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Signal Peptide Peptidase Forms a Homodimer That Is Labeled by an Active Site-directed γ-Secretase Inhibitor

2004; Elsevier BV; Volume: 279; Issue: 15 Linguagem: Inglês

10.1074/jbc.m309305200

ISSN

1083-351X

Autores

Andrew C. Nyborg, Anna Y. Kornilova, Karen Jansen, Thomas B. Ladd, Michael S. Wolfe, Todd E. Golde,

Tópico(s)

Cellular transport and secretion

Resumo

Presenilin (PS) is the presumptive catalytic component of the intramembrane aspartyl protease γ-secretase complex. Recently a family of presenilin homologs was identified. One member of this family, signal peptide peptidase (SPP), has been shown to be a protease, which supports the hypothesis that PS and presenilin homologs are related intramembrane-cleaving aspartyl proteases. SPP has been reported as a glycoprotein of ∼45 kDa. Our initial characterization of SPP isolated from human brain and cell lines demonstrated that SPP is primarily present as an SDS-stable ∼95-kDa protein on Western blots. Upon heating or treatment of this ∼95-kDa SPP band with acid, a ∼45-kDa band could be resolved. Co-purification of two different epitope-tagged forms of SPP from a stably transfected cell line expressing both tagged versions demonstrated that the ∼95-kDa band is a homodimer of SPP. Pulse-chase metabolic labeling studies demonstrated that the SPP homodimer assembles rapidly and is metabolically stable. In a glycerol velocity gradient, SPP sedimented from ∼100–200 kDa. Significantly the SPP homodimer was specifically labeled by an active site-directed photoaffinity probe (III-63) for PS, indicating that the active sites of SPP and PS/γ-secretase are similar and providing strong evidence that the homodimer is functionally active. Collectively these data suggest that SPP exists in vivo as a functional dimer. Presenilin (PS) is the presumptive catalytic component of the intramembrane aspartyl protease γ-secretase complex. Recently a family of presenilin homologs was identified. One member of this family, signal peptide peptidase (SPP), has been shown to be a protease, which supports the hypothesis that PS and presenilin homologs are related intramembrane-cleaving aspartyl proteases. SPP has been reported as a glycoprotein of ∼45 kDa. Our initial characterization of SPP isolated from human brain and cell lines demonstrated that SPP is primarily present as an SDS-stable ∼95-kDa protein on Western blots. Upon heating or treatment of this ∼95-kDa SPP band with acid, a ∼45-kDa band could be resolved. Co-purification of two different epitope-tagged forms of SPP from a stably transfected cell line expressing both tagged versions demonstrated that the ∼95-kDa band is a homodimer of SPP. Pulse-chase metabolic labeling studies demonstrated that the SPP homodimer assembles rapidly and is metabolically stable. In a glycerol velocity gradient, SPP sedimented from ∼100–200 kDa. Significantly the SPP homodimer was specifically labeled by an active site-directed photoaffinity probe (III-63) for PS, indicating that the active sites of SPP and PS/γ-secretase are similar and providing strong evidence that the homodimer is functionally active. Collectively these data suggest that SPP exists in vivo as a functional dimer. Presenilins (PSs) 1The abbreviations used are: PS, presenilin; SPP, signal peptide peptidase; I-CLiP, intramembrane cleaving protease; (Z-LL)2-ketone, 1,3-di-(N-carboxybenzoyl-l-leucyl-l-leucyl)amino acetone; HEK, human embryonic kidney 293; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid; PI, protease inhibitor; NTF, NH2-terminal fragment; CTF, COOH-terminal fragment; NTA, nitrilotriacetic acid; wt, wild type; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. were first identified through genetic studies demonstrating that mutations in them caused Alzheimer's disease (1Levy-Lahad E. Wasco W. Poorkaj P. Romano D.M. Oshima J. Pettingell W.H. Yu C.E. Jondro P.D. Schmidt S.D. Wang K. Crowley A.C. Fu Y.-H. Guenette S.Y. Galas D. Nemens E. Wijsman E.M. Bird T.D. Schellenberg G.D. Tanzi R.E. Science. 1995; 269: 973-977Crossref PubMed Scopus (2241) Google Scholar, 2Sherrington R. Rogaev E.I. Liang Y. Rogaeva E.A. Levesque G. Ikeda M. Chi H. Lin C. Li G. Holman K. Tsuda T. Mar L. Foncin J.