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• W2137023074 abstract "BACE is a transmembrane protease with β-secretase activity that cleaves the amyloid precursor protein (APP). After BACE cleavage, APP becomes a substrate for γ-secretase, leading to release of amyloid-β peptide (Aβ), which accumulates in senile plaques in Alzheimer disease. APP and BACE are co-internalized from the cell surface to early endosomes. APP is also known to interact at the cell surface and be internalized by the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytic and signaling receptor. Using a new fluorescence resonance energy transfer (FRET)-based assay of protein proximity, fluorescence lifetime imaging (FLIM), and co-immunoprecipitation we demonstrate that the light chain of LRP interacts with BACE on the cell surface in association with lipid rafts. Surprisingly, the BACE-LRP interaction leads to an increase in LRP C-terminal fragment, release of secreted LRP in the media and subsequent release of the LRP intracellular domain from the membrane. Taken together, these data suggest that there is a close interaction between BACE and LRP on the cell surface, and that LRP is a novel BACE substrate. BACE is a transmembrane protease with β-secretase activity that cleaves the amyloid precursor protein (APP). After BACE cleavage, APP becomes a substrate for γ-secretase, leading to release of amyloid-β peptide (Aβ), which accumulates in senile plaques in Alzheimer disease. APP and BACE are co-internalized from the cell surface to early endosomes. APP is also known to interact at the cell surface and be internalized by the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytic and signaling receptor. Using a new fluorescence resonance energy transfer (FRET)-based assay of protein proximity, fluorescence lifetime imaging (FLIM), and co-immunoprecipitation we demonstrate that the light chain of LRP interacts with BACE on the cell surface in association with lipid rafts. Surprisingly, the BACE-LRP interaction leads to an increase in LRP C-terminal fragment, release of secreted LRP in the media and subsequent release of the LRP intracellular domain from the membrane. Taken together, these data suggest that there is a close interaction between BACE and LRP on the cell surface, and that LRP is a novel BACE substrate. BACE 1The abbreviations used are: BACE, β site of APP-cleaving enzyme; Aβ, amyloid-β; APP, amyloid precursor protein; EEA1, early endosome antigen 1; FLIM, fluorescence lifetime imaging; FRET, fluorescence resonance energy transfer; LC, LRP light chain; LRP, low density lipoprotein receptor-related protein; mLRP, mini-LRP; Ab, antibody; GFP, green fluorescent protein; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; SEAP, secretory alkaline phosphatase; CM, caveolae mambrane; NCM, noncaveolae membrane; DMEM, Dulbecco's modified Eagle's medium; ER, endoplasmic reticulum; siRNA, short interfering RNA; ICD, intracellular domain; CTF, C-terminal fragment; LDL, low density lipoprotein. (β site of APP-cleaving enzyme) is a type I membrane-associated aspartyl protease that cleaves APP (1Vassar 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. Jarosinski M.A. Biere A.L. Curran E. Burgess T. Louis J.C. Collins F. Treanor J. Rogers G. Citron M. Science. 1999; 286: 735-741Crossref PubMed Scopus (3308) Google Scholar, 2Sinha 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. 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. John V. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1482) Google Scholar, 3Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashier J.R. Stratman N.C. Mathews W.R. Buhl A.E. Carter D.B. Tomasselli A.G. Parodi L.A. Heinrikson R.L. Gurney M.E. Nature. 