Abstract
The regulated secretory pathway of neurons is the major source of extracellular Aβ that accumulates in Alzheimer's disease (AD). Extracellular Aβ secreted from that pathway is generated by β-secretase processing of amyloid precursor protein (APP). Previously, cysteine protease activity was demonstrated as the major β-secretase activity in regulated secretory vesicles of neuronal chromaffin cells. In this study, the representative cysteine protease activity in these secretory vesicles was purified and identified as cathepsin B by peptide sequencing. Immunoelectron microscopy demonstrated colocalization of cathepsin B with Aβ in these vesicles. The selective cathepsin B inhibitor, CA074, blocked the conversion of endogenous APP to Aβ in isolated regulated secretory vesicles. In chromaffin cells, CA074Me (a cell permeable form of CA074) reduced by about 50% the extracellular Aβ released by the regulated secretory pathway, but CA074Me had no effect on Aβ released by the constitutive pathway. Furthermore, CA074Me inhibited processing of APP into the COOH-terminal β-secretase-like cleavage product. These results provide evidence for cathepsin B as a candidate β-secretase in regulated secretory vesicles of neuronal chromaffin cells. These findings implicate cathepsin B as β-secretase in the regulated secretory pathway of brain neurons, suggesting that inhibitors of cathepsin B may be considered as therapeutic agents to reduce Aβ in AD.
References
Barrett, A.J., Rawlings, N.D., Woessner, J.R. (1998). Handbook of Proteolytic Enzymes (San Diego, USA: Academic Press), pp. 609–617.Search in Google Scholar
Benjannet, S., Savaria, D., Laslop, A., Munzer, J.S., Chretien, M., Marcinkiewicz, M., Seidah, N.G. (1997). α1-Antitrypsin Portland inhibits processing of precursors mediated by proprotein convertases primarily within the constitutive secretory pathway. J. Biol. Chem. 272 , 26210 –26218.10.1074/jbc.272.42.26210Search in Google Scholar
Bernstein, H.G., Kirschke, H., Wiederanders, B., Schmidt, D., Rinne, A. (1990). Antigenic expression of cathepsin B in aged human brain. Brain Res. Bull. 24 , 543 –549.10.1016/0361-9230(90)90157-USearch in Google Scholar
Brion, C., Miller, S.G., and Moore, H.P. (1982). Regulated and constitutive secretion, differential effects of protein synthesis arrest on transport of glycosaminoglycan chains to the two secretory pathways. J. Biol. Chem. 267 , 1477 –1483.Search in Google Scholar
Cai, H., Wang, Y., McCarthy, D., Wen, H., Corchelt, D.R., Price, D.L., and Wong, P.C. (2001). BACE 1 is the major β-secretase for generation of Aβ by neurons. Nat. Neurosci. 4 , 233 –234.10.1038/85064Search in Google Scholar
Cataldo, A.M. and Nixon, R.A. (1990). Enzymatically active lysosomal proteases are associated with amyloid deposits in Alzheimer brain. Proc. Natl. Acad. Sci. USA 87 , 3861 –3865.10.1073/pnas.87.10.3861Search in Google Scholar
Cataldo, A.M., Paskevich, P.A., Kominami, E., and Nixon, R.A. (1991). Lysosomal hydrolases of different classes are abnormally distributed in brains of patients with Alzheimer's disease. Proc. Natl. Acad. Sci. USA 88 , 10998 –11002.10.1073/pnas.88.24.10998Search in Google Scholar
