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• W2000392076 endingPage "11995" @default.
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• W2000392076 abstract "Ectodomain shedding and intramembrane proteolysis of the amyloid precursor protein (APP) by α-, β- and γ-secretase are involved in the pathogenesis of Alzheimer disease (AD). Increased proteolytic processing and secretion of another membrane protein, the interleukin-1 receptor II (IL-1R2), have also been linked to the pathogenesis of AD. IL-1R2 is a decoy receptor that may limit detrimental effects of IL-1 in the brain. At present, the proteolytic processing of IL-1R2 remains little understood. Here we show that IL-1R2 can be proteolytically processed in a manner similar to APP. IL-1R2 expressed in human embryonic kidney 293 cells first undergoes ectodomain shedding in an α-secretase-like manner, resulting in secretion of the IL-1R2 ectodomain and the generation of an IL-1R2 C-terminal fragment. This fragment undergoes further intramembrane proteolysis by γ-secretase, leading to the generation of the soluble intracellular domain of IL-1R2. Intramembrane cleavage of IL-1R2 was abolished by a highly specific inhibitor of γ-secretase and was absent in mouse embryonic fibroblasts deficient in γ-secretase activity. Surprisingly, the β-secretase BACE1 and its homolog BACE2 increased IL-1R2 secretion resulting in C-terminal fragments nearly identical to the ones generated by the α-secretase-like cleavage. This suggests that both proteases may act as alternative α-secretase-like proteases. Importantly, BACE1 and BACE2 did not cleave several other membrane proteins, demonstrating that both proteases do not contribute to general membrane protein turnover but only cleave specific proteins. This study reveals a similar proteolytic processing of IL-1R2 and APP and may provide an explanation for the increased IL-1R2 secretion observed in AD. Ectodomain shedding and intramembrane proteolysis of the amyloid precursor protein (APP) by α-, β- and γ-secretase are involved in the pathogenesis of Alzheimer disease (AD). Increased proteolytic processing and secretion of another membrane protein, the interleukin-1 receptor II (IL-1R2), have also been linked to the pathogenesis of AD. IL-1R2 is a decoy receptor that may limit detrimental effects of IL-1 in the brain. At present, the proteolytic processing of IL-1R2 remains little understood. Here we show that IL-1R2 can be proteolytically processed in a manner similar to APP. IL-1R2 expressed in human embryonic kidney 293 cells first undergoes ectodomain shedding in an α-secretase-like manner, resulting in secretion of the IL-1R2 ectodomain and the generation of an IL-1R2 C-terminal fragment. This fragment undergoes further intramembrane proteolysis by γ-secretase, leading to the generation of the soluble intracellular domain of IL-1R2. Intramembrane cleavage of IL-1R2 was abolished by a highly specific inhibitor of γ-secretase and was absent in mouse embryonic fibroblasts deficient in γ-secretase activity. Surprisingly, the β-secretase BACE1 and its homolog BACE2 increased IL-1R2 secretion resulting in C-terminal fragments nearly identical to the ones generated by the α-secretase-like cleavage. This suggests that both proteases may act as alternative α-secretase-like proteases. Importantly, BACE1 and BACE2 did not cleave several other membrane proteins, demonstrating that both proteases do not contribute to general membrane protein turnover but only cleave specific proteins. This study reveals a similar proteolytic processing of IL-1R2 and APP and may provide an explanation for the increased IL-1R2 secretion observed in AD. Regulated intramembrane proteolysis (RIP) 2The abbreviations