1999; 402: 533-537Crossref PubMed Scopus (1339) Google Scholar, 4Hussain 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. Christie G. Mol. Cell Neurosci. 1999; 14: 419-427Crossref PubMed Scopus (1001) Google Scholar). Besides APP, the few BACE substrates that have been identified include the APP homologues APLP1 and -2 (5Li Q. Sudhof T.C. J. Biol. Chem. 2004; 279: 10542-10550Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), P-selectin glycoprotein ligand-1 (PSGL-1), and a membrane-bound sialyltransferase (6Kitazume S. Tachida Y. Oka R. Shirotani K. Saido T.C. Hashimoto Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13554-13559Crossref PubMed Scopus (232) Google Scholar). Post-translational processing of BACE involves N-glycosylation, removal of its prodomain by a furin-like protease, and further complex glycosylation (7Bennett B.D. Denis P. Haniu M. Teplow D.B. Kahn S. Louis J.C. Citron M. Vassar R. J. Biol. Chem. 2000; 275: 37712-37717Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 8Capell A. Steiner H. Willem M. Kaiser H. Meyer C. Walter J. Lammich S. Multhaup G. Haass C. J. Biol. Chem. 2000; 275: 30849-30854Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 9Creemers J.W. Ines Dominguez D. Plets E. Serneels L. Taylor N.A. Multhaup G. Craessaerts K. Annaert W. De Strooper B. J. Biol. Chem. 2001; 276: 4211-4217Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). After glycosylation, BACE co-traffics with APP and is rapidly transported to the Golgi apparatus and distal secretory pathway (9Creemers J.W. Ines Dominguez D. Plets E. Serneels L. Taylor N.A. Multhaup G. Craessaerts K. Annaert W. De Strooper B. J. Biol. Chem. 2001; 276: 4211-4217Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Measurable amounts of APP and BACE are present on the plasma membrane (10Kinoshita A. Fukumoto H. Shah T. Whelan C.M. Irizarry M.C. Hyman B.T. J. Cell Sci. 2003; 116: 3339-3346Crossref PubMed Scopus (226) Google Scholar, 11Huse J.T. Pijak D.S. Leslie G.J. Lee V.M. Doms R.W. J. Biol. Chem. 2000; 275: 33729-33737Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 12Riddell D.R. Christie G. Hussain I. Dingwall C. Curr. Biol. 2001; 11: 1288-1293Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar) and in lipid rafts (12Riddell D.R. Christie G. Hussain I. Dingwall C. Curr. Biol. 2001; 11: 1288-1293Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 13Ehehalt R. Keller P. Haass C. Thiele C. Simons K. J. Cell Biol. 2003; 160: 113-123Crossref PubMed Scopus (926) Google Scholar, 14Cordy J.M. Hussain I. Dingwall C. Hooper N.M. Turner A.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11735-11740Crossref PubMed Scopus (311) Google Scholar). BACE and APP are internalized from the cell surface to early endosomes and cycle between the cell membrane and endosomes (10Kinoshita A. Fukumoto H. Shah T. Whelan C.M. Irizarry M.C. Hyman B.T. J. Cell Sci. 2003; 116: 3339-3346Crossref PubMed Scopus (226) Google Scholar, 11Huse J.T. Pijak D.S. Leslie G.J. Lee V.M. Doms R.W. J. Biol. Chem. 2000; 275: 33729-33737Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 15Walter J. Fluhrer R. Hartung B. Willem M. Kaether C. Capell A. Lammich S. Multhaup G. Haass C. J. Biol. Chem. 2001; 276: 14634-14641Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). The low density lipoprotein receptor-related protein, LRP, is a type I integral membrane protein with a 515-kDa extracellular α-chain non-convalently bound to the 85 kDa membrane-spanning β-chain. It is also found on the cell surface and cycles between the cell membrane and endosomes. Multiple intracellular adaptor and scaffolding proteins bind the LRP 100 amino acid cytoplasmic tail (16Herz J. Strickland D.K. J. Clin. Investig. 