Citron, M., Oltersdorf, T., Haass, C., McConlogue, L., Hung, A.Y., Seubert, P., Vigo-Pelfrey, C., Lieverburg, I., and Selkoe, D.J. (1992). Mutation of the β-amyloid precursor protein in familial Alzheimer's disease increases β-protein production. Nature 360 , 672 –674.10.1038/360672a0Search in Google Scholar
Efthimiopoulos, S., Vassilacopoulou, D., Rippellino, J.A., Tezap-sidis, N., and Robakis, N.K. (1996). Cholinergic agonists stimulate secretion of soluble full-length amyloid precursor protein in neuroendocrine cells. Proc. Natl. Acad. Sci. USA 93 , 8046 –8050.10.1073/pnas.93.15.8046Search in Google Scholar
Gensberg, K., Jan, S., and Matthews, G.M. (1998). Subtilisin-related serine proteases in the mammalian constitutive secretory pathway. Semin. Cell Dev. Biol. 9 , 11 –17.10.1006/scdb.1997.0196Search in Google Scholar
Greenbaum, D., Medzihradszky, I.K.F., Burlingame, A., and Bogyo, M. (2000). Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools. Chem. Biol. 7 , 569 –581.10.1016/S1074-5521(00)00014-4Search in Google Scholar
Greenbaum, D., Baruch, A., Hayrapetian, L., Darula, Z., Burlingame, A., Medzihradszky, K.F., and Bogyo, M. (2002a). Chemical approaches for functionally probing the proteome. Mol. Cell. Proteomics 1 , 60 –68.10.1074/mcp.T100003-MCP200Search in Google Scholar PubMed
Greenbaum, D.C., Arnold, W.D., Lu, F., Hayrapetian, L., Baruch, A., Krumrine, J., Toba, S., Chehade, K., Bromme, D., Kuntz, I.D., and Bogyo, M. (2002b). Small molecule affinity fingerprinting. A tool for enzyme family sub classification, target identification, and inhibitor design. Chem. Biol. 9 , 1085 –1094.10.1016/S1074-5521(02)00238-7Search in Google Scholar
Gumbiner, G. and Kelly, R.B. (1982). Two distinct intracellular pathways transport secretory and membrane glycoproteins to the surface of pituitary tumor cells. Cell 28 , 51 –55.10.1016/0092-8674(82)90374-9Search in Google Scholar
Hill, P.A., Buttle, D.J., Jones, S.J., Boyde, A., Murata, M., Reynolds, J.J., and Meikle, M.C. (1994). Inhibition of bone resorption by selective inactivators of cysteine proteinases. J. Cell Biochem. 56 , 118 –130.10.1002/jcb.240560116Search in Google Scholar PubMed
Hook, V.Y.H. and Reisine, T.D. (2003). Cysteine proteases are the major β-secretase in the regulated secretory pathway that provides most of the β-amyloid in Alzheimer's disease: role of BACE 1 in the constitutive secretory pathway. J. Neurosci. Res. 74 , 393 –405.10.1002/jnr.10784Search in Google Scholar PubMed
Hook, V.Y.H., Toneff, T., Aaron, W., Yasothorsnrikul, S., Bundey, R., and Reisine, T. (2002). β-Amyloid peptide in regulated secretory vesicles of chromaffin cells: evidence for multiple cysteine proteolytic activities in distinct pathways for β-secretase activity in chromaffin vesicles. J. Neurochem. 81 , 237 –256.10.1046/j.1471-4159.2002.00794.xSearch in Google Scholar PubMed
Hook, V., Yasothornsrikul, S., Greenbaum, D., Medzihradszky, K.F., Troutner, K., Toneff, T., Bundey, R., Reinheckel, T., Peters, C., and Bogyo, M. (2004). Cathepsin L and Arg/Lys aminopeptidase: a distinct prohormone processing pathway for the biosynthesis of peptide neurotransmitters and hormones. Biol. Chem. 385 , 473 –480.10.1515/BC.2004.055Search in Google Scholar PubMed
