Purification and characterization of the human γ-secretase activating protein

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.     

Intracellular trafficking of the β-secretase and processing of amyloid precursor protein

The main component of the amyloid plaques found in the brains of those with Alzheimer's disease (AD) is a polymerized form of the β-amyloid peptide (Aβ) and is considered to play a central role in the pathogenesis of this neurodegenerative disorder. Aβ is derived from the proteolytic processing of the amyloid precursor protein (APP). Beta site APP-cleaving enzyme, BACE1 (also known as β-secretase) is a membrane-bound aspartyl protease responsible for the initial step in the generation of Aβ peptide and is thus a prime target for therapeutic intervention. Substantive evidence now indicates that the processing of APP by BACE1 is regulated by the intracellular sorting of the enzyme and, moreover, perturbations in these intracellular trafficking pathways have been linked to late-onset AD. In this review, we highlight the recent advances in the understanding of the regulation of the intracellular sorting of BACE1 and APP and illustrate why the trafficking of these cargos represent a key issue for understanding the membrane-mediated events associated with the generation of the neurotoxic Aβ products in AD.     

Highly polymorphic repeat marker within the β-amyloid precursor protein gene

We have identified a polymorphic compound dinucleotide repeat sequence in intron 1 of the -amyloid precursor protein (APP) gene on chromosome 21. Using polymerase chain reaction (PCR) amplification of the locus, designated APPivsl, we detected 13 alleles in the CEPH family members (heterozygosity = 0.69). Lod score analysis showed complete linkage of the marker to the loci D21S210 and D21221.     

Structure and mechanism of the γ-secretase intramembrane protease complex

γ-Secretase is a membrane protein complex that proteolyzes within the transmembrane domain of >100 substrates, including those derived from the amyloid precursor protein and the Notch family of cell surface receptors. The nine-transmembrane presenilin is the catalytic component of this aspartyl protease complex that carries out hydrolysis in the lipid bilayer. Advances in cryoelectron microscopy have led to the elucidation of the structure of the γ-secretase complex at atomic resolution. Recently, structures of the enzyme have been determined with bound APP- or Notch-derived substrates, providing insight into the nature of substrate recognition and processing. Molecular dynamics simulations of substrate-bound enzymes suggest dynamic mechanisms of intramembrane proteolysis. Structures of the enzyme bound to small-molecule inhibitors and modulators have also been solved, setting the stage for rational structure-based drug discovery targeting γ-secretase.     

Metabolism of amyloid precursor protein in COS cells transfected with a β-secretase candidate

Thimet oligopeptidase (TOP) is a thiol- andmetallo-dependent peptidase and has been shown to beone of the -secretase candidates. TOPexpressed in COS cells cleaved amyloid precursorprotein (APP) at the -secretase site, and wefound a proteolytic product of APP called secretedform of APP by -secretase (sAPP) in theconditioned media. Here we demonstrate thatsAPP was increased in conditioned media whenTOP was coexpressed in COS cells with APP and treatedwith an ADAM inhibitor SI-27. In addition, althoughTOP expressed in COS cell was localized at nuclei orGolgi apparatus, it exclusively colocalized at Golgiapparatus when APP was coexpressed with TOP.     

Substrate sequence influences γ-secretase modulator activity, role of the transmembrane domain of the amyloid precursor protein

A subset of non-steroidal anti-inflammatory drugs modulates the γ cleavage site in the amyloid precursor protein (APP) to selectively reduce production of Aβ42. It is unclear precisely how these γ-secretase modulators (GSMs) act to preferentially spare Aβ40 production as well as Notch processing and signaling. In an effort to determine the substrate requirements in NSAID/GSM activity, we determined the effects of sulindac sulfide and flurbiprofen on γ-cleavage of artificial constructs containing several γ-secretase substrates. Using FLAG-tagged constructs that expressed extracellularly truncated APP, Notch-1, or CD44, we found that these substrates have different sensitivities to sulindac sulfide. γ-Secretase cleavage of APP was altered by sulindac sulfide, but CD44 and Notch-1 were either insensitive or only minimally altered by this compound. Using chimeric APP constructs, we observed that the transmembrane domain (TMD) of APP played a pivotal role in determining drug sensitivity. Substituting the APP TMD with that of APLP2 retained the sensitivity to γ-cleavage modulation, but replacing TMDs from Notch-1 or ErbB4 rendered the resultant molecules insensitive to drug treatment. Specifically, the GXXXG motif within APP appeared to be critical to GSM activity. Consequently, the modulatory effects on γ-cleavage appears to be substrate-dependent. We hypothesize that the substrate present in the γ-secretase complex influences the conformation of the complex so that the binding site of GSMs is either stabilized or less favorable to influence the cleavage of the respective substrates.     

