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• W2034796667 abstract "The familial Alzheimer's disease gene products, presenilin-1 and presenilin-2, have been reported to be functionally involved in amyloid precursor protein processing, notch receptor signaling, and programmed cell death or apoptosis. However, the molecular mechanisms by which presenilins regulate these processes remain unknown. With regard to the latter, we describe a molecular link between presenilins and the apoptotic pathway. Bcl-XL, an anti-apoptotic member of the Bcl-2 family was shown to interact with the carboxyl-terminal fragments of PS1 and PS2 by the yeast two-hybrid system. In vivo interaction analysis revealed that both PS2 and its naturally occurring carboxyl-terminal products, PS2short and PS2Ccas, associated with Bcl-XL, whereas the caspase-3-generated amino-terminal PS2Ncas fragment did not. This interaction was corroborated by demonstrating that Bcl-XL and PS2 partially co-localized to sites of the vesicular transport system. Functional analysis revealed that presenilins can influence mitochondrial-dependent apoptotic activities, such as cytochrome c release and Bax-mediated apoptosis. Together, these data support a possible role of the Alzheimer's presenilins in modulating the anti-apoptotic effects of Bcl-XL. The familial Alzheimer's disease gene products, presenilin-1 and presenilin-2, have been reported to be functionally involved in amyloid precursor protein processing, notch receptor signaling, and programmed cell death or apoptosis. However, the molecular mechanisms by which presenilins regulate these processes remain unknown. With regard to the latter, we describe a molecular link between presenilins and the apoptotic pathway. Bcl-XL, an anti-apoptotic member of the Bcl-2 family was shown to interact with the carboxyl-terminal fragments of PS1 and PS2 by the yeast two-hybrid system. In vivo interaction analysis revealed that both PS2 and its naturally occurring carboxyl-terminal products, PS2short and PS2Ccas, associated with Bcl-XL, whereas the caspase-3-generated amino-terminal PS2Ncas fragment did not. This interaction was corroborated by demonstrating that Bcl-XL and PS2 partially co-localized to sites of the vesicular transport system. Functional analysis revealed that presenilins can influence mitochondrial-dependent apoptotic activities, such as cytochrome c release and Bax-mediated apoptosis. Together, these data support a possible role of the Alzheimer's presenilins in modulating the anti-apoptotic effects of Bcl-XL. familial Alzheimer's disease programmed cell death amyloid precursor protein presenilin-1 presenilin-2 amino acid polyacrylamide gel electrophoresis green fluorescent protein monoclonal antibody dithiobispropionimidate·2 HCl Alzheimer's disease, a progressive neurodegenerative disorder of late life, is characterized by deposition of β-amyloid plaques, accumulation of intracellular neurofibillary tangles, and neuronal cell loss (1Price D.L. Sisodia S.S. Annu. Rev. Neurosci. 1998; 21: 479-505Crossref PubMed Scopus (509) Google Scholar). Approximately 10% of Alzheimer's disease cases are familial (FAD)1 and co-segregate with autosomal dominant inheritance (2Tanzi R.E. Kovacs D.M. Kim T.-W. Moir R.D. Guenette S.Y. Wasco W. Neurobiol. Dis. 1996; 3: 159-168Crossref PubMed Scopus (239) Google Scholar). The majority of FAD is linked to mutations in genes encoding presenilin-1 (PS1) (3Sherrington 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 (3585) Google Scholar) and presenilin-2 (PS2) (4Levy-Lahad E. Wasco W. Poorkaj P. Romano D.M. Oshima J. Pettingell W.H., Yu, C. 