摘要
朊病毒疾病是一种致死性中枢神经系统退化性疾病,包括了人的库鲁病、吉-斯综合症(GSS)、致死性家族失眠症(FFI)、克雅氏病(CJD)和变异性克雅氏病(vCJD),以及动物的牛海绵状脑病(BSE)和羊瘙痒症(Scrapie)。错误折叠的朊蛋白在大脑特定区域内沉积,会引起朊病毒病。在所有的朊病毒疾病中,致病的关键因素是细胞内正常PrPc蛋白高级构象变化,转变成致病性的PrPSc。
Calnexin (CNX)是内质网内Ⅰ型膜蛋白,它可以帮助新生N糖基化蛋白的正确折叠以及内质网质量控制。前人的研究表明,凝集素分子伴侣CNX和糖蛋白底物的结合有两种模式,只依赖与凝集素位点的结合模式以及依赖凝集素位点和多肽结合位点的双重结合模式。
在本研究中,我们构建了CNX和PrP的真核和原核表达载体。在随后的试验中,通过非变性凝胶电泳(N-PAGE)和免疫共沉淀(Co-ip)的方法,首次证明了CNX可以和PrP在体内体外相互作用。由于体外大肠杆菌表达出来的PrP是没有糖基化的,因此,实验结果可以推测CNX和PrP的结合是依赖双重结合模式。
Williams等人的体外实验结果表明,CNX可以抑制很多蛋白的热聚集。为了证实CNX能否抑制PrP的热聚集,我们采用了浊度法。浊度实验结果表明CNX能够很有效地抑制PrP的热聚集,当CNX和PrP的摩尔比为2比1的时候,PrP的热聚集基本上被完全抑制了。接下来,原子力显微镜(AFM)、分子筛液相色谱(SEC)和静态光散射等实验也进一步验证了这一结果。
为了进一步弄清CNX能否降低PrP引起的细胞毒性,我们采用了MTT细胞毒性实验。从实验结果可以看出,单独转染PrP的细胞存活率和空白对照相比有明显的下降,但是PrP和CNX共转染的细胞存活率和空白对照相比却几乎没有变化,说明CNX可以抑制PrP引起的细胞毒性。为了进一步了解CNX抑制细胞毒性是不是由于抑制了PrP诱导的细胞凋亡,运用了流式细胞仪测定转染细胞的凋亡,结果证明CNX可以抑制PrP诱导的细胞凋亡,随着CNX的量的增加,凋亡的抑制效果也越来越明显。
对PrP和CNX的相互作用的研究可能可以给朊病毒疾病或其他一些蛋白质构象病诸如老年痴呆症(AD)、帕金森病(Parkinson's disease)和亨廷顿病(Huntington's disease)等由蛋白质错误折叠和聚集引起的疾病提供潜在的治疗手段。
Prion diseases are fatal, neurodegenerative diseases that include scrapie in sheep; bovine spongiform encephalopathy (BSE) in cattle; Kuru, Gerstmann Straussler Scheinker disease (GSS), fatal familial insomnia (FFI), Creutzfeldt-Jakob disease (CJD), and variant Creutzfeldt-Jakob disease (vCJD)in human. Misfolding and aggregation of prion protein deposit on selected regions of the brain can result in prion diseases. A key event in all prion diseases appears to be the supreme structure changes of normal cellular form of prion protein (PrPc) into the infectious scrapie isoform (PrPSc).
Calnexin(CNX) is a type I integral membrane protein in the endoplasmic reticulum (ER) that ensures the proper folding and quality control of newly synthesized N-linked glycoproteins. Previous studies showed us that the lectin chaperone CNX had two mechanisms of association with glycoproteins:lectin-binding only or lectin and polypeptide binding.
In this work, we constructed prokaryotic and eukaryotic expression vectors of calnexin (CNX) and prion protein (PrP). Our findings for the first time confirmed that calnexin interacts with PrP. The immunoprecipitation and native polyacrylamide-gel electrophoresis results indicate that calnexin could bind PrP in vivo and in vitro. The E. coli expressed PrP was non-glycosylated, we guess the interaction of PrP with CNX maybe depend on polypeptide binding site.
Some researchs demonstrated that CNX could suppresses thermal aggregation of non-glycosylated polypeptides in vitro. To assess whether CNX is capable of supressing aggregation of PrP in vitro, the turbidity assay was employed. Consistent with a molecular chaperone function, CNX effectively suppressed the aggregation of PrP in a concentration-dependent manner with maximal suppression occurring at a molar ratio of one CNX molecule to two PrP molecules. We can see at this molar ratio, the aggregation of PrP was almost suppressed. Then the atom force microscopy (AFM), light scattering and size exclusion chromatograph (SEC) assays proved our conclusion further.
To investigate whether CNX could inhibit cytotoxicity induced by PrP, we employed MTT assay. The result showed that the viability of the cell expressed PrP dropped remarkably, while viability of the cells expressed both CNX and PrP kept unchanged compared with that blank control, when the concentration of CNX is the same as PrP, the viability of the cell did not changed significantly. In order to investigate whether CNX could inhibit the cell apoptosis induced by PrP in SK-N-SH cells, we employed flow cytometry analyses. The result showed CNX effectively suppressed the apoptosis induced by PrP in a concentration-dependent manner.