2001; 108: 779-784Crossref PubMed Scopus (888) Google Scholar, 17Li Y. Cam J. Bu G. Mol. Neurobiol. 2001; 23: 53-67Crossref PubMed Scopus (116) Google Scholar); its four extracellular binding domains mediate endocytosis of a wide array of ligands, including several of potential importance for Alzheimer disease pathophysiology: APP, apolipoprotein E and α2-macroglobulin (16Herz J. Strickland D.K. J. Clin. Investig. 2001; 108: 779-784Crossref PubMed Scopus (888) Google Scholar, 17Li Y. Cam J. Bu G. Mol. Neurobiol. 2001; 23: 53-67Crossref PubMed Scopus (116) Google Scholar, 18Kounnas M.Z. Moir R.D. Rebeck G.W. Bush A.I. Argraves W.S. Tanzi R.E. Hyman B.T. Strickland D.K. Cell. 1995; 82: 331-340Abstract Full Text PDF PubMed Scopus (447) Google Scholar). The LRP ligand binding domains interact with KPI-containing forms of APP. In addition, an interaction between the C-terminal domain of APP and LRP, mediated by the cytoplasmic adaptor protein Fe65, impacts APP internalization (18Kounnas M.Z. Moir R.D. Rebeck G.W. Bush A.I. Argraves W.S. Tanzi R.E. Hyman B.T. Strickland D.K. Cell. 1995; 82: 331-340Abstract Full Text PDF PubMed Scopus (447) Google Scholar, 19Ulery P.G. Beers J. Mikhailenko I. Tanzi R.E. Rebeck G.W. Hyman B.T. Strickland D.K. J. Biol. Chem. 2000; 275: 7410-7415Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 20Pietrzik C.U. Busse T. Merriam D.E. Weggen S. Koo E.H. EMBO J. 2002; 21: 5691-5700Crossref PubMed Scopus (176) Google Scholar, 21Rebeck G.W. Moir R.D. Mui S. Strickland D.K. Tanzi R.E. Hyman B.T. Brain Res. Mol. Brain Res. 2001; 87: 238-245Crossref PubMed Scopus (53) Google Scholar, 22Kinoshita A. Whelan C.M. Smith C.J. Mikhailenko I. Rebeck G.W. Strickland D.K. Hyman B.T. J. Neurosci. 2001; 21: 8354-8361Crossref PubMed Google Scholar, 23Trommsdorff M. Borg J.P. Margolis B. Herz J. J. Biol. Chem. 1998; 273: 33556-33560Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar). In addition to its role in endocytosis, LRP has an interesting pattern of proteolysis that parallels APP in some ways. Ectodomain shedding of LRP has been described (24Quinn K.A. Pye V.J. Dai Y.P. Chesterman C.N. Owensby D.A. Exp. Cell Res. 1999; 251: 433-441Crossref PubMed Scopus (77) Google Scholar) and proteolysis of LRP by matrix metalloproteases was recently reported (25Rozanov D.V. Hahn-Dantona E. Strickland D.K. Strongin A.Y. J. Biol. Chem. 2004; 279: 4260-4268Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar); MT1-MMP also cleaves APP (26Higashi S. Miyazaki K. Biochemistry. 2003; 42: 6514-6526Crossref PubMed Scopus (38) Google Scholar) and the postulated α-secretases of the ADAM family are also metalloproteinases. Furthermore, and as with APP, γ-secretase cleavage of LRP leads to release of the LRP intracellular domain (LRP-ICD), which can translocate to the nucleus and interact with Tip60 (27Kinoshita A. Shah T. Tangredi M.M. Strickland D.K. Hyman B.T. J. Biol. Chem. 2003; 278: 41182-41188Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 28May P. Reddy Y.K. Herz J. J. Biol. Chem. 2002; 277: 18736-18743Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Given that BACE and APP interact and traffic with one another, and that APP interacts with and traffics with LRP, we now examined whether LRP interacts with BACE. Using both a FRET-based assay of protein proximity and co-immunoprecipitation, we demonstrate that the LRP-ICD interacts with BACE and that this interaction seems to take place in lipid rafts on the cell surface. Surprisingly, the BACE-LRP interaction does not appear to enhance BACE endocytosis from the cell surface. Instead BACE induces LRP extracellular domain cleavage and subsequent