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 (2230) Google Scholar, 5Rogaev E.I. Sherrington R. Rogaeva E.A. Levesque G. Ikeda M. Liang Y. Chi H. Lin C. Holman K. Tsuda T. Mar L. Sorbl S. Nacmias B. Piacentini S. Amaducci L. Chumakov I. Cohen D. Lannfelt L. Fraser P.E. Rommens J.M. St. George-Hyslop P.H. Nature. 1995; 376: 775-778Crossref PubMed Scopus (1791) Google Scholar), which are highly penetrant and have been shown to influence amyloid precursor protein (APP) by increasing the production of the neurotoxic form of β-amyloid, β-amyloid-42/-43 (6Duff K. Eckman C. Zehr C., Yu, X. Prada C.-M. Perez-tur J. Hutton M. Buee L. Harigaya Y. Yager D. Morgan D. Gordon M.N. Holcomb L. Refolo L. Zenk B. Hardy J. Younkin S. Nature. 1996; 383: 710-713Crossref PubMed Scopus (1320) Google Scholar, 7Qian S. Jiang P. Guan X.-M. Singh G. Trumbauer M.E., Yu, H. Chen H.Y. Van der Ploeg L.H.T. Zheng H. Neuron. 1998; 20: 611-617Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Structurally, PS1 and PS2 gene products are multipass membrane proteins consisting of 6–8 spanning regions with a large hydrophilic loop at the carboxyl terminus (8Doan A. Thinakaran G. Borchelt D.R. Slunt H.H. Ratovitsky T. Podlisny M. Selkoe D.J. Seeger M. Gandy S.E. Sisodia S.S. Neuron. 1996; 7: 1023-1030Abstract Full Text Full Text PDF Scopus (355) Google Scholar, 9De Strooper B. Beullens M. Contreras B. Levesque L. Craessaerts K. Cordell B. Moechars D. Bollen M. Fraser P. St. George-Hyslop P. Van Leuven F. J. Biol. Chem. 1997; 272: 3590-3598Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 10Lehmann S. Chiesa R. Harris D.A. J. Biol. Chem. 1997; 272: 12047-12051Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 11Dewji N.N. Singer S.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14025-14030Crossref PubMed Scopus (48) Google Scholar). Immunolocalization studies have demonstrated that these ubiquitously expressed molecules, primarily localized to the endoplasmic reticulum and the Golgi apparatus (9De Strooper B. Beullens M. Contreras B. Levesque L. Craessaerts K. Cordell B. Moechars D. Bollen M. Fraser P. St. George-Hyslop P. Van Leuven F. J. Biol. Chem. 1997; 272: 3590-3598Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 12Cook D.G. Sung J.C. Golde T.E. Felsenstein K.M Wojczyk B.S. Tanzi R.E. Trojanowski J.Q. Lee V.M.-Y. Doms R.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9223-9228Crossref PubMed Scopus (182) Google Scholar), are also found on nuclear and plasma membranes (13Li J. Xu M. Zhou H. Ma J. Potter H. Cell. 1997; 90: 917-927Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 14Dewj N.N. Singer S.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9926-9931Crossref PubMed Scopus (61) Google Scholar). At the amino acid level, these proteins are ∼67% identical and exhibit homology to two Caenorhabitis elegans gene products, SEL-12 and HOP1, both of which facilitate notch receptor-mediated signaling, thus suggesting a role for presenilins in this process (15Levitan D. Greenwald I. Nature. 1995; 351: 351-354Crossref Scopus (628) Google Scholar, 16Li X. Greenwald I. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12204-12209Crossref PubMed Scopus (137) Google Scholar). In addition to their roles in APP processing and notch receptor signaling, extensive evidence suggests presenilins involvement in programmed cell death (PCD) or apoptosis. ALG-3, a truncated mouse homologue of PS2, corresponding to the last 103 amino acids, rescues cells from T cell receptor-induced apoptosis by inhibiting Fas-meditated death signal (17Vito P. Lacan· E. D'Adamio L. Science. 