secretase (BACE) within its catalytic domain: A

Endoproteolysis of E-secretase (BACE) within its catalytic domain: A potential mechanism for regulation Jason T. Huse*, Damani Byant¶, Yaxiong Yang¶, Donald S. Pijak*, Ian D’Souza ¶, James. J. Lah §, Virginia M.-Y. Lee†, Robert W. Doms*, and David G. Cook¶‡

*Department of Microbiology and †Center for Neurodegenerative Disease Research, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; ¶ Veterans Affairs Puget Sound

Department of Medicine, University of Washington School of Medicine, Seattle WA 98108; § Department of Neurology, Emory University School of Medicine, Atlanta, GA 30322.

‡ To whom correspondence should be addressed: David G. Cook, Ph.D. VAPSHCS, GRECC (182B) 1660 S. Columbian Way Seattle, WA 98108 206-768-5437 (phone) 206 764-2569 (FAX) [email protected] Running title: Endoproteolysis of BACE within its catalytic domain Key words: Alzheimer's disease, BACE, E-secretase, endoproteolysis, tissue differentiation, trans-Golgi network, amyloid

Downloaded from www.jbc.org by guest, on March 21, 2013

Health Care System, Geriatric Research Education and Clinical Center (GRECC) and

Huse et. al.

Summary Sequential proteolysis of the amyloid precursor protein (APP) by E- and J-secretase activities yields the AE peptide that is widely deposited in the brains of individuals with Alzheimer's disease (AD). The membrane anchored aspartyl protease BACE (E-site APPcleaving enzyme) exhibits all of the characteristics of a E-secretase and has been shown to cleave APP at its E-site in vitro and in vivo. We found that BACE undergoes cleavage on a surface exposed alpha helix between amino acid residues Leu228 and Ala229, generating stable

efficiency of BACE endoproteolysis was observed to depend heavily on cell and tissue type. In contrast to brain where holoprotein was predominant, BACE was found primarily as endoproteolyzed fragments in pancreas, liver, and muscle. In addition, we observed a marked up-regulation of BACE endoproteolysis in C2 myoblasts upon differentiation into multinucleated myotubes, a well-established model system of muscle tissue specification. As in liver, BACE exists as endoproteolyzed fragments in the hepatic cell line, HepG2. We found that HepG2 cells are capable generating AE, suggesting that endoproteolyzed BACE retains measurable E-secretase activity. We also found that BACE endoproteolysis occurs only after export from the endoplasmic reticulum (ER), is enhanced in the trans-Golgi network (TGN), and is sensitive to inhibitors of vesicular acidification. The membrane bound proteases TACE (tumor necrosis factor D converting enzyme) and furin were not found to be responsible for this cleavage nor was BACE observed to mediate its own endoproteolysis by an autocatalytic mechanism. Thus, we characterize a specific processing event for that may serve to regulate the enzymatic activity of BACE on a post-translational level.

2


N- and C-terminal fragments that remain covalently associated via a disulfide bond. The

Huse et. al.

Introduction Alzheimer's disease (AD)1 is characterized pathologically by the florid accumulation of insoluble amyloid E peptide (AE) in the central nervous system (CNS) (1). A causative role for AE in AD progression has been accepted by many for some time (2,3), and recent evidence from mouse models has underscored the importance of this peptide in the process of cognitive decline (4-6). AE is produced by the sequential processing of the amyloid precursor protein (APP) by two proteolytic activities that have historically been referred to as E- and J-secretase.

protein precludes the generation of the peptide (7). Numerous studies have identified the membrane anchored aspartyl protease BACE as the E-secretase (8-12), while evidence continues to mount that J-secretase activity is mediated at least in part by the presenilin proteins (PS1 and PS2) (13-17). A group of metalloprotease disintegrins, including tumor necrosis factor-D converting enzyme (TACE), appears to be responsible for at least a significant fraction of cellular D-secretase activity (18,19). The central role of AE in AD pathogenesis requires careful study of the proteins involved in APP/AE processing and metabolism. The D-secretase activity of TACE, for instance, may depend on C-terminal phosphorylation of the enzyme by protein kinase C (PKC) (18,19). The presenilins have also been demonstrated to undergo phosphorylation (20-22), although the functional impact of these modifications remains unclear. More thoroughly characterized is the endoproteolysis of PS1 and PS2, an event that generates distinct presenilin 1

The Abbreviations used are: AD, Alzheimer's disease; Aß, amyloid ß; APP, amyloid precursor protein; BACE, ß-site APP-cleaving enzyme; BACE-Ct, BACE C-terminal fragment; BACE-Nt, BACE N-terminal fragment; TACE, tumor necrosis factor a converting enzyme; PS1, presenilin 1; PS2, presenilin 2; ER, endoplasmic reticulum; TGN, trans-Golgi network; HA, influenza hemaglutinin; BC-1, BACE C-terminal

3


Alternatively, cleavage of APP by so-called D-secretase activity within the AE region of the

Huse et. al. N- and C-terminal fragments which are thought to represent the biologically active forms of the proteins. At endogenous levels the cleavage fragments of PS1 and PS2 predominate, while under conditions of over expression, the vast majority of the presenilins exist as holoproteins, presumably due to the saturation of the endoproteolytic pathway (23-27). Despite its fairly recent discovery, BACE has already been the subject of numerous investigations designed to elucidate its basic cell biology. During maturation in the secretory pathway, BACE undergoes glycosylation at 3-4 N-linked sites and is separated from its propeptide domain (28-30). This latter processing step is mediated by a member of the

cysteine residues within its cytoplasmic domain and C-terminally phosphorylated (33,34). Mature BACE has a t1/2 of 12-16 hours and cycles between the cell surface, the endosomal system, and the TGN multiple times through the course of its life span (29,30,34). The cleavage site specificity of BACE for its APP substrate has been shown to depend heavily on the intracellular localization of the protease (35). In this study, we describe a specific endoproteolytic event that may have a significant effect on BACE activity. While the existence of BACE breakdown products has been documented (10), the protein fragments themselves have not been well characterized and the process by which they are generated remains unclear. We demonstrate BACE endoproteolysis occurs between amino acid residues Leu228 and Ala229, generating stable N- and C-terminal fragments that remain associated in a heterodimeric complex stabilized in part by a disulfide bond. This endoproteolytic process is regulated in a cell type and tissue type dependent fashion. We show that BACE endoproteolysis does not appear to take place in neurons. However, the

antisera 1; BME, ß-mercaptoethanol; FBS, fetal bovine serum; PBS, phosphate buffered saline; RIPA, radioimmunoprecipitation assay

4


proprotein convertase family of proteases (31,32). In addition, BACE is palmitoylated at

Huse et. al. processing event occurs robustly in a variety of native tissues and may play an important role in the differentiation of muscle cells. We also demonstrate that endoproteolyzed BACE appears to retain at least some E-secretase activity. Finally, we investigate the cell biological determinants and molecular mechanisms underlying the cleavage of BACE. Our results indicate that BACE, like the presenilins, undergoes an endoproteolytic cleavage event with intriguing functional implications.


5

Huse et. al.

