γ-Secretase Is a Functional Component of Phagosomes
2005; Elsevier BV; Volume: 280; Issue: 43 Linguagem: Inglês
10.1074/jbc.m504069200
ISSN1083-351X
AutoresIsabelle Jutras, Annie Laplante, Jonathan Boulais, Sylvain Brunet, Gopal Thinakaran, Michel Desjardins,
Tópico(s)Invertebrate Immune Response Mechanisms
Resumoγ-Secretase is a high molecular mass protein complex that catalyzes the intramembrane cleavage of its protein substrates. Two proteins involved in phagocytosis, CD44 and the low density lipoprotein receptor-related protein, are γ-secretase substrates, suggesting that this complex might regulate some aspects of phagocytosis. Our results indicate that the four components of γ-secretase, viz. presenilin, nicastrin, APH-1, and PEN-2, are present and enriched on phagosome membranes from both murine macrophages and Drosophila S2 phagocytes. The γ-secretase components form high molecular mass complexes in lipid microdomains of the phagosome membrane with the topology expected for the functional enzyme. In contrast to the majority of the phagosome proteins studied so far, which appear to associate transiently with this organelle, γ-secretase resides on newly formed phagosomes and remains associated throughout their maturation into phagolysosomes. Finally, our results indicate that interferon-γ stimulates γ-secretase-dependent cleavages on phagosomes and that γ-secretase activity may be involved in the phagocytic response of macrophages to inflammatory cytokines. γ-Secretase is a high molecular mass protein complex that catalyzes the intramembrane cleavage of its protein substrates. Two proteins involved in phagocytosis, CD44 and the low density lipoprotein receptor-related protein, are γ-secretase substrates, suggesting that this complex might regulate some aspects of phagocytosis. Our results indicate that the four components of γ-secretase, viz. presenilin, nicastrin, APH-1, and PEN-2, are present and enriched on phagosome membranes from both murine macrophages and Drosophila S2 phagocytes. The γ-secretase components form high molecular mass complexes in lipid microdomains of the phagosome membrane with the topology expected for the functional enzyme. In contrast to the majority of the phagosome proteins studied so far, which appear to associate transiently with this organelle, γ-secretase resides on newly formed phagosomes and remains associated throughout their maturation into phagolysosomes. Finally, our results indicate that interferon-γ stimulates γ-secretase-dependent cleavages on phagosomes and that γ-secretase activity may be involved in the phagocytic response of macrophages to inflammatory cytokines. γ-Secretase catalyzes the cleavage of selected type I transmembrane protein substrates within their membrane-spanning domains, an atypical hydrolytic event considering that it occurs in the hydrophobic conditions of the lipid bilayer (1Xia W. Wolfe M.S. J. Cell Sci. 2003; 116: 2839-2844Crossref PubMed Scopus (78) Google Scholar). This type of intramembrane proteolysis was discovered as an essential step in the release of β-amyloid peptides from the amyloid precursor protein (APP), 3The abbreviations used are:APPamyloid precursor proteinLRPlow density lipoprotein receptor-related proteinPSpresenilinNTFN-terminal fragmentCTFC-terminal fragmentpAbspolyclonal antibodiesIFN-γinterferon-γTNF-αtumor necrosis factor-αDAPTN-(N-(3,5-difluorophenacetyl-l-alanyl))-S-phenylglycine t-butyl esterPBSphosphate-buffered salineCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidBNBlue nativeTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineCHAPSO3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid 3The abbreviations used are:APPamyloid precursor proteinLRPlow density lipoprotein receptor-related proteinPSpresenilinNTFN-terminal fragmentCTFC-terminal fragmentpAbspolyclonal antibodiesIFN-γinterferon-γTNF-αtumor necrosis factor-αDAPTN-(N-(3,5-difluorophenacetyl-l-alanyl))-S-phenylglycine t-butyl esterPBSphosphate-buffered salineCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidBNBlue nativeTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineCHAPSO3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid a pivotal process in the development of Alzheimer disease pathology (2Selkoe D. Kopan R. Ann. Rev. Neurosci. 