1996; 271: 521-525Crossref PubMed Scopus (456) Google Scholar). Overexpression of PS2 increases apoptosis induced by a number of apoptotic stimuli (18Vito P. Wolozin B. Ganjei J.K. Katsunori I. Lacan· E. D'Adamio L. J. Biol. Chem. 1996; 271: 31025-31028Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), whereas FAD-associated PS1 and PS2 mutations generate molecules with constitutive pro-apoptotic activity (19Wolozin B. Iwaski K. Vito P. Ganjei J.K. Lacan· E. Sunderland T. Zhao B. Kusiak J.W. Wasco W. D'Adamio L. Science. 1996; 274: 1710-1713Crossref PubMed Scopus (392) Google Scholar, 20Guo Q. Sopher B.L. Furukawa K. Pham D.G. Robinson N. Martin G.M. Mattson M.P. J. Neurosci. 1997; 17: 4212-4222Crossref PubMed Google Scholar, 21Janicki S. Monteiro M.J. J. Cell Biol. 1997; 139: 485-495Crossref PubMed Scopus (114) Google Scholar). Complementary studies have demonstrated that depletion of PS2 protein levels by antisense RNA protected cells against apoptosis induced by a number of cell death-inducing apoptotic stimuli (18Vito P. Wolozin B. Ganjei J.K. Katsunori I. Lacan· E. D'Adamio L. J. Biol. Chem. 1996; 271: 31025-31028Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 19Wolozin B. Iwaski K. Vito P. Ganjei J.K. Lacan· E. Sunderland T. Zhao B. Kusiak J.W. Wasco W. D'Adamio L. Science. 1996; 274: 1710-1713Crossref PubMed Scopus (392) Google Scholar). Strikingly, physiological ALG-3-like counterparts have also been identified, exhibiting similar anti-apoptotic properties. For example, PS2Ccas, which represents a 119-amino acid carboxyl-terminal fragment of PS2 generated by caspase-3 cleavage (22Vito P. Ghayur T. D'Adamio L. J. Biol. Chem. 1997; 272: 28315-28320Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 23Loetscher H. Deuschle U. Brockhaus M. Reinhardt D. Nelboeck P. Mous J. Grünberg J. Haass C. Jacobsen H. J. Biol. Chem. 1997; 272: 20655-20659Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 24Kim T.-W. Pettingell W.H. Jung Y.-K. Kovacs D.M. Tanzi R.E. Science. 1997; 277: 373-376Crossref PubMed Scopus (328) Google Scholar), protects cells from various apoptotic stimuli (22Vito P. Ghayur T. D'Adamio L. J. Biol. Chem. 1997; 272: 28315-28320Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). In addition, PS2short (PS2 s), a molecule generated either by alternative transcription or by proteolysis, exhibits similar anti-apoptotic property (22Vito P. Ghayur T. D'Adamio L. J. Biol. Chem. 1997; 272: 28315-28320Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). To characterize better the molecular mechanisms by which presenilins and the apoptotic machinery are linked, we have tried to identify proteins interacting with both PS1 and PS2. Here we show that Bcl-XL, an anti-apoptotic member of the Bcl-2 family of protein (25Boise L.K. Gonzalez-Garcia M. Postema C.E. Ding L. Lindsten T. Turka L.A. Mao X. Núñez G. Thompson C.B. Cell. 1993; 74: 597-608Abstract Full Text PDF PubMed Scopus (2929) Google Scholar), associates with both PS1 and PS2. Moreover, we observed that presenilins can act upon the mitochondria by influencing cytochrome c release and by augmenting the proapoptotic effects of Bax, a Bcl-2 family member that opposes Bcl-XLfunction. Human kidney 293 T fibroblasts and COS-7 cells were grown in Dulbecco's modified Eagle's media supplemented with 10% fetal calf serum (Biofluids; Rockville MD) and glutamine (2 mm). The human Jurkat T-cell line was cultured in RPMI 1640 media containing 10% fetal calf serum (Biofluids), glutamine (2 mm), β-mercaptoethanol (25 nm), and the antibiotics streptomycin (10 μg/ml), penicillin (10 units/ml), and gentamicin (10 μg/ml) (Life