摘要
可传播性海绵状脑病(Transmissible spongiform encephalopathies, TSEs)即朊病毒病(prion diseases),是一类罕见的致死性神经系统退行性疾病,包括人类的克雅氏病(Creutzfeldt-Jakob disease, CJD)、GSS综合症(Gerstmann-Straussler-Scheinker syndrome, GSS)、Kuru病、家族性致死性失眠症(fatal familial insomnia, FFI)、绵羊和山羊的瘙痒病(scrapie),牛及其它动物的海绵状脑病等,根据疾病的来源不同可分为自发形成(散发型)、遗传型和医源型三种形式。本文分为三部分,第一部分我们利用原核表达系统表达了全长人PrP以及各种PrP八肽重复区突变体,采用蛋白酶消化、沉淀实验、聚集实验以及圆二色谱(CD)等方法探讨了锰离子对野生型PrP以及各中八肽重复突变体生物学特性的影响。第二部分采用pull-down实验和免疫共沉淀方法探讨了PrP51-91和微管蛋白之间的相互作用。并且我们通过共沉淀法、比浊法及免疫荧光检测PrP51-91与铜离子对微管蛋白聚合的影响。此外,再通过共聚焦显微镜以及MTT法检测PrP51-91与铜离子对细胞中微管以及细胞毒性的影响。第三部分我们在分子水平证实了细胞内PrP (CytoPrP)与微管间的相互作用,并进一步确定CytoPrP对微管的影响能导致的细胞凋亡。
第一部分：锰离子对PrP生化特性的影响研究
锰离子可能在朊病毒的发病过程中起一定作用。我们利用锰离子处理野生型PrP和PrP八肽重复序列突变体后。通过PK消化、透射电镜、沉淀实验以及圆二色谱法分析锰离子对各种重组PrP生化特性的影响情况。为了检测锰离子能否影响野生型PrP和PrP八肽重复序列突变体的PK抗性,我们利用不同浓度的PK酶对经锰离子处理后的PrP进行消化,通过Western blot进行PK抗性检测。结果显示,经锰离子处理后,野生型PrP和PrP突变体的PK抗性均增强,并且PrP八肽重复序列突变体增强程度要强于野生型PrP。为了检测锰离子能否影响野生型PrP和PrP八肽重复序列突变体聚集,我们利用透射电镜、比浊法以及沉淀实验对其进行检测。结果显示,经锰离子处理后,野生型PrP和PrP突变体更容易发生聚集,并且PrP八肽重复序列突变体比野生型PrP更容易发生聚集。为了检测锰离子对野生型PrP和PrP八肽重复序列突变体二级结构的影响。我们利用圆二色谱法对其进行观察。结果显示,经锰离子处理后,野生型PrP和PrP突变体p-折叠的含量明显增加,并且PrP八肽重复序列突变体p-折叠的含量增加幅度明显高于野生型PrP增加的幅度。这些结果提示,锰离子能增强PrP的PK抗性,使其更容易发生聚集,使其由以α螺旋为主变为以β-折叠为主,而且锰离子对PrP的作用强度与八肽重复区的数目有关。锰离子可能作为一种辅助因子参与PrPC到PrPSc的转变。
第二部分：PrP51-91促进微管聚集并拮抗铜离子的微管解聚作用
我们通过免疫共沉淀以及pull down实验显示原核表达的PrP八肽重复序列能与微管蛋白形成复合物,提示PrP八肽序列能与微管蛋白发生相互作用。为了检测PrP八肽重复序列与tubulin的相互作用能否影响微管的聚集,我们利用体外微管聚集实验、共沉淀实验对其进行检测。结果显示,PrP八肽重复序列能促进微管发生聚集。为了检测PrP八肽重复序列能否抵抗铜离子导致的微管解聚作用,我们利用比浊法、免疫荧光对其进行检测。结果显示,铜离子能使微管发生解聚作用,PrP八肽重复序列则能抵抗铜离子所引起的微管解聚作用。为了检测PrP八肽重复序列以及铜离子对细胞中微管以及细胞活性的影响,我们利用共聚焦显微镜以及MTT法对其进行观察。结果显示,铜离子能使细胞中微管发生解聚,而PrP八肽重复序列则能抵抗铜离子所引起的微管解聚。此外,铜离子对细胞的毒性可能与其促进微管解聚有关,PrP八肽重复序列先于或者同时与铜离子作用细胞能拮抗铜离子引起的细胞毒性作用。
第三部分：CytoPrP能使微管发生解聚而导致细胞凋亡
我们将HeLa细胞转染CytoPrP,利用免疫共沉淀实验确定在细胞中CytoPrP能与微管发生相互作用。我们再利用共聚焦显微镜进一步确定CytoPrP能与微管在细胞中能发生共定位。为了检测CytoPrP与tubulin的相互作用能否影响微管的聚集,我们利用共聚焦显微镜以及Western blot对其进行检测。结果显示,CytoPrP能抑制细胞内微管发生聚集,并且使细胞内微管蛋白含量减少。为了检测CytoPrP所引起的细胞凋亡是通过微管解聚引起的,我们利用MTT以及Annexin V/PI双染方法对其进行检测。结果显示,CytoPrP能使细胞发生凋亡并且使其发生凋亡是通过促进微管解聚引起的。这些结果提示,CytoPrP与微管发生相互作用后,促进微管发生解聚,从而导致细胞凋亡。
Transmissible spongiform encephalopathies (TSEs), prion diseases, are rare degenerative neurological disorders that afflict human beings, including Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), Kuru, and fatal familial insomnia (FFI), sheep and goat (scrapie), cattle (bovine spongiform encephalopathy, BSE), and other animals. They may have a sporadic, inherited or acquired origin. This study contains three individual parts, including investigation the effect of PrP on the biochemical characteristics of the recombinant wild-type and mutated PrPs, the analyses of PrP51-91 on microtubule polymerization and the study of the mechanism of apoptosis caused by CytoPrP.
Part I:The effects of manganese on the biochemical characteristics of PrP