2003; 26: 565-597Crossref PubMed Scopus (560) Google Scholar). γ-Secretase activity was later found to function in Notch signaling by releasing the Notch intracellular domain, which translocates to the nucleus and activates the transcription of target genes (2Selkoe D. Kopan R. Ann. Rev. Neurosci. 2003; 26: 565-597Crossref PubMed Scopus (560) Google Scholar). Several other γ-secretase substrates, including CD44, Notch ligands, and the low density lipoprotein receptor-related protein (LRP; also known as CD91), have been identified, and all share a common type I transmembrane protein topology (3Koo E.H. Kopan R. Nat. Med. 2004; 10: S26-S33Crossref PubMed Scopus (151) Google Scholar). γ-Secretase cleavage of these substrates is preceded by proteolysis within their extracellular domains (3Koo E.H. Kopan R. Nat. Med. 2004; 10: S26-S33Crossref PubMed Scopus (151) Google Scholar). Although the biological function of γ-secretase cleavage remains to be deciphered for many substrates, recent evidence indicates that the role of γ-secretase in signal transduction may not be limited to the Notch pathway. Other γ-secretase substrates, including APP, CD44, and LRP, have indeed been shown to generate transcriptionally active C-terminal fragments (4Cao X. Südhof T.C. J. Biol. Chem. 2004; 279: 24609-24611Google Scholar, 5Lammich S. Okochi M. Takeda M. Kaether C. Capell A. Zimmer A.K. Edbauer D. Walter J. Steiner H. Haass C. J. Biol. Chem. 2002; 277: 44754-44759Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 6May P. Reddy Y.K. Herz J. J. Biol. Chem. 2002; 277: 18736-18743Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Conversely, cleavage by γ-secretase terminates intracellular signaling mediated by the netrin receptor DCC (deleted in colorectal cancer) (7Parent A.T. Barnes N.Y. Taniguchi Y. Thinakaran G. Sisodia S.S. J. Neurosci. 2005; 25: 1540-1549Crossref PubMed Scopus (58) Google Scholar). Taken together, these studies suggest that γ-secretase-mediated cleavage may be generally involved in cell signaling. amyloid precursor protein low density lipoprotein receptor-related protein presenilin N-terminal fragment C-terminal fragment polyclonal antibodies interferon-γ tumor necrosis factor-α N-(N-(3,5-difluorophenacetyl-l-alanyl))-S-phenylglycine t-butyl ester phosphate-buffered saline 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Blue native N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid amyloid precursor protein low density lipoprotein receptor-related protein presenilin N-terminal fragment C-terminal fragment polyclonal antibodies interferon-γ tumor necrosis factor-α N-(N-(3,5-difluorophenacetyl-l-alanyl))-S-phenylglycine t-butyl ester phosphate-buffered saline 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Blue native N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid Significant research efforts have recently been invested in characterizing the biochemistry of γ-secretase. Cumulative evidence indicates that γ-secretase consists of a high molecular mass complex composed of at least four different proteins, viz. presenilin (PS)-1 or the less abundant homolog PS2, nicastrin, APH-1, and PEN-2, all of which are integral membrane proteins (8Iwatsubo T. Curr. Opin. Neurobiol. 2004; 14: 379-383Crossref PubMed Scopus (152) Google Scholar). Current models propose that PS1 contains the catalytic site of γ-secretase, an unusual aspartyl protease with two aspartates located in transmembrane domains 6 and 7 of PS1, respectively (9Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1685) Google Scholar). Nicastrin and APH-1 are suggested to function in stabilizing and trafficking full-length PS1 through the secretory pathway (10LaVoie M.J. Fraering P.C. Ostaszewski B.L. Ye W. Kimberly W.T. Wolfe M.S. Selkoe D.J. J. Biol. Chem. 2003; 278: 37213-37222Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). PS1 undergoes endoproteolysis in its cytoplasmic loop, located between transmembrane domains 6 and 7, generating the N-terminal (NTF) and C-terminal (CTF) fragments, which remain associated in the complex (11Thinakaran G. Borchelt D.R. Lee M.K. Slunt H.H. Spitzer L. Kim G. Rotovitsky T. Davenport F. Nordstedt C. Seeger M. Hardy J. Levey A.I. Gandy S.E. Jenkins N.A. Copeland N.G. Price D.L. Sisodia S.S. Neuron. 