-F. Bruni A.C. Montesi M.P. Sorbi S. Rainero I. Pinessi L. Nee L. Chumakov I. Pollen D. Brookes A. Sanseau P. Polinsky R.J. Wasco W. Da Silva H.A.R. Haines J.L. Pericak-Vance M.A. Tanzi R.E. Roses A.D. Fraser P.E. Rommens J.M. St. George-Hyslop P.H. Nature. 1995; 375: 754-760Crossref PubMed Scopus (3599) Google Scholar). PSs are integral membrane proteins with multiple membrane-spanning domains that are thought to be the catalytic components of a high molecular mass complex referred to as γ-secretase that carries out intramembrane cleavage of multiple type I integral membrane proteins (3Golde T.E. Younkin S.G. Trends Mol. Med. 2001; 7: 264-269Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 4Wolfe M.S. De Los Angeles J. Miller D.D. Xia W. Selkoe D.J. Biochemistry. 1999; 38: 11223-11230Crossref PubMed Scopus (185) Google Scholar). Because γ-secretase catalyzes the final cleavage that releases amyloid β from its precursor protein, it is a target of emerging Alzheimer's disease therapeutic compounds (3Golde T.E. Younkin S.G. Trends Mol. Med. 2001; 7: 264-269Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Recent studies have provided a more complete picture of the γ-secretase complex. In addition to PS1 or PS2, three other proteins are required for activity: Nicastrin, APH-1, and PEN-2 (5Yu G. Nishimura M. Arawaka S. Levitan D. Zhang L. Tandon A. Song Y.Q. Rogaeva E. Chen F. Kawarai T. Supala A. Levesque L. Yu H. Yang D.S. Holmes E. Milman P. Liang Y. Zhang D.M. Xu D.H. Sato C. Rogaev E. Smith M. Janus C. Zhang Y. Aebersold R. Farrer L.S. Sorbi S. Bruni A. Fraser P. St. George-Hyslop P. Nature. 2000; 407: 48-54Crossref PubMed Scopus (827) Google Scholar, 6Francis 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. Curtis D. Dev. Cell. 2002; 3: 85-97Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar, 7Edbauer D. Winkler E. Haass C. Steiner H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8666-8671Crossref PubMed Scopus (218) Google Scholar, 8Takasugi N. Tomita T. Hayashi I. Tsuruoka M. Niimura M. Takahashi Y. Thinakaran G. Iwatsubo T. Nature. 2003; 422: 438-441Crossref PubMed Scopus (789) Google Scholar, 9Kimberly W.T. LaVoie M.J. Ostaszewski B.L. Ye W. Wolfe M.S. Selkoe D.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6382-6387Crossref PubMed Scopus (684) Google Scholar). Both inhibitor studies and mutational analysis suggest that PSs are intramembrane cleaving aspartyl proteases (aspartyl I-CLiPs) (4Wolfe M.S. De Los Angeles J. Miller D.D. Xia W. Selkoe D.J. Biochemistry. 1999; 38: 11223-11230Crossref PubMed Scopus (185) Google Scholar). Active site-directed aspartyl protease inhibitors as well as other γ-secretase inhibitors have been shown to bind PS (10Esler W.P. Kimberly W.T. Ostaszewski B.L. Diehl T.S. Moore C.L. Tsai J.Y. Rahmati T. Xia W. Selkoe D.J. Wolfe M.S. Nat. Cell Biol. 2000; 2: 428-434Crossref PubMed Scopus (508) Google Scholar, 11Li 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.K. Shearman M.S. Smith A.L. Shi X.P. Yin K.C. Shafer J.A. Gardell S.J. Nature. 2000; 405: 689-694Crossref PubMed Scopus (867) Google Scholar, 12Seiffert D. Bradley J.D. Rominger C.M. Rominger D.H. Yang F. Meredith Jr., J.E. Wang Q. Roach A.H. Thompson L.A. Spitz S.M. Higaki J.N. Prakash S.R. Combs A.P. Copeland R.A. Arneric S.P. Hartig P.R. Robertson D.W. Cordell B. Stern A.M. Olson R.E. Zaczek R. J. Biol. Chem. 2000; 275: 34086-34091Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 13Evin G. Sharples R.A. Weidemann A. Reinhard F.B. Carbone V. Culvenor J.G. Holsinger R.M. Sernee M.F. Beyreuther K. Masters C.L. Biochemistry. 2001; 40: 8359-8368Crossref PubMed Scopus (43) Google Scholar). Moreover mutation of either of two conserved aspartates present in adjacent transmembrane domain results in dominant negative PSs that inhibit γ-secretase activity (14Wolfe M.S. Xia W.M. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1699) Google Scholar). Despite these recent advances in the understanding of γ-secretase, the multiprotein complex mediating this activity poses significant problems for studies that would inevitably lead to a more definitive structural and mechanistic understanding of this protease. Although the identification of additional members of the γ-secretase complex has enabled reconstitution of γ-secretase activity in a heterologous system (15Edbauer D. Winkler E. Regula J.T. Pesold B. Steiner H. Haass C. Nat. Cell Biol. 2003; 5: 486-488Crossref PubMed Scopus (781) Google Scholar), to date the active complex has not been purified to homogeneity. Shortly after the identification of human PSs, close homologs were recognized in plants, invertebrates, and vertebrates. Some of these homologs such as those found in Caenorhabditis elegans and Drosophila have been extensively studied and shown to function in high molecular mass complexes like human PSs (for a review, see Ref. 16Golde T.E. Eckman C.B. Sci. STKE. 2003; http://stke.sciencemag.org/cgi/content/full/OC_sigtrans;2003/172/re4PubMed Google Scholar). More recently, other proteins with less obvious homology to PSs have been recognized by data base searching (17Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar, 18Ponting C.P. Hutton M. Nyborg A. Baker M. Jansen K. Golde T.E. Hum. Mol. Genet. 2002; 11: 1037-1044Crossref PubMed Scopus (154) Google Scholar, 19Grigorenko A.P. Moliaka Y.K. Korovaitseva G.I. Rogaev E.I. Biochemistry (Mosc.). 2002; 67: 826-835Crossref PubMed Scopus (56) Google Scholar). These proteins have been referred to by various names. Herein they will be referred to as presenilin homologs/signal peptide peptidase (SPP). All of these proteins are predicted to be integral membrane proteins with multiple membrane-spanning regions and contain both the conserved transmembrane aspartates and the PAL motif near the COOH terminus (16Golde T.E. Eckman C.B. Sci. STKE. 2003; http://stke.sciencemag.org/cgi/content/full/OC_sigtrans;2003/172/re4PubMed Google Scholar, 20Weihofen A. Martoglio B. Trends Cell Biol. 2003; 13: 71-78Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Shortly after its initial in silico identification, one of the presenilin homologs/SPP was identified as an aspartyl I-CliP (17Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar). This protein has been termed signal peptide peptidase as it has been shown to carry out the intramembrane cleavage of signal peptides of major histocompatibility complex class I molecules and hepatitis C virus polyprotein following the initial cleavage of these type II membrane proteins by signal peptidase. SPP-mediated cleavage of major histocompatibility complex class I appears to play an important role in normal immune surveillance as HLA E epitopes are produced from the signal peptide of major histocompatibility complex class I by SPP cleavage (21Lemberg M.K. Bland F.A. Weihofen A. Braud V.M. Martoglio B. J. Immunol. 2001; 167: 6441-6446Crossref PubMed Scopus (153) Google Scholar). These epitopes are presented to natural killer cells at the cell surface. Such presentation is thought to indicate that the probed cell is healthy (22Karre K. Hansson M. Kiessling R. Immunol. Today. 1991; 12: 343-345Abstract Full Text PDF PubMed Scopus (30) Google Scholar). SPP cleavage of an internal signal sequence in the hepatitis C virus polyprotein appears to be essential for proper maturation of the viral core protein. SPP was originally identified using an inhibitor labeling approach with a compound termed TBL4K, which is a photoaffinity probe, based on an inhibitor of SPP activity, (Z-LL)2-ketone (17Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar). In contrast to human PS1, human SPP activity can be reconstituted in yeast without co-expression of other protein cofactors. Moreover mutation of the second of the conserved transmembrane aspartates (Asp-265) does not alter labeling with TBL4K but does block catalytic activity. More recent studies demonstrate that some γ-secretase inhibitors can inhibit SPP activity and that relatively high concentrations of (Z-LL)2-ketone SPP inhibitors can inhibit γ-secretase activity (23Weihofen A. Lemberg M.K. Friedmann E. Rueeger H. Schmitz A. Paganetti P. Rovelli G. Martoglio B. J. Biol. Chem. 2003; 278: 16528-16533Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 24Kornilova A.Y. Das C. Wolfe M.S. J. Biol. Chem. 