Research on PrP interacts with CNX may have clinical benefits in prion-related diseases and other protein conformation diseases such as Alzheimer's, Parkinson's and Huntington's diseases, and other diseases especially resulted from protein misfolding and accumulation.
引文
参考文献
1. Prusiner, S. B. (1982). Novel proteinaceous infectious particles cause scrapie. Science 216(4542),136-44.
2. Delasnerie-Laupretre, N., Poser, S., Pocchiari, M., Wientjens, D. P., and Will, R. (1995). Creutzfeldt-Jakob disease in Europe. Lancet 346(8979),898.
3. Cuille, J., and Chelle, R. L. (1939) Experimental transmission of trembling to the goat CR. Seances Acard Sci 208,1058-60.
4. Aguzzi, A., and Polymenidou, M. (2004). Mammalian prion biology:one century of evolving concepts. Cell 116(2),313-27.
5. Gajdusek, D. C. (1977). Unconventional viruses and the origin and disappearance of kuru. Science 197(4307),943-60.
6. Gajdusek, D. C., Gibbs, C. J., Asher, D. M., and David, E. (1968) Transmission of experimental Kuru to the spider monkey (Ateles geoffreyi). Science 162(854), 699-94.
7. Griffith, J. S. (1967). Self-replication and scrapie. Nature 215(105),1043-4.
8. Prusiner, S. B. (1988). Molecular structure, biology, and genetics of prions. Adv Virus Res 35,83-136.
9. Bolton, D. C., Meyer, R. K., and Prusiner, S. B. (1985). Scrapie PrP 27-30 is a sialoglycoprotein. J Viro 153(2),596-606.
10. Meyer, R. K., McKinley, M. P., Bowman, K. A., Braunfeld, M. B., Barry, R. A., and Prusiner, S. B. (1986). Separation and properties of cellular and scrapie prion proteins. Proc Natl Acad Sci USA 83(8),2310-4
11. Basler, K., Oesch, B., Scott, M., Westaway, D., Walchli, M., Groth, D. F., McKinley, M. P., Prusiner, S. B., and Weissmann, C. (1986). Scrapie and cellular
PrP isoforms are encoded by the same chromosomal gene. Cell 46(3),417-28.
12. Aguzzi, A., and Polymenidou, M. (2004). Mammalian prion biology:one century
of evolving concepts. Cell 116(2),313-27.
13. Soto, C., and Castilla, J. (2004). The controversial protein-only hypothesis of
prion propagation. Nat Med 10 Suppl, S63-7.
14. Aiken, J. M., and Marsh, R. F. (1990). The search for scrapie agent nucleic acid. Microbiol Rev 54(3),242-46.
15. Bieschke, J., Weber, P., Sarafoff, N., Beekes, M., Giese, A., and Kretzschmar, H. (2004). Autocatalytic self-propagation of misfolded prion protein. Proc Natl Acad Sci USA 101(33),12207-11.
16. Deleault, N. R., Harris, B. T., Rees, J. R., and Supattapone, S. (2007). Formation of native prions from minimal components in vitro. Proc Natl Acad Sci U S A 104(23),9741-6.
17. Brown, D. R., and Sassoon, J. (2002). Copper-dependent functions for the prion protein. Mol Biotechnol 22(2),165-78.
18. van Rheede, T., Smolenaars, M. M., Madsen, O., and de Jong, W. W. (2003). Molecular evolution of the mammalian prion protein. Mol Biol Evol 20(1),111-21.
19. Gasset, M., Baldwin, M. A., Lloyd, D. H., Gabriel, J. M., Holtzman, D. M., Cohen, F., Fletterick, R., and Prusiner, S. B. (1992). Predicted alpha-helical regions of the prion protein when synthesized as peptides form amyloid. Proc Natl Acad Sci U S A 89(22),10940-4.
20. Huang, Z., Gabriel, J. M., Baldwin, M. A., Fletterick, R. J., Prusiner, S. B., and Cohen, F. E. (1994). Proposed three-dimensional structure for the cellular prion protein. Proc Natl Acad Sci USA 91(15),7139-43.
21. Zhang, H., Kaneko, K., Nguyen, J. T., Livshits, T. L., Baldwin, M. A., Cohen, F. E., James, T. L., and Prusiner, S. B. (1995). Conformational transitions in peptides containing two putative alpha-helices of the prion protein. J Mol Biol 250(4), 514-26.
22. James, T. L., Liu, H., Ulyanov, N. B., Farr-Jones, S., Zhang, H., Donne, D. G., Kaneko, K., Groth, D., Mehlhorn, I., Prusiner, S. B., and Cohen, F. E. (1997). Solution structure of a 142-residue recombinant prion protein corresponding to the infectious fragment of the scrapie isoform. Proc Natl Acad Sci U S A 94(19), 10086-91.