Manganese may play some roles in the pathogenesis of prion disease. In this study, recombinant wild-type PrP (WT-PrP) and PrP mutants with deleted or inserted octarepeats were exposed to manganese, and then their biochemical and biophysical characteristics were evaluated by proteinase K (PK) digestion, sedimentation experiments, transmission electron microscopy and circular dichroism (CD). To test whether manganese can influence the PK resistance of various PrPs, different concentrations of PK were used to digest the PrPs. Western blots showed that there were remarkable PrP-specific signals in Mn2+-treated PrPs after treated with higher doses of PK. Moreover, compared with WT-PrP, PrP mutants are more resistant to PK digestion after treatment with manganese. To see the effect of manganese on aggregation of various PrPs, sedimentation experiments, turbidimetry and transmission electron microscopy were performed. Our results showed that the treatment of manganese can efficiently induce aggregations of PrPs, moreover, PrP mutants seem to be easier to cause aggregation then the WT-PrP after incubation with manganese.
To investigate the effect of manganese on the structures of various PrPs, the secondary structures of refolded Mn+-treated PrPs were comparably examined by CD analysis. Our results showed that an obvious conversion from a predominant a-helix to a more extensiveβ-sheet formation was observed in the CD spectrums of each PrP preparation treated with Mn2+. Moreover, PrP mutants can cause conformational conversion easier than the WT-PrP after treatment with manganese. Our study provides the evidence that the increase of PK-resistance of PrPs after incubation with Mn2+correlates well with its more aggregation and higherβ-sheet content. Moreover, the effect of manganese on PrP seems to associate with the octapeptide repeats number. It strongly highlights that manganese may play an important role in the conversion from PrPC to PrPSc and the pathogenesis of prion disease.
PartⅡ:PrP51-91 enhances the formation of microtubule and antagonizes Cu2+-induced microtubule-disrupting activity
Using pull-down and co-immunoprecipitation assay, PrP51-91 can form complex with tubulin form hamster brain homogenate. It suggests that a remarkable interaction between the PrP51-91 and tubulin was identified. To test whether the interaction between PrP and tubulin influenced the assembling of microtubules from tubulin in vitro, microtubule assembly assay and sedimentation test were performed. Our results showed that PrP51-91 can induce tubulin polymerization. To see whether PrP51-91 can antagonize Cu2+-induced inhibition of microtubule assembly, microtubule assembly assay and immunofluorescence staining were performed. Our results showed that Cu2+has an inhibitive effect on microtubule formation, whereas PrP51-91 efficiently protects against Cu2+-induced microtubule-disrupting activity. To identify that PrP51-91 can protect against Cu2+toxicity on the culture cells and stabilizes cellular microtubules. Confocal microscopy and MTT assay were performed. Our results showed that Cu2+can inhibit microtubule of cell polymerization, whereas PrP51-91 can antagonize Cu2+-induced microtubule-disrupting activity. Moreover, PrP51-91 can efficiently protect against Cu2+-induced cytotoxicity.