Hussain, I., Powell, D., Howlett, D.R., Tew, D.G., Meek, T.D., Chapman, C., et al. (1999). Identification of a novel aspartic protease (Asp 2) as β-secretase. Mol. Cell. Neurosci. 14 , 419 –427.10.1006/mcne.1999.0811Search in Google Scholar PubMed
Iverson, L.L., Mortishire-Smith, R.J., Pollack, S.J., and Shearman, M.S. (1995). The toxicity in vitro of β-amyloid protein. Biochem. J. 311 , 1 –16.10.1042/bj3110001Search in Google Scholar PubMed PubMed Central
Jolly-Tornetta, C., Gao, Z.Y., Lee, V.M., and Wolf, B.A. (1998). Regulation of amyloid precursor protein secretion by glutamate receptors in human Ntera 2 neurons. J. Biol. Chem. 272 , 140015 –140021.10.1074/jbc.273.22.14015Search in Google Scholar PubMed
Lin, X., Koelsch, G., Wu, S., Downs, D., Dashti, A., and Tang, J. (2000). Human aspartic protease memapsin 2 cleaves the β-secretase site of β-amyloid precursor protein. Proc. Natl. Acad. Sci. USA 97 , 1456 –1460.10.1073/pnas.97.4.1456Search in Google Scholar PubMed PubMed Central
Lodish, H., Berk, A., Zipursky, S.L., Matsudaira, P., Baltimore, D., and Darnell, J. (1999). Molecular Cell Biology, 4th ed. (New York, USA: W.H. Freeman), pp. 691–726.Search in Google Scholar
Luo, Y., Bolon, B., Kahn, S., Bennet, B.D., Babu-Khan, S., Denis, P., Fan, W., Kha, H., Ahang, J., Gong, Y., et al. (2001). Mice deficient in BACE 1, the Alzheimer's β-secretase, have normal phenotype and abolished β-amyloid generation. Nat. Neurosci. 4 , 231 –232.10.1038/85059Search in Google Scholar
Nakamura, N.Y., Takeda, M., Suzuki, H., Hattori, H., Tada, K., Hariguchi, S., Hashimoto, S., and Nishimura, T. (1991). Abnormal distribution of cathepsins in the brain of patients with Alzheimer's disease. Neurosci. Lett. 130 , 195 –198.10.1016/0304-3940(91)90395-ASearch in Google Scholar
Nakanishi, H., Tominaga, K., Amano, T., Hirotsu, I., Inoue, T., and Yamamoto, K. (1994). Age-related changes in activities and localizations of cathepsins D, E, B, and L in rat brain tissues. Exp. Neurol. 126 , 119 –128.10.1006/exnr.1994.1048Search in Google Scholar PubMed
Nitsch, R.M., Slack, B.E., Wurtman, R.J., and Growdon, J.H. (1992). Release of Alzheimer's amyloid precursor derivatives by activation of muscarinic acetylcholine receptors. Science 258 , 304 –307.10.1126/science.1411529Search in Google Scholar PubMed
Nitsch, R.J., Farber, S.A., Growdon, J.H., and Wurtman, R.J. (1993). Release of amyloid beta-protein precursor derivatives by electrical depolarization of rat hippocampal slices. Proc. Natl. Acad. Sci. USA 90 , 5191 –5193.10.1073/pnas.90.11.5191Search in Google Scholar PubMed PubMed Central
Roberds, S.L., Anderson, J., Basi, G., Bienkowski, M.J., Branstetter, D.G., Chen, K.S., Freedman, S.B., Frigon, N.L., Games, D., Hu, K., et al. (2001). BACE knockout mice are healthy despite lacking the primary β-secretase activity in the brain: implications for Alzheimer's disease therapeutics. Hum. Mol. Genet. 10 , 1317 –1324.10.1093/hmg/10.12.1317Search in Google Scholar PubMed
Selkoe, D.J. (2001). Alzheimer's disease: genes, proteins, therapy. Physiol. Rev. 81 , 741 –761.10.1152/physrev.2001.81.2.741Search in Google Scholar PubMed
Sinha, S., Anderson, J.P., Barbour, R., Basi, G.S., Caccavello, R., et al. (1999). Purification and cloning of amyloid precursor protein β-secretase from human brain. Nature 402 , 537 –540.10.1038/990114Search in Google Scholar PubMed