1996; 17: 181-190Abstract Full Text Full Text PDF PubMed Scopus (939) Google Scholar, 12Capell A. Grunberg J. Pesold B. Diehlmann A. Citron M. Nixon R. Beyreuther K. Selkoe D.J. Haass C. J. Biol. Chem. 1998; 273: 3205-3211Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). PEN-2 apparently promotes this cleavage event, a key step in the activation of γ-secretase (13Takasugi N. Tomita T. Hayashi I. Tsuruoka M. Niimura M. Takahashi Y. Thinakaran G. Iwatsubo T. Nature. 2003; 422: 438-441Crossref PubMed Scopus (783) Google Scholar). Several reports have demonstrated that the coexpression of all four proteins is required to produce functional γ-secretase (13Takasugi N. Tomita T. Hayashi I. Tsuruoka M. Niimura M. Takahashi Y. Thinakaran G. Iwatsubo T. Nature. 2003; 422: 438-441Crossref PubMed Scopus (783) Google Scholar, 14Kimberly 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 (676) Google Scholar, 15Edbauer D. Winkler E. Regula J.T. Pesold B. Steiner H. Haass C. Nat. Cell Biol. 2003; 5: 486-488Crossref PubMed Scopus (775) Google Scholar, 16Hu Y. Fortini M.E. J. Cell Biol. 2003; 161: 685-690Crossref PubMed Scopus (127) Google Scholar, 17Kim S.H. Ikeuchi T. Yu C. Sisodia S.S. J. Biol. Chem. 2003; 278: 33992-34002Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Although the assembly of a functional γ-secretase complex can occur in the endoplasmic reticulum (18Kim S.H. Yin Y.I. Li Y.-M. Sisodia S.S. J. Biol. Chem. 2004; 279: 48615-48619Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), γ-secretase probably meets its substrates in late compartments of the secretory pathway and in compartments of the endosomal/lysosomal pathway (19Pasternak S.H. Bagshaw R.D. Guiral M. Zhang S. Ackerley C.A. Pak B.J. Callahan J.W. Mahuran D.J. J. Biol. Chem. 2003; 278: 26687-26694Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 20Gupta-Rossi N. Six E. LeBail O. Logeat F. Chastagner P. Olry A. Israël A. Brou C. J. Cell Biol. 2004; 166: 73-83Crossref PubMed Scopus (182) Google Scholar). It remains unclear whether γ-secretase cleavage events occur at the plasma membrane, given that the endocytosis of Notch has recently been described as a prerequisite for its processing by γ-secretase (20Gupta-Rossi N. Six E. LeBail O. Logeat F. Chastagner P. Olry A. Israël A. Brou C. J. Cell Biol. 2004; 166: 73-83Crossref PubMed Scopus (182) Google Scholar). Phagocytosis is a process used by a wide range of organisms, from amoeba to vertebrates, to internalize large particles, typically the size of 1 μm or more (21Underhill D.M. Ozinsky A. Annu. Rev. Immunol. 2002; 20: 825-852Crossref PubMed Scopus (836) Google Scholar). Newly formed phagosomes engage in a maturation process that involves fusion with endosomes of increasing age and ultimately with lysosomes. The killing of phagocytosed microbes in mammalian professional phagocytes such as macrophages forms the first line of defense against infectious diseases. Microbe degradation in the phagosome produces protein antigens, which are presented at the cell surface to activate specific lymphocytes and elicit appropriate immune responses. Phagosome-associated functions are thus important participants in the immune system. At least two acknowledged substrates of γ-secretase, CD44 and LRP (also known as CD91) (3Koo E.H. Kopan R. Nat. Med. 2004; 10: S26-S33Crossref PubMed Scopus (151) Google Scholar), are involved in phagocytosis and are known residents of phagosome membranes (22Leemans J.C. Florquin S. Heikens M. Pals S.T. van der Neut R. van der Poll T. J. Clin. Investig. 