release of the LRP intracellular domain from the membrane. Taken together, these data suggest a close interaction between BACE and LRP on the cell surface and suggest that LRP is processed by BACE in a fashion analogous to APP processing. Generation of Expression Constructs of LRP and BACE and BACE siRNA—The generation of the LRP light chain with two copies of Myc at its N terminus (amino acids 3844–4525) (Myc-LC), the minireceptor mLRP1-Myc that encodes the N-terminal cluster of ligand binding repeats fused to the light chain of LRP and tagged with Myc at its C terminus has been described previously (29Mikhailenko I. Battey F.D. Migliorini M. Ruiz J.F. Argraves K. Moayeri M. Strickland D.K. J. Biol. Chem. 2001; 276: 39484-39491Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). These constructs were used instead of full-length LRP because of its functional similarity to and better expression than full-length LRP. To make mLRP1-GFP, mLRP1 was PCRed into pEGFP-N1 (Clontech). To create the LC-LDLR chimera, a unique KpnI restriction site was introduced into the cytoplasmic portion of LC downstream of the transmembrane domain using the QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA). PCR-generated sequences encoding cytoplasmic domains of the human LDL receptor were then inserted in place of the LRP sequence. To make the LRP165-Myc construct, mLRP1-Myc was digested, and the band containing the vector and the N-terminal 14 amino acids and the C-terminal 165 amino acids of LRP was extracted and self-ligated as described (27Kinoshita A. Shah T. Tangredi M.M. Strickland D.K. Hyman B.T. J. Biol. Chem. 2003; 278: 41182-41188Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). LRP light chain (amino acid 4148–4544) was fused C-terminally to the Gal4-VP16 synthetic transcription factor, and subcloned into pcDNA3 expression vector (Invitrogen). The leader peptide from LRP was fused to a hemagglutinin epitope, which was then fused to the N terminus of the fusion protein in the construct. Secretory alkaline phosphatase (SEAP) was fused by PCR to the N terminus of LC (amino acids 4018–4525) in pSecTagB (Invitrogen); the SEAP-cDNA was kindly provided by S. F. Lichtenthaler (A. Butenandt-Institut, Munich, Germany). The Fe65-Myc clone and BACE with N-terminal Myc and C-terminal V5 tags have been described previously (10Kinoshita A. Fukumoto H. Shah T. Whelan C.M. Irizarry M.C. Hyman B.T. J. Cell Sci. 2003; 116: 3339-3346Crossref PubMed Scopus (226) Google Scholar, 22Kinoshita A. Whelan C.M. Smith C.J. Mikhailenko I. Rebeck G.W. Strickland D.K. Hyman B.T. J. Neurosci. 2001; 21: 8354-8361Crossref PubMed Google Scholar) as have the phosphorylation site (15Walter J. Fluhrer R. Hartung B. Willem M. Kaether C. Capell A. Lammich S. Multhaup G. Haass C. J. Biol. Chem. 2001; 276: 14634-14641Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar) and dileucine mutants of BACE (S498D, S498A, L499A/L500A), BACE-GFP, and the catalytically inactive BACE construct (D93A/D289A) (30von Arnim C.A. Tangredi M.M. Peltan I.D. Lee B.M. Irizarry M.C. Kinoshita A. Hyman B.T. J. Cell Sci. 2004; 117: 5437-5445Crossref PubMed Scopus (97) Google Scholar). pcDNA3.1-mDab1 was a generous gift from Dr. J. Herz (University of Texas, Dallas, TX). Authenticity of the PCR-generated constructs was confirmed by sequencing. The constructs used in this study are summarized in Fig. 1. An siRNA corresponding to the BACE1 gene was designed as described in Kao et al. (31Kao S-C. Krichevsky A.M. Kosik K.S. Tsai L.