23. Liu, H., Farr-Jones, S., Ulyanov, N. B., Llinas, M., Marqusee, S., Groth, D., Cohen, F. E., Prusiner, S. B., and James, T. L. (1999). Solution structure of Syrian hamster prion protein rPrP(90-231). Biochemistry 38(17),5362-77.
24. Lopez Garcia, F., Zahn, R., Riek, R., and Wuthrich, K. (2000). NMR structure of the bovine prion protein. Proc Natl Acad Sci USA 97(15),8334-9.
25. Riek, R., Hornemann, S., Wider, G., Billeter, M., Glockshuber, R., and Wuthrich, K. (1996). NMR structure of the mouse prion protein domain PrP(121-321). Nature 382(6587),180-2
26. Riek, R., Hornemann, S., Wider, G., Glockshuber, R., and Wuthrich, K. (1997). NMR characterization of the full-length recombinant murine prion protein, mPrP(23-231). FEBS Lett 413(2),282-8.
27. Riek, R., Wider, G., Billeter, M., Hornemann, S., Glockshuber, R., and Wuthrich, K. (1998). Prion protein NMR structure and familial human spongiform encephalopathies. Proc Natl Acad Sci U S A 95(20),11667-72.
28. Christen, B., Perez, DR., Hornemann, S,. Wuthrich, K. (2008). NMR structure of the bank vole prion protein at 20 degrees C contains a structured loop of residues 165-171. J Mol Biol 383(2),306-12.
29. Li, J., Mei, FH., Xiao, GF., Guo, CY., Lin, DH. (2007).1H,13C and 15N resonance assignments of rabbit prion protein (91-228). J Biomol NMR 38(2),181.
30. Bueler, H., Aguzzi, A., Sailer, A., Greiner, R. A., Autenried, P., Aguet, M., and Weissmann, C. (1993). Mice devoid of PrP are resistant to scrapie. Cell 73(7), 1339-47.
31. Brown, D. R., and Sassoon, J. (2002). Copper-dependent functions for the prion protein. Mol Biotechnol 22(2),165-78.
32. Brown, D. R., Schulz-Schaeffer, W. J., Schmidt, B., and Kretzschmar, H. A. (1997). Prion protein-deficient cells show altered response to oxidative stress due to decreased SOD-1 activity. Exp Neurol 146(1),104-12.
33. Brown, D. R., Qin, K., Herms, J. W, Madlung, A., Manson, J., Strome, R., Fraser, P. E., Kruck, T., von Bohlen, A., Schulz-Schaeffer, W., Giese, A., Westaway, D., and Kretzschmar, H. (1997). The cellular prion protein binds copper in vivo. Nature 390(6661),684-7.
34. Viles, J. H., Cohen, F. E., Prusiner, S. B., Goodin, D. B., Wright, P. E., and Dyson, H. J. (1999). Copper binding to the prion protein:structural implications of four identical cooperative binding sites. Proc Natl Acad Sci U S A 96(5),2042-7.
35. Wong, B. S., Chen, S. G., Colucci, M., Xie, Z., Pan, T., Liu, T., Li, R., Gambetti, P., Sy, M. S., and Brown, D. R. (2001). Aberrant metal binding by prion protein in human prion disease. JNeurochem 78(6),1400-8.
36. Wong, B. S., Pan, T., Liu, T., Li, R., Petersen, R. B., Jones, I. M., Gambetti, P., Brown, D. R., and Sy, M. S. (2000). Prion disease:A loss of antioxidant function? Biochem Biophys Res Commun 275(2),249-52.
37. Rossi, L., Arciello, M., Capo, C., and Rotilio, G. (2006). Copper imbalance and oxidative stress in neurodegeneration. Ital J Biochem 55(3-4),212-21.
38. Moore, R. C., Mastrangelo, P., Bouzamondo, E., Heinrich, C., Legname, G., Prusiner, S. B., Hood, L., Westaway, D., DeArmond, S. J., and Tremblay, P. (2001). Doppel induced cerebellar degeneration in transgenic mice. Proc Nat Acad Sci USA 98(26),15288-93.
39. Li, R., Liu, D., Zanusso, G., Liu, T., Fayen, J. D., Huang, J. H., Petersen, R. B., Gambetti, P., and Sy, M. S. (2001). The expression and potential function of cellular prion protein in human lymphocytes. Cell Immunol 207(1),49-58.
40. Pietri, M., Caprini, A., Mouillet-Richard, S., Pradines, E., Ermonval, M., Grassi, J., Kellermann, O., and Schneider, B. (2006). Overstimulation of PrPc signaling pathways by prion peptide 106-126 causes oxidative injury of bioaminergic neuronal cells. JBiol Chem 281(38),28470-9.
41. Krebs, B., Dorner-Ciossek, C., Schmalzbauer, R., Vassallo, N., Herms, J., and Kretzschmar, H. A. (2006). Prion protein induced signaling cascades in monocytes. Biochem Biophys Res Commun 340(1),13-22.