Experimental Procedures Plasmid Construction and Expression—The generation of BACE, BACE-KK, and BACETGN constructs has been described (30,35). TACE and m-furin-HA were expressed in pcDNA3.1 (19,36). Expression of DNA constructs in cultured cells was obtained using Geneporter transfection reagent (Gene Therapy Systems, San Diego, CA) with a DNA/Geneporter ratio of 1 µg/10 µl. For some experiments BACE and APP were expressed using a replication-defective Semliki Forest Virus (SFV) vector expressing wild-type human BACE and wild-type APP695. The vectors were generated by cloning cDNA into the pSFV-1

Antibodies—Several C-terminal antibodies were used in the recognition of BACE and BACECt. BC-1 is a rabbit polyclonal antisera directed against C-terminus of BACE (30). Polyclonal rabbit antisera (88.6) recognizing the carboxyl-terminus of BACE was generated by immunizing rabbits with the 17 amino acid synthetic peptide Cys-Leu-Arg-Gln-Gln-His-AspAsp-Phe-Ala-Asp-Asp-Ile-Ser-Leu-Leu-Lys conjugated to Keyhole Limpet Hemocyanin (KLH). Antibody pre-adsorption experiments were carried out with non-conjugated synthetic peptides. The specific competing peptide was the BACE carboxyl-terminus sequence listed above. The nonspecific competitor peptide was a 17 amino acid peptide sequence in the BACE amino-terminus (Glu-Thr-Asp-Glu-Glu-Pro-Glu-Glu-Pro-Gly-Arg-Arg-Gly-Ser-Phe-Val-Glu). Another affinity-purified rabbit polyclonal recognizing the BACE C-terminus was obtained from Affinity Bioreagents (ABR). HA-tagged BACE constructs were recognized with either a monoclonal antibody (mAb HA11; Covance, Richmond, CA) or a rabbit polyclonal antisera (rbHA11; Covance) directed against the HA epitope. The N-terminus of APP was recognized with the goat polyclonal anitsera Karen (38). APP C-terminus was detected with a rabbit polyclonal

6


expression plasmid. Virions were produced and titered as described elsewhere (37).

Huse et. al. antibody (Zymed) and AE was detected with 4g8 and 6e10 (Signet). Immunoprecipitations and western blots were performed using standard methods. Radiolabeled gels were exposed to Phosphorimager screens which were then read 24-48 hours later on a Storm 860 Scanner (Molecular Dynamics) and analyzed in ImageQuant version 1.2 (Molecular Dynamics). Cell Culture and Chemical Reagents—Human embryonic kidney 293 cells, HepG2, and Baby Hamster Kidney (BHK) cells were cultured in Dulbecco's modified Eagle medium (DMEM, Life Technologies, Inc.) supplemented with 10% fetal bovine serum (FBS). C2 cells (39) were cultured in DMEM supplemented with 10% FBS. The C2 cells were induced to differentiate by

additionally supplemented with 1% penicillin/streptomycin. In certain experimental situations, additional chemical reagents were utilized at the following concentrations: phorbol 12-myristate 13-acetate (SIGMA)-10 µM; bafilomycin A1 (SIGMA)-1 µM; chloroquine (SIGMA)-100 µM. Metabolic Labeling and Pulse-Chase Experiments—Cells expressing BACE were preincubated in methionine/cysteine free DMEM supplemented with 10% dialyzed FBS (starve medium) for 30 min after which they were incubated in the same medium containing 0.5 mCi of 35

S cysteine/methionine per well for a predetermined time period. The cells were then washed

once in phosphate buffered saline (PBS), lysed in radioimmunoprecipitation assay (RIPA) buffer (1% sodium deoxycholate, 0.1% SDS, 1% TX-100, 5 mM EDTA, 50 mM Tris pH 8, 150 mM NaCl) and quantitatively immunoprecipitated with the desired antibody. For pulse-chase experiments, cells were metabolically labeled as described above for 30 min after which they were quenched with starve medium containing a 100 molar excess of cold methionine/cysteine. The cells were then chased in normal growth medium for time periods ranging from 1 to 24 h, washed in PBS, lysed in RIPA, and immunoprecipitated with the desired antibody.

7


switching to DMEM, 1% FBS, and 1Pg/ml insulin (Sigma) for 4 days. All media were

Huse et. al. In some experiments wild-type human APP 695 or wild-type human BACE were expressed using recombinant, replication-defective Semliki-Forest virus (SFV) vectors (37). In these experiments the cells were incubated with virus using a multiplicity of infection (MOI) of 2-10 for one hour in DMEM without serum. Cells were rinsed and incubated in normal growth medium for approximately 3-4 hours prior to metabolic 35S-methionine labeling as described above. For experiments expressing APP695wt in HepG2 cells SFV-infected cells were metabolically labeled for 6 hours following a 4 hour incubation period. N-terminal Radiosequencing—Custom DMEM deficient in either methionine or isoleucine

10% dialyzed FBS. 293 cells expressing HA-tagged BACE were preincubated in either the methionine free or isoleucine free medium for 30 min followed by an 18 h incubation in the same media supplemented with either 35S methionine or 3H isoleucine, respectively. The cells were then washed in PBS, lysed in RIPA, and immunoprecipitated with mAb HA11. Immunoprecipitates were eluted in 1X Laemmli sample buffer plus 0.34 M E-mercaptoethanol, electrophoresed on 8% polyacrilamide gels, and transferred to Immobilon™-P PVDF membrane (Millipore). Bands corresponding to BACE-Ct were then cut from the membrane and sequenced by automated Edman degradation collecting each amino acid separately. The amino acids were assayed for radioactivity in a Beckman LS 6500 scintillation counter. Fluorescence Microscopy—Antibody staining of HeLa cells was performed as described previously (30). Briefly, cells were fixed and permeablized in ice cold methanol followed by incubation in primary antibody diluted in PBS supplemented with 2% FBS and 0.02% saponin. BACE constructs were recognized using rb-HA11 while a monoclonal antibody directed against GM-130 (Transduction Laboratories) was used to detect Golgi. Cells were then incubated in

8


was prepared using a MEM-D Selectamine kit (Life Technologies, Inc.) and supplemented with

Huse et. al. fluorescent-conjugated secondary antibodies diluted in the same solution as the primaries. All micrographs were taken with a Nikon E600 microscope utilizing UV illumination.


9

Huse et. al.

Results BACE is endoproteolytically processed to generate stable N- and C-terminal fragments. While characterizing the biochemical properties of BACE under conditions of overexpression in cell culture, we repeatedly noticed the presence of a ~37 kD band by SDSPAGE whose immunoreactivity closely paralleled that of the BACE holoprotein. This species could be readily demonstrated in cell lysates by western blot using a variety of antibodies directed against the C-terminus of BACE (Fig. 1B-C). Furthermore, peptide competition experiments confirmed that the band was specific and not a background artifact (Fig. 1C). We

endoproteolysis generating a distinct ~37 kD C-terminal fragment (BACE-Ct), a mechanism by which the protein's activity could be regulated (Fig. 1A). We observed significant levels of BACE endoproteolysis in a variety of cell types including human embryonic kidney 293 cells (Fig. 1B), baby hamster kidney (BHK) cells (Fig. 1C), chinese hamster ovary (CHO) cells, HeLa cells, and N2a cells (data not shown). Using metabolic pulse-chase analysis, we investigated the kinetics of BACE endoproteolysis. 293 cells expressing BACE were radiolabeled with 35S-methionine/cysteine for 30 minutes and chased in non-radioactive media for various times up to 24 hours, after which the cells were lysed and subjected to immunoprecipitation. In these experiments, BACECt first emerged 4 hours into the chase after which the fragment accumulated slightly, reaching a maximum at the 12 hour time point, before being slowly turned over (Fig. 2A). The initial appearance of BACE-Ct several hours after synthesis is characteristic of a physiological breakdown product and is not consistent with BACE-Ct being a post-lysis artifact. In addition, our data indicates that endoproteolyzed BACE is stable, and that the cleavage event itself does