-H. J. Biol. Chem. 2004; 279: 1942-1949Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) and synthesized by Dharmacon (Lafayette, CO). The following sense sequence was used: 5′-gctttgtggagatggtgga-3′. Cell Culture Conditions and Transient Transfection—H4 cells derived from human neuroglioma cells, mouse neuroblastoma N2a, pac1, LRP-deficient CHO cells (13-5-1), and HEK293 cells are used in this study. Transient transfection of the cells was performed using a liposome-mediated method (FuGENE 6; Roche Applied Science) according to the manufacturer's instruction. Cells were passaged 24 h prior to transfection and harvested or stained 24 h post-transfection. Primary cortical neurons were prepared as described previously (32Berezovska O. Frosch M. McLean P. Knowles R. Koo E. Kang D. Shen J. Lu F.M. Lux S.E. Tonegawa S. Hyman B.T. Brain Res. Mol. Brain Res. 1999; 69: 273-280Crossref PubMed Scopus (62) Google Scholar). Cortical neurons were isolated from embryonic day 16 CD1 mice (Charles River). Staining was performed 8 days after preparation. For experiments requiring delivery of siRNA, cells were transfected by electroporation per the manufacturer's instructions (AMAXA, Gaithersburg, MD). Specific knockdown of >70% of overexpressing BACE was observed starting 24 h after transfection by Western blot and immunostaining (data not shown). Immunocytochemistry and Antibodies—Cells were fixed in 4% paraformaldehyde, permeabilized by 0.5% Triton X-100 in TBS, and blocked with 1.5% normal goat serum. For surface staining Triton-X treatment was omitted. The following antibodies were used: the Golgi organelle marker GM130 mAb and the FITC-conjugated endosomal marker EEA1 mAb (BD Transduction Laboratories, San Diego, CA). The tag antibodies used were rabbit anti-Myc Ab (Upstate Biotechnology, Lake Placid, NY), anti-Myc mAb, anti-V5 mAb (both from Invitrogen), and rabbit anti-V5 Ab (Abcam, Cambridge, MA). Antibodies raised in rabbit against the N (46Kawarabayashi T. Shoji M. Younkin L.H. Wen-Lang L. Dickson D.W. Murakami T. Matsubara E. Abe K. Ashe K.H. Younkin S.G. J. Neurosci. 2004; 24: 3801-3809Crossref PubMed Scopus (295) Google Scholar–56) and C termini (487–501) of BACE were obtained from Calbiochem. A hybridoma secreting an mAb to the LRP intracellular domain (11H4) was obtained from the American Type Culture Collection. Alexa-555-labeled cholera toxin B (CTx-B, Molecular Probes, Eugene, OR) was used to visualize lipid rafts. Secondary antibodies used were labeled with FITC and Cy3 (Jackson Immunoresearch, West Grove, PA) or Alexa 488 (Molecular Probes). Immunostained cells were coverslipped and mounted for confocal or two photon microscopic imaging. The immunostained cells were observed with the appropriate filters by confocal microscopy using a Bio-Rad 1024 confocal 3-channel instrument. Isolation of Caveolae and Noncaveolae Membrane (CM and NCM, respectively)—Caveolae and noncaveolae membrane fractions were isolated from rat smooth muscle cells (pac1) using the method of Smart et al. (33Smart E.J. Ying Y.S. Mineo C. Anderson R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Crossref PubMed Scopus (676) Google Scholar). Briefly, 70% confluent rat smooth muscle cells were collected and Dounce-homogenized in a hypertonic buffer containing protease and phosphatase inhibitors. First, the plasma membrane were isolated and sonicated. CM and NCM fractions were prepared using an optiprep gradient with the sonicated plasma membrane samples. 