used are: RIP, regulated intramembrane proteolysis; APP, amyloid precursor protein; AD, Alzheimer disease; IL-1R2, interleukin-1 receptor II; ADAM, a disintegrin and metalloprotease; CTF, C-terminal fragment; sCTF, short CTF; lCTF, long CTF; BACE, β-site APP-cleaving enzyme; HA, hemagglutinin; PBS, phosphate-buffered saline; NTF, N-terminal fragment; TGF, transforming growth factor; TNF, tumor necrosis factor; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; PMA, phorbol 12-myristate 13-acetate; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid; SFV, Semliki Forest virus; GPI, glycosylphosphatidylinositol; AP, alkaline phosphatase; ICD, intracellular domain; IL-1, interleukin-1; PS1, presenilin 1.2The abbreviations used are: RIP, regulated intramembrane proteolysis; APP, amyloid precursor protein; AD, Alzheimer disease; IL-1R2, interleukin-1 receptor II; ADAM, a disintegrin and metalloprotease; CTF, C-terminal fragment; sCTF, short CTF; lCTF, long CTF; BACE, β-site APP-cleaving enzyme; HA, hemagglutinin; PBS, phosphate-buffered saline; NTF, N-terminal fragment; TGF, transforming growth factor; TNF, tumor necrosis factor; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; PMA, phorbol 12-myristate 13-acetate; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid; SFV, Semliki Forest virus; GPI, glycosylphosphatidylinositol; AP, alkaline phosphatase; ICD, intracellular domain; IL-1, interleukin-1; PS1, presenilin 1. is a two-step proteolytic cleavage mechanism required for signal transduction and the degradation of membrane proteins (reviewed in Refs. 1Brown M.S. Ye J. Rawson R.B. Goldstein J.L. Cell. 2000; 100: 391-398Abstract Full Text Full Text PDF PubMed Scopus (1141) Google Scholar, 2Kopan R. Ilagan M.X. Nat. Rev. Mol. Cell Biol. 2004; 5: 499-504Crossref PubMed Scopus (491) Google Scholar). The first cleavage is referred to as ectodomain shedding and occurs within the ectodomain at a peptide bond very close to the transmembrane domain. This results in the secretion of most of the ectodomain (reviewed in Refs. 3Blobel C.P. Nat. Rev. Mol. Cell Biol. 2005; 6: 32-43Crossref PubMed Scopus (909) Google Scholar, 4Huovila A.P. Turner A.J. Pelto-Huikko M. Karkkainen I. Ortiz R.M. Trends Biochem. Sci. 2005; 30: 413-422Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar) and the generation of a membrane-bound stub, which can undergo a second cleavage within its transmembrane domain, called intramembrane proteolysis (reviewed in Ref. 5Weihofen A. Martoglio B. Trends Cell Biol. 2003; 13: 71-78Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Numerous type I membrane proteins, including Notch, CD44, and the amyloid precursor protein (APP), undergo RIP. The ectodomain shedding is carried out by members of the ADAM (a disintegrin and metalloprotease) family and additionally by matrix metalloproteases and to a lower extent by the aspartyl proteases BACE1 and BACE2 (β-site APP-cleaving enzymes 1 and 2) (for a review see Ref. 6Blobel C.P. Curr. Opin. Cell Biol. 2000; 12: 606-612Crossref PubMed Scopus (224) Google Scholar). The subsequent intramembrane proteolysis is catalyzed by the γ-secretase protease complex, consisting of the essential proteins PS1 and PS2 (presenilin 1 or 2), nicastrin, Pen-2, and Aph-1 (for a review see Ref. 7Haass C. EMBO J. 2004; 23: 483-488Crossref PubMed Scopus (479) Google Scholar). The presenilins are assumed to constitute the active site of γ-secretase by providing two aspartic acid residues that are critical for γ-secretase activity (8Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1676) Google Scholar). Nicastrin functions as a receptor for γ-secretase substrates (9Shah S. Lee S.F. Tabuchi K. Hao Y.H. Yu C. LaPlant Q. Ball H. Dann III, C.E. Sudhof