Loss of cleavage at β'-site contributes to apparent increase in β-amyloid peptide (Aβ) secretion by β-secretase (BACE1)-glycosylphosphatidylinositol (GPI) processing of amyloid precursor protein

Several lines of evidence implicate lipid raft microdomains in Alzheimer disease-associated β-amyloid peptide (Aβ) production. Notably, targeting β-secretase (β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1)) exclusively to lipid rafts by the addition of a glycosylphosphatidylinositol (GPI) anchor to its ectodomain has been reported to elevate Aβ secretion. Paradoxically, Aβ secretion is not reduced by the expression of non-raft resident S-palmitoylation-deficient BACE1 (BACE1-4C/A (C474A/C478A/C482A/C485A)). We addressed this apparent discrepancy in raft microdomain-associated BACE1 processing of APP in this study. As previously reported, we found that expression of BACE1-GPI elevated Aβ secretion as compared with wild-type BACE1 (WTBACE1) or BACE1-4C/A. However, this increase occurred without any difference in the levels of APP ectodomain released following BACE1 cleavage (soluble APPβ), arguing against an overall increase in BACE1 processing of APP per se. Further analysis revealed that WTBACE1 cleaves APP at β- and β'-sites, generating +1 and +11 β-C-terminal fragments and secreting intact as well as N-terminally truncated Aβ. In contrast, three different BACE1-GPI chimeras preferentially cleaved APP at the β-site, mainly generating +1 β-C-terminal fragment and secreting intact Aβ. As a consequence, cells expressing BACE1-GPI secreted relatively higher levels of intact Aβ without an increase in BACE1 processing of APP. Markedly reduced cleavage at β'-site exhibited by BACE1-GPI was cell type-independent and insensitive to subcellular localization of APP or the pathogenic KM/NL mutant. We conclude that the apparent elevation in Aβ secretion by BACE1-GPI is mainly attributed to preferential cleavage at the β-site and failure to detect +11 Aβ species secreted by cells expressing WTBACE1.     

Contribution of the γ-secretase subunits to the formation of catalytic pore of presenilin 1 protein

γ-Secretase is an intramembrane-cleaving protease related to the etiology of Alzheimer disease. γ-Secretase is a membrane protein complex composed of presenilin (PS) and three indispensable subunits: nicastrin, Aph-1, and Pen-2. PS functions as a protease subunit forming a hydrophilic catalytic pore structure within the lipid bilayer. However, it remains unclear how other subunits are involved in the pore formation. Here, we show that the hydrophilic pore adopted with an open conformation has already been formed by PS within the immature γ-secretase complex. The binding of the subunits induces the close proximity between transmembrane domains facing the catalytic pore. We propose a model in which the γ-secretase subunits restrict the arrangement of the transmembrane domains of PS during the formation of the functional structure of the catalytic pore.     

MEK inhibitors suppress β-amyloid production by altering the level of a β-C-terminal fragment of amyloid precursor protein in neuronal cells

β-Amyloid peptide (Aβ) is generated via sequential proteolysis of amyloid precursor protein (APP) by β- and γ-secretases. Cell-based screening experiments disclosed that the MEK (MAP kinase kinase) inhibitors, U0126 and PD184352, suppress Aβ secretion from human neuronal SH-SY5Y cells expressing Swedish mutant APP. These inhibitors did not affect the cellular levels of APP but significantly reduced those of the APP β-C-terminal fragment (β-CTF). Additionally, β-CTF levels were markedly reduced by these inhibitors in cells expressing the fragment in a γ-secretase-independent and proteasome-dependent manner. Our results suggest that MEK inhibitors reduce Aβ generation via secretase-independent alteration of β-CTF levels.     

Adaptor protein 2-mediated endocytosis of the β-secretase BACE1 is dispensable for amyloid precursor protein processing

The β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease that catalyzes the proteolytic processing of APP and other plasma membrane protein precursors. BACE1 cycles between the trans-Golgi network (TGN), the plasma membrane, and endosomes by virtue of signals contained within its cytosolic C-terminal domain. One of these signals is the DXXLL-motif sequence DISLL, which controls transport between the TGN and endosomes via interaction with GGA proteins. Here we show that the DISLL sequence is embedded within a longer [DE]XXXL[LI]-motif sequence, DDISLL, which mediates internalization from the plasma membrane by interaction with the clathrin-associated, heterotetrameric adaptor protein 2 (AP-2) complex. Mutation of this signal or knockdown of either AP-2 or clathrin decreases endosomal localization and increases plasma membrane localization of BACE1. Remarkably, internalization-defective BACE1 is able to cleave an APP mutant that itself cannot be delivered to endosomes. The drug brefeldin A reversibly prevents BACE1-catalyzed APP cleavage, ruling out that this reaction occurs in the endoplasmic reticulum (ER) or ER-Golgi intermediate compartment. Taken together, these observations support the notion that BACE1 is capable of cleaving APP in late compartments of the secretory pathway.     