2003; 111: 681-689Crossref PubMed Scopus (104) Google Scholar, 23Ogden C.A. de Cathelineau A. Hoffmann P.R. Bratton D. Ghebrehiwet B. Fadok V.A. Henson P.M. J. Exp. Med. 2001; 194: 781-795Crossref PubMed Scopus (945) Google Scholar). These findings prompted us to investigate if the γ-secretase complex localizes to phagosomes and hence if γ-secretase cleavage events occur on this compartment. In this study, we show that all of the components of the γ-secretase reside on phagosomes of both murine macrophages and Drosophila phagocytic cells. The biochemical characterization of γ-secretase on phagosomes demonstrates that γ-secretase associates with lipid rafts on phagosome membranes, is present predominantly as a high molecular mass complex of 440 kDa, and catalyzes γ-secretase-specific cleavages on phagosomes. Our results show that the γ-secretase complex is an abundant, stable, and conserved component of phagosomes, suggesting that γ-secretase actively regulates some of the functions of this highly specialized organelle. Antibodies—The rat anti-Lamp1 luminal monoclonal antibody ID4B was from the Developmental Studies Hybridoma Bank. The rabbit anti-Rab5a and Rab7 polyclonal antibodies (pAbs) and the goat anti-LRP pAb were from Santa Cruz Biotechnology, Inc. The mouse monoclonal antibody raised against the nicastrin N terminus and anti-flotillin monoclonal antibody were from BD Biosciences. The rabbit anti-PS1 NTF and anti-PS1 CTF pAbs were raised against residues 1-65 and 263-407, respectively, of PS1 (11Thinakaran G. Borchelt D.R. Lee M.K. Slunt H.H. Spitzer L. Kim G. Rotovitsky T. Davenport F. Nordstedt C. Seeger M. Hardy J. Levey A.I. Gandy S.E. Jenkins N.A. Copeland N.G. Price D.L. Sisodia S.S. Neuron. 1996; 17: 181-190Abstract Full Text Full Text PDF PubMed Scopus (939) Google Scholar, 24Thinakaran G. Regard J.B. Bouton C.M. Harris C.L. Price D.L. Borchelt D.R. Sisodia S.S. Neurobiol. Dis. 1998; 4 (C. M. L.): 438-453Crossref PubMed Scopus (171) Google Scholar). The rabbit anti-nicastrin pAb was raised against residues 62-93 of nicastrin (25Leem J.Y. Vijayan S. Han P. Cai D. Machura M. Lopes K.O. Veselits M.L. Xu H. Thinakaran G. J. Biol. Chem. 2002; 277: 19236-19240Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). The rabbit anti-PEN-2 pAb was raised against residues 1-26 of PEN-2 (26Luo W.J. Wang H. Li H. Kim B.S. Shah S. Lee H.J. Thinakaran G. Kim T.W. Yu G. Xu H. J. Biol. Chem. 2003; 278: 7850-7854Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Rabbit pAb 369 was raised against the C-terminal domain of APP (27Xu H. Gouras G.K. Greenfield J.P. Vincent B. Naslund J. Mazzarelli L. Fried G. Jovanovic J.N. Seeger M. Relkin N.R. Liao F. Checler F. Buxbaum J.D. Chait B.T. Thinakaran G. Sisodia S.S. Wang R. Greengard P. Gandy S. Nat. Med. 1998; 4: 447-451Crossref PubMed Scopus (514) Google Scholar). Cell Culture and Organelle Isolation—J774 murine macrophages were cultured in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% heat-inactivated fetal bovine serum, 1% glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in 5% CO2. Drosophila melanogaster S2 cells were cultured in Schneider's medium (Sigma) supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 26 °C. For caspase inhibition, J774 macrophages were incubated overnight in the presence of benzyloxycarbonyl-VAD-fluoromethyl ketone (Calbiochem). For cytokine-treated cells, 200 units/ml interferon-γ (IFN-γ) alone or with 5 ng/ml tumor necrosis factor-α (TNF-α) (Calbiochem) was added in the medium 24 h prior to the isolation of phagosomes. For treatment with γ-secretase inhibitors, N-(N-(3,5-difluorophenacetyl-l-alanyl))-S-phenylglycine t-butyl ester (DAPT) or L-685,458 (γ-secretase inhibitor IX and X, respectively; Calbiochem), was added in the medium 24 h prior to the isolation of phagosomes. Phagosomes were formed by the internalization of 0.8-μm blue-dyed latex beads (Estapor® Microspheres) (28Desjardins M. Huber L.A. Parton R.G. Griffiths G. J. Cell Biol. 1994; 124: 677-688Crossref PubMed Scopus (558) Google Scholar). Cells were allowed to internalize the beads for various periods as indicated in the figure legends. When the internalization was followed by a chase period, the cells were washed with ice-cold phosphate-buffered saline (PBS), and new medium was added. The cells were then further incubated for various periods as indicated. For observations under a electron microscope, cells were fixed and processed as described previously (28Desjardins M. Huber L.A. Parton R.G. Griffiths G. J. Cell Biol. 