T. Yu G. Cell. 2005; 122: 435-447Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). As a result of γ-secretase cleavage, the C-terminal fragments (CTFs) of type I membrane proteins are processed to two smaller fragments. A small peptide is secreted into the extracellular space, whereas the intracellular domain is released into the cytosol. For some of these proteins, such as the cell surface receptor Notch and the cell adhesion proteins N- and E-cadherin, the liberated intracellular domain may participate in signal transduction through different mechanisms (2Kopan R. Ilagan M.X. Nat. Rev. Mol. Cell Biol. 2004; 5: 499-504Crossref PubMed Scopus (491) Google Scholar), whereas for other proteins, such as APP, the intracellular domain may be degraded without a prior role in signaling (10Hebert S.S. Serneels L. Tolia A. Craessaerts K. Derks C. Filippov M.A. Muller U. De Strooper B. EMBO Rep. 2006; 7: 739-745Crossref PubMed Scopus (167) Google Scholar).One of the proteins, for which RIP has been studied in much detail, is APP. In contrast to several other proteins undergoing RIP, the ectodomain cleavage of APP is not only catalyzed by one but by three different proteases, which cleave at distinct peptide bonds. Shedding of APP mainly occurs by an ADAM metalloprotease, which is also referred to as α-secretase and cleaves within the Aβ sequence (reviewed in Ref. 11Allinson T.M. Parkin E.T. Turner A.J. Hooper N.M. J. Neurosci. Res. 2003; 74: 342-352Crossref PubMed Scopus (375) Google Scholar). Additionally, BACE1 (also referred to as β-secretase) and its homolog BACE2 cleave in the ectodomain of APP. BACE1 cleaves APP at the N terminus of the Aβ peptide domain, thus catalyzing the first step in the generation of the Aβ peptide (reviewed in Ref. 12Citron M. Trends Pharmacol. Sci. 2004; 25: 92-97Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), which is deposited in the brain of patients suffering from Alzheimer disease (AD) (reviewed in Ref. 13Selkoe D.J. Schenk D. Annu. Rev. Pharmacol. Toxicol. 2003; 43: 545-584Crossref PubMed Scopus (737) Google Scholar). BACE2 cleaves APP close to the α-secretase cleavage site within the Aβ domain and thus acts as an alternative α-secretase (14Yan R. Munzner J.B. Shuck M.E. Bienkowski M.J. J. Biol. Chem. 2001; 276: 34019-34027Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), preventing the release of an intact Aβ peptide.An additional substrate for ectodomain shedding is the type II receptor for interleukin-1 (IL-1), which is widely expressed, including neurons (15French R.A. VanHoy R.W. Chizzonite R. Zachary J.F. Dantzer R. Parnet P. Bluthe R.M. Kelley K.W. J. Neuroimmunol. 1999; 93: 194-202Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). IL-1 is a potent inflammatory and immunoregulatory cytokine and is a key factor in the events leading to neurodegeneration following brain trauma, stroke, and brain inflammation. IL-1 can bind to two types of receptors (for an overview see Ref. 16Mantovani A. Locati M. Vecchi A. Sozzani S. Allavena P. Trends Immunol. 2001; 22: 328-336Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). Binding to the type I receptor (IL-1R1) initiates a signaling cascade that finally leads to NFκB activation. In contrast, the type II receptor (IL-1R2) acts as a decoy receptor, which is capable of binding IL-1 but is incapable of signaling to NFκB and thus acts as a “ligand sink” preventing IL-1 from binding to IL-1R1. IL-1R2 is a single-span membrane protein that contains a large extracellular ligand-binding domain, followed by a transmembrane domain and a short cytoplasmic domain of 29 amino acids. This cytoplasmic domain lacks the Toll/IL-1R domain found in the type I receptor, which would be required for the signal transduction by binding and recruiting cytosolic adaptors and kinases, such as MyD88 and IRAK. IL-1 and its signaling have been implicated in multiple ways in AD (reviewed in Ref. 17Griffin W.S. Nicoll J.A. Grimaldi L.M. Sheng J.G. Mrak R.E. Exp. Gerontol. 