Modulation of β-amyloid precursor protein trafficking and processing by the low density lipoprotein receptor family

Selenium is an essential micronutrient that function through selenoproteins. Selenium deficiency results in lower concentrations of selenium and selenoproteins. The brain maintains it's selenium better than other tissues under low-selenium conditions. Recently, the selenium-containing protein selenoprotein P (Sepp) has been identified as a possible transporter of selenium. The targeted disruption of the selenoprotein P gene (Sepp1) results in decreased brain selenium concentration and neurological dysfunction, unless selenium intake is excessive However, the effect of selenoprotein P deficiency on the processes of memory formation and synaptic plasticity is unknown. In the present studies Sepp1(-/-) mice and wild type littermate controls (Sepp1(+/+)) fed a high-selenium diet (1 mg Se/kg) were used to characterize activity, motor coordination, and anxiety as well as hippocampus-dependent learning and memory. Normal associative learning, but disrupted spatial learning was observed in Sepp1(-/-) mice. In addition, severe alterations were observed in synaptic transmission, short-term plasticity and long-term potentiation in hippocampus area CA1 synapses of Sepp1(-/-) mice on a 1 mg Se/kg diet and Sepp1(+/+) mice fed a selenium-deficient (0 mg Se/kg) diet. Taken together, these data suggest that selenoprotein P is required for normal synaptic function, either through presence of the protein or delivery of required selenium to the CNS.     

Reduction of histone cytotoxicity by the Alzheimer β-amyloid peptide precursor

In a search for Alzheimer β-amyloid peptide precursor ligands, Potempska et al. (Arch. Biochem. Biophys. (1993) 304, 448) found that histones bind with high affinity and specificity to the secreted precursor. Because exogenous histones can be cytotoxic, we compared the effects of histones on the viability of cells which produce little β-amyloid peptide precursor (U-937) to those on cells that produce twenty times as much precursor (COS-7). Addition of purified histones caused necrosis of U-937 cells (histone H4, LD₅₀=1.5 μM). Extracellular Aβ precursor in the submicromolar range prevented histone-induced U-937 cell necrosis. Cell-surface precursor also reduced histone toxicity: COS-7 cells were less sensitive to the toxic effects of histone H4 (LD₅₀=5.4 μM). COS-7 cells in which the expression of an APP mRNA-directed ribozyme reduced the synthesis of the protein by up to 80% were more sensitive to histone H4 (LD₅₀=3.2 μM) than cells that expressed the vector alone. Histone H4 binds to cell-associated Aβ precursor. Cells expressing the Aβ precursor-directed ribozyme bound less ¹²⁵I-labeled histone H4 than those expressing the vector alone. In the limited extracellular space of tissues in vivo, both secreted and cell-surface Aβ precursor protein may play significant roles in trapping chromatin or histones and removing them from the extracellular milieu.     

Determination of the proteolytic cleavage sites of the amyloid precursor-like protein 2 by the proteases ADAM10, BACE1 and γ-secretase

Regulated intramembrane proteolysis of the amyloid precursor protein (APP) by the protease activities α-, β- and γ-secretase controls the generation of the neurotoxic amyloid β peptide. APLP2, the amyloid precursor-like protein 2, is a homolog of APP, which shows functional overlap with APP, but lacks an amyloid β domain. Compared to APP, less is known about the proteolytic processing of APLP2, in particular in neurons, and the cleavage sites have not yet been determined. APLP2 is cleaved by the β-secretase BACE1 and additionally by an α-secretase activity. The two metalloproteases ADAM10 and ADAM17 have been suggested as candidate APLP2 α-secretases in cell lines. Here, we used RNA interference and found that ADAM10, but not ADAM17, is required for the constitutive α-secretase cleavage of APLP2 in HEK293 and SH-SY5Y cells. Likewise, in primary murine neurons knock-down of ADAM10 suppressed APLP2 α-secretase cleavage. Using mass spectrometry we determined the proteolytic cleavage sites in the APLP2 sequence. ADAM10 was found to cleave APLP2 after arginine 670, whereas BACE1 cleaves after leucine 659. Both cleavage sites are located in close proximity to the membrane. γ-secretase cleavage was found to occur at different peptide bonds between alanine 694 and valine 700, which is close to the N-terminus of the predicted APLP2 transmembrane domain. Determination of the APLP2 cleavage sites enables functional studies of the different APLP2 ectodomain fragments and the production of cleavage-site specific antibodies for APLP2, which may be used for biomarker development.     