1994; 124: 677-688Crossref PubMed Scopus (558) Google Scholar). To isolate phagosomes, cells were washed and scraped in ice-cold PBS. The cells were lysed in ice-cold homogenization buffer containing 250 mm sucrose, 3 mm imidazole (pH 7.4), and protease inhibitors (Roche Applied Science) using a 22-gauge 112-inch needle. The post-nuclear supernatant was adjusted to 40% sucrose; placed on a cushion of 62% sucrose; and layered with a sucrose gradient composed of 35, 25, and 10% layers. After centrifugation at 24,000 rpm for 1 h, the isolated phagosomes were collected at the 10-25% sucrose interface. To isolate the total cell membranes, post-nuclear supernatants were centrifuged at 120,000 × g for 1 h onto a 62% sucrose cushion. Mass Spectrometry Analysis—Phagosome proteins were separated by SDS-PAGE, and 1-mm-wide bands were excised and trypsin-digested. The resulting tryptic peptides were extracted with 0.2 m urea in 50% aqueous acetonitrile and analyzed by nanoscale liquid chromatography/quadrupole time-of-flight tandem mass spectrometry using a Waters capillary liquid chromatograph coupled to a Q-Tof Ultima mass spectrometer. SDS-PAGE and Western Blotting—Purified phagosomes or total cell membranes were resuspended in Laemmli lysis buffer prior to SDS-PAGE and Western blotting. The amount of proteins and latex beads in the samples was quantified using an EZQ™ protein quantification kit (Molecular Probes, Inc.) and by spectrophotometry, respectively. Deglycosylation Assay—The glycosylation pattern of nicastrin was examined by treating phagosome protein extracts with endoglycosidase H or peptide N-glycosidase F (New England Biolabs Inc.) according to the manufacturer's protocol. Immunofluorescence—Phagosomes were formed by the internalization of 3-μm latex beads (Estapor® microspheres) for 1 h, and purified phagosomes were incubated with the appropriate primary antibody in PBS containing 0.5% bovine serum albumin (PBS/bovine serum albumin) for 1 h at 4°C. The phagosomes were washed with PBS/bovine serum albumin and further incubated with Alexa Fluor® 488-conjugated anti-rabbit or anti-rat antibody (Molecular Probes, Inc.) for 30 min at 4 °C. The immunolabeled phagosomes were washed with PBS/bovine serum albumin and observed using a Leica SP2 confocal microscope. Pronase Assay—To determine the sensitivity of phagosome-associated proteins to Pronase, purified phagosomes were incubated for 1 h at 37 °C with Pronase (Sigma), which consists of a mixture of proteases, as described previously (29Dermine J.-F. Duclos S. Garin J. St. Louis F. Rea S. Parton R.G. Desjardins M. J. Biol. Chem. 2001; 276: 18507-18512Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). Equivalent amounts of phagosomes were treated in parallel with or without Pronase and subjected to SDS-PAGE prior to Western blotting. Lipid Raft Isolation—Lipid rafts were isolated from purified phagosomes as described previously (29Dermine J.-F. Duclos S. Garin J. St. Louis F. Rea S. Parton R.G. Desjardins M. J. Biol. Chem. 2001; 276: 18507-18512Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). Briefly, phagosome membranes were incubated in 0.5% Triton X-100 or 0.5% CHAPS for 30 min on ice. Lipid rafts were separated from the solubilized membranes by floatation on an OptiPrep™ gradient, from which seven equivalent fractions were collected. The proteins in each fraction were precipitated with methanol/chloroform and resuspended in Laemmli lysis buffer. Blue Native (BN) PAGE—For BN-PAGE, the γ-secretase protein complexes were isolated from purified phagosomes by detergent solubilization in 0.5% n-dodecyl β-d-maltoside. The solubilized fractions were subjected to BN-PAGE as described (30Schagger H. Methods Cell Biol. 2001; 65: 231-244Crossref PubMed Google Scholar). After electrophoresis, either the gel was transferred directly to a polyvinylidene difluoride membrane for Western blotting, or a lane of the gel was subjected to Tricine/SDS-PAGE prior to Western blotting. γ-Secretase Activity—To assess the generation of the APP CTFγ (the C-terminal product of APP cleavage by γ-secretase (43Yu C. Kim S.H. Ikeuchi T. Xu H. Gasparini L. Wang R. Sisodia