2000; 35: 481-487Crossref PubMed Scopus (64) Google Scholar), but the underlying molecular mechanisms are not yet well understood. For example, brain trauma, which is a risk factor for AD, enhances IL-1 expression. Likewise, increased IL-1 expression has been observed in AD (reviewed in Ref. 18Mrak R.E. Griffin W.S. Neurobiol. Aging. 2001; 22: 903-908Crossref PubMed Scopus (300) Google Scholar). In turn, overnight stimulation of astrocytes with IL-1 strongly stimulates translation of APP mRNA, leading to increased APP protein levels (19Rogers J.T. Leiter L.M. McPhee J. Cahill C.M. Zhan S.S. Potter H. Nilsson L.N. J. Biol. Chem. 1999; 274: 6421-6431Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). Potentially, this may result in increased Aβ peptide generation. However, short term treatment of neuroglioma cells with IL-1 has also been shown to stimulate α-secretase cleavage of APP (20Ma G. Chen S. Wang X. Ba M. Yang H. Lu G. J. Neurosci. Res. 2005; 80: 683-692Crossref PubMed Scopus (23) Google Scholar), which may prevent Aβ generation and increase the secretion of a soluble APP form that is neuroprotective and neurotrophic (reviewed in Ref. 21Vardy E.R. Catto A.J. Hooper N.M. Trends Mol. Med. 2005; 11: 464-472Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Genetic analyses have linked polymorphisms in the two IL-1 genes, IL-1α and IL-1β, to an increased risk of AD. Interestingly, both polymorphisms appear to increase IL-1 production in vitro or in vivo (reviewed in Ref. 18Mrak R.E. Griffin W.S. Neurobiol. Aging. 2001; 22: 903-908Crossref PubMed Scopus (300) Google Scholar). An additional connection between IL-1 and AD is the finding that the soluble, secreted form of IL-1R2 is elevated in cerebrospinal fluid of patients with mild to moderate AD but not in the late stages of the disease (22Lindberg C. Chromek M. Ahrengart L. Brauner A. Schultzberg M. Garlind A. Neurochem. Int. 2005; 46: 551-557Crossref PubMed Scopus (47) Google Scholar, 23Garlind A. Brauner A. Hojeberg B. Basun H. Schultzberg M. Brain Res. 1999; 826: 112-116Crossref PubMed Scopus (75) Google Scholar). Given that secreted IL-1R2 binds IL-1 and acts as a sink for IL-1, the increase in IL-1R2 shedding may be a way of trying to limit detrimental consequences of increased IL-1 expression and activity in the brain. However, the molecular mechanisms governing the proteolytic processing of IL-1R2 are little understood, but the processing seems to depend at least partly on a metalloprotease activity (24Cui X. Rouhani F.N. Hawari F. Levine S.J. J. Immunol. 2003; 171: 6814-6819Crossref PubMed Scopus (153) Google Scholar, 25Orlando S. Sironi M. Bianchi G. Drummond A.H. Boraschi D. Yabes D. Mantovani A. J. Biol. Chem. 1997; 272: 31764-31769Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), which may be the ADAM protease TNF-converting enzyme (ADAM17) (26Reddy P. Slack J.L. Davis R. Cerretti D.P. Kozlosky C.J. Blanton R.A. Shows D. Peschon J.J. Black R.A. J. Biol. Chem. 2000; 275: 14608-14614Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). A more detailed knowledge of IL-1R2 processing may help to better understand the role of IL-1R2 secretion and of IL-1 in the AD brain. Here we show that IL-1R2 undergoes a similar set of proteolytic cleavages as APP.EXPERIMENTAL PROCEDURESReagents—The following antibodies were used: anti-HA epitope antibody HA.11 (Covance); anti-FLAGM2 antibody, anti-FLAGM2-agarose, anti-HA-9658 antibody, anti-BACE1-NT