2003; 278: 16470-16473Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Although the topography of SPP has not been definitively established, some evidence suggests that the transmembrane regions containing the proposed catalytic aspartates have an inverted topology relative to PS. If true, this topology would be consistent with the cleavage of type II membrane proteins by SPP and type I membrane proteins by PS (17Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar). Collectively these bioinformatic and experimental studies of SPP and PS1 support the notion that these proteins are members of a family of biomedically important aspartyl I-CliPs. Because both enzymes are potential targets for therapeutic intervention, PS for the treatment of Alzheimer's disease and SPP for anti-hepatitis C virus therapy, it will be important to identify compounds and conditions for the selective inhibition of each protease. In the present study, we set out to characterize a ∼95-kDa species of SPP. Intact SPP is theoretically a ∼42-kDa protein that, when N-glycosylated, has been reported to migrate at ∼45 kDa (17Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar). Here we demonstrated that the ∼95-kDa species is a homodimer and that the vast majority of SPP in mammalian cells and brain exists in this dimeric form. This dimer was SDS-stable but was partially heat- and acid-labile. Significantly a photoactivable active site-directed γ-secretase inhibitor specifically labeled the dimeric form of SPP. These results suggest that SPP functions as a homodimer and provide additional evidence that the active sites of SPP and PS are structurally similar. SPP Constructs—Full-length SPP was cloned by amplifying a 1134-bp sequence from a human brain cDNA library. The wild type SPP cDNA contains the entire coding sequence of SPP and 17 bases of 5′ untranslated sequence and 10 bases of 3′ untranslated sequence and was cloned into HindIII (5′) and XbaI (3′) sites of the pAG3 expression vector (25Murphy M.P. Hickman L.J. Eckman C.B. Uljon S.N. Wang R. Golde T.E. J. Biol. Chem. 1999; 274: 11914-11923Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Full-length SPP was also cloned into the pcDNA6-V5-his vector (Invitrogen) to generate a carboxyl-terminally V5 + His6 epitope-tagged SPP (SPP-CTV5his) and into pFLAG-CMV-2 (Sigma) to generate an amino-terminally FLAG epitope-tagged SPP (SPP-NTFLAG). In SPP-CTV5his, SPP was cloned into HindIII (5′) and XbaI (3′) of cDNA6-V5-his vector such that a vector-derived six-amino acid spacer (SRGPFE) exists between the end of the SPP coding sequence and the V5 tag. In SPP-NTFLAG, full-length SPP was cloned into the HindIII (5′) and XhoI (3′) sites of pFLAG-CMV-2 such that a vector-derived three-amino acid spacer (LLA) separates the FLAG tag from the amino terminus of SPP. All constructs were verified by sequencing. Generation of Pooled Stable Lines—Chinese hamster ovary, human embryonic kidney 293 (HEK), or human neuroglioma (H4) cells were transfected with 2 μg of plasmid DNA preincubated with 8 μl of Fu-GENE 6 transfection reagent (Roche Applied Science) in serum-free OptiMEM (Invitrogen) overnight. Media were then replaced with either Ham's F-12 (Chinese hamster ovary cells) or Dulbecco's modified Eagle's medium (HEK cells and H4 cells) supplemented with 10% fetal bovine serum (Hyclone) and 2.5 μg/ml blasticidin S (Calbiochem). Following selection, greater than 500 colonies were observed in each pooled stable line. Expression levels were monitored periodically by immunoblotting throughout the course of the experiments and remained stable through multiple passages. The HEK cell line that co-expresses the SPP-CTV5his and SPP-NTFLAG constructs was made by mixing 2 μg of SPP-NTFLAG and 0.2 μg of the SPP-CTV5his DNA in the transfection mixture. Stable cell lines were selected by selecting for blasticidin S encoded by pCDNA-6-SPPCTV5his. Cell lines were maintained at 37 °C under 5% CO2. Antibodies, Western Blotting, and Immunostaining—Anti-peptide antisera were raised in rabbits to the amino- (anti-SPPnt, residues 1–20) and carboxyl (anti-SPPct, residues 358–377)-terminal domains of human SPP (Covance). Peptides corresponding