42. Vey, M., Pilkuhn, S., Wille, H., Nixon, R., DeArmond, S. J., Smart, E. J., Anderson, R. G., Taraboulos, A., and Prusiner, S. B. (1991). Subcellular colocalization of the cellular and scrapie prion protein in caveolae-like mencbrnous somains. Proc Natl Acad Sci U S A 93(25),14945-9.
43. Milhavet, O., and Lehmann, S. (2002). Oxidative stress and the prion protein in transmissible spongiform encephalopathies. Brain Res Rev 38(3),328-39.
44. Pauly, P. C., and Harris, D. A. (1998). Copper stimulates endocytosis of the prion protein. JBiol Chem 273(50),33107-10.
45. Lee, K. S., Magalhaes, A. C., Zanata, S. M., Brentani, R. R., Martins, V. R., and Prado, M. A. (2001). Internalization of mammalian fluorescent cellular prion protein and N-terminal deletion mutants in living cells. JNeurochem 79(1),79-87.
46. Toni, M., Massimino, M. L., Griffoni, C., Salvato, B., Tomasi, V., and Spisni, E. (2005). Extracellular copper ions regulate cellular prion protein (prpc) expression and metabolism in neuronal cells. FEBS Lett 579(3),741-4.
47. Weissmann, C. (2004). The state of the prion. Nat Rev Microbiol 2(11),861-71.
48. Hornemann, S., and Glockshuber, R. (1998). A scrapie-like unfolding intermediate of the prion protein domain PrP(121-231) induced by acidic pH. Proc Natl Acad Sci U S A 95(11),6010-4.
49. Swietnicki, W., Petersen, R., Gambetti, P., and Surewicz, W. K. (1997). pH-dependent stability and conformation of the recombinant human prion protein PrP(90-231). J Biol Chem 272(44),27517-20.
50. Vanik, D. L., and Surewicz, W. K. (2002). Disease-associated F198S mutation increases the propensity of the recombinant prion protein for conformational conversion to scrapie-like form. JBiol Chem 277(50),49065-70.
51. Foguel, D., and Silva, J. L. (2004). New insights into the mechanisms of protein misfolding and aggregation in amyloidogenic diseases derived from pressure studies. Biochemistry 43(36),11361-70.
52. Borchelt, D. R., Scott, M., Taraboulos, A., Stahl, N., and Prusiner, S. B. (1990). Scrpie and cellular prion poteins differ in their kinetics of synthesis and topology in cultured cells. J Cell Biol 110(3),743-52.
53. Hovinfellner, J. A., and Budka, H. (1996). Immunomophology of human prion disease. Neuropathol Appl Neurobiol 23(3),264-8.
54. Brown, P., Kenney, K., Little, B., Ironside, J., Will, R., Cervenakova, L., Bjork, R. J., San Martin, R. A., Safar, J., and Roos, R. (1995). Intracerabral distribution of infectious amyloid protein in spongiform encephalopathy. Ann Neurol 38(2), 245-53.
55. Monari, L., Chen, S. G., Brown, P., Parchi, P., Petersen, R. B., Mikol, J., Gray, F., Cortelli, P., Montagna, P., and Ghetti, B. (1992). Fatal Familial Insomnia and Familial Creutzfeldt-Jakob disease:disease phenotype detemined by a DNA polymorphism. Science 258(5083),806-8.
56. Warren, J. D., Schott, J. M., Fox, N. C., Thom, M., Revesz, T., Holton, J. L., Scaravilli, F., Thomas, D. G., Plant, G. T., Rudge, P., and Rossor, M. N. (2005). Brain biopsy in dementia. Brain 128(9),2016-25.
57. Di Fede, G., Giaccone, G., Limido, L., Mangieri, M., Suardi, S., Puoti, G., Morbin, M., Mazzoleni, G., Ghetti, B., and Tagliavini, F. (2007). The epsilon isoforof 14-3-3 protein is a component of the prion protein amyloid deposits of Gerstmann-Straussler-Scheinker disease. J Neuropathol Exp Neurol 66(2),124-30.
58. Shiga, Y., Wakabayashi, H., Miyazawa, K., Kido, H., and Itoyama, Y. (2006). 14-3-3 protein levels and isoform patterns in the cerebrospinal fluid of Creutzfddvqakob disease patients in the progressive and terminal stages. J Clin Neurosci 13(6),661-5.
59. Aguzzi, A., and Glatzel, M. (2004). vCJD tissue distribution and transmission by transfusion:a worst-case scenario coming true? Lancet 363(11),411-2.
60. Chesebro, B. (1998). BSE and prions:uncertainties about the agent. Science 279(5347),42-3.
61. Weissmann, C., and Aguzzi, A. (2005). Approaches to therapy of prion diseases. Annu Rev Med 56,321-344.
62. Horiuchi, M., Baron, G. Sl, Xiong, L. W., and Caughey, B. (2001). Inhibition of interactions and interconversions of prion protein isoforms by peptide fragments from the C-terminal folded domain. JBiol Chem 76(18),15489-97.
63. Supattapone, S., Wille, H., Uyechi, L., Safar, J., Tremblay, P., Szoka, F. C., Cohen, F. E., Prusiner, S. B., and Scott, M. R. (2001). Branched polyamines cure prion-infected neuroblastoma cells. J Virol 75(7),3453-61.