10


interpreted these findings to suggest that a portion of the BACE holoprotein undergoes

Huse et. al. not simply promote BACE degradation. Pulse-chase experiments were also performed using an antibody directed against the N-terminus of BACE (Fig. 2B). These studies demonstrated the accumulation of a ~37 kD N-terminal fragment (BACE-Nt) with kinetics parallel to those of BACE-Ct. Thus, BACE endoproteolysis results in the production of stable N- and C-terminal fragments. BACE endoproteolysis occurs between Leu228 and Ala229, and the resulting fragments remain associated after cleavage. The exact site of BACE endoproteolysis was determined by N-terminal radiosequencing of BACE-Ct after metabolic labeling with either 35S-

acid residues Leu228 and Ala229. Analysis of the position of these residues in the published BACE crystal structure (40) revealed that both lie in a solvent exposed D-helix connecting the two extracellular lobes of the protein (Fig. 3B-C). Cleavage at this site would split apart the BACE catalytic domains, and could potentially inactivate the enzyme. However, structural studies have indicated the existence of a disulfide bond between residues Cys216 and Cys420 (Fig. 3C) (29,40). Such a connection could serve to maintain the association of BACE-Ct and BACE-Nt even after endoproteolysis. In light of this possibility, we asked whether a distinct BACE C-terminal fragment could be visualized under non-reducing conditions that should preserve the integrity of disulfide bonds. TX-100 lysates from 293 cells expressing BACE were analyzed by western blot after incubation at either 37 or 55 °C in the presence or the absence of E-mercaptoethanol (BME). Under non-reducing conditions, the BACE holoprotein migrated at a lower apparent molecular weight than under reducing conditions, suggestive of intramolecular disulfide bonding and consistent with previous results (Fig. 3D) (30). While BACE-Ct was readily apparent in the

11


methionine or 3H-isoleucine (Fig. 3A), which demonstrated cleavage to occur between amino

Huse et. al. presence of EME, it could not be detected in the absence of reducing agent, consistent with the notion that the BACE N- and C-terminal fragments remain covalently associated by a disulfide bond even after endoproteolysis (Fig. 3D). BACE endoproteolysis occurs readily in several different nonneuronal tissues and may play a role in cellular differentiation. To determine whether BACE endoproteolysis takes place at significant levels in vivo, we assayed for the presence of BACE-Ct in a variety of native tissue samples derived from rat. In lysates from pancreas, liver, and muscle, western blot demonstrated high levels of a C-terminal BACE fragment that co-migrated with BACE-Ct

By contrast, samples from cerebral cortex, cerebellum, diencephelon, and brain stem exhibited negligible amounts of BACE-Ct, while the holoprotein was clearly evident (Fig. 4A). Analysis of human tissue yielded similar results. Samples from cerebral cortex and cerebellum revealed only BACE holoprotein, while lysates from muscle tissue exhibited a large band co-migrating with BACE-Ct. The specificity of the antibody was confirmed by peptide blocking (Fig. 4B). To determine whether BACE-Nt is also present at significant levels in native tissue we performed western blots on rat muscle lysates using a BACE N-terminal antibody (Fig. 4C). Although the N-terminal BACE antibody (left panel) was less sensitive than the C-terminal BACE antibody (right panel), the N-terminal antibody still detected a ~35-37 kD band comigrating with BACE-Ct. The similar size of the Nt- and Ct-BACE fragments in muscle is consistent with data showing that immunoprecipitations using both Nt- and Ct-BACE antibodies detected apparently identical, closely spaced doublet bands at approximately 37kD in size (Fig. 2B). This would be expected under immunoprecipitating conditions because BACE Nt and Ct fragments remain attached via a common disulfide bond. These findings are also in

12


derived from BHK cells (Fig. 4A). BACE holoprotein was not readily apparent in these tissues.

Huse et. al. keeping with the expected size of the fragments based upon the position of the endoproteolytic cleavage. Thus these results indicate that N- and C- terminal cleavage products of BACE endoproteolysis selectively accumulate in cell lines and in vivo. The existence of highly stable, BACE cleavage fragments suggests that this form of BACE may have a specific physiological function in certain tissue and cell types. We further investigated the endoproteolytic processing of BACE in muscle tissue using C2 murine myoblasts which can be induced to differentiate into multinucleated myotubes (39). We found that differentiation dramatically altered the processing of endogenously expressed

but not BACE-Ct (Fig. 4D). However, upon differentiation into myotubes this expression pattern was reversed. While BACE holoprotein was down-regulated (note that four times the number of cells were loaded in the C2 lane compared to the C2(Diff) lane in order to visualize the holoprotein), BACE-Ct expression was markedly up-regulated (Fig. 4D). The specificity of the BACE antibody was confirmed by BACE-Ct peptide antibody preadsorbtion (Fig 4D). Pulse chase analysis of BACE metabolism in differentiated C2 myotube cultures (Fig. 4E) confirmed the existence of a precursor-product relationship between BACE and BACE-Ct in muscle tissue with kinetics similar to those observed in other non-neuronal cell types (Fig. 2A-B). These findings demonstrate that endoproteolytic conversion of BACE is a marked feature attending muscle differentiation, suggesting a physiological role in the specification of muscle cells, and perhaps, more broadly, in tissue development. Cell types containing endoproteolyzed BACE retain E-secretase activity. We next asked if E-secretase activity and AE production can occur in cells where endogenous BACE is expressed in the endoproteolyzed form. Because BACE-Ct fragments are highly expressed in

13


BACE. Undifferentiated C2 myoblasts exhibited relatively low levels of BACE holoprotein,

Huse et. al. liver we examined BACE expression in the human hepatic cell line, HepG2. Western blot analysis of HepG2 cells revealed high levels of BACE-Ct with virtually no detectable BACE holoprotein (Fig. 5A). The specificity of the antibody for BACE was confirmed by peptide blocking (Fig. 5A). These findings indicate that stable levels of endogenous BACE exist almost exclusively as endoproteolyzed fragments in HepG2 cells. APP processing was examined by metabolically labeling HepG2 cells expressing Semliki Forest virus (SFV)induced wild-type human APP695 . Cells expressing SFV-E-galactosidase (lacZ) served as controls. Immunoprecipitation with antibodies directed against the C-terminal domain of APP

APP cleavage (Fig 5B). Immunoprecipitation of the conditioned medium with 6e10, a monoclonal antibody recognizing amino acid residues 1-10 of AEreadily detected AE (Fig. 5B). The same experimental approach also detected the production of AE in differentiated C2 myotube cultures (data not shown). These results strongly suggest that endoproteolyzed BACE retains at least some APP E-site cleaving enzyme activity. Cell biological characteristics of BACE endoproteolysis. To more accurately determine the cellular compartment(s) where the BACE endoproteolytic event takes place, we used BACE constructs targeted to specific organelles. Placing a dilysine motif on the Cterminus of BACE (BACE-KK) effectively retains the protein in the ER (30), while replacing the cytoplasmic tail of BACE with the intracellular domain of the proprotein convertase furin targets the protease to the TGN (BACE-TGN) (Fig. 1A) (35). The cellular distributions of BACE-KK and BACE-TGN were confirmed by immunofluourescence (Fig. 6A). When expressed in HeLa cells, BACE-KK exhibited a fine reticular staining pattern, consistent with ER localization and identical to previous results (30), while BACE-TGN displayed positive