to these domains of SPP were synthesized and coupled to keyhole limpet hemocyanin prior to immunization. Lysates were prepared by lysis in either 2% CHAPSO in 50 mm HEPES (CHAPSO) and 1× Complete PI or 1% Triton X-100 in Tris-buffered saline with 1× Complete PI (TX-100). Except where indicated in the figure legends, sample loading buffer was added to each sample such that the sample contained a final concentration of 10% glycerol, 2% SDS, and 2.5% β-mercaptoethanol. Samples were run on 10–20% Tris-HCl polyacrylamide gels (Bio-Rad) and then transblotted to Immobilon-P membranes (Millipore). Blots were probed with rabbit anti-SPPct at a 1:1000 dilution to detect the carboxyl-terminal 20 amino acids of SPP or rabbit anti-SPPnt at a 1:1000 dilution to detect the amino-terminal 20 amino acids of SPP. PS1 was detected with a 1:1000 mixture of antibodies that detect PS1 CTF (PS490, gifts of E. Koo) and PS1 amino terminus (852B3). Anti-V5 antibody (Invitrogen) and anti-FLAG (Sigma) were used at a 1:1000 dilution. Peptide preabsorption was performed by adding excess peptide dissolved in water and incubating it for 1 h at 37 °C. The preabsorbed anti-serum was then centrifuged at 20,800 × g for 2 min. Trichloroacetic Acid Precipitation—Trichloroacetic acid precipitation was performed as described previously (17Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar). Briefly 10% trichloroacetic acid was added to sucrose gradient fractionation samples. Samples were then centrifuged at 20,800 × g for 2 min, and the supernatant was discarded. Pellets were washed twice in acetone and then allowed to dry. Pellets were resuspended in 0.5% CHAPSO, 50 mm HEPES, 150 mm NaCl, and 1× PI. Immunocytochemistry—Immunocytochemistry was carried out as described previously (26Lah J.J. Levey A.I. Mol. Cell. Neurosci. 2000; 16: 111-126Crossref PubMed Scopus (82) Google Scholar). Briefly HEK cells coexpressing SPP-CTV5his and SPP-NTFLAG were plated on 4-well chamber slides. Cells were fixed in 2% paraformaldehyde in phosphate-buffered saline and permeabilized in 0.05% saponin. Anti-V5 rabbit polyclonal antibody (Sigma) was used at a 1:500 dilution, and anti-FLAG mouse monoclonal (Sigma) was used at a 1:1000 dilution. Secondary goat anti-rabbit IgG (568) and goat anti-mouse IgG (488) were used sequentially at a 1:2000 dilution. Imaging was performed on a confocal Olympus microscope with Fluoview software system. Ni2+ Affinity Purification—HEK cells (one to two confluent 150-mm plates) stably expressing the SPP-CTV5his construct were lysed in CHAPSO lysis buffer with 1× EDTA-free PI. Lysates were centrifuged at 3220 × g for 5 min and the supernatant was diluted to 1% CHAPSO, 50 mm HEPES, and 20 mm imidazole and rocked with 100 μl of nickel NTA slurry overnight at 4 °C. Nickel NTA beads (Qiagen) were washed two times in 50 bed volumes of cold 50 mm HEPES, 1% CHAPSO, 150 mm NaCl, 20 mm imidazole, and 1× EDTA-free PI. Two consecutive elutions were collected in 50 mm HEPES, 1% CHAPSO, 150 mm NaCl, and 200 mm imidazole. All buffers were at pH 7 and had 1× EDTA-free PI. Gradient Fractionation—Sucrose gradients were run as described previously (27Wahrle S. Das P. Nyborg A.C. McLendon C. Shoji M. Kawarabayashi T. Younkin L.H. Younkin S.G. Golde T.E. Neurobiol. Dis. 2002; 9: 11-23Crossref PubMed Scopus (365) Google Scholar). Glycerol velocity gradients were run as described previously (28Yu 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. Fraser P.E. J. Biol. Chem. 1998; 273: 16470-16475Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). Briefly HEK wild type (wt) or HEK cells stably expressing the SPP-CTV5his construct (five confluent 150-mm plates) were lysed in either CHAPSO or TX-100. Lysates were spun at 3220 × g to remove nuclei, insoluble material, and cellular debris, and 1 ml of supernatant was loaded on to the top of an 11-ml 10–40% linear glycerol gradient with either 0.5% CHAPSO, 150 mm NaCl, and 25 mm HEPES or 0.1% TX-100 in Tris-buffered saline. The samples were then centrifuged in an SW 41 rotor for 15 h at 110,000 × g at 4 °C. One-milliliter fractions were collected from the top and analyzed by SDS-PAGE for SPP or PS1 proteins. Control gradients were