64. Helenius, A., and Aebi, M. (2004). Roles of N-linked glycans in the endoplasmic reticulum. Annu Rev Biochem 73,1019-49.
65. Hammond, C., Braakman, I., and Helenius, A. (1994). Role of N-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control. Proc Natl Acad Sci USA 91(3),913-7.
66. Ware, F. E., Vassilakos, A., Peterson, P. A., Jackson, M. R., Lehrman, M. A., and Williams, D. B. (1995). The molecular chaperone calnexin binds Glc1Man9GlcNAc2 oligosaccharide as an initial step in recognizing unfolded glycoproteins. JBiol Chem 270(9),4697-704.
67. Spiro, R. G., Zhu, Q., Bhoyroo, V., and Soling, H. D. (1996). Definition of the lectin-like properties of the molecular chaperone, calreticulin, and demonstration of its copurification with endomannosidase from rat liver Golgi. JBiol Chem 271(19), 11588-94.
68. Wada, I., Rindress, D., Cameron, P. H., Ou, W. J., Doherty, J. J.,2nd, Louvard, D., Bell, A. W., Dignard, D., Thomas, D. Y. and Bergeron, J. J. (1991). SSR alpha and associated calnexin are major calcium binding proteins of the endoplasmic reticulum membrane. JBiol Chem 266(29),19599-610.
69. Fliegel, L., Burns, K., MacLennan, D. H., Reithmeier, R. A. and Michalak, M. (1989). Molecular cloning of the high affinity calcium-binding protein (calreticulin) of skeletal muscle sarcoplasmic reticulum. JBiol Chem 264(36),21522-8.
70. Hammond, C., Braakman, I. and Helenius, A. (1994). Role of N-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control. Proc Natl Acad Sci USA 91(3),913-917.
71. Ou, W. J., Cameron, P. H., Thomas, D. Y. and Bergeron, J. J. (1993). Association of folding ntermediates of glycoproteins with calnexin during protein maturation. Nature 364(6440),771-6.
72. Spiro, R. G., Zhu, Q., Bhoyroo, V. and Soling, H. D. (1996). Definition of the lectin-like properties of the molecular chaperone, calreticulin, and demonstration of its copurification with endomannosidase from rat liver Golgi. JBiol Chem 271(19), 11588-94.
73. Ware, F. E., Vassilakos, A., Peterson, P. A., Jackson, M. R., Lehrman, M. A. and Williams, D. B. (1995). The molecular chaperone calnexin binds Glc1Man9GlcNAc2 oligosaccharide as an initial step in recognizing unfolded glycoproteins. JBiol Chem 270(9),4697-04.
74. Baksh, S., Spamer, C., Heilmann, C. and Michalak, M. (1995). Identification of the Zn2+ binding region in calreticulin. FEBSLett 376(1-2),53-7.
75. Corbett, E. F., Michalak, K. M., Oikawa, K., Johnson, S., Campbell, I. D., Eggleton, P., Kay, C. and Michalak, M. (2000). The conformation of calreticulin is influenced by the endoplasmic reticulum luminal environment. JBiol Chem 275(35),27177-85.
76. Oliver, J. D., van der Wal, F. J., Bulleid, N. J. and High, S. (1997). Interaction of the thiol-dependent reductase ERp57 with nascent glycoproteins. Science 275(5296),86-8.
77. Schrag, J. D., Bergeron, J. J., Li, Y., Borisova, S., Hahn, M., Thomas, D. Y. and Cygler, M. (2001). The structure of calnexin, an ER chaperone involved in quality control of protein folding. Mol Cell 8(3),633-44.
78. Ellgaard, L., Riek, R., Herrmann, T., Guntert, P., Braun, D., Helenius, A. and Wuthrich, K. (2001). NMR structure of the calreticulin P-domain. Proc Natl Acad Sci USA 98(6),3133-8.
79. Vassilakos, A., Michalak, M., Lehrman, M. A. and Williams, D. B. (1998). Oligosaccharide binding characteristics of the molecular chaperones calnexin and calreticulin. Biochemistry 37(10),3480-90.
80. Leach, M. R., Cohen-Doyle, M. F., Thomas, D. Y. and Williams, D. B. (2002). Localization of the lectin, ERp57 binding, and polypeptide binding sites of calnexin and calreticulin. JBiol Chem 277(33),29686-97.
81. Kapoor, M., Ellgaard, L., Gopalakrishnapai, J., Schirra, C., Gemma, E., Oscarson, S., Helenius, A. and Surolia, A. (2004). Mutational analysis provides molecular insight into the carbohydrate-binding region of calreticulin:pivotal roles of tyrosine-109 and aspartate-135 in carbohydrate recognition. Biochemistry 43(1), 97-106.
82. Thomson, S. P. and Williams, D. B. (2005). Delineation of the lectin site of the molecular chaperone calreticulin. Cell Stress Chaperones 10(3),242-51.
83. Leach, M. R. and Williams, D. B. (2004). Lectin-deficient calnexin is capable of binding class I histocompatibility molecules in vivo and preventing their degradation. JBiol Chem 279(10),9072-9.