Effect of ionophores on the processing of theβ-amyloid precursor protein in different cell lines

Summary 1. Alzheimer's disease is characterized by the deposition in the brain of extracellular amyloid plaques and vascular deposits consisting mostly of amyloid-peptide (A). A, a polypeptide of 39–43 amino acids (M _r, 4 kDa), is derived proteolytically from a family of proteins of 695–770 amino acids (M _r, 110–140 kDa) called-amyloid precursor protein (APP).2.APP, an integral membrane glycoprotein, is extensively posttranslationally modified within the endoplasmic reticulum (ER) and various Golgi compartments.APP is cleaved by proteases in either the trans-Golgi network or the post-Golgi apparatus and then secreted as a truncated soluble form into the conditioned media of cultured cells and cerebrospinal fluid samples from human subjects.APP can be processed either by an antiamyloidogenic secretory pathway or by an endosomal/lysosomal pathway.3. I studied the effect of two ionophores on the processing ofAPP in cultured cells. Monensin and, in some cases, ammonium chloride increase the intracellular accumulation ofAPP in several cell lines and may alter its processing. Monensin, which had the most consistent effects, also inhibited secretion ofAPP in a differentiated (growth factor mediated) cell line. Nigericin, with greater K⁺ selectivity, was less able to alter the accumulation and possible processing of the protein.4. These results suggest that the increase in the accumulation of intracellularAPP observed after treating cells with ionophores has some specificity. The selective effect of these ionophores on the metabolism ofAPP may provide a model system to analyze the pathways for studying maturation, secretion, and degradation ofAPP.     

The type III TGF-β receptor betaglycan transmembrane-cytoplasmic domain fragment is stable after ectodomain cleavage and is a substrate of the intramembrane protease γ-secretase

The Type III TGF-β receptor, betaglycan, is a widely expressed proteoglycan co-receptor for TGF-β superfamily ligands. The full-length protein undergoes ectodomain cleavage with release of a soluble ectodomain fragment. The fate of the resulting transmembrane-cytoplasmic fragment, however, has never been explored. We demonstrate here that the transmembrane-cytoplasmic fragment is stable in transfected cells and in cell lines expressing endogenous betaglycan. Production of this fragment is inhibited by the ectodomain shedding inhibitor TAPI-2. Treatment of cells with inhibitors of the intramembrane protease γ-secretase stabilizes this fragment, suggesting that it is a substrate of γ-secretase. Expression of the transmembrane-cytoplasmic fragment as well as γ-secretase inhibitor stabilization are independent of TGF-β1 or -β2 and are unaffected by mutation of the cytoplasmic domain serines that undergo phosphorylation. γ-Secretase inhibition or the expression of a transmembrane-cytoplasmic fragment in HepG2 cells blunted TGF-β2 signaling. Our findings thus suggest that the transmembrane-cytoplasmic fragment remaining after betaglycan ectodomain cleavage is stable and a substrate of γ-secretase, which may have significant implications for the TGF-β signaling response.     

Purification and characterization of novel organic-solvent-tolerant β-amylase and serine protease from a newly isolated Salimicrobium halophilum strain LY20

A halophilic isolate Salimicrobium halophilum strain LY20 producing extracellular amylase and protease was isolated from Yuncheng, China. Production of both enzymes was synchronized with bacterial growth and reached a maximum level during the early-stationary phase. The amylase and protease were purified to homogeneity with molecular weights of 81 and 30?kDa, respectively. Optimal amylase activity was observed at 70?°C, pH 10.0% and 10% NaCl. Complete inhibition by EDTA, diethyl pyrocarbonate (DEPC), and phenylarsine oxide (PAO) indicated that the amylase was a metalloenzyme with histidine and cysteine residues essential for its catalysis. Maltose was the main product of starch hydrolysis, indicating an β-amylase activity. The purified protease from LY20 showed highest activity at 80?°C, pH 10.0% and 12.5% NaCl. Complete inhibition was shown by phenylmethylsulfonyl fluoride, DEPC, and PAO, indicating that the enzyme probably belonged to the subclass of the serine proteases with histidine and cysteine residues essential for catalysis. Furthermore, both enzymes were highly stable over broad temperature (30-80?°C), pH (6.0-12.0) and NaCl concentration (2.5-20%) ranges, showing excellent thermostable, alkalistable, and halotolerant nature. The surfactants (SDS, Tween 80, and Triton X-100) did not affect their activities. In addition, both enzymes from LY20 displayed remarkable stability in the presence of water-soluble organic solvents with log P(ow) (?) ≤?-0.24.