Sisodia, S.S. (1999). Alzheimer's disease: perspectives for the new millennium. J. Clin. Invest. 104 , 1169 –1170.10.1172/JCI8508Search in Google Scholar PubMed PubMed Central
Tezapsidis, N., Li, H.G., Rippellino, J.A., Efthimiopoulos, S., Vassilacopoulou, D., Sambamurti, K., Toneff, T., Yasothornsrikul, S., Hook, V.Y.H., and Robakis, N.K. (1998). Release of non-transmembrane, full-length Alzheimer's amyloid precursor protein (APP) from the lumenar surface of chromaffin granule membranes. Biochemistry 37 , 1274 –1282.10.1021/bi9714159Search in Google Scholar PubMed
Thomas, G. (2002). Furin at the cutting edge: from protein traffic to embryogenesis and disease. Nat. Rev. Mol. Cell Biol. 3 , 753 –766.10.1038/nrm934Search in Google Scholar
Vassar, R., Bennet, B.D., Babu-Khan, S., Mendiaz, E.A., Denis, P., Teplow, D.B., Ross, S., Amarante, P., Loeloff, R., Luo, Y., et al. (1999). β-Secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE. Science 286 , 735 –741.10.1126/science.286.5440.735Search in Google Scholar
Vassilacopoulou, D,. Ripellino, J.A., Tezapsidis, N., Hook, V.Y.H., and Robakis, N.K. (1995). Full-length and truncated APP in chromaffin granules: solubilization of granule membrane APP by a proteolytic mechanism. J. Neurochem. 64 , 2140 –2146.Search in Google Scholar
Yamamoto, A., Kaji, T., Tomoo, K., Ishida, T., Inoue, M., Murata, M., and Kitamura, K. (1992). Crystallization and preliminary X-ray study of the cathepsin B complexed with CA074, a selective inhibitor. J. Mol. Biol. 227 , 942 –944.10.1016/0022-2836(92)90234-BSearch in Google Scholar
Yan, R., Bienkowski, M.J., Shuck, M.E., Miao, H., Tory, M.C., Pauley, A.M., Brashier, J.R., Stratment, N.C., Mathews, W.R., Buhl, A.E., et al. (1999). Membrane-anchored aspartyl protease with Alzheimer's disease β-secretase activity. Nature 402 , 533 –537.10.1038/990107Search in Google Scholar
Yasothornsrikul, S., Aaron, W., Toneff, T., and Hook, V.Y.H. (1999). Evidence for the proenkephalin processing enzyme ‘prohormone thiol protease’ (PTP) as a multicatalytic cysteine protease complex; activation by glutathione localized to secretory vesicles. Biochemistry 38 , 7421 –7430.10.1021/bi990239wSearch in Google Scholar
Yasothornsrikul, S., Greenbaum, D., Medzihradszky, K.F., Toneff, T., Bundey, R., Miller, R., Schilling, B., Petermann, I., Dehnert, J., Logvinova, A. et al. (2003). Cathepsin L in secretory vesicles functions as a processing enzyme for production of the enkephalin peptide neurotransmitter. Proc. Natl. Acad. Sci. USA 100 , 9590 –9595.10.1073/pnas.1531542100Search in Google Scholar
Yoshiyama, Y., Arai, K., Oki, T., and Hattori, T. (2000). Expression of invariant chain and pro-cathepsin L in Alzheimer's brain. Neurosci. Lett. 290 , 125 –128.10.1016/S0304-3940(00)01326-4Search in Google Scholar
Zhang, J., Goodlettt, D.R., Quinn, J. Peskind, E., Kaye, J.A., Zhou, Y., Pan, C., Yi, E., Eng, J., Wang, Q., Aebersold, R.H., and Montine, T.J. (2005). Quantitative proteomics of cerebrospinal fluid from patients with Alzheimer disease. J. Alzheimer's Disease 7 , 125 –133.10.3233/JAD-2005-7205Search in Google Scholar PubMed
©2005 by Walter de Gruyter Berlin New York
