Abstract
PSEN1 and PSEN2 encode polytopic membrane proteins, termed presenilin 1 (PS1) and presenilin 2 (PS2) that play an essential role in intramembranous (“γ-secretase”) proteolysis of selected type I membrane proteins, that include Notch1 and β-amyloid precursor protein (APP). In order to gain insights into biochemical mechanisms underlying γ-secretase processing of Notch1 and APP, we have developed a novel in vitro assay in which γ-secretase-mediated generation of S3/NICD and APP-CTFγ can be readily detected in isolated membrane fractions derived from immortalized PS1+/− mouse embryonic fibroblasts; production of the APP and Notch1 derivatives are inhibited by a highly selective and potent γ-secretase inhibitor, L-685,458, with a IC50 of ∼50 pM. In membranes prepared from PS1-deficient fibroblasts, we detected APP-CTFγ, albeit at low levels. Unexpectedly, and despite the presence of endogenous PS2 in membranes prepared from PS1-deficient fibroblasts, production of the Notch derivatives, S3/NICD, was nearly undetectable in these reactions. Moreover, S3/NICD production is neither detected in detergent-solubilized membrane preparations from PS1-deficient cells, nor in reactions containing PS1-containing membranes that were co-solubilized with membranes from PS −/− cells expressing a chimeric Notch 1 species. These findings strongly suggest that the factors responsible for intramembranous, γ-secretase proteolysis of APP and Notch1 are neither equivalent, nor exchangeable.
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Artavanis-Tsakonas S., Rand M. D., and Lake R. J. (1999) Notch signaling: cell fate control and signal integration in development. Science 284, 770–776.
Capell A., Steiner H., Romig H., Keck S., Baader M., Grim M.G., Baumeister R., and Haass, C. (2000) Presenilin-1 differentially facilitates endoproteolysis of the β-amyloid precursor protein and Notch. Nat. Cell Biol. 2, 205–211.
De Strooper B., Saftig P., Craessaerts K., Vanderstichele H., Guhde G., Annaert W., et al. (1998) Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein. Nature 391, 387–390.
De Strooper B., Annaert W., Cupers P., Saftig P., Craessaerts K., Mumm J. S., et al. (1999) A presenilin-1-dependent γ-secretase-like protease mediates release of Notch intracellular domain. Nature 398, 518–522.
Evan G. I., Lewis G. K., Ramsay G., and Bishop J. M. (1985) Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol. Cell. Biol. 5, 3610–3616.
Herreman A., Serneels L., Annaert W., Collen D., Schoonjans L., and De Strooper B. (2000) Total inactivation of γ-secretase activity in presenilin-deficient embryonic stem cells. Nat. Cell Biol. 2, 461–462.
Kim S.-H., Lah J. J., Thinakaran G., Levey A., and Sisodia S. S. (2000) Subcellular localization of presenilin: association with a unique membrane pool in cultured cells. Neurobiol. Dis. 7, 99–117.
Koo E. H. and Squazzo S. L. (1994) Evidence that production and release of amyloid beta-protein involves the endocytic pathway. J. Biol. Chem. 269, 17,386–17,389.
Kulic L., Walter J., Multhaup G., Teplow D. B., Baumeister R., Romig H., et al. (2000) Separation of presenilin function in amyloid β-peptide generation and endoproteolysis of Notch. Proc. Natl Acad. Sci. USA 23, 5913–5918.
Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
Levy-Lahad E., Wasco W., Poorkaj P., Romano D. M., Oshima J., Pettingell W. H., et al. (1995) Candidate gene for the chromosome 1 familial Alzheimer’s disease locus. Science 269, 973–977.
Li 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., et al. (2000a) Photoactivated γ-secretase inhibitors directed to the active site covalently label presenilin 1. Nature 405, 689–694.
Li Y-M., Lai M.-T., Xu M., Huang Q., DiMuzio-Mower J., Sardana M. K., et al. (2000b) Presenilin 1 is linked with γ-secretase activity in the detergent solubilized state. Proc. Natl Acad. Sci. USA 97, 6138–6143.
Lichtenthaler S. F., Wang R., Grimm H., Uljon S. N., Masters C. L., and Beyreuther K. (1999) Mechanism of the cleavage specificity of Alzheimer’s disease γ-secretase identified by phenylalamine-scanning mutagenesis of the transmembrane domain of the amyloid precursor protein. Proc. Natl Acad. Sci. USA 96, 3053–3058.
Lo A. C., Haass C., Wagner S. L., Teplow D. B., and Sisodia S. S. (1994) Metabolism of the “Swedish” amyloid precursor protein variant in Madin-Darby canine kidney cells. J. Biol. Chem. 269, 30,966–30,973.
McLendon C., Xin T. P., Ziani-Cherif C., Murphy M. P., Findlay K. A., Lewis P. A., et al. (2000) Cell-free assays for γ-secretase activity. FASEB J. 14, 2383–2386.