S.S. J. Biol. Chem. 2001; 276: 43756-43760Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar)), purified phagosomes were resuspended in PBS containing 5 mm EDTA and 0.5 mm 1,10-phenanthroline, and aliquots were incubated at either 4 °C or 37 °C for 2 h. The reactions were terminated by the addition of Laemmli lysis buffer, and proteins were separated by SDS-PAGE. The presence of APP CTFs was analyzed by Western blotting. γ-Secretase enzyme activity was also monitored with a fluorescent polymer superquenching-based assay using a peptide containing the sequence GVVIATVK flanked by biotin and a fluorescence quencher (QTL Lightspeed™ γ-secretase assay, QTL Biosystems) according to the manufacturer's protocol. The protein contents in total cell membranes or in purified 1-h-old phagosomes were quantified in purified phagosomes or in total cell membranes. Increasing amounts of membranes or of purified phagosomes were incubated with the peptide substrate for 3 h at 37°C in the absence or presence of 1 μm L-685,458 (γ-secretase inhibitor X), and the fluorescence was measured using a SpectraMax GeminiEM instrument (Molecular Devices Corp.). In a recent proteomic analysis of purified latex bead-containing phagosomes, we identified >500 proteins on this organelle by mass spectrometry (31Garin J. Diez R. Kieffer S. Dermine J.-F. Duclos S. Gagnon E. Sadoul R. Rondeau C. Desjardins M. J. Cell Biol. 2001; 152: 165-180Crossref PubMed Scopus (606) Google Scholar, 32Brunet S. Thibault P. Gagnon E. Kearney P. Bergeron J.J. Desjardins M. Trends Cell Biol. 2003; 13: 629-638Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). This analysis revealed the presence of four of the components of the γ-secretase complex, viz. PS1, PS2, nicastrin, and APH-1 (TABLE ONE). To assess the significance of these observations, the relative abundance of γ-secretase components in phagosomes compared with a total cell lysate and a total cell membrane preparation was determined. As demonstrated in Fig. 1A, PS1, nicastrin, and PEN-2 were highly enriched on phagosome membranes. The PS1 NTF and CTF were clearly the predominant forms of PS1 compared with full-length PS1 (Fig. 1A), indicating that mainly the processed form of PS1 localizes to phagosomes. Likewise, the deglycosylation profile of nicastrin indicated that nicastrin was present in its mature glycosylated form (Fig. 1B). The slight shift in molecular mass observed after treatment with endoglycosidase H is expected of highly glycosylated proteins such as nicastrin (33Yang D.S. Tandon A. Chen F. Yu G. Yu H. Arawaka S. Hasegawa H. Duthie M. Schmidt S.D. Ramabhadran T.V. Nixon R.A. Mathews P.M. Gandy S.E. Mount H.T.J. St. George-Hyslop P. Fraser P.E. J. Biol. Chem. 2002; 277: 28135-28142Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). The PS1 CTF on phagosomes was present in three distinct bands, possibly corresponding to a previously characterized phosphorylated form of the PS1 CTF (highest band) and a product of PS1 CTF cleavage by caspase-3 (lowest band) (see Fig. 6B) (34Saura C.A. Tomita T. Soriano S. Takahashi M. Leem J.Y. Honda T. Koo E.H. Iwatsubo T. Thinakaran G. J. Biol. Chem. 2000; 275: 17136-17142Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Nicastrin and PS1 were also detected by immunofluorescent labeling of isolated phagosomes (Fig. 1C). In contrast to the uniform Lamp1 labeling of the phagosome surface, PS1 and nicastrin labeling was often observed in patches on phagosomes (Fig. 1C, insets), a pattern that may reflect the association of γ-secretase components with lipid microdomains (see Fig. 4).TABLE ONEPeptides of the γ-secretase protein complex identified on the phagosomes of murine J774 macrophages and Drosophila S2 cells by MS-MS analysisProteinSwiss-Prot accession no.PeptidesJ774 macrophagesAPH-1Q8BVF7KADEGLASLSEDGRADEGLASLSEDGRPresenilin-1P49769MLVETAQERAAVQELSGSILTSEDPEERKDGQLIYTPFTEDTETVGQRNicastrinP57716LENIDSFVELGQVALRSGAGVPEVVLRLLYGFLVRMVYDMENGKFPVRNQVEDLLATLEKALANVATVLARNVMFVFFQGETFDYIGSSRAPDVTTLSRFPVRLENIDSFVELGQVALRLAQSQALPPSSLQRDrosophila S2 cellsPresenilin homologO02194ATQNSLASPEAAAASGQRLTGGGGGSGGPPTNEMEEEQGLKNicastrin homologQ9VC27LLGYLLSQLQPDIHRAASYPGSQLTNLPPMRYISVLGGSQESSGYTYRCFQDFNNHNYETHALRNVYATLYPRSLNPDSLQMK Open table in a new tab FIGURE 6IFN-γ increases the γ-secretase cleavage of LRP on phagosomes. A, the presence of the 12-kDa C-terminal (C-term) fragment of LRP, a product of γ-secretase processing, and of the γ-secretase components was assessed in J774 macrophages incubated with IFN-γ for 24 h prior to phagosome formation. 