Technologies, Inc.). Cells were cultured at 37 °C in a 5% CO2 humidifying chamber. Plasmids for the interactor trap were generously provided by Dr. Roger Brent (Harvard University, Boston), except for the pEG202-LexA-RFHM7.3, −12 baits, and pJG4–5-B42-Cdi3 prey control plasmids, which were kindly provided by Dr. R. F. Finley (Wayne State University, Detroit, MI). The pcDNA3-FLAG-Bcl-XL (Dr. G. Nunez, University of Michigan, Ann Arbor, MI) and the pSFFV-Bax (Dr. S. Korsymeyer, Washington University, St. Louis, MO) constructs were gifts. The pcDNA3-FLAG-AIP1 has been previously described (26Vito P. Pellegrini L. Guiet C. D'Adamio L. J. Biol. Chem. 1999; 274: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). PS1 and PS2 fragments were amplified by polymerase chain reaction and cloned into pcDNA3. Bcl-XL was cloned into the pEGFP-N1 construct (CLONTECH; Palo Alto, CA). The rabbit anti-human PS2n antibody was generated against the carboxyl-terminal region (a.a. 341–377) of the hydrophilic loop (22Vito P. Ghayur T. D'Adamio L. J. Biol. Chem. 1997; 272: 28315-28320Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The 2972 polyclonal antibody (27Walter J. Capell A. Grünberg J. Pesold B. Schindzielorz A. Prio R. Podlisny M.B. Fraser P. St. George-Hyslop P.S. Selkoe D.J. Haass C. Mol. Med. 1996; 2: 673-691Crossref PubMed Google Scholar) was raised against the amino terminus (a.a. 2–81) of PS2 and was generously provided by Dr. C. Haass (University of Mannheim, Germany). The negative control mouse IgG and anti-human Bcl-XL monoclonal antibody (mAb) was purchased from Southern Biotechnology Inc. (Birmingham, AL). The carboxyl-terminal regions of PS1 (a.a. 264–467, PS1CT) and PS2 (a.a.330–448, PS2Ccas) were fused to the LexA DNA-binding domain in pEG202. Full-length Bcl-XL cDNA was cloned into the galactose-inducible pJG4-5 vector containing the B42-activation domain. The yeast two-hybrid system was performed as described previously (28Finley Jr., R. Brent R. DNA Cloning, Expression Systems: A Practical Approach. Oxford Universal Press, Oxford1995: 169-203Google Scholar). Briefly, the yeast strain RFY231 (MAT α trp1Δ::hisGhis3ura3–1 leu2::3 Lexop -LEU2) (29Kolonin M.G. Finley Jr., R.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14266-14271Crossref PubMed Scopus (87) Google Scholar) harboring either pEG202-LexA-PS1CT or -PS2Ccas and the lacZ reporter plasmid, pSH18–34, were transformed by the lithium acetate method with the pJG4–5-B42-Bcl-XL construct. Presenilin and Bcl-XL interactions were assayed for growth on the basis of leucine prototrophy on Gal Ura−His−Trp−Leu−plates. Subsequent testing for β-galactosidase activity was performed on Gal Ura−His−Trp− plates containing 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside. 2 × 105 293 T cells were transfected by the CaPO4method with 5 μg of each of the indicated constructs. Eight h post-transfection, cells were washed once in phosphate-buffered saline (Biofluids), plated in fresh media, and cultured overnight. Overexpressed proteins were cross-linked by resuspending cells in phosphate-buffered saline containing 10 mmdimethyl-3,3′-dithiobispropionimidate·2 HCl (Pierce) and incubated on ice for 40 min. Cells were washed and solubilized in 1% Nonidet P-40 (Calbiochem) lysing buffer containing 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, and protease inhibitors including aprotinin (1%), leupeptin (1 mm), pepstatin (2 mm) (Sigma), and 4-(2-aminoethyl)benzenesulfonyl fluoride) (2 mm) (ICN, Aurora, OH). After centrifugation (750 ×g; 30 min; 4 °C), cell lysates were rotated with bovine serum albumin-preabsorbed