prepared identically but included a combination of commercially available, characterized, molecular mass standards (Serva). Metabolic Labeling and Immunoprecipitation of SPP—HEK wt or SPP-CTV5his stably expressing cells were labeled for 2 h with 1 mCi/ml [35S]methionine. SPP was immunoprecipitated with either anti-SPPnt or anti-V5 antibodies from TX-100 lysates. Each analysis was performed in duplicate. Immunoprecipitated proteins were separated on 10–20% Tris-HCl gels (Bio-Rad). Phosphorimaging analysis of the dried gels was performed. ImageQuant was used to quantitate the SPP in each sample and determine the half-life of SPP. Covalent Labeling of SPP by Photoactivable and Biotinylated Active Site-directed γ-Secretase Inhibitor—Photoprobe III-63 and analog III-31-C were synthesized as described previously (29Esler W.P. Das C. Campbell W.A. Kimberly W.T. Kornilova A.Y. Diehl T.S. Ye W. Ostaszewski B.L. Xia W. Selkoe D.J. Wolfe M.S. Nat. Cell Biol. 2002; 4: E110-E112Crossref PubMed Scopus (38) Google Scholar). The photolabeling was performed as described previously (24Kornilova A.Y. Das C. Wolfe M.S. J. Biol. Chem. 2003; 278: 16470-16473Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Briefly 500 nm compound III-63 was incubated with CHAPSO lysates for 1 h in the absence or presence of a parent compound, III-31-C (20 μm), or an SPP inhibitor, (Z-LL)2-ketone (1 μm). Samples were irradiated for 45 min at 350 nm. Biotinylated proteins were precipitated with immobilized streptavidin and detected by SDS-PAGE using anti-V5 antibody for SPP-CTV5his-expressing cells or anti-SPPct antibody for the endogenous SPP in wt cells. Antibodies to SPP Recognize an ∼95-kDa Band—Anti-peptide antisera were raised in rabbits to the amino (anti-SPPnt, residues 1–20) and carboxyl (anti-SPPct, residues 358–377) terminal domains of human SPP. Both of these antibodies recognized a band of ∼95 kDa in human brain and in human cell lines indicating that this band is likely to contain full-length SPP (Fig. 1a). In HEK and H4 cells, a second band of ∼45 kDa was also recognized by both anti-SPP antibodies (Fig. 1, a and d). In human brain, this band was not detected by either antibody even upon longer exposure (not shown). Preabsorption of the antibody with peptide completely blocked detection of both the ∼95- and ∼45-kDa bands (Fig. 1a), and neither band was present in preimmune sera (Fig. 1d). Variability in the relative amounts of the two bands could be seen depending on how the sample was handled post-lysis. For example, samples that had been manipulated for long periods of time (Ni2+ affinity purification or overnight fractionation) or heated excessively had more of the ∼45-kDa band relative to the ∼95-kDa band (e.g. Fig. 1d). Although in most lysates the ∼95-kDa band was always present, lower and more variable levels of the ∼45-kDa band were seen. The ∼95-kDa species was observed from lysates of cells that stably overexpress SPP with a V5 and His6 epitope tag at its COOH terminus (SPP-CTV5his) or a FLAG epitope tag at the NH2 terminus (SPP-NTFLAG) (Fig. 1b). The COOH-terminal V5his tag on SPP was placed at the very COOH terminus after the putative KKXX endoplasmic reticulum retrieval signal. In some cases, placement of an epitope tag beyond the endoplasmic reticulum retrieval signal can alter the fate of the protein. With SPP, we found no evidence for any effect of the epitope tags on the characteristics of the protein that we analyzed in this study. Except for expected shifts in molecular mass, identical results were obtained with COOH-terminally tagged SPP, NH2-terminally tagged SPP, and endogenous SPP. Upon longer exposure of the blots, a minor ∼45-kDa band was also detected by anti-V5 and anti-FLAG antibodies in immunoblots of SPP-CTV5his-transfected HEK or SPP-NTFLAG-transfected HEK cells (not shown). Ni2+ affinity purification of SPP-CTV5his from HEK cells stably expressing this protein resulted in purification of a ∼45- and a ∼95-kDa band (Fig. 1c). Both ∼45- and ∼95-kDa bands were detected by the anti-SPPnt and anti-SPPct antibodies in SPP lysates that had a higher relative proportion of the ∼45-kDa species than typically seen; nevertheless these