PartⅢ:Cytosolic PrP induces apoptosis of cell by disrupting microtubule assembly
In the present study, the Hela cells were transfected with pcDNA3.1-CytoPrP. The molecular interaction between cytosolic PrP and tubulin was confirmed using immunoprecipitation. Moreover, the confocal microscopy was used to identify that CytoPrP can co-localize with tubulin in the HeLa cells. To test whether the interaction between CytoPrP and tubulin influenced the assembling of microtubules from tubulin, confocal microscopy and Western blot assay were performed. Our results showed that CytoPrP can inhibit the polymerization of tubulin and decrease the quality of microtubule protein. In order to identify that the inhibition of microtubule polymerization caused by cytosolic PrP was able to lead to the apoptosis of cells, immunofluorescent staining and the annexin V/PI double-staining assays were performed. Our results demonstrated that the inhibition of microtubule polymerization caused by cytosolic PrP was able to lead to the apoptosis of cells. The association of apoptosis with microtubule-disrupting activity caused by cytosolic PrP may further provide insight into the unresolved biological function of PrP in the neurons.
引文
1. Prusiner SB. Prions. Proc Natl Acad Sci.1998; 95:13363-13383.
2. Aguzzi A, Montrasio F, Kaeser PS. Prions:health scare and biological challenge. Nat Rev Mol Cell Biol,2001; 2(2):118-126.
3. Harris DA. Cellular biology of prion diseases. Clin Microbiol Rev.1999; 12(3): 429-444.
4. Prusiner SB, Scott MR, DeArmond SJ, Cohen FE. Prion protein biology. Cell. 1998; 93(3):337-348.
5. Glatzel M, Stoeck K, Seeger H, Luhrs T, Aguzzi A. Human prion diseases: molecular and clinical aspects. Arch Neurol.2005; 62(4):545-552.
6. Aguzzi A, Polymenidou M. Mammalian prion biology:one century of evolving concepts. Cell.2004; 116(2):313-327.
7. Glatzel M, Ott PM, Linder T, Gebbers JO, Gmur A, Wust W, Huber G, Moch H, Podvinec M, Stamm B, Aguzzi A. Human prion diseases:epidemiology and integrated risk assessment. Lancet Neurol.2003; 2(12):757-763.
8. Collins SJ, Lawson VA, Masters CL. Transmissible spongiform encephalopathies. Lancet.2004; 363(9402):51-61.
9. Gambetti P, Kong Q, Zou W, Parchi P, Chen SG Sporadic and familial CJD: classification and characterisation. Br Med Bull.2003; 66:213-239.
10. Legname G, Baskakov IV, Nguyen HO, Riesner D, Cohen FE, DeArmond SJ, Prusiner SB. Synthetic mammalian prions. Science.2004; 305(5684):673-676.
11.Priola SA, Chesebro B. Abnormal properties of prion protein with insertional mutations in different cell types. J Biol Chem.1998; 273(19):11980-119855.
12. Bueler H, Aguzzi A, Sailer A, Greiner RA, Autenried P, Aguet M, Weissmann C. Mice devoid of PrP are resistant to scrapie. Cell.1993; 73(7):1339-1347.
13. Fischer MB, Roeckl C, Parizek P, Schwarz HP, Aguzzi A. Binding of disease-associated prion protein to plasminogen. Nature.2000; 408(6811): 479-483.
14. Drisaldi B, Stewart RS, Adles C, Stewart LR, Quaglio E, Biasini E, Fioriti L, Chiesa R, Harris DA. Mutant PrP is delayed in its exit from the endoplasmic reticulum, but neither wild-type nor mutant PrP undergoes retrotranslocation prior to proteasomal degradation. J Biol Chem.2003; 278(24):21732-21743.
15. Glatzel M, Abela E, Maissen M, Aguzzi A. Extraneural pathologic prion protein in sporadic Creutzfeldt-Jakob disease. N Engl J Med.2003; 349(19):1812-1820.
16. Goldfarb LG, Brown P, McCombie WR, Goldgaber D,.Swergold GD, Wills PR, Cervenakova L, Baron H, Gibbs CJ, Gajdusek DC. Transmissible familial Creutzfeldt-Jakob disease associated with five, seven, and eight extra octapeptide coding repeats in the PRNP gene. Proc Natl Acad Sci USA.1991; 88: 10926-10930.