1-h-old phagosomes were purified, and equal amounts of phagosomes from control (-) or IFN-γ-treated (+) cells were loaded in each lane. Western blotting was performed for the indicated proteins. The cleavage product of the PS1 CTF by caspase-3 is indicated by the asterisk. B, J774 cells were incubated overnight in the absence (Control) or presence of a caspase inhibitor (benzyloxycarbonyl-VAD-fluoromethyl ketone (Z-VAD)) prior to the internalization of latex beads for 1 h and phagosome isolation. Equal amounts of phagosomes were separated, and the PS1 CTF was revealed by Western blotting. The cleavage product of the PS1 CTF by caspase-3 is indicated by the asterisk.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4γ-Secretase components are associated with phagosome lipid rafts. Purified 1-h-old phagosomes from J774 cells were solubilized in 0.5% Triton X-100 (A) or 0.5% CHAPS (B). Following the floatation of the detergent-resistant membranes (rafts) on an OptiPrep gradient, seven fractions were collected from the top of the gradient. Equivalent volumes from each fraction were loaded in each lane, and Western blotting was performed for the indicated proteins. Lamp1 and flotillin are shown as a controls for non-raft- and raft-associated proteins, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) γ-Secretase components are highly conserved proteins from worms to mammals (1Xia W. Wolfe M.S. J. Cell Sci. 2003; 116: 2839-2844Crossref PubMed Scopus (78) Google Scholar). To determine whether the localization of γ-secretase to phagosomes was conserved as well, phagosomes were purified from Drosophila S2 cells, known to exhibit efficient phagocytic properties (35Rämet M. Manfruelli P. Pearson A. Mathey-Prevot B. Ezekowitz R.A. Nature. 2002; 416 (R. A. B.): 644-648Crossref PubMed Scopus (586) Google Scholar). γ-Secretase protein homologs were identified on Drosophila S2 phagosomes both by mass spectrometry (TABLE ONE) and by Western blotting (Fig. 1D). Similar to the distribution of γ-secretase in mammalian macrophages (Fig. 1A), γ-secretase components were enriched on phagosomes from S2 cells compared with a total membrane preparation (Fig. 1D), suggesting that the function of γ-secretase on phagosomes has been evolutionarily conserved. Phagolysosome biogenesis and maturation involves complex changes in the protein composition of phagosomes (36Desjardins M. Nat. Rev. Immunol. 2003; 3: 280-291Crossref PubMed Scopus (182) Google Scholar). For instance, Rab5 rapidly associates with nascent phagosomes but dissociates from maturing phagosomes, whereas maturing phagosomes progressively recruit Lamp1 (Fig. 2). In contrast to Rab5 and Lamp1, the proteins of the γ-secretase complex remained abundant and stable on phagosomes throughout their maturation into phagolysosomes (Fig. 2). The dynamics of γ-secretase association with phagosomes also demonstrate that the complex is not targeted to phagosomes simply to be degraded. These results indicate an enrichment of the γ-secretase complex on late phagosome membranes. In contrast, nicastrin displayed a much greater enrichment than PS1 on purified lysosomes (19Pasternak S.H. Bagshaw R.D. Guiral M. Zhang S. Ackerley C.A. Pak B.J. Callahan J.W. Mahuran D.J. J. Biol. Chem. 2003; 278: 26687-26694Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). As late phagosomes and lysosomes may interact with different intracellular compartments, the cellular origin of the γ-secretase complex found on these distinct organelles could differ, and γ-secretase components may thus be present in different proportions. Purified latex bead-containing phagosomes constitute a unique tool to determine the membrane topology of resident proteins. The
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