84. Frickel, E. M., Riek, R., Jelesarov, I., Helenius, A., Wuthrich, K. and Ellgaard, L. (2002). TROSY-NMR reveals interaction between ERp57 and the tip of the calreticulin P-domain. Proc Natl Acad Sci U S A 99(4),1954-9.
85. Pollock, S., Kozlov, G., Pelletier, M. F., Trempe, J. F., Jansen, G., Sitnikov, D., Bergeron, J. J., Gehring, K., Ekiel, I. and Thomas, D. Y. (2004). Specific interaction of ERp57 and calnexin determined by NMR spectroscopy and an ER two-hybrid system. EMBO J 23(5),1020-9.
86. Baksh, S. and Michalak, M. (1991). Expression of calreticulin in Escherichia coli and identification of its Ca2+binding domains. JBiol Chem 266(32),21458-65.
87. Corbett, E. F., Michalak, K. M., Oikawa, K., Johnson, S., Campbell, I. D., Eggleton, P., Kay, C. and Michalak, M. (2000). The conformation of calreticulin is influenced by the endoplasmic reticulum luminal environment. JBiol Chem 275(35),27177-85.
88. Li, Z., Stafford, W. F. and Bouvier, M. (2001). The metal ion binding properties of calreticulin modulate its conformational flexibility and thermal stability. Biochemistry 40(37),11193-201.
89. Ihara, Y., Cohen-Doyle, M. F., Saito, Y. and Williams, D. B. (1999). Calnexin discriminates between protein conformational states and functions as a molecular chaperone in vitro. Mol Cell 4(3),331-41.
90. Ou, W. J., Bergeron, J. J., Li, Y., Kang, C. Y. and Thomas, D. Y. (1995). Conformational changes induced in the endoplasmic reticulum luminal domain of calnexin by Mg-ATP and Ca2+. JBiol Chem 270(30),18051-59.
91. Saito, Y., Ihara, Y., Leach, M. R., Cohen-Doyle, M. F. and Williams, D. B. (1999). Calreticulin functions in vitro as a molecular chaperone for both glycosylated and nonglycosylated proteins. EMBO J 18(23),6718-29.
92. David, V., Hochstenbach, F., Rajagopalan, S. and Brenner, M. B. (1993). Interaction with newly synthesized and retained proteins in the endoplasmic reticulum suggests a chaperone function for human integral membrane protein IP90 (calnexin). JBiol Chem 268(13),9585-92.
93. Van Leeuwen, J. E. and Kearse, K. P. (1996). The related molecular chaperones calnexin and calreticulin differentially associate with nascent T cell antigen receptor proteins within the endoplasmic reticulum. JBiol Chem 271(41),25345-9.
94. Chen, W., Helenius, J., Braakman, I. and Helenius, A. (1995). Cotranslational folding and calnexin binding during glycoprotein synthesis. Proc Natl Acad Sci U S A 92(14),6229-33.
95. Anderson, K. S. and Cresswell, P. (1994). A role for calnexin (IP90) in the assembly of class Ⅱ MHC molecules. EMBO J 13(3),675-82.
96. Degen, E., Cohen-Doyle, M. F. and Williams, D. B. (1992). Efficient dissociation of the p88 chaperone from major histocompatibility complex class I molecules requires both beta 2-microglobulin and peptide. J Exp Med 175(6),1653-61.
97. Danilczyk, U. G. and Williams, D. B. (2001). The lectin chaperone calnexin utilizes polypeptide-based interactions to associate with many of its substrates in vivo. JBiol Chem 276(27),25532-40.
98. Parodi, A. J. (2000). Role of N-oligosaccharide endoplasmic reticulum processing reactions in glycoprotein folding and degradation. Biochem J 348(1),1-13.
99. Molinari, M., Eriksson, K. K., Calanca, V., Galli, C., Cresswell, P., Michalak, M. and Helenius, A. (2004). Contrasting functions of calreticulin and calnexin in glycoprotein folding and ER quality control. Mol Cell 13(1),125-35.
100. Gao, B., Adhikari, R., Howarth, M., Nakamura, K., Gold, M. C., Hill, A. B., Knee, R., Michalak, M. and Elliott, T. (2002). Assembly and antigen-presenting function of MHC class I molecules in cells lacking the ER chaperone calreticulin. Immunity 16(1),99-109.
101. Jannatipour, M. and Rokeach, L. A. (1995). The Schizosaccharomyces pombe homologue of the chaperone calnexin is essential for viability. JBiol Chem 270(9), 4845-53.
102. Parlati, F., Dignard, D., Bergeron, J. J. and Thomas, D. Y. (1995). The calnexin homologue cnxl+in Schizosaccharomyces pombe, is an essential gene which can be complemented by its soluble ER domain. EMBO J 14(13),3064-72.
103. Muller-Taubenberger, A., Lupas, A. N., Li, H., Ecke, M., Simmeth, E. and Gerisch, G. (2001). Calreticulin and calnexin in the endoplasmic reticulum are important for phagocytosis. EMBO J 20(23),6772-82.