anti-FLAG M2 affinity gel (Sigma), for 1 h at room temperature. Protein-bound beads were extensively washed, and immunoabsorbed molecules were dissociated by the addition of Laemmli's sample buffer (30Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207227) Google Scholar) and resolved under reducing conditions (100 mm dithiothreitol) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12%). Proteins were blotted onto nitrocellulose (Schleicher & Schuell) and probed with anti-PS2 antibodies, followed by an anti-rabbit IgG-horseradish peroxidase antibody (Promega, Madison, WI) and visualized by enhanced chemiluminescence (ECL) (Pierce). 4 × 106 Jurkat cells (107/ml) were transfected by electroporation with the indicated plasmids. All transfections were normalized with pcDNA3 (Invitrogen, Carlsbad, CA). Cells were allowed to recover by incubation for 1 h 30 min at 37 °C in a humidifying chamber. Cells were layered onto a Ficoll-Paque (Amersham Pharmacia Biotech) cushion and centrifuged (750 × g; 20 min; 25 °C). The cell layer at the interface was removed, washed, and placed into culture. Cell samples were taken at various times, and nuclei were stained by the addition of an equal volume of a 2× hypotonic propidium iodide solution (50 μg/ml). Cell death was assessed by measuring the percentage of fragmented DNA by flow cytometry (31Nicoletti I. Migliorati G. Pagliacci M.C. Grignani F. Riccardi C. J. Immunol. Methods. 1991; 139: 271-279Crossref PubMed Scopus (4426) Google Scholar). For analysis of cytochrome release, 24–30 h after transfection, 293 cells were resuspended in 500 μl of buffer A (20 mmHepes, pH 7.5, 10 mm KCl, 1.5 mmMgCl2, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol) containing 250 mm sucrose and a mixture of protease inhibitors. Cells were homogenized with a glass Pyrex homogenizer; nuclei and unbroken cells were removed by centrifugation at 1,000 × g for 10 min at 4 °C. The resulting supernatant was subjected to 10,000 × g centrifugation for 20 min at 4 °C, and the pellet fraction, containing the mitochondria, was washed in buffer A/sucrose and solubilized in TNC buffer (10 mm Tris, pH 8, 0.5% Nonidet P-40, 5 mm CaCl2). The supernatant fraction was further centrifuged at 100,000 × g for 1 h at 4 °C to generate cytosol. Equal amount of lysate (10 μg) from each fraction was separated by SDS-PAGE (10%) and blotted onto nitrocellulose membranes and subsequently probed with either anti-cytochrome c (PharMingen, San Diego, CA) or anti-cytochrome oxidase subunit IV mAb (Molecular Probes, Eugene, OR). COS-7 cells were plated at ∼40% confluence the day before transfection. Transfections were carried out using a total of 1 μg of DNA by the LipofectAMINE method (Life Technologies, Inc.) according to the manufacturer's instructions. Twenty-four hours after transfection, cells were trypsinized and plated onto 15-mm round coverslips. Cells were fixed (2% paraformaldehyde), washed in phosphate-buffered saline, and incubated with the anti-PS2n antibody followed by a Texas Red-conjugated secondary antibody (Jackson Laboratories, West Grove, PA). Fluorescence analysis of GFP-tagged Bcl-XL and immunolabeled PS2 was performed on a Bio-Rad MRC1024 confocal microscope. cDNA corresponding to the capase-3-generated carboxyl-terminal fragment of PS2Ccas was fused to the LexA DNA-binding domain and used as “bait” in the yeast two-hybrid system. This carboxyl-terminal region, which includes a portion of the large hydrophilic loop, was chosen as bait because (i) it is cytoplasmically exposed and therefore likely to interact with other molecules, (ii) it exists as a physiological