300-μl membrane fractions (PM, CM, NCM) were solubilized with 100 μlof4× lysis buffer (4% Nonidet P-40, 200 mm Tris, and 600 mm NaCl) to attain final concentration of 1× (1% Nonidet P-40, 50 mm Tris, and 150 mm NaCl). Following the addition of protease and phosphatase inhibitors, CM and NCM lysates were precleared and immunoprecipitated with anti-LRP monoclonal IgG (5A6)-protein G complex overnight. CM and NCM immunoprecipitates along with PM lysate were separated on 4–12% SDS-PAGE under nonreducing conditions and transferred onto nitrocellulose membrane. The membranes were probed with 125I-labeled 11H4 and exposed to Biomax MR film (Kodak). Cholesterol Depletion—For cholesterol depletion, H4 cells were grown for 24 h in Opti-MEM with 10% fetal bovine serum and then for 24 h in either DMEM supplemented with 2 mm l-glutamine, 10% delipidated fetal bovine serum (Cocalico Biologicals), 20 μm lovastatin (Calbiochem), and 0.5 mm mevalonate (mevalonolactone, Sigma) or DMEM with 10% complete fetal bovine serum (34Liscum L. Arnio E. Anthony M. Howley A. Sturley S.L. Agler M. J. Lipid Res. 2002; 43: 1708-1717Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Prior to experimentation, cells were incubated for 10 min in DMEM with 10 mm methyl β-cyclodextrin (Mβ-CD, Sigma) or DMEM alone. Tissue Staining—The Massachusetts Alzheimer Disease Research Center Brain Bank provided temporal cortex. Tissue staining was performed with 11H4 labeled by Alexa-488 and BACE-CT (Calbiochem) labeled by Cy3. Co-immunoprecipitation—Immunoprecipitation experiments were carried out with BioMag beads conjugated to goat anti-mouse IgG (PerSeptive Biosystems, Framingham, MA). The magnetic beads were incubated overnight at 4 °C with anti-V5 or anti-Myc mAb or TBS alone. Lysates from H4 cells co-transfected with BACE-V5 and mLRP1-Myc or pure lysis buffer were added to the bead-antibody complex for 2 h at 4 °C. After the supernatants were collected, the beads were washed in lysis buffer and then boiled with 2× Tris-glycine SDS sample buffer (Invitrogen) for 3 min. The supernatants were loaded onto 10–20% Tris-glycine polyacrylamide gels (Novex, San Diego, CA) under denaturing and reducing conditions. The proteins were transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA) and blocked in 5% nonfat dried milk. mLRP1-Myc was detected by rabbit anti-Myc Ab. BACE was detected by rabbit anti-BACE-NT Ab. Secondary antibodies conjugated to horseradish peroxidase were applied and visualized by chemiluminescence. The Massachusetts Alzheimer Disease Research Center Brain Bank provided temporal cortex. Our protein solubilization procedure was adapted from previously reported studies (35Orlando L.R. Dunah A.W. Standaert D.G. Young A.B. Neuropharmacology. 2002; 43: 161-173Crossref PubMed Scopus (31) Google Scholar) with minor modifications. The tissue was homogenized at 1 ml/100 mg tissue in ice-cold TEVP-sucrose buffer (containing 10 mm Tris, pH 7.4, 5 mm NaF, 1 mm Na3VO4, 1 mm EDTA, 1 mm EGTA, and 320 mm sucrose). The homogenates were centrifuged at 4 °C, and the supernatants were removed. The pellets were resuspended in 800 μl of TEVP with 1% SDS, sonicated for 10 s, and then boiled for 5 min. The samples were centrifuged, and the supernatant was collected for immunoprecipitation after the protein concentration was determined by protein assay (Bio-Rad). Co-immunoprecipitation in human brain tissue was performed as described above with rabbit anti-BACE-CT as pull-down Ab and probed with 11H4 mAb. FRET Measurements using Fluorescence Lifetime Imaging Microscopy (FLIM)—FRET is observed when two fluorophores are in very close proximity, i.e. <0 nm. FRET measurements using FLIM relies on the observation that fluorescence lifetimes (the time of fluorophore emission after brief excitation, measured in picoseconds) are shorter in the presence of a FRET acceptor. We have utilized a new FLIM technique that can detect protein-protein proximity using multiphoton microscopy (36Berezovska O. Ramdya P. Skoch J. Wolfe M.S. Bacskai B.J. Hyman B.T. J. Neurosci. 