104. Balow, J. P., Weissman, J. D. and Kearse, K. P. (1995). Unique expression of major histocompatibility complex class I proteins in the absence of glucose trimming and calnexin association. JBiol Chem 270(48),29025-9.
105. Knee, R., Ahsan, I., Mesaeli, N., Kaufman, R. J. and Michalak, M. (2003). Compromised calnexin function in calreticulin-deficient cells. Biochem Biophys Res Commun 304(4),661-6.
106. Molinari, M., Eriksson, K. K., Calanca, V., Galli, C., Cresswell, P., Michalak, M. and Helenius, A. (2004). Contrasting functions of calreticulin and calnexin in glycoprotein folding and ER quality control. Mol Cell 13(1),125-35.
107. Mesaeli, N., Nakamura, K., Zvaritch, E., Dickie, P., Dziak, E., Krause, K. H., Opas, M., MacLennan, D. H. and Michalak, M. (1999). Calreticulin is essential for cardiac development. J Cell Biol 144(5),857-68.
108. Guo, L., Nakamura, K., Lynch, J., Opas, M., Olson, E. N., Agellon, L. B. and Michalak, M. (2002). Cardiac-specific expression of calcineurin reverses embryonic lethality in calreticulin-deficient mouse. J Biol Chem 277(52),50776-9.
109. Nakamura, K., Zuppini, A., Arnaudeau, S., Lynch, J., Ahsan, I., Krause, R., Papp, S., De Smedt, H., Parys, J. B., Muller-Esterl, W. et al. (2001). Functional specialization of calreticulin domains. J Cell Biol 154(5),961-72.
110. Arnaudeau, S., Frieden, M., Nakamura, K., Castelbou, C., Michalak, M. and Demaurex, N. (2002). Calreticulin differentially modulates calcium uptake and release in the endoplasmic reticulum and mitochondria. JBiol Chem 277(48), 46696-705.
111. John, L. M., Lechleiter, J. D. and Camacho, P. (1998). Differential modulation of SERCA2 isoforms by calreticulin. J Cell Biol 142(4),963-73.
112. Lynch, J. and Michalak, M. (2003). Calreticulin is an upstream regulator of calcineurin. Biochem Biophys Res Commun 311(4),1173-9.
113. Denzel, A., Molinari, M., Trigueros, C., Martin, J. E., Velmurgan, S., Brown, S., Stamp, G. and Owen, M. J. (2002). Early postnatal death and motor disorders in mice congenitally deficient in calnexin expression. Mol Cell Biol 22(21),7398-404.
114. McCracken, A. A. and Brodsky, J. L. (2003). Evolving questions and paradigm shifts in endoplasmic-reticulum-associated degradation (ERAD). BioEssays 25(9), 868-77.
115. Jakob, C. A., Bodmer, D., Spirig, U., Battig, P., Marcil, A., Dignard, D., Bergeron, J. J., Thomas, D. Y. and Aebi, M. (2001). Htmlp, a mannosidase-like protein, is involved in glycoprotein degradation in yeast. EMBO Rep 2(5),423-30.
116. Hosokawa, N., Wada, I., Hasegawa, K., Yorihuzi, T., Tremblay, L. O., Herscovics, A. and Nagata, K. (2001). A novel ER alpha-mannosidase-like protein accelerates Erassociated degradation. EMBO Rep 2(5),415-22.
117. Boyce, M., and Yuan, J. (2006). Cellular response to endoplasmic reticulum stress:a matter of life or death. Cell Death Differ 13(3),363-73.
118. Szegezdi, E., Logue, S. E., Gorman, A. M., and Samali, A. (2006). Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep.7(9),880-5.
119. Ron, D., and Walter, P. (2007). Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev 8(7),519-29.
120. Caramelo, J. J., Castro, O. A., de Prat-Gay, G. and Parodi, A. J. (2004). The endoplasmic reticulum glucosyltransferase recognizes nearly native glycoprotein folding intermediates. JBiol Chem 279(44),46280-5.
121. Ritter, C., Quirin, K., Kowarik, M. and Helenius, A. (2005). Minor folding defects trigger local modification of glycoproteins by the ER folding sensor GT. EMBO J 24(9),1730-8.
122. Taylor, S. C., Ferguson, A. D., Bergeron, J. J. and Thomas, D. Y. (2004). The ER protein folding sensor UDP-glucose glycoprotein-glucosyltransferase modifies substrates distant to local changes in glycoprotein conformation. Nat Struct Mol Biol 11(2),128-34.
123. Arunachalam, B. and Cresswell, P. (1995). Molecular requirements for the interaction of class II major histocompatibility complex molecules and invariant chain with calnexin. JBiol Chem 270(6),2784-90.
124. Zhang, Q., Tector, M. and Salter, R. D. (1995). Calnexin recognizes carbohydrate and protein determinants of class I major histocompatibility complex molecules. JBiol Chem 270(8),3944-8.
125. Jorgensen, C. S., Heegaard, N. H., Holm, A., Hojrup, P. and Houen, G. (2000). Polypeptide binding properties of the chaperone calreticulin. Eur J Biochem 267(10),2945-54.