14


revealed the presence of APP C-terminal fragments consistent with D- and E-secretase mediated

Huse et. al. immunoreactivity in a perinuclear distribution that colocalized well with the Golgi marker GM130. The BACE targeting mutants were expressed in 293 cells and immunoprecipitations of radiolabeled protein were performed to assay for endoproteolysis. While BACE-Ct was undetectable in cells expressing BACE-KK, the fragment accumulated at roughly twice the wild type level in cells expressing BACE-TGN (Fig. 6B). These findings indicated that endoproteolysis occurs only after BACE is exported from the ER and that the efficiency of the cleavage event itself is enhanced in the TGN. The larger size of BACE-Ct in cells expressing BACE-TGN simply reflects the increased length of the furin cytoplasmic tail relative to its

To further define the cell biology of BACE endoproteolysis, we asked whether the generation of BACE-Ct requires an acidic microenvironment. A variety of proteases, including BACE, are optimally active at acidic pH (10,12). We addressed this issue using two pharmacological reagents. Chloroquine, a weak base, accumulates in acidic vesicles where it counteracts the established pH gradient (41,42), while bafilomycin A1 selectively blocks vacuolar-type H+-ATPases (43), protein complexes that pump protons into a variety of cellular compartments including endosomes and the TGN (44). 293 cells expressing BACE were labeled with 35S cysteine/methionine for 16 hours in the presence of either chloroquine or bafilomycin A1. This treatment eliminated the production of BACE-Ct, as visualized by immunoprecipitation of radiolabeled protein (Fig. 6C). Furthermore, metabolic pulse-chase analysis revealed that bafilomycin A1 also blocks BACE-Ct production in C2 myotubes, a cell type where endoproteolyzed BACE is highly expressed (Fig. 6D). These experiments indicate that BACE endoproteolysis most likely occurs in an acidic domain of the cell, such as the TGN or the endosomal/lysosomal system.

15


wild-type BACE counterpart.

Huse et. al. BACE is not cleaved by either furin or TACE and does not appear to autocatalyze its own endoproteolysis. We next sought to determine what enzyme(s) mediate the endoproteolytic processing of BACE. In light of our results implicating the TGN as a likely site for BACE endoproteolysis, we considered two proteases as good candidates. The previously mentioned D-secretase TACE was originally found to be involved with the cleavage of cell surface proteins (45,46). However, more recent evidence has implicated TACE in protein kinase C (PKC) regulated D-secretase activity within the TGN, where the protein competes with E-secretase for APP substrate (19). In addition to TACE, furin is known to be active primarily

or a furin-like protease has also been demonstrated to cleave BACE at its propeptide site (31,32), demonstrating that these two proteins interact in cells. Arguing against a role for furin, however, is the fact that the BACE endoproteolysis site is not marked by the consensus sequence, Arg-Xaa-(Lys/Arg)-Arg, typically associated with furin cleavage (47). The impact of TACE and furin on the endoproteolysis of BACE was determined by coexpression studies. 293 cells were transfected with either BACE alone, BACE and TACE, or BACE and mfurin-HA (an HA tagged murine furin construct). The cells were then labeled with 35S cysteine/methionine for 8 hours, lysed, and immunoprecipitated with mAb HA11. Analogous experiments using BACE-TGN were also performed. We found that coexpression of TACE or mfurin-HA with either BACE-HA or BACE-TGN did not lead to an appreciable increase in the accumulation of BACE-Ct (Fig. 7A, C). We also attempted to upregulate TACE activity by treating the cells with the PKC activator phorbol 12-myristate 13-acetate (PMA) 4 hours into the 35S cysteine/methionine labeling period. No effect on BACE-Ct levels was observed (Fig. 7A). TACE activity was confirmed by co-transfection with APP in 293 cells. A

16


in the TGN where it mediates the processing of several different precursor proteins (47). Furin

Huse et. al. 30 minute 35S cysteine/methionine-label followed by PMA stimulation lead to an increase in secreted APPD derived from both endogenous APP751 and over-expressed APP695 (Fig. 7B), consistent with reported results (19). An adequate level of furin-HA expression was also determined by western blot with (Fig. 7D). Thus, neither TACE nor furin was found to mediate the cleavage of BACE. We also considered whether an autocatalytic mechanism might be responsible for BACE endoproteolysis. Several enzymes, including furin and possibly the presenilins, undergo autocatalytic processing events that significantly impact the activity of the proteases themselves

microenvironment correlates well with the documented acidic pH optimum for BACE. To address this possibility, we generated a BACE construct whose second active site aspartate residue (Asp289) mutated to asparagine (BACE-D289N). This mutation has been demonstrated to eliminate completely the protease activity of BACE (8) and, in our hands, co-expression of BACE-D289N with APP in 293 cells did not lead to the typically observed increase in AE secretion (data not shown). If BACE does autocatalyze its own endoproteolysis, one would expect BACE-Ct not to be produced in cells expressing BACE-D289N, or to be produced only at very low levels due to the presence of endogenous BACE. We transfected BACE-D289N and BACE into 293 cells and assayed for endoproteolysis by immunoprecipitation of radiolabeled protein. No significant differences were found between the BACE-Ct levels of cells expressing either BACE-D289N or BACE (Fig. 7E). Our findings, therefore, are not consistent with BACE directly mediating its own endoproteolysis. Nevertheless, an autocatalytic mechanism cannot be completely excluded (see Discussion).

17


(13,15,47). In addition, the dependence of BACE endoproteolysis on an acidic

Huse et. al.

Discussion The proteolysis of APP by BACE is a crucial step in the generation of AE in brain and represents an important potential target of therapeutic strategies aimed at slowing or even halting the progression AD. Consequently, a better understanding of the cellular mechanisms regulating E-secretase activity is of obvious importance. We and others have found that BACE is a stable protein with a t1/2 of 12-16 h (29,30). This long life span strongly implies that, in order for cells to effectively exert tight control over E-secretase activity, regulatory mechanisms must exist at a posttranslational level. We have described an endoproteolytic processing

kD in size. BACE endoproteolysis appears to be a normal cellular metabolic process resulting in the generation of a highly stable form of the protein. In this regard BACE may be similar to a wide variety of proteins that are known to be regulated by endoproteolysis including zymogens, clotting factors, complement proteins, and hormones (48,49). In addition, several membrane bound proteases such as the ADAM (A Disintegrin And Metalloprotease) and proprotein convertase families require specific cleavage events during the maturation process to obtain biological activity (47,50,51). Structural analysis of BACE endoproteolysis revealed that cleavage occurs on a solvent exposed D-helix bridging the two extracellular lobes of the protein. Analogous helices are absent from soluble aspartyl proteases like pepsin (40,52), consistent with the fact that these enzymes have not been shown to undergo endoproteolysis at similar positions in their amino acid sequences (53,54). Typically, cleavage at this site would be expected to inactivate BACE by dissociating the two catalytic aspartic acid residues. However, we found that the BACE-Nt and -Ct remain covalently associated after proteolysis via a common disulfide bridge. This