fragment generated by proteolysis, and (iii) it inhibits some forms of PCD. Sequence analysis of a specific interactor obtained from the yeast two-hybrid hunt prompted us to investigate whether Bcl-XL, a member of the Bcl-2 family (25Boise L.K. Gonzalez-Garcia M. Postema C.E. Ding L. Lindsten T. Turka L.A. Mao X. Núñez G. Thompson C.B. Cell. 1993; 74: 597-608Abstract Full Text PDF PubMed Scopus (2929) Google Scholar), interacts with PS2Ccas. TableI summarizes the results of the interaction between PS2Ccas and Bcl-XL. By day 3, as determined by both yeast growth and β-galactosidase activity, interaction between LexA-PS2Ccas and B42-Bcl-XL was undetected, whereas growth and β-galactosidase activity for two different positive controls (LexA-PS2Ccas and B42-cl.19; LexA-HM12 and B42-Cdi3) were observed. B42-cl.19 represents a novel molecule identified in the interactor trap, 2L. Pellegrini, B. Passer, and L. D'Adamio, unpublished observations. whereas the LexA-HM12 and B42-Cid3 interaction has been previously described (32Finley Jr., R.L. Brent R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12980-12984Crossref PubMed Scopus (241) Google Scholar). Interestingly, by day 6, growth was observed by yeast containing LexA-PS2Ccas and B42-Bcl-XL. Of importance, negative controls remained unchanged by this time. Taken together, this observation suggested the possibility that PS2 may interact with Bcl-XL.Table IYeast two-hybrid analysis reveals an interaction between PS2Ccas and Bcl-XLDay 3Day 6Cdi3Cl.19Bcl-xLCdi3Cl.19Bcl-XLβ-GalGrowthβ-GalGrowthβ-GalGrowthβ-GalGrowthβ-GalGrowthβ-GalGrowthPS2Ccas−−++−−−−++++RFHM 7.3−−−−−−−−−−−−RFHM 12++−−−−++−−−−RFY231 yeast containing pEG202 expressing the indicated LexA-tagged proteins and the lacZ reporter plasmid, pSH18–34, was transformed with pJG4–5 B42-tagged construct expressing either Cdi3, C1.19, or Bcl-XL. Yeast were streaked onto the appropriate selection plates (see “Materials and Methods”). Growth and β-galactosidase (β-Gal) activity was scored on days 3 and 6. Note that for PS2Ccas and Bcl-XL interactions, growth and β-galactosidase activity was not observed until day 6. LexA-HM7.3 and LexA-HM12 were used as negative control “baits,” whereas LexA-HM12 was used as a positive control with B42-Cdi3. Open table in a new tab RFY231 yeast containing pEG202 expressing the indicated LexA-tagged proteins and the lacZ reporter plasmid, pSH18–34, was transformed with pJG4–5 B42-tagged construct expressing either Cdi3, C1.19, or Bcl-XL. Yeast were streaked onto the appropriate selection plates (see “Materials and Methods”). Growth and β-galactosidase (β-Gal) activity was scored on days 3 and 6. Note that for PS2Ccas and Bcl-XL interactions, growth and β-galactosidase activity was not observed until day 6. LexA-HM7.3 and LexA-HM12 were used as negative control “baits,” whereas LexA-HM12 was used as a positive control with B42-Cdi3. To verify the interaction observed in yeast, we performed an in vivo interaction analysis. 293 T cells were transiently co-transfected with plasmids expressing either the negative control FLAG-AIP1 (26Vito P. Pellegrini L. Guiet C. D'Adamio L. J. Biol. Chem. 1999; 274: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) or FLAG-Bcl-XL products in combination with either PS2Ccas or the amino-terminal product of caspase-3 cleavage, PS2Ncas. Western blot analysis of protein complexes, initially chemically cross-linked in vivo and immunoprecipitated with mouse anti-FLAG antibodies, revealed that PS2Ccas, but not PS2Ncas, was co-precipitated by FLAG-Bcl-XL. (Fig. 1, A and B, respectively). In contrast, the FLAG-AIP1 