antibody, and HA-agarose (Sigma); horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit secondary antibody (DAKO); Alexa 488-coupled anti-rabbit and Alexa 594 anti-mouse antibodies (Molecular Probes); anti-BACE1 and BACE2 (ProSci, against their C termini). Antibodies 6687 (against APP C terminus) (27Steiner H. Kostka M. Romig H. Basset G. Pesold B. Hardy J. Capell A. Meyn L. Grim M.L. Baumeister R. Fechteler K. Haass C. Nat. Cell Biol. 2000; 2: 848-851Crossref PubMed Scopus (250) Google Scholar) and 3027 (against PS1 loop) (28Walter J. Grunberg J. Capell A. Pesold B. Schindzielorz A. Citron M. Mendla K. George-Hyslop P.S. Multhaup G. Selkoe D.J. Haass C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5349-5354Crossref PubMed Scopus (101) Google Scholar) were described previously. Antibody 22C11 (anti APP ectodomain) was provided by Konrad Beyreuther. Nicastrin antibody N1660 (Sigma), monoclonal presenilin NTF (29Capell A. Saffrich R. Olivo J.C. Meyn L. Walter J. Grunberg J. Mathews P. Nixon R. Dotti C. Haass C. J. Neurochem. 1997; 69: 2432-2440Crossref PubMed Scopus (74) Google Scholar), Pen-2 1638 (30Steiner H. Winkler E. Edbauer D. Prokop S. Basset G. Yamasaki A. Kostka M. Haass C. J. Biol. Chem. 2002; 277: 39062-39065Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar), and Aph-1 433G (31Prokop S. Shirotani K. Edbauer D. Haass C. Steiner H. J. Biol. Chem. 2004; 279: 23255-23261Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) antibodies were described before. PMA was obtained from Sigma. The metalloprotease inhibitor TAPI-1 was purchased from Peptides International. Dodecyl maltoside was purchased from Calbiochem. CHAPSO was purchased from Biomol, and the BACE inhibitor C3 was from Calbiochem (β-secretase inhibitor IV). BACE2-Fc fusion protein was kindly provided by Regina Fluhrer.Plasmid Construction—Generation of vector peak12 expressing BACE1, BACE2, and HA-tagged alkaline phosphatase (AP) (HA-AP, soluble AP) has been described (32Lichtenthaler S.F. Dominguez D.I. Westmeyer G.G. Reiss K. Haass C. Saftig P. De Strooper B. Seed B. J. Biol. Chem. 2003; 278: 48713-48719Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). The cDNAs of all shedding substrates are of human origin. Plasmid peak12/PSGL-1 3tag was described previously (32Lichtenthaler S.F. Dominguez D.I. Westmeyer G.G. Reiss K. Haass C. Saftig P. De Strooper B. Seed B. J. Biol. Chem. 2003; 278: 48713-48719Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar) and encodes the signal peptide of CD5, followed by an HA epitope tag and the coding sequence of PSGL-1 with an AU1 tag in the extracellular domain and a FLAG tag in the cytoplasmic domain. The PSGL-1 sequence can be cut out using an XbaI and a NotI restriction site. Peak12/HA-Xba-FLAG was generated by PCR and encodes an HA tag between the HindIII and the XbaI site and a FLAG tag between the XbaI and the NotI site. To obtain peak12/CD5-HA-Xba-FLAG, the HindIII/XbaI fragment of peak12/PSGL-1 3tag was cloned into peak12/HA-Xba-FLAG and encodes the CD5 signal peptide followed by an HA and a FLAG tag. The plasmids encoding full-length IL-1R2 or its deletion mutant (lacking part of the ectodomain) (peak12/IL-1R2, peak12/Δ334-IL-1R2 and peak12/Δ322-IL-1R2) carry the CD5 signal peptide (MPMGSLQPLATLYLLGMLVASVLG), an N-terminal HA tag (YPYDVPDYA followed by the linker sequence SGGGGGLE or SGGGGGLD for the Δ334 mutant), and a C-terminal FLAG tag and were generated by PCR using appropriate primers. Peak12/Δ329-IL-1R2 was generated in the same way but has no N-terminal HA tag. The PCR fragments (lacking the native signal peptide sequence of IL-1R2) were cloned into the XbaI site of peak12/CD5-HA-Xba-FLAG. Thus, the first amino acid of the IL-1R2 sequence is