18


pathway that cleaves BACE into distinct N- and C- terminal fragments, each approximately 37

Huse et. al. finding suggests the possibility that such post-translational regulation may alter and/or attenuate, rather than abolish, the enzymatic activity of BACE. Although BACE endoproteolysis occurs readily in a variety of cultured cell types, only a relatively small proportion of the holoprotein appears to undergo cleavage when overexpressed. By contrast, endoproteolyzed BACE accumulates at high levels endogenously in muscle, liver, and pancreas, coupled with undetectable or very low levels of holoprotein. This dichotomy is reminiscent of the regulated endoproteolysis of the presenilins, which exist primarily as holoproteins when over-expressed, and yet are found almost entirely as N- and C-terminal

be assigned to BACE endoproteolysis at this time, this post-translational event appears to be a normal, regulated metabolic process yielding stable cleavage products that accumulate in certain native tissues. Moreover, the pattern of endoproteolyzed BACE expression is generally consistent with BACE mRNA levels in different tissue types. For instance, pancreas exhibits robust BACE mRNA expression (10) and is among the tissues harboring the highest levels of BACE cleavage fragments. In addition, we have shown that an upregulation of BACE endoproteolysis is associated with myogenic differentiation in C2 cells, a well-established model of muscle development. Together, these findings strongly suggest that endoproteolyzed BACE has a biologically significant, but as yet undefined, function(s) in a number of different cell types, particularly in cells of non-neuronal lineage. We attempted to localize BACE endoproteolysis by targeting the protein to different sites within the secretory pathway. These studies revealed that the cleavage event takes place only after the protein is exported from the ER and appears to be enhanced in the TGN. We also found BACE endoproteolysis to be sensitive to inhibitors of vesicular acidification, a result

19


cleavage fragments endogenously (23,24,55,56). While a specific physiological purpose cannot

Huse et. al. consistent with a TGN and/or endosomal localization for BACE cleavage. Multiple lines of evidence implicate the TGN in BACE endoproteolysis. Thus, we considered two membrane bound proteases, TACE and furin, both of which are active in the TGN, to be excellent candidate enzymes. However, neither enzyme upregulated the production of BACE-Ct when co-expressed with BACE in 293 cells. We also demonstrated that BACE-Ct could be generated from a catalytically inactive BACE (BACE-D289N), suggesting that BACE does not cleave itself. However, this finding does not exclude autocatalysis as a mechanism for BACE endoproteolysis. If BACE cleavage occurs via an intermolecular (trans) mechanism, as

D289N could have been mediated in our studies by endogenous BACE. The potential impact of BACE endoproteolysis on APP processing is intriguing since Esecretase activity is known to be quite low in nonneuronal tissues such as pancreas that express high levels of BACE mRNA (9,10). Some have argued that a predominance of alternatively spliced BACE variants with reduced APP processing activity may account for this discrepancy (57,58). However, more recent data indicate that the most abundant BACE mRNA species in pancreas correspond to full length protein, implying that down regulation of E-secretase activity most likely occurs at a posttranslational level (59). We have shown that BACE exists primarily as endoproteolytic fragments in pancreas, muscle, and liver, while holoprotein predominates in neuronal tissue. This would imply that BACE endoproteolysis significantly reduces E-secretase activity in these tissue types. We observed definitive E-secretase activity and subsequent AE production in HEPG2 cells, where virtually all detectable BACE protein exists as endoproteolytic fragments. Although we cannot completely exclude the possibility that trace amounts of full length BACE mediated E-secretase activity in these studies, it appears far more

20


opposed to an intramolecular (cys) mechanism, the endoproteolysis of the recombinant BACE-

Huse et. al. likely that the readily measurable levels of AEwere generated by the highly abundant endoproteolyzed BACE species. These findings, therefore, in conjunction with previous work (9,10) suggest that endoproteolysis may significantly attenuate, but most likely does not abolish, the E-secretase activity of BACE in certain nonneuronal cell types. An improved understanding of this posttranslational event could aid considerably in the crafting of methods to reduce cerebral amyloid deposition by specifically activating BACE endoproteolysis in neural tissue. Finally, the effects of BACE endoproteolysis may extend beyond APP cleavage. For instance, BACE processing could modulate the ability of the protease to act on other substrates,

cleavage of the sialyltransferase, ST6Gal-1 (60). Much work remains to be done to better understand what will likely emerge as numerous functions for BACE, an enzyme that is widely expressed in a diverse array of cell and tissue types.

21


a possibility made all the more feasible by recent findings implicating the protease in the

Huse et. al.

Acknowledgments We thank Dr. David W. McCourt at Midwest Analytical, Inc. for his help in the radiosequencing of BACE-Ct. We also thank Mark Biscone for his assistance in the design of structural figures. As always, we appreciate the thoughtful comments and insights provided by the members of the Doms, Lee, and Schellenberg labs. This work was supported by National Institutes of Health Grant NIH-PO1 AG11542 (RWD), a HHMI Predoctoral Fellowship (J.T.H.), NIA-R01 AG11762 (G. Schellenberg), a VA Merit Award (DGC), and NIA AG05136 Alzheimer’s Disease Research Center/Project (DGC). Downloaded from www.jbc.org by guest, on March 21, 2013

22

Huse et. al.

References 1.

Glenner, G. G., and Wong, C. W. (1984) Biochem Biophys Res Comm 120(3), 885-890

2.

Price, D. L., and Sisodia, S. S. (1998) Annu Rev Neurosci 21, 479-505

3.

Selkoe, D. J. (1999) Nature 399 (Suppl), A23-A31

4.

Chen, G., Chen, K. S., Knox, J., Inglis, J., Bernard, A., Martin, S. J., Justice, A., McConlogue, L., Games, D., Freedman, S. B., and Morris, R. G. (2000) Nature 408(6815), 975-979 Janus, C., Pearson, J., McLaurin, J., Mathews, P. M., Jiang, Y., Schmidt, S. D., Chishti, M. A., Horne, P., Heslin, D., French, J., Mount, H. T., Nixon, R. A., Mercken, M., Bergeron, C., Fraser, P. E., St George-Hyslop, P., and Westaway, D. (2000) Nature 408(6815), 979-982

6.

Morgan, D., Diamond, D. M., Gottschall, P. E., Ugen, K. E., Dickey, C., Hardy, J., Duff, K., Jantzen, P., DiCarlo, G., Wilcock, D., Connor, K., Hatcher, J., Hope, C., Gordon, M., and Arendash, G. W. (2000) Nature 408(6815), 982-985

7.

Esch, F. S., Keim, P. S., Beattie, E. C., Blacher, R. W., Culwell, A. R., Oltersdorf, T., McClure, D., and Ward, P. J. (1990) Science 248(4959), 1122-1124

8.

Hussain, I., Powell, D., Howlett, D. R., Tew, D. G., Meek, T. D., Chapman, C., Gloger, I. S., Murphy, K. E., Southan, C. D., Ryan, D. M., Smith, T. S., Simmons, D. L., Walsh, F. S., Dingwall, C., and Christie, G. (1999) Mol Cell Neurosci 14(6), 419-427

9.