126. Nair, S., Wearsch, P. A., Mitchell, D. A., Wassenberg, J. J., Gilboa, E. and Nicchitta, C. V. (1999). Calreticulin displays in vivo peptide-binding activity and can elicit CTL responses against bound peptides. J Immunol 162(11),6426-32.
127. Culina, S., Lauvau, G., Gubler, B. and van Endert, P. M. (2004). Calreticulin promotes folding of functional human leukocyte antigen class I molecules in vitro. JBiol Chem 279(52),54210-5.
128. Rizvi, S. M., Mancino, L., Thammavongsa, V., Cantley, R. L. and Raghavan, M. (2004). A polypeptide binding conformation of calreticulin is induced by heat shock, calcium depletion, or by deletion of the C-terminal acidic region. Mol Cell 15(6),913-23.
129. Thammavongsa, V., Mancino, L. and Raghavan, M. (2005). Polypeptide substrate recognition by calnexin requires specific conformations of the calnexin protein. JBiol Chem 280(39),33497-505.
130. Jackson, G. S., Murray, I., Hosszu, L. L., Gibbs, N., Waltho, J. P., Clarke, A. R., and Collinge, J. (2001). Location and properties of metal-binding sites on the human prion protein. Proc Natl Acad Sci U S A 98(15),8531-5.
131. Rezaei, H., Marc, D., Choiset, Y., Takahashi, M., Hui Bon Hoa, G., Haertle, T., Grosclaude, J., and Debey, P. (2000). High yield purification and physico-chemical properties of full-length recombinant allelic variants of sheep prion protein linked to scrapie susceptibility. Eur J Biochem 267(10),2833-9.
132. Yin, S. M., Zheng, Y, and Tien, P. (2003). On-column purification and refolding of recombinant bovine prion protein:using its octarepeat sequences as a natural affinity tag. Protein Expr Purif 32(1),104-9.
133. Hahn, M., Borisova, S., Schrag, J. D., Tessier, D. C., Zapun, A., Tom, R., Kamen, A. A., Bergeron, J. J., Thomas, D. Y., and Cygler, M. (1998). Identification and crystallization of a protease-resistant core of calnexin that retains biological activity. Struct Biol 123(3),260-4.
134. Sun G, Guo M, Shen A, Mei F, Peng X, Gong R, Guo D, Wu J, Tien P, and Xiao G. (2005). Bovine PrPC directly interacts with alpha B-crystalline. FEBS Lett 579(24),5419-24.
135. Soto, C., and Estrada, L. D. (2008). Protein misfolding and neurodegeneration. Arch Neurol 65,184-189.
136. Prusiner, S. B. (1998) The prion diseases, Brain Pathol 8(3),499-513.
137. Ellgaard, L., Molinari, M., and Helenius, A. (1999). Setting the standards: quality control in the secretory pathway. Science 286(5446),1882-8.
138. Zapun, A., Petrescu, S. M., Rudd, P. M., Dwek, R. A., Thomas, D. Y., and Bergeron, J. J. (1997). Conformation-independent binding of monoglucosylated ribonuclease B to calnexin. Cell 88(1),29-38.
139. Wetzel, R. (2006). On amyloid assemble and structure. Acc Chem Res 39, 567-679.
140. Ma J., Wollmann, R., and Lindquist, S. (2002). Neurotoxicity and neurodegeneration when PrP accumulates in the cytosol. Science 298(5599), 1781-5.
141. Ma J., and Lindquist, S. (2001). Wild-type PrP and a mutant associated with prion disease are subject to retrograde transport and proteasome degradation. Proc Natl Acad Sci USA 98(26),14955.
142. Suzuki, T., Park, H., Hollingsworth, N. M., Sternglanz, R. and Lennarz, W. J. (2000). PNG1, a yeast gene encoding a highly conserved peptide:N-glycanase. J Cell Biol 149(5),1039-52.
143. Lehmann, S. and Harris, D. A. (1997). Blockade of glycosylation promotes acquisition of scrapie-like properties by the prion protein in cultured cells.J Biol Chem 272(34),21479-87.
144. Hebert, D. N., and Molinari, M. (2007). In and out of the ER:protein folding, quality control, degradation, and related human diseases. Physiol Rev 87(4), 1377-408.
145. Soto, C. (2003). Unfolding the role of protein misfolding in neurodegenerative diseases. Nat Rev Neurosci 4(1),49-60.
146. Hetz, C., Russelakis-Carneiro, M., Walchli, S., Carboni, S., Vial-Knecht, E., Maundrell, K., Castilla, J., and Soto, C. (2005). The disulfide isomerase Grp58 is a protective factor against prion neurotoxicity. J Neurosci 25(11),2793-802.
147. Jin, T., Gu, Y., Zanusso, G., Sy, M., Kumar, A., Cohen, M., Gambetti, P., and Singh, N. (2000). The chaperone protein BiP binds to a mutant prion protein and mediates its degradation by the proteasome. JBiol Chem 275(49),38699-704.