amino acid Gly-23 of full-length IL-1R2 (numbering corresponding to protein accession number NP_004624 in the NCBI data base). Amino acid numbers of the IL-1R2 deletion mutants (Δ322, Δ329, and Δ334) indicate that the deletions stop before the given amino acid number, which refers to its position within the HA-IL-1R2-FLAG full-length sequence (counting without the CD5 signal peptide). Vector peak12/MMP-IL-1R2 was used for retroviral generation and was obtained by cloning the Hin-dIII/NotI fragment of peak12/IL-1R2 into the HindIII/NotI sites of the peak12/MMP-KilA vector (33Randow F. Seed B. Nat. Cell Biol. 2001; 3: 891-896Crossref PubMed Scopus (289) Google Scholar). An additional IRES-GFP cassette from peak12/MMP-TK-IRES-GFP (obtained from Felix Randow; IRES is of encephalomyocarditis virus origin) was cloned into the NotI site of peak12/MMP-IL-1R2 to yield peak12/MMP-IL-1R2-IRES-GFP. Vectors pMDtet.G and pMD.gagpol were described previously (34Ory D.S. Neugeboren B.A. Mulligan R.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11400-11406Crossref PubMed Scopus (796) Google Scholar). The coding sequences of CD14 (GPI-anchored protein), CD16 (GPI-anchored protein), and P-selectin (all three lacking the native signal peptide sequence) were amplified by PCR, digested with XbaI and NotI, and cloned into plasmid peak12/PSGL-1 3tag, which was cut with XbaI and NotI. The resulting plasmids encode the CD5 signal peptide followed by an HA epitope tag and the corresponding protein sequence. As templates, pCDM12/CD14 and CD16 (obtained from Brian Seed) and pCMV/P-selectin (obtained from Denisa Wagner) were used. Peak12/pro-TGFα-HA encodes human pro-TGFα with an HA tag inserted between amino acids His-43 and Phe-44, which is four amino acids C-terminal to the Ala-Val propeptide cleavage site. Thus, after signal peptide and propeptide cleavage, the mature pro-TGFα and the soluble TGFα retain the HA tag. Peak12/HA-pro-TGFα-FLAG contains an additional FLAG tag at the C terminus of pro-TGFα and was generated by cloning the HindIII/XbaI-digested PCR fragment of HA-tagged pro-TGFα into the HindIII and XbaI sites of peak12/CD5-HA-XBA-FLAG. TGFα and TNFα cDNAs were from ATCC. Peak12/FLAG-TNFα-HA was created by cloning a PCR fragment of FLAG-TNFα containing a 5′-HindIII site and a 3′-XbaI site that was then inserted into HindIII/XbaI peak vector, which has an HA tag between the XbaI and the NotI site. The N-terminal, cytoplasmic FLAG tag was added to TNFα by PCR and suitable primers and was cloned into the HindIII/NotI sites of the peak12 vector, resulting in peak12/FLAG-TNFα. For expression in neurons and glial cells IL-1R2, TGFα and CD16 were cut out from the corresponding peak12 plasmids using HindIII and NotI, blunt-ended, and ligated into the SmaI site of the Semliki Forest virus (SFV) type 1. The identity of all constructs obtained by PCR was confirmed by DNA sequencing.Cell Culture, Transfections, Western Blot—Human embryonic kidney 293-EBNA cells (HEK293) were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum (Hyclone) as described (35Neumann S. Schobel S. Jager S. Trautwein A. Haass C. Pietrzik C.U. Lichtenthaler S.F. J. Biol. Chem. 2006; 281: 7583-7594Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Cells stably expressing wild-type PS1 or its catalytically inactive mutant PS1 D385N are HEK293 cells without the EBNA gene (36Capell A. Steiner H. Romig H. Keck S. Baader M. Grim M.G. Baumeister R. Haass C. Nat. Cell Biol. 2000; 2: 205-211Crossref PubMed Scopus (138) Google Scholar). These cells were cultured as above with an additional 200 μg/ml Zeocin (Invitrogen). G418 was added to culture murine embryonic fibroblasts PS1/2-/- knock-out cells. Transfections were carried out using