Sinha, S., Anderson, J. P., Barbour, R., Basi, G. S., Caccavello, R., Davis, D., Doan, M., Dovey, H. F., Frigon, N., Hong, J., Jacobson-Croak, K., Jewett, N.,

23


5.

Huse et. al. Keim, P., Knops, J., Lieberburg, I., Power, M., Tan, H., Tatsuno, G., Tung, J., Schenk, D., Seubert, P., Suomensaari, S. M., Wang, S., Walker, D., Zhao, J., McConlogue, L., and John, V. (1999) Nature 402, 537-540 10.

Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E. A., Denis, P., Teplow, D. B., Ross, S., Amarante, P., Loeloff, R., Luo, Y., Fisher, S., Fuller, J., Edenson, S., Lile, J., Jaronsinski, M. A., Biere, A. L., Curran, E., Burgess, T., Louis, J.-C., Collins, F., Treanor, J., Rogers, G., and Citron, M. (1999) Science 286(5440), 735-741 Yan, R., Bienkowski, M. J., Shuck, M. E., Miao, H., Tory, M. C., Pauley, A. M., Brashler, J. R., Stratman, N. C., Mathews, W. R., Buhl, A. E., Carter, D. B., Tomasselli, A. G., Parodi, L. A., Heinrikson, R. L., and Gurney, M. E. (1999) Nature 402, 533-537

12.

Lin, X., Koelsch, G., Wu, S., Downs, D., Dashti, A., and Tang, J. (2000) Proc Natl Acad Sci USA 97(4), 1456-1460

13.

Wolfe, M. S., Xia, W., Ostaszewski, B. L., Diehl, T. S., Kimberly, W. T., and Selkoe, D. J. (1999) Nature 398(6727), 513-517

14.

Esler, W. P., Kimberly, W. T., Ostaszewski, B. L., Diehl, T. S., Moore, C. L., Tsai, J. Y., Rahmati, T., Xia, W., Selkoe, D. J., and Wolfe, M. S. (2000) Nat Cell Biol 2(7), 428-434

15.

Kimberly, W. T., Xia, W., Rahmati, T., Wolfe, M. S., and Selkoe, D. J. (2000) J Biol Chem 275(5), 3173-3178

16.

Li, Y. M., Xu, M., Lai, M. T., Huang, Q., Castro, J. L., DiMuzio-Mower, J., Harrison, T., Lellis, C., Nadin, A., Neduvelil, J. G., Register, R. B., Sardana, M.

24


11.

Huse et. al. K., Shearman, M. S., Smith, A. L., Shi, X. P., Yin, K. C., Shafer, J. A., and Gardell, S. J. (2000) Nature 405(6787), 689-694 17.

Francis, R., McGrath, G., Zhang, J., Ruddy, D. A., Sym, M., Apfeld, J., Nicoll, M., Maxwell, M., Hai, B., Ellis, M. C., Parks, A. L., Xu, W., Li, J., Gurney, M., Myers, R. L., Himes, C. S., Hiebsch, R., Ruble, C., Nye, J. S., and Curtis, D. (2002) Dev Cell 3(85-97)

18.

Buxbaum, J. D., Liu, K. N., Luo, Y., Slack, J. L., Stocking, K. L., Peschon, J. J., Johnson, R. S., Castner, B. J., Cerretti, D. P., and Black, R. A. (1998) J Biol Chem

19.

Skovronsky, D. M., Moore, D. B., Milla, M. E., Doms, R. W., and Lee, V. M.-Y. (2000) J Biol Chem 275(4), 2568-2575

20.

De Strooper, B., Beullens, M., Contreras, B., Levesque, L., Craessaerts, K., Cordell, B., Moechars, D., Bollen, M., Fraser, P., St. George-Hyslop, P., and Van Leuven, F. (1997) J Biol Chem 272(6), 3590-3598

21.

Seeger, M., Nordstedt, C., Petanceska, S., Kovacs, D. M., Gouras, G. K., Hahne, S., Fraser, P., Levesque, L., Czernik, A. J., St George-Hyslop, P., Sisodia, S. S., Thinakaran, G., Tanzi, R. E., Greengard, P., and Gandy, S. (1997) Proc Natl Acad Sci USA 94, 5090-5094

22.

Walter, J., Grunberg, J., Capell, A., Pesold, B., Schindzielorz, A., Citron, M., Mendla, K., St. George-Hyslop, P., Multhaup, G., Selkoe, D. J., and Haass, C. (1997) Proc Natl Acad Sci USA 94, 5349-5354

23.

Thinakaran, G., Borchelt, D. R., Lee, M. K., Slunt, H. H., Spitzer, L., Kim, G., Ratovitsky, T., Davenport, F., Nordstedt, C., Seeger, M., Hardy, J., Levey, A. I.,

25


273(43), 27765-27767

Huse et. al. Gandy, S. E., Jenkins, N. A., Copeland, N. G., Price, D. L., and Sisodia, S. S. (1996) Neuron 17, 181-190 24.

Kim, T.-W., Pettingell, W. H., Hallmark, O. G., Moir, R. D., Wasco, W., and Tanzi, R. E. (1997) J Biol Chem 272(17), 11006-11010

25.

Thinakaran, G., Harris, C. L., Ratovitski, T., Davenport, F., Slunt, H. H., Price, D. L., Borchelt, D. R., and Sisodia, S. S. (1997) J Biol Chem 272(45), 28415-28422

26.

Capell, A., Grunberg, J., Pesold, B., Diehlmann, A., Citron, M., Nixon, R., Beyreuther, K., Selkoe, D. J., and Haass, C. (1998) J Biol Chem 273(6), 3205-

27.

Yu, G., Chen, F., Levesque, G., Nishimura, M., Zhang, D. M., Levesque, L., Rogaeva, E., Xu, D., Liang, Y., Duthie, M., St George-Hyslop, P. H., and Fraser, P. E. (1998) J Biol Chem 273(26), 16470-16475

28.

Capell, A., Steiner, H., Willem, M., Kaiser, H., Meyer, C., Walter, J., Lammich, S., Multhaup, G., and Haass, C. (2000) J Biol Chem 275(40), 30849-30854

29.

Haniu, M., Denis, P., Young, Y., Mendiaz, E. A., Fuller, J., Hui, J., Bennett, B. D., Kahn, S., Ross, S., Burgess, T., Katta, V., Rogers, G., Vassar, R., and Citron, M. (2000) J Biol Chem 275(14), 21099-21106

30.

Huse, J. T., Pijak, D. S., Leslie, G. J., Lee, V. M.-Y., and Doms, R. W. (2000) J Biol Chem 275(43), 33729-33737

31.

Bennett, B. D., Denis, P., Haniu, M., Teplow, D. B., Kahn, S., Louis, J. C., Citron, M., and Vassar, R. (2000) J Biol Chem

26


3211

Huse et. al. 32.

Creemers, J. W., Dominguez, D. I., Plets, E., Serneels, L., Taylor, N. A., Multhaup, G., Craessaerts, K., Annaert, W., and De Strooper, B. (2001) J Biol Chem 276(6), 4211-4217

33.

Benjannet, S., Elagoz, A., Wickham, L., Mamarbachi, M., Munzer, J. S., Basak, A., Lazure, C., Cromlish, J. A., Sisodia, S. S., Checler, F., Cretien, M., and Seidah, N. G. (2001) J Biol Chem 14, 10879-10887

34.