Martys-Zage J. L., Kim S.-H., Berechid B., Bingham S. J., Chu S., Sklar J., et al. (2000) Requirement for presenilin 1 in facilitating Jagged 2-mediated endoproteolysis and signaling of Notch 1. J. Mol. Neuroscience 15, 189–204.
Murphy M. P., Hickman L. J., Eckman C. B., Uljon S. N., Wang R., and Golde T. E. (1999) γ-secretase, evidence for multiple proteolytic activities and influence of membrane positioning of substrate on generation of amyloid β peptides of varying length. J. Biol. Chem. 274, 11,914–11,923.
Naruse S., Thinakaran G., Luo J.J., Kusiak J.W., Tomita T., Iwatsubo T., et al. (1998) Effects of PS1 deficiency on membrane protein trafficking in neurons. Neuron 21, 1213–1221.
Petit A., Bihel F., da Costa C. A., Pourquie O., Checler F., and Kraus J. L. (2001) New protease inhibitors prevent γ-secretase-mediated production of Aβ40/42 without affecting Notch cleavage. Nat. Cell Biol. 3, 507–511.
Pinnix I., Musunuru U., Tun H., Sridharan A., Golde T., Echman C., et al. (2001) A novel γ-secretase assay based on detection of the putative C-terminal fragment-γ of amyloid β protein precursor. J. Biol. Chem. 276, 481–487.
Rand M. D., Grimm L. M., Artavanis-Tsakonas S., Patriub V., Blacklow S. C., Sklar J., and Aster J. C. (2000) Calcium depletion dissociates and activates heterodimeric Notch receptors. Mol. Cell. Biol. 20, 1825–1835.
Rogaev E. I., Sherrington R., Rogaev E. A., Levesque G., Ikeda M., Liang Y., et al. (1995) Familial Alzheimer’s disease in kindreds with missence mutations in a gene on chromosome 1 related to the Alzheimer’s disease type 3 gene. Nature 376, 775–778.
Schroeter E. H., Kisslinger J. A., and Kopan R. (1998) Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain. Nature 393, 382–386.
Selkoe D.J. (2000) Notch and presenilins in vertebrates and invertebrates: implications for neuronal development and degeneration. Curr. Opin. Neurobiol. 10, 50–57.
Shearman M. S., Beher D., Clarke E. E., Lewis H. D., Harrison T., Hunt P., et al. (2000) L-685,458, an aspartyl protease transition state mimic, is a potent inhibitor of amyloid β-protein precursor γ-secretase activity. Biochemistry 39, 8698–8704.
Sherrington R., Rogaev E. I., Liang Y., Rogaeva E. A., Levesque G., Ikeda M., et al. (1995) Cloning of a gene bearing missense mutations in early-onset familial Alzheimer’s disease. Nature 375, 754–760.
Sisodia S. S., Koo E. H., Hoffman P. N., Perry G., and Price D. L. (1993) Identification and transport of full-length amyloid precursor proteins in rat peripheral nervous system. J. Neurosci. 13, 3136–3142.
Smart E. J. Ying Y.-S., Mineo C., and Anderson R. G. W. (1995) A detergent-free method for purifying caveolae membrane from tissue culture cells. Proc. Natl. Acad. Sci. USA 92, 10,104–10,108.
Struhl G. and Adachi A. (1998) Nuclear access and action of Notch in vivo. Cell 93, 649–660.
Struhl G. and Adachi A. (2000) Requirements for presenilin-dependent cleavage of notch and other transmembrane proteins. Mol. Cell 6, 625–636.
Thinakaran G., Harris C. L., Ratovitsky T., Davenport F., Slunt H. H., Price D. L., et al. (1997) Evidence that levels of presenilins (PS1 and PS2) are coordinately regulated by competition for limiting cellular factors. J. Biol. Chem. 272, 28,415–28,422.
Wong P., Zheng H., Chen H., Becher M. W., Sirinathsinghji D. J. S., Trumbauer M. E., et al. (1997) Presenilin 1 is required for Notch1 and Dll1 expression in the paraxial mesoderm. Nature 387, 288–292.
Yu G., Nishimura M., Arawaka S., Levitan D., Zhang L., Tandon A., et al. (2000) Nicastrin modulates presenilin-mediated notch/glp-1 signal transduction and βAPP processing. Nature 407, 48–54.
Zhang Z., Nadeau P., Song W., Donoviel D., Yuan M., Bernstein A., and Yanker B. A. (2000) Presenilins are required for γ-secretase cleavage of β-APP and transmembrane cleavage of Notch1. Nat. Cell Biol. 2, 463–465.
Zheng H., Jiang M.-H., Trumbauer M. E., Sirinathsinghje D. J. S., Hopkins R., Smith D. W., et al. (1995) β-amyloid precursor protein-deficient mice show reactive gliosis and decreased locomotor activity. Cell 81, 525–531.
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Ikeuchi, T., Sisodia, S.S. Cell-free generation of the Notch1 intracellular domain (NICD) and APP-CTFγ. Neuromol Med 1, 43–54 (2002). https://doi.org/10.1385/NMM:1:1:43
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DOI: https://doi.org/10.1385/NMM:1:1:43