Lipofectamine 2000 (Invitrogen). One day after transfection, the medium was replaced with fresh medium. After overnight incubation, conditioned medium and cell lysate (in 50 mm Tris, pH 7.5, 150 mm NaCl, 1% Nonidet P-40) were collected.To analyze the effect of PMA and TAPI-1 on shedding, cells were treated as described previously (32Lichtenthaler S.F. Dominguez D.I. Westmeyer G.G. Reiss K. Haass C. Saftig P. De Strooper B. Seed B. J. Biol. Chem. 2003; 278: 48713-48719Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). For the detection of secreted and cellular APP by immunoblot, the protein concentration in the cell lysate was measured, and corresponding aliquots of lysate or conditioned medium were directly loaded onto an electrophoresis gel. For transient transfections of IL-1R2, TGFα, CD14, and CD16 either together with BACE1 or BACE2, alkaline phosphatase (AP; plasmid HA-AP) was cotransfected as a transfection efficiency control. AP activity was measured as described previously (32Lichtenthaler S.F. Dominguez D.I. Westmeyer G.G. Reiss K. Haass C. Saftig P. De Strooper B. Seed B. J. Biol. Chem. 2003; 278: 48713-48719Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 37Schobel S. Neumann S. Seed B. Lichtenthaler S.F. Int. J. Dev. Neurosci. 2006; 24: 141-148Crossref PubMed Scopus (23) Google Scholar). Aliquots of the conditioned medium were treated for 30 min at 65 °C to heat-inactivate the endogenous alkaline phosphatase activity. Corresponding aliquots of lysate or conditioned medium were loaded onto the gel. Immunoblot detection was carried out using the indicated antibodies.Infection of Primary Neurons and Glial Cells with SFV—Cortical neurons and glial cells were prepared from E14 mouse embryos from BACE1-deficient and BACE1, BACE2 double-deficient mice as described (38De Strooper B. Saftig P. Craessaerts K. Vanderstichele H. Guhde G. Annaert W. Von Figura K. Van Leuven F. Nature. 1998; 391: 387-390Crossref PubMed Scopus (1541) Google Scholar, 39Dominguez D. Tournoy J. Hartmann D. Huth T. Cryns K. Deforce S. Serneels L. Camacho I.E. Marjaux E. Craessaerts K. Roebroek A.J. Schwake M. D'Hooge R. Bach P. Kalinke U. Moechars D. Alzheimer C. Reiss K. Saftig P. De Strooper B. J. Biol. Chem. 2005; 280: 30797-30806Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). The BACE knock-outs were verified by Northern and Western blot detection and by functional analysis demonstrating that APP cleavage by BACE was virtually eliminated in the neurons (39Dominguez D. Tournoy J. Hartmann D. Huth T. Cryns K. Deforce S. Serneels L. Camacho I.E. Marjaux E. Craessaerts K. Roebroek A.J. Schwake M. D'Hooge R. Bach P. Kalinke U. Moechars D. Alzheimer C. Reiss K. Saftig P. De Strooper B. J. Biol. Chem. 2005; 280: 30797-30806Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar).Preparation of recombinant SFV stocks has been described previously (40De Strooper B. Simons M. Multhaup G. Van Leuven F. Beyreuther K. Dotti C.G. EMBO J. 1995; 14: 4932-4938Crossref PubMed Scopus (161) Google Scholar). Virus was diluted 1:100 in conditioned culture medium and added to 4-day-old neurons. Two hours post-infection, cells were labeled with 100 μCi/ml [35S]methionine/cysteine for 6 h and lysed in immunoprecipitation buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS in TBS buffer). IL-1R2, TGFα, and CD16 full-length and CTFs were immunoprecipitated using anti-FLAG antibody. Immunoprecipitated material was separated by SDS-PAGE, and dried gels were exposed to a PhosphorImager (Amersham Biosciences).Retroviral Transduction—To produce retroviral supernatants (replication-deficient Moloney murine leukemia virus), plasmids pMDtet.G and pMD.gagpol and either peak12/MMP-GFP or peak12/MMP-HA-IL-1R2-FLAG-IRES-GFP