negative control did not precipitate either PS2Ccas or PS2Ncas. We then tested whether PS2 associated with Bcl-XL. PS2 typically runs as a prominent band of ∼50 kDa, consistent with its predicted size, in addition to a high molecular mass smear due to aggregation. As shown in Fig. 2, PS2 was detected in FLAG-Bcl-XL but not in FLAG-AIP1 immunocomplexes. Overexpression studies of PS2 have been previously shown to result in the generation of PS2s, a proteolytically derived product of ∼30 kDa (22Vito P. Ghayur T. D'Adamio L. J. Biol. Chem. 1997; 272: 28315-28320Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 23Loetscher H. Deuschle U. Brockhaus M. Reinhardt D. Nelboeck P. Mous J. Grünberg J. Haass C. Jacobsen H. J. Biol. Chem. 1997; 272: 20655-20659Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). As expected, a band migrating at ∼30 kDa was observed in total cell lysates overexpressing PS2. Moreover, this cleaved product was also detected in FLAG-Bcl-XL but not in FLAG-AIP1 immunoprecipitating conditions. The observation that FLAG-Bcl-XL associated with the proteolytically generated PS2 s fragment prompted us to assess further this interaction. In these experiments, plasmids expressing FLAG-Bcl-XL and untagged PS2s were co-transfected in 293 T cells and analyzed for interactions. Fig. 3 confirms a specific interaction between FLAG-Bcl-XL and PS2s. Overall, these results demonstrate that Bcl-XL binds both full-length and its natural occurring carboxyl-terminal fragments of PS2 but not the amino-terminal PS2Ncas product and, thus, maps the site of Bcl-XL interaction to the carboxyl terminus of PS2.Figure 3Bcl-XL co-immunoprecipitates PS2s with and without cross-linker. Lysates from 293 T cells were transiently transfected with the indicated plasmids, and immunoblot analysis was performed with the anti-PS2n antibody. Detection of Bcl-XL and AIP1 in total cell lysates by immunoblotting with anti-FLAG (left panel). Detection of PS2s in total cell lysates and analysis of anti-FLAG immunocomplexes in the presence (+) or absence (−) of the chemical cross-linker DTBP probed with the anti-PS2n antibody (right panel).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The reducible chemical cross-linker DTBP was used in the above studies to assess the interactions between Bcl-XL and various forms of PS2. To rule out the possibility that DTBP captures two molecules only in close proximity and not in an actual association, we tested Bcl-XL and PS2s interactions without DTBP. Fig. 3illustrates that FLAG-Bcl-XL readily precipitated PS2s in the absence of DTPB, as detected by the anti-PS2n antibody. Thus, these results argue against the possibility that these two molecules are only found in proximity and do not interact. Finally, we addressed whether endogenous PS2 could associate with Bcl-XL. Cell lysates derived from 293 T cells overexpressing Bcl-XL were incubated with either an isotype-matched IgG negative control (Fig. 4, lane 2) or the anti-Bcl-XL mAb (Fig. 4, lane 3) coupled to protein G-agarose. Immunoabsorbed material was analyzed for the presence of PS2 by Western blot using the anti-PS2n antibody. Fig.4 shows that the anti-Bcl-XLmAb immunoprecipitated a species of ∼50 kDa, which corresponds to the expected mass of full-length PS2. Verification of overexpressed Bcl-XL in total cell lysates is also shown (Fig. 4, lane 1). The above studies establish an in vivo interaction between Bcl-XL and presenilins. As a step toward validating the relevance of this association, co-localization studies were carried out. COS-7 cells were co-transfected with GFP-tagged Bcl-XLand untagged PS2 