Walter, J., Fluhrer, R., Hartung, B., Willem, M., Kaether, C., Capell, A., Lammich, S., Multhaup, G., and Haass, C. (2001) J Biol Chem 276(18), 14634-

35.

Huse, J. T., Liu, K., Pijak, D. S., Lee, V. M.-Y., and Doms, R. W. (2002) J Biol Chem 277, 16278-16284

36.

Bosshart, H., Humphrey, J., Deignan, E., Davidson, J., Drazba, J., Yuan, L., Oorschot, V., Peters, P. J., and Bonifacino, J. S. (1994) J Cell Biol 126(5), 11571172

37.

Cook, D. G., Sung, J. C., Golde, T. E., Felsenstein, K. M., Wojczyk, B. S., Tanzi, R. E., Trojanowski, J. Q., Lee, V. M.-Y., and Doms, R. W. (1996) Proc Natl Acad Sci 93, 9223-9228

38.

Chyung, A. S. C., Greenberg, B. D., Cook, D. G., Doms, R. W., and Lee, V. M.Y. (1997) J Cell Biol 138(3), 671-680

39.

Yaffe, D., and Saxel, O. (1977) Nature 270, 725-727

40.

Hong, L., Koelsch, G., Lin, X., Wu, S., Terzyan, S., Ghosh, A., Zhang, X. C., and Tang, J. (2000) Science 290, 150-153

41.

Lie, S. O., and Schofield, B. (1973) Biochem Pharm 22, 3109-3114

27


14641

Huse et. al. 42.

Krogstad, D. J., Schlesinger, P. H., and Gluzman, I. Y. (1985) J Cell Biol 101, 2302-2309

43.

Drose, S., and Altendorf, K. (1997) J Exp Biol 200, 1-8

44.

Nelson, N. (1991) Trends Pharmacol Sci 12, 71-75

45.

Black, R. A., Rauch, C. T., Kozlosky, C. J., Peschon, J. J., Slack, J. L., Wolfson, M. F., Castner, B. J., Stockling, K. L., Reddy, P., Srinivasan, S., Nelson, N., Boiani, N., Schooley, K. A., Gerhart, M., Davis, R., Fitzner, J. N., Johnson, R. S., Paxton, R. J., March, C. J., and Cerretti, D. P. (1997) Nature 385, 729-733 Moss, M. L., Jin, S.-L. C., Milla, M. E., Burkhart, W., Carter, H. L., Chen, W.-J., Clay, W. C., Didsbury, J. R., Hassler, D., Hoffman, C. R., Kost, T. A., Lambert, M. H., Leesnitzer, M. A., McCauley, P., McGeehan, G., Mitchell, J., Moyer, M., Pahel, G., Rocque, W., Overton, L. K., Schoenen, F., Seaton, T., Su, J.-L., Warner, J., D., W., and Becherer, J. D. (1997) Nature 385, 733-736

47.

Nakayama, K. (1997) Biochem J 327, 625-635

48.

Neurath, H. (1986) J Cellular Biochem 32, 35-49

49.

Khan, A. R., and James, M. N. G. (1998) Protein Science 7, 815-836

50.

Wolfsberg, T. G., Primakoff, P., Myles, D. G., and White, J. M. (1995) J Cell Biol 131, 275-278

51.

Blobel, C. P. (1997) Cell 90, 589-592

52.

Bennett, B. D., Babu-Khan, S., Loeloff, R., Louis, J.-C., Curran, E., Citron, M., and Vassar, R. (2000) J Biol Chem 275(27), 20647-20651

53.

Takahashi, K., Tanji, M., Yakabe, E., Hirasawa, A., Athauda, S. B. P., and Kageyama, T. (1995) Adv Exp Med Biol 362, 53-65

28


46.

Huse et. al. 54.

Yamamoto. (1995) Adv Exp Med Biol 362, 223-229

55.

Ratovitski, T., Slunt, H. H., Thinakaran, G., Price, D. L., Sisodia, S. S., and Borchelt, D. R. (1997) J Biol Chem 272(39), 24536-24541

56.

Steiner, H., Capell, A., Pesold, B., Citron, M., Kloetzel, P. M., Selkoe, D. J., Romig, H., Mendla, K., and Haass, C. (1998) J Biol Chem 273(48), 32322-32331

57.

Bodendorf, U., Fischer, F., Bodian, D., Multhaup, G., and Paganetti, P. (2001) J Biol Chem 276, 12019-12023 Tanahashi, H., and Tabira, T. (2001) Neurosci Lett 307, 9-12

59.

Ehehalt, R., Michel, B., De Pietri Tonelli, D., Zacchetti, D., Simons, K., and Keller, P. (2002) Biochem Biophys Res Comm 293, 30-37

60.

Kitazume, S., Tachida, Y., Oka, R., Shirotani, K., Saido, T. C., and Hashimoto, Y. (2001) Proc Natl Acad Sci USA 98, 13554-13559

29


58.

Huse et. al.

Figure Legends

Fig. 1. A, schematic showing topological models of BACE constructs used. Lumenal and cytoplasmic domains are indicated. White circles denote the positions of the catalytic aspartic acid residues. The cytoplasmic domain of murine furin used to design BACETGN is shown in blue. Endoproteolysis of BACE at Ala229 is also demonstrated, a process that generates the BACE cleavage products BACE-Nt (green) and BACE-Ct (gray). B, Lysates from 293 cells expressing a C-terminally HA-tagged BACE construct

denotes BACE-Ct. C, Lysates from BHK cells expressing untagged wild-type BACE were analyzed by western blot with 88.6 (rabbit polyclonal antisera recognizing BACE C-terminus). Blots were probed with antibody preadsorbed with specific “S”, nonspecific “NS”, or no preadsorption “-“ (See Experimental Procedures).

Fig. 2. Pulse-chase analysis of BACE endoproteolysis. A, 293 cells expressing BACE were metabolically labeled for 30 min with 35S-cysteine/methionine and chased in cold medium for the indicated amount of time. Cells were then lysed and immunoprecipitated with the C-terminal antibody BC-1. B, BHK cells infected with SFV-expressing wild type BACE were metabolically labeled for 45 min as in panel A. Lysates for each time point were divided in half and immunoprecipitated with BACE-Ct (88.6) or BACE-Nt antibodies. Arrows denote BACE endoproteolytic fragments.

30


“B” or mock transfected “M”, were subjected to western blot with mAb HA11. Arrow

Huse et. al. Fig. 3. A, Graph showing the results of N-terminal radiosequencing performed on BACE-Ct (see Experimental Procedures). The positioning of radiolabeled methioinine and isoleucine residues identified the BACE endoproteolysis site at Ala229. B-C, The BACE endoproteolysis site shown from two different views in the context of the published crystal structure (40). The N-terminal and C-terminal extracellular lobes of BACE are shown in green and gray, respectively while the protein's catalytic aspartic acid residues are indicated in red. The endoproteolysis site (white asterisk) lies in a solvent exposed D-helix connecting the two extracellular lobes of the protein (blue).

linking the two extracellular domains of BACE. D, Lysates from 293 cells expressing HA-tagged BACE were treated with an appropriate amount of Laemmli sample buffer with or without 2 M EME and incubated at either 37 or 55 °C for 10 min. Lysates from mock transfected 293 cells were also included as negative controls (-). The samples were then subjected to SDS-PAGE and western blot with mAb HA11. In the presence of BME, BACE-Ct could be readily visualized (arrow), while in the absence of reducing agent the fragment was undetectable.