were transfected into HEK293 cells by calcium phosphate precipitation. Medium was changed after 24 h, collected after 48 h, and filtered through a sterile filter. The retroviral transductions were carried out using Polybrene (Sigma).In Vitro Generation of IL-1R2-ICD—293E cells transiently expressing full-length IL-1R2 were incubated for 4 h with PMA prior to membrane preparation. Membrane preparations were generated as described (41Sastre M. Steiner H. Fuchs K. Capell A. Multhaup G. Condron M.M. Teplow D.B. Haass C. EMBO Rep. 2001; 2: 835-841Crossref PubMed Scopus (424) Google Scholar) and were resuspended in citrate buffer (pH 6.4, 5 mm EDTA, inhibitor mixture from Roche Applied Science) with or without 1 μm DAPT and then incubated either at 4 or 37 °C for 2 h. After incubation the supernatant and membranes were separated via ultracentrifugation. HEK293 cells (without EBNA) stably expressing wild-type PS1 or PS1 D385N and additionally transiently expressing peak12/Δ329-IL-1R2-FLAG were directly subjected to membrane preparation without prior PMA incubation and then treated as above.Coimmunoprecipitation of γ-Secretase Complex with IL-1R2 Substrate—HA-IL-1R2-FLAG was retrovirally transduced into HEK293 cells stably expressing either PS1 WT or the catalytic inactive mutant PS1 D385N. For immunoprecipitation, one 10-cm dish of each cell line was lysed in standard STE (150 mm NaCl, 50 mm Tris, 2 mm EDTA) + 1% CHAPSO buffer followed by a clarifying spin at 13,000 rpm with a Heraeus cryocentrifuge and a further purification step at 55,000 rpm in a Beckman ultracentrifuge with a TLA-55 rotor. Prior to immunoprecipitation of CTFs with FLAG M2 affinity agarose, the lysates were immunoprecipitated with HA affinity agarose for 2 h leading to a depletion of IL-1R2 full-length protein in the lysate. Subsequently, after a 2-h incubation with the lysate, FLAG M2-agarose was washed two times each with 0.1% CHAPSO wash buffer and STE buffer and afterward eluted with 100 μg of FLAG peptide and subjected to SDS-PAGE. Immunoblot detection was carried out for IL-1R2 CTF and the γ-secretase complex components nicastrin, presenilin 1 NTF, Aph-1, and Pen-2.Mass Spectrometry of IL-1R2 Cleavage Sites—For analysis of α-, β-, and γ-cleavage of secreted IL-1R2 peptides, HEK293 cells were transfected with peak12/IL-1R2, peak12/Δ334-IL-1R2, or peak12/Δ322-IL-1R2. 48 h after transfection, fresh medium was incubated for 4 h and subsequently put on ice. In case of peak12-Δ322-IL-1R2, medium was supplemented with 1 mmol of DAPT during incubation to prevent turnover by γ-secretase. Protease inhibitor mixture (Sigma) was added at a dilution of 1:100. Medium was then subjected to a clarifying spin by centrifugation. Afterward medium was subjected to immunoprecipitation with HA-agarose beads for 4 h in the case of Δ334-IL1R2 construct and for 2 h in the case of Δ322-IL1R2 construct. Bound peptides were eluted either with a mixture of 0.3% trifluoroacetic acid, 50% acetonitrile, H2O saturated with α-cyano matrix, or in the case of the IL-1R2 ectodomain with HA peptide (50 μg) in 300 mm NaCl for MALDI-TOF analysis (Voyager DESTR, Applied Biosystems) and with 0.1% formic acid, 50% methanol, H2O for nanoelectrospray ionization mass spectrometry analysis (Q-STAR Applied Biosystems). MALDI-TOF spectra were recorded in the linear mode and analyzed with Data Explorer™ (Applied Biosyste" @default.
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• W2000392076 date "2007-04-01" @default.
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• W2000392076 title "Regulated Intramembrane Proteolysis of the Interleukin-1 Receptor II by α-, β-, and γ-Secretase" @default.
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