expression constructs, and 24 h after transfection, cells were analyzed by confocal microscopy. Single color analysis of PS2 (Texas Red; Fig. 5, left panel) and GFP-Bcl-XL (Fig. 5, right panel) co-transfectants revealed that the expressed proteins appeared similar in their localization patterns, reminiscent of the vesicular transport system (Fig. 5). Simultaneous dual color analysis (Fig. 5, middle panel) confirmed the partial co-localization (yellow) of Bcl-XL and PS2, possibly to the endoplasmic reticulum. PS1 is both structurally and functionally homologous to PS2. They display ∼67% homology at the amino acid level, exhibit similar membrane topologies, and are endoproteolytically processed in a similar fashion (23Loetscher H. Deuschle U. Brockhaus M. Reinhardt D. Nelboeck P. Mous J. Grünberg J. Haass C. Jacobsen H. J. Biol. Chem. 1997; 272: 20655-20659Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 33Thinakaran G. Borchelt D.R. Lee M.K. Slunt H.H. Spitzer L. Kim G. Ratovitsky T. Davenport F. Nordstedt C. Seeger M. Neuron. 1996; 17: 181-190Abstract Full Text Full Text PDF PubMed Scopus (940) Google Scholar, 34Podlisny M.B. Citron M. Amarante P. Sherrington R. Xia W. Zhang J. Diehl T. Levesque G. Fraser P. Haass C. Koo E.H.M. Seubert P. St. George-Hyslop P. Teplow D.B. Selkoe D.J. Neurobiol. Dis. 1997; 3: 325-337Crossref PubMed Scopus (273) Google Scholar, 35Walter J. Grünberg J. Capell A. Pesold B. Schindzielorz A. Citron M. Mendla K. St George-Hyslop P. Multhaup G. Selkoe D.J. Haass C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5349-5354Crossref PubMed Scopus (101) Google Scholar). PS1 and PS2 also participate in APP processing, notch receptor signaling, and PCD. Given these similarities, we investigated whether PS1 also associated with Bcl-XL in the yeast two-hybrid system. By using the carboxyl-terminal portion of PS1CT, which included the entire hydrophilic loop, we obtained results similar to those found with LexA-PS2Ccas. As compared with the positive control interaction of LexA-PS1CT and B42-cl.69, growth and β-galactosidase activity was delayed, not appearing until day 6 (TableII). B42-cl.69 was originally identified as a LexA-PS1CT interactor in a yeast two-hybrid screen 3B. Passer, L. Pellegrini, and L. D'Adamio, unpublished observations. ; however, for these studies, it was used as a positive control. Thus, like LexA-PS2Cas, LexA-PS1CT binds Bcl-XL.Table IIPS1CT and Bcl-XL interaction by the yeast two-hybrid systemDay 3Day 6Cdi3Cl.69Bcl-XLCdi3Cl.69Bcl-XLβ-GalGrowthβ-GalGrowthβ-GalGrowthβ-GalGrowthβ-GalGrowthβ-GalGrowthPS1CT−−++−−−−++++RFHM 7.3−−−−−−−−−−−−RFHM 12++−−−−++−−−−Yeast transformants harboring the indicated LexA expression plasmids together with the lacZ, reporter plasmid, pSH18–34, and either Cdi3, Cl.69, or Bcl-XL fused to the B42 activation domain were assayed for growth and β-galactosidase (β-Gal) activity. Note that for PS1CT and Bcl-XL interactions, growth and β-galactosidase activity was not observed until day 6. Open table in a new tab Yeast transformants harboring the indicated LexA expression plasmids together with the lacZ, reporter plasmid, pSH18–34, and either Cdi3, Cl.69, or Bcl-XL fused to the B42 activation domain were assayed for growth and β-galactosidase (β-Gal) activity. Note that for PS1CT and Bcl-XL interactions, growth and β-galactosidase activity was not observed until day 6. Because presenilins can interact with Bcl-XL and have been previously shown to sensitize cells to apoptotic stimuli, it is plausible that presenilins can modulate PCD through its int" @default.
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