Fig. 4. A, Western blot with affinity purified BACE-Ct antibody shows relative amounts of BACE holoprotein and BACE-Ct in lysates with equal protein content derived from rat frontal cortex, cerebellum, diencephalon. brain stem, pancreas, muscle, and liver (two lanes for each tissue correspond to 25 µg and 12 µg protein). Transfected BHK cell lysate serves as positive control. B, Western blot carried out as in panel A shows BACE expression in lysates with equal protein content from human tissue (cortex, cerebellum,

31


Also shown is a disulfide bond between residues Cys216 and Cys420 (yellow) covalently

Huse et. al. and muscle). For peptide block, antibody pre-adsorbed with BACE-Ct peptide was used on an equivalent companion blot (right panel). C, Western blot of endogenous BACE-Nt (left panel) and BACE-Ct (right panel) fragments from rat muscle (duplicate lanes). “B” indicates recombinant BACE over-expressed in BHK cells. D, Western blot with affinity purified BACE-Ct antibody shows differentiated mouse C2 cells “C2 Diff” endogenously express large amounts of BACE-Ct in comparison to undifferentiated C2 myoblasts “C2”. In order to make low levels of BACE holoprotein visible 8 X 105 undifferentiated cells were loaded per lane as compared to 2 X 105 differentiated cells per

positive control (note lack of BACE-Ct fragments). Peptide block control was done with an equivalent companion blot. E, Differentiated C2 cells expressing SFV-derived wt BACE were metabolically labeled for 30 min with 35S-cysteine/methionine and chased in cold medium for the indicated amount of time. Cells were then lysed and immunoprecipitated with the C-terminal antibody, 88.6. Lower panel is darker exposure of the portion of the upper panel showing BACE-Ct. Arrows Indicate BACE-Ct.

Fig. 5. A. Western blot shows expression of BACE-Ct, but not holoprotein in human hepatic, HepG2 cells. Equivalent western blot was treated with antibodies preadsorbed with BACE-Ct peptides to show specificity of antibody for BACE. “BHK” indicates BACE over-expressed in BHK cells. B. HepG2 cells generate APP C-terminal fragments consistent with E- and D-secretase activity and also generate AE. HepG2 cells expressing SFV-mediated APP695 or SFV-lacZ were metabolically labeled with 35Smethionine/cystine, lysed, and subjected to immunoprecipitation with an antibody

32


lane. “N” indicates endogeneous BACE from primary rat cortical neurons used as a

Huse et. al. recognizing APP C-terminal fragments. Conditioned medium was immunoprecipitated with 6E10 to reveal secreted AE.

Fig. 6. A, Immunofluorescence micrographs of HeLa cells expressing BACE, BACEKK, or BACE-TGN. All cells were fixed and permeablized with methanol prior to BACE staining (see Experimental Procedures). The lower row of panels indicates the extent of colocalization (yellow) between BACE-TGN (green) and the Golgi resident protein GM-130 (red). B, 293 cells expressing either BACE, BACE-KK, or BACE-TGN

Arrows denote BACE-Ct derived from either BACE-HA or BACE-TGN. C, 293 cells expressing BACE or BACE-TGN were pretreated with either normal growth medium (-), growth medium containing 100 µM chloroquine (Chl), or growth medium containing 1 µM bafilomycin A1 (Baf) for 3 h. The cells were then radiolabeled with 35Scysteine/methionine for 18 h with chloroquine or bafilomycin A1 included as before, lysed, and immunoprecipitated. Arrows indicate BACE-Ct. D, Bafilomycin (Baf) blocked the endoproteolysis of BACE in differentiated C2 cells expressing SFV-mediated wt BACE. “NI” indicates non-infected controls. Cells were metabolically labeled and then chased in “cold” medium for the indicated time (hours). Lysates were immunprecipated with BACE-Ct antibodies. Lower panel is darker exposure of a portion of the upper panel. Arrows indicate BACE-Ct.

Fig. 7. A, 293 cells in 6 well plates expressing either BACE alone or BACE and TACE were radiolabeled with 35S-cysteine/methionine for 8 h, lysed and immunoprecipitated

33


were radiolabeled with 35S-cysteine/methionine for 18 h, lysed, and immunoprecipitated.

Huse et. al. (lanes 2-5). In half of the wells, TACE/ADAM activity was stimulated with 10 µM PMA 4 h into the labeling period (lanes 3,5,7, and 9). Analogous experiments were also performed using BACE-TGN (lanes 6-9) and mock transfected cells were employed as a negative control (lane 1). Arrows indicate BACE-Ct. B, As a control for TACE activity, 293 cells expressing APP and TACE were radiolabeled with 35S-cysteine/methionine for 30 min after which they were either left untreated or incubated with 10 µM PMA for another 30 min. Conditioned media was then collected and immunoprecipitated for secreted APPD (see Experimental procedures). APPD species derived from APP751 and

mfurin-HA in 293 cells (lanes 2-5). Mock transfected cells provided a negative control (lane 1). Radiolabeling for 18 h followed by cell lysis and immunoprecipitation did not reveal any increase in the production of BACE-Ct (arrows) resulting from furin coexpression. D, The presence of mfurin-HA was verified by western blot. E, 293 cells expressing either BACE-D289N, BACE or mock transfected were radiolabeled for 18 h, lysed and immunoprecipitated. BACE-Ct was effectively generated from both BACEHA and BACE-D289N (arrow).

34


APP695 are indicated. C, BACE and BACE-TGN were either expressed alone or with








secretase (BACE) within its catalytic domain: A

secretase (BACE) within its catalytic domain: A

Suggest Documents

Dropping the BACE: Beta Secretase (BACE1) as an ... - InTechOpen

A Conserved Telomerase Motif within the Catalytic Domain of ...

Serotonin derivatives as inhibitors of -secretase (BACE 1)

New Aminoimidazoles as Î²-Secretase (BACE-1) Inhibitors Showing ...

Gram-Scale Synthesis of a Î²-Secretase 1 (BACE 1 ... - ACS Publications

Release of a membrane-bound death domain by Î³-secretase ...

Decrease in catalytic capacity of Î³-secretase can facilitate

Decrease in catalytic capacity of Î³-secretase can facilitate ...

Fragment Binding to Î²-Secretase 1 without Catalytic Aspartate

SiO2 Composite and Its Catalytic

preparation and its catalytic applications

Domain cross-talk within a bifunctional enzyme

Material and Its Catalytic Activity

A second catalytic domain in the Elp3 histone acetyltransferases: a ...

Intact MutY and its catalytic domain differentially contact with A/8-oxoG ...

The -secretase-generated intracellular domain of ... - Semantic Scholar

SH2-Catalytic Domain Linker Heterogeneity Influences Allosteric ...

Catalytic Domain of Mutans Streptococcal Glucosyltransferase

Minimal catalytic domain of N-acetylglucosaminyltransferase V

Mechanism of Diphtheria Toxin Catalytic Domain ... - BioMedSearch

Minimal catalytic domain of N ... - Oxford Academic

Autoantibodies against the Catalytic Domain of

A fully active catalytic domain of bovine aspartyl (asparaginyl ... - NCBI

Processing of metacaspase into a cytoplasmic catalytic domain ...