed (approximately 4.5 h). The sample was then centrifuged to remove any remaining insoluble particulates. Ni-NTA resin (1.2 mL/g of cells) was equilibrated with buffer A [40 mM HEPES and 300 mM NaCl (pH 7.8)]. The supernatant was tumbled with the resin for 1 h at room temperature. The resin was loaded into a column and sequentially washed with buffer A containing 3% (v/v) Empigen and buffer A containing 30 mM imidazole and 1.5% (v/v) Empigen, to elute all non-His10-tagged proteins from the resin. Empigen was then exchanged for the detergent ndodecylphosphocholine (DPC) by re-equilibrating the column with 12 column volumes of 20 mM phosphate (pH 7.2) containing 0.5% (w/v) DPC. GSAP was eluted from the column with 250 mM imidazole containing 0.5% (w/v) DPC (pH 7.8). Purification was monitored by A280. After purification, 2 mM DTT was added to the sample to reduce disulfide bonds. The purification process was monitored by SDS−PAGE. Electrophoresis experiments were conducted using an Invitrogen (Grand Island, NY) Novex-Mini Gel system and NuPAGE 4 to 12% Bis-Tris polyacrylamide gels and MES running buffer. Purification of the Thioredoxin-His6-GSAP Fusion Protein. The purification of Trx-His6-GSAP was mentioned but not described in the original paper8 but is similar to the His10-GSAP purification strategy described above (personal communication). Trx-His6-GSAP-expressing cells were grown, harvested, and lysed as described above. After lysis, the supernatant was collected and bound to Ni-NTA resin equilibrated in 50 mM phosphate and 500 mM NaCl (pH 7.8). The slurry was tumbled for 1 h at room temperature. The resin was rinsed with 50 mM phosphate and 500 mM NaCl (pH 7.8) and then washed with 50 mM phosphate, 500 mM NaCl, and 40 mM imidazole (pH 7.8) to remove any remaining nonspecifically bound protein. Trx-His6-GSAP was eluted from the column with 50 mM phosphate, 500 mM NaCl, and 300 mM imidazole (pH 7.8). The eluate was concentrated to a volume of