results again demonstrated that each band contained full-length SPP (Fig. 1d). The Ni2+ affinity-purified ∼95-kDa band was recognized by both anti-SPPnt and anti-SPPct antibodies demonstrating that the antibodies were recognizing both tagged and untagged versions of SPP (Fig. 1e). Notably the flow-through of the Ni2+ affinity purification had a significant amount of untagged SPP, as detected by anti-SPPnt and anti-SPPct antibodies, that was not purified and ran at a slightly lower molecular mass relative to the eluted SPP-CTV5his (Fig. 1e, lane 2 versus lanes 3 and 4). SPP Is an SDS-stable Dimer—Based on these immunoblotting results, we postulated that the ∼95-kDa form of SPP is either an SDS-stable complex with another protein or a homodimer. To investigate the relationship between the two forms we conducted several experiments. First we attempted to dissociate the ∼95-kDa form using various detergents for lysis. Lysis of HEK cells that overexpress SPP-CTV5his in 1% Triton X-100, 2% CHAPSO, 0.1% SDS, 1× radioimmune precipitation assay buffer, or 6 m urea did not significantly alter the relative levels of the ∼95- and ∼45-kDa bands detected after immunoblotting with anti-V5 antibody (not shown). In all instances, the ∼95-kDa band was the predominant species. To obtain partially purified SPP, we performed sucrose gradient fractionation and collected the buoyant fractions 4 and 5, which contained detergent-resistant membranes and associated proteins. We reported previously that these detergent-resistant membranes contain γ-secretase activity (27Wahrle S. Das P. Nyborg A.C. McLendon C. Shoji M. Kawarabayashi T. Younkin L.H. Younkin S.G. Golde T.E. Neurobiol. Dis. 2002; 9: 11-23Crossref PubMed Scopus (365) Google Scholar). Like PS1, SPP was enriched in these detergent-resistant membranes as well (not shown). Using the detergent-resistant membranes, we performed a number of experiments to determine whether we could disassociate the ∼95-kDa band into the smaller 45-kDa band. Heating the sample without loading buffer did very little to the ratio of the 45- to 95-kDa bands (Fig. 2a, lane 2). However, heating the sample at 65 °C for 20 min after loading buffer (SDS and β-mercaptoethanol) was added made a large difference in the amount of the 45-kDa band relative to the 95-kDa band. Additionally trichloroacetic acid precipitation of the sample generated large quantities of the 45-kDa species (Fig. 2a, lane 4). The combination of trichloroacetic acid precipitation and heating at 65 °C for 20 min after loading buffer was added caused all the ∼95-kDa species to be converted to the ∼45-kDa species (Fig. 2a, lane 5). These are the same conditions (trichloroacetic acid precipitation and heated Western samples) that Weihofen et al. (17Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar) used when SPP was initially characterized as a ∼45-kDa protein. Integral membrane proteins with multiple transmembrane domains are often difficult to recover if cell lysates are heated excessively prior to SDS-PAGE; therefore, in the experiments shown previously, the lysates were only heated at 37 °C for 10 min prior to gel loading. To further determine whether heating alters the relative levels of the two bands observed for SPP, we altered the incubation temperature of TX-100 lysates prior to PAGE. These results showed that as SPP-CTV5his-overexpressing HEK cell lysates were heated from 37 to 65 °C for 20 min, the amount of the ∼95-kDa species decreased, and the ∼45-kDa species increased (Fig. 2b). Above 65 °C the amount of both species decreased, and high molecular mass aggregates became predominant (Fig. 2b). Lysates heated above 85 °C contained primarily an aggregated species that barely entered the gel at all (not shown). Similar results were obtained for CHAPSO-lysed SPP-CTV5his-overexpressing HEK cells, TX-100-lysed SPP-NTFLAG-overexpressing HEK cells, and endogenous SPP in HEK cells (not shown). To confirm that the ∼45-kDa species was a product of the heated ∼95-kDa species, CHAPSO lysates from HEK cells stably overexpressing SPP-CTV5his were separated by SDS-PAGE, and a gel slice in the 90–100-kDa m

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