17. Poulter M, Baker HF, Frith CD, Leach M, Lofthouse R, Ridley RM, Shah T, Owen F, Collinge J, Brown J. Inherited prion disease with 144 base pair gene insertion.1. Genealogical and molecular studies. Brain.1992; 115:675-685.
18. Owen F, Poulter M, Shah T, Collinge J, Lofthouse R, Baker H, Ridley R. Mcvey J, Crow Tj. An in-frame insertion in the prion protein gene in familial Creutzfeldt-Jakob disease. Brain Res Mol Brain Res.1990;7(3):273-276.
19. Goldfarb LG, Brown P, Little BW, Cervenakova L, Kenney K, Gibbs CJ, Gajdusek DC. A new (two-repeat) octapeptide coding insert mutation in Creutzfeldt-Jakob disease. Neurology.1993;43:2392-2394.
20. Cochran EJ, Bennett DA, Cervenakova L, Kenney K, Bernard B, Foster NL, Benson MD, Goldfarb LG, Brown P. Familial Creutzfeldt-Jakob disease with a five-repeat octapeptide insert mutation. Neurology.1996;47:727-733.
21. Capellari S, Vital C, Parchi P, Petersen RB, Ferrer X, Jarnier D, Pegoraro E, Gambetti P, Julien J. Familial prion disease with a novel 144-bp insertion in the prion protein gene in a Basque family. Neurology.1997;49:133-141.
22. Rossi G, Giaccone G, Giampaolo L, lussich S, Puoti G, Frigo M, Cavaletti G, Frattola L, Bugiani O, Tagliavini F. Creutzfeldt-Jakob disease with a novel four extra-repeat insertional mutation in the PrP gene. Neurology.2000; 55:405-410.
23. Skworc KH, Windl O, Schulz-Schaeffer WJ, Giese A, Bergk J, Nagele A, Vieregge P, Zerr I, Poser S, Kretzschmar HA. Familial Creutzfeldt-Jakob disease with a novel 120-bp insertion in the prion protein gene. Ann Neurol.1999; 46: 693-700.
24. Oda T, Kitamoto T, Tateishi J, Mitsuhashi T, Iwabuchi K, Haga C, Oguni E, Kato Y, Tominaga I, Yanai K, Kashima H, Kogure T, Hori K, Ogino K. Prion disease with 144 base pair insertion in a Japanese family line. Acta Neuropathol (Berl). 1995; 90:80-86.
25. King A, Doey L, Rossor M, Mead S, Collinge J, Lantos P. Phenotypic variability in the brains of a family with a prion disease characterized by a 144-base pair insertion in the prion protein gene. Neuropathol Appl Neurobiol.2003; 29: 98-105.
26. Nicholl D, Windl O, de Silva R, Sawcer S, Dempster M, Ironside JW, Estibeiro JP, Yuill GM, Lathe R, Will RG. Inherited Creutzfeldt-Jakob disease in a British family associated with a novel 144 base pair insertion of the prion protein gene. J Neurol Neurosurg Psychiatry.1995; 58:65-69.
27. Krasemann S, Zerr I, Weber T, Poser S, Kretzschmar H, Hunsmann G, Bodemer W. Prion disease associated with a novel nine octapeptide repeat insertion in the PRNP gene. Brain Res Mol Brain Res.1995; 34:173-176.
28. Beck JA, Mead S, Campbell TA, Dichinson A, Wientjens DPMW, Croes EA, Van Duijn CM, Collinge J. Two-octapeptide repeat deletion of prion protein associated with rapidly progressive dementia. Neurology.2001;57:354-356.
29. Chiesa R, Drisaldi B, Quaglio E, Migheli A, Piccardo P, Ghetti B, Harris DA. Accumulation of protease-resistant prion protein (PrP) and apoptosis of cerebellar granule cells in transgenic mice expressing a PrP insertional mutation. Proc Natl Acad Sci U S A.2000;97:5574-5579.
30. Viles JH, Cohen FE, Prusiner SB, Goodin DB, Wright PE, Dyson HJ. Copper binding to the prion protein:structural implications of four identical cooperative binding sites. Proc Natl Acad Sci U S A.1999; 96:2042-2047.
31. Stockel J, Safar J, Wallace AC, Cohen FE, Prusiner SB. Prion protein selectively binds copper (Ⅱ) ions. Biochemistry.1998;37:7185-7193.
32. Miura T, Hori-i A, Takeuchi H. Metal-dependent alpha-helix formation promoted by the glycine-rich octapeptide region of prion protein. FEBS Lett.1996; 396: 248-252.
33. Miura T, Hori-i A, Mototani H, Takeuchi H. Raman spectroscopic study on the copper (Ⅱ) binding mode of prion octapeptide and its pH dependence. Biochemistry.1999; 38:11560-11569.
34. Hormshaw MP, McDermott JR, Candy JM, Lakey JH. Copper binding to the N-terminal tandem repeat region of mammalian and avian prion protein:structural studies using synthetic peptides. Biochem. Biophys. Res. Commun.1995; 214: 993-999.
35. Hormshaw MP, McDermott JR, Candy JM, Lakey JH. Copper binding to the N-terminal tandem repeat regions of mammalian and avian prion protein. Biochem Biophys Res Commun. Biochem. Biophys. Res. Commun.1995; 207: 621-629.
36. Brown DR, Qin KF, Herms JW, Madlung A, Manson J, Strome R, Fraser PE, Kruck T, Bohlen AV, Schaeffer WS, Giese A, Westaway D, Kretzschmar H. The cellular prion protein binds copper in vivo. Nature.1997; 390:684-687.
37. Vassallo N, Herms J. Cellular prion protein function in copper homeostasis and redox signaling at the synapsse. J. Neurochem.2003; 86:538-544.
38. Perera WS, Hooper NM. Ablation of the metal ion-induced endocytosis of the prion protein by disease-associated mutation of the octarepeat region. Curr Biol. 2001;11:519-523.
39. Hesketh S, Sassoon J, Knight R, Hopkins J, Brown DR. Elevated manganese levels in blood and central nervous system occur before onset of clinical signs in scrapie and bovine spongiform encephalopathy. J Anim Sci.2007;85:1596-1609.
40. Brown DR, HaWz F, Glassmith LL, Wong BS, Jones IM, Clive C, Haswell SJ. Consequences of manganese replacement of copper for prion protein function and proteinase resistance. EMBO J.2000; 19:1180-1186.
41. Gao JM, Gao C, Han J, Zhou XB, Xiao XL, Zhang J, Chen L, Zhang BY, Hong T, Dong XP. Dynamic analyses of PrP and PrPSc in brain tissues of golden hamsters infected with scrapie strain 263K revealed various PrP forms. Biomed Environ Sci. 2004; 17:8-20.
42. Yang S, Thackray AM, Fitzmaurice TJ, Bujdoso R. Copperinduced structural changes in the ovine prion protein are inXuenced by a polymorphism at codon 112. Biochim Biophys Acta.2008; 1784:683-692.
43. Thackray AM, Knight R, Haswell SJ, Bujdoso R, Brown DR. Metal imbalance and compromised antioxidant function are early changes in prion disease. Biochem J.2002; 362:253-258.
44. Wong BS, Brown DR, Pan T, Whiteman M, Liu T, Bu X, Li R, Gambetti P, Olesik J, Rubenstein R, Sy MS. Oxidative impairment in scrapie-infected mice is associated with brain metals perturbations and altered antioxidant activities. J Neurochem.2001; 79:689-698.
45. Dong CF, Huang YX, An R, Chen JM, Wang XF, Shan B, Lei YJ, Han J, Zhang BY, Dong XP. Sensitive detection of PrPSc by Western blot assay based on streptomycin sulphate precipitation. Zoonoses Public Health.2007; 54:328-336.
46. Lasmezas CI. Putative functions of PrP (C), Br Med Bull.2003; 66:61-70.
47. Ye X, Scallet AC, Kascsak RJ, Carp RI. Astrocytosis and amyloid deposition in scrapie-infected hamsters. Brain Res.1998; 809(2):277-287.
48. Wadsworth JD, Joiner S, Hill AF, Campbell TA, Desbruslais M, Luthert PJ, Collinge J. Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay. Lancet. 2001; 358:171-180.
49. Ingrosso L, Vetrugno V, Cardone F, Pocchiari M. Molecular diagnostics of transmissible spongiform encephalopathies. Trends Mol. Med.2002; 8:273-280.
50. Moussa A., Coleman AW, Bencsik A, Leclere E, Perret F, Martin A, Perron H. Use of streptomycin for precipitation and detection of proteinase K resistant prion protein (PrPSc) in biological samples. Chem Commun (Camb).2006; 9:973-985.
51. Chen SG, Teplow DB, Parchi P, Teller JK, Gambetti P, Autilio-Gambetti L Truncated forms of the human prion protein in normal brain and in prion disease. J Biol Chem.1995; 270:19173-19180.
52. Gao JM, Wan YZ, Wang XF, Han J, Chen L, Zhou W, Dong XP. Analyses of some biochemical features of the recombinant human PrP proteins with mutations in the octapeptide repeats region within N-terminus. Chin J Virol.2005; 21: 376-383 (in Chinese).
53. Donne DG, Viles JH, Groth D, Mehlhorn I, James TL, Cohen FE, Prusiner SB, Wright PE, Dyson HJ. Structure of the recombinant full-length hamster prion protein PrP (29-231):the N terminus is highly flexible. Proc Natl Acad Sci U S A. 1997; 94(25):13452-13457.
54. Riek R, Hornemann S, Wider G, Glockshuber R, Wuthrich K. NMR characterization of the full-length recombinant murine prion protein, mPrP (23-231). FEBS Lett.1997; 413(2):282-288.
55. Capellari S, Parchi P, Wolff BD, Campbell J, Atkinson R, Posey DM, Petersen RB, Gambetti P. Creutzfeldt-Jakob disease associated with a deletion of two repeats in the prion protein gene. Neurology.2002; 59(10):1628-1630.
56. Origin of extra prion repeat units [online]. Available at:http://www.mad-cow.org/ prion_repeat_insertions.html.
57. Atarashi R, Nishida N, Shigematsu K, Goto S, Kondo T, Sakaguchi S. Deletion of N-terminal Residues 23-88 from Prion Protein (PrP) Abrogates the Potential to Rescue PrP-deficient Mice from PrP-like Protein/Doppel-induced Neurodegeneration. J Biol Chem.2003; 278:28944-28949.
58. Bounhar Y, Zhang Y, Goodyer C, Gand LeBlanc A. Prion protein protects human neurons against Bax-mediated apoptosis. J Biol Chem.2001; 276,39145-39149.
59. Dong CF, Shi S, Wang XF, An R, Li P, Chen JM, Wang X, Wang GR, Shan B, Zhang BY, Han J, Dong XP. The N-terminus of PrP is responsible for interacting with tubulin and fCJD related PrP mutants possess stronger inhibitive eVect on microtubule assembly in vitro. Arch Biochem Biophys.2008; 470:83-92.
60. Brazier MW, Davies P, Player E, Marken F, Viles JH, Brown DR. Manganese binding to the prion protein. J Biol Chem.2008; 283:12831-12839.
1. Prusiner SB, Scott MR, DeArmond SJ, Cohen FE. Prion protein biology. Cell. 1998; 93(3):337-48.
2. Glatzel M, Stoeck K, Seeger H, Luhrs T, Aguzzi A. Human prion diseases: molecular and clinical aspects. Arch Neurol.2005; 62(4):545-552.
3. Aguzzi A, Montrasio F, Kaeser PS. Prions:health scare and biological challenge. Nat Rev Mol Cell Biol.2001; 2(2):118-126.
4. Castilla J, Saa P, Hetz C, Soto C. In vitro generation of infectious scrapie prions. Cell.121:195-206. Commented in Cell.2005; 121:155-162.
5. Legname G, Baskakov IV, Nguyen HO, Riesner D, Cohen FE, DeArmond SJ, Prusiner SB. Synthetic mammalian prions. Science.2004; 305(5684):673-676.
6. Gambetti P, Kong Q, Zou W, Parchi P, Chen SG. Sporadic and familial CJD: classification and characterisation. Br Med Bull.2003; 66:213-239.
7. Capellari S, Parchi P, Wolff BD, Campbell J, Atkinson R, Posey DM, Petersen RB, Gambetti P. Creutzfeldt-Jakob disease associated with a deletion of two repeats in the prion protein gene. Neurology.2002; 59(10):1628-1630.
8. Goldfarb LG, Brown P, McCombie WR, Goldgaber D, Swergold GD, Wills PR, Cervenakova L, Baron H, Gibbs CJ Jr, Gajdusek DC. Transmissible familial Creutzfeldt-Jakob disease associated with five, seven, and eight extra octapeptide coding repeats in the PRNP gene. Proc Natl Acad Sci USA.1991; 88(23): 10926-30.
9. Drisaldi B, Stewart RS, Adles C, Stewart LR, Quaglio E, Biasini E, Fioriti L, Chiesa R, Harris DA. Mutant PrP is delayed in its exit from the endoplasmic reticulum, but neither wild-type nor mutant PrP undergoes retrotranslocation prior to proteasomal degradation. J Biol Chem.2003; 278(24):21732-21743.
10. Glatzel M, Abela E, Maissen M, Aguzzi A. Extraneural pathologic