2003; 23: 4560-4566Crossref PubMed Google Scholar, 37Bacskai B.J. Skoch J. Hickey G.A. Allen R. Hyman B.T. J. Biomed. Opt. 2003; 8: 368-375Crossref PubMed Scopus (140) Google Scholar). A mode-locked Ti-sapphire laser (Spectra Physics) sends a ∼100-fs pulse every ∼12.5 ns to excite the fluorophore. Images were acquired using a Bio-Rad Radiance 2000 multiphoton microscope. We used a high speed Hamamatsu MCP detector (MCP5900; Hamamatsu, Ichinocho, Japan) and hardware/software from Becker and Hickl (SPC 830, Berlin, Germany) to measure fluorescence lifetimes on a pixel-by-pixel basis. Excitation at 800 nm was empirically determined to excite GFP, Alexa 488 and FITC, but not Cy3. Donor fluorophore (GFP, Alexa 488, or FITC) lifetimes were fit to two exponential decay curves to calculate the fraction of fluorophores within each pixel that interact with an acceptor. As a negative control, GFP, Alexa 488, or FITC lifetimes were measured in the absence of acceptor (Cy3), which showed lifetimes equivalent to GFP, Alexa 488-IgG, or FITC IgG alone, in solution or with co-transfection with an empty vector (pEGFP) measured in the presence of Cy3-labeled BACE-V5 or LC-Myc. No bleedthrough or mis-excitation of Cy3 was observed under these conditions. Statistical testing was performed by Student's t test. Internalization Assay—To quantitate BACE internalization we modified a previously reported protocol (38Sever S. Damke H. Schmid S.L. J. Cell Biol. 2000; 150: 1137-1148Crossref PubMed Scopus (194) Google Scholar). CHO 13-5-1 (LRP-null cells) were grown to 70% confluency in 6-well plates and transiently transfected with Myc-BACE and either empty vector or LC-GFP. Cells were then washed once with ice-cold PBS containing 1 mm CaCl2 and 1 mm MgCl2, 0.2% bovine serum albumin, and 5 mm glucose (PBS++++) and 0.4 μg/ml biotinylated Myc-mAb (Upstate Biotechnologies) in PBS++++ was applied for 30 min on ice. After that the cells were allowed to endocytose at 37 °C for the indicated times. Returning the plates to ice stopped endocytosis. Surface biotin was masked with streptavidin (Roche Applied Science) for 1 h on ice. Avidin was quenched with 0.5 mg/ml biocytin (Sigma). Cells were harvested in blocking buffer (1% Triton X-100, 0.1% SDS, 0.2% bovine serum albumin, 50 mm NaCl, 1 mm Tris, pH 7.4) and incubated on IgG-coated 96-well plates at 4 °C overnight. After three washes in PBS, the plates were incubated in streptavidin-peroxidase 1:5000 (Roche Applied Science) in blocking buffer for 1 h. After another wash cycle 3× in PBS, the plates were incubated with 200 μl of 10 mg of o-phenyldiamine HCl (Sigma), 10 μl of 30% H2O2 (Sigma) in 25 ml of 50 mm Na2HPO4, 27 mm citrate, pH 5.0. The reaction was terminated by the addition of 50 μl of H2SO4 and the A490 was read. BACE internalization was then graphed as the percentage of internalized Myc-BACE of total surface Myc-BACE. LRP Ectodomain Secretion Assay—HEK cells passaged into 12-well plates were transfected with a β-galactosidase reporter, LRPβ-fused N-terminally to secreted alkaline phosphatase and either empty vector, BACE, or a catalytically inactive BACE mutant. Each condition was transfected in triplicate except for siRNA experiments, which were transfected in duplicate. Media was changed 24 h later, and then collected after another 24 h. Measurement of SEAP activity in the conditioned media was carried out in triplicate by chemiluminescent assay (Roche Applied Science) according to the manufacturer's instructions. SEAP activity was normalized to β-galactosidase activity, which was measured by hydrolysis of o-nitrophenyl-β-d-galactopyranoside in cells lysed with reporter lysis buffer (Promega). Pharmacologic inhibition of LRP cleavage was assessed after overnight treatment with vehicle (Me2SO) or a cell-permeable, peptidomimetic inhibitor of BACE (Calbiochem) (39Abbenante G. Kovacs D.M. Leung D.L. Craik D.J. Tanzi R.E. Fairlie D.P. Biochem. Biophys. Res. Commun. 2000; 268: 133-135Crossref PubMed Scopus (39) Google Scholar). Western Blotting—N2a cells co-transfected with LC-Myc and either empty vector, BACE, or a catalytically inactive BACE mutant and treated with 1 μm γ-secretase inhibitor DAPT (40Kornilova A.Y. Das C. Wolfe M.S. J. Biol. Chem. 2003; 278: 16470-16473Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) for 12 h (a generous gift from M. Wolfe, Brigham and Women's Hospital, Boston, MA) were lysed in 1% Triton X-100 in TBS buffer and proteinase inhibitor tablets (Roche Applied Science) and then loaded onto 4–20% Tris-glycine polyacrylamide gels (Novex) under denaturing and reducing conditions. The proteins were transferred to polyvinylidene difluoride membrane and LRP light chain was detected by rabbit anti-Myc Ab with Alexa 680 (Molecular Probes) goat anti-rabbit secondary and visualized on a Licor Odyssey near-infrared gel reader (Lincoln, Nebraska). Luciferase Assay—HEK293 cells were transfected with LRP-Gal4-VP16 (LRP-GV) in the absence or presence of BACE and relative luciferase activity determined (28May P. Reddy Y.K. Herz J. J. Biol. Chem. 2002; 277: 18736-18743Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Activity relative to β-galactosidase is shown and averaged for triplicate transfection. In all cases transfection was confirmed by immunoblotting. Localization of BACE and LRP Constructs—We first tested the localization of BACE and LRP in co-expressing H4 cells. When expressed individually, both mLRP1 and BACE were localized mainly in punctate structures in the cells. mLRP1-positive structures largely overlapped with BACE-positive structures when they were co-expressed. To determine the subcellular distribution we immunostained co-expressing H4 cells with organelle markers or, in cell surface stained without Triton X-100 treatment, Alexa555-labeled CTx-B as a raft marker. mLRP1 and BACE co-localized in the endosomal compartments stained by EEA1 (Fig. 2A). To a lesser extent, the Golgi marker GM130 also overlapped with mLRP1 and BACE (Fig. 2B). On the cell surface Myc-LC and BACE are partly co-localized with one another in lipid rafts. The results of the immunocytochemistry suggest that LC and BACE are co-localized in distinct compartments of the cell including lipid rafts (Fig. 2C), Golgi and prominently in the endosomal compartment. To confirm that LRP localizes to lipid rafts we prepared total membrane and separated CM and NCM fractions using an optiprep gradient. LRP was present in caveolae as well as in noncaveolae fractions (Fig. 2D), which is in accordance with our confocal data showing partial overlap with the lipid raft marker CTx-B. We then looked for co-localization under physiological conditions. By staining human brain sections, including the hippocampal formation, we were able to observe similar results in neurons expressing endogenous levels of LRP and BACE (Fig. 3D). Co-immunoprecipitation of BACE and LRP in Human Brain Tissue—From the immunohistochemical experiments that showed robust co-localization in both transfected cells and human" @default.
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• W2137023074 date "2005-05-01" @default.
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• W2137023074 title "The Low Density Lipoprotein Receptor-related Protein (LRP) Is a Novel β-Secretase (BACE1) Substrate" @default.
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• W2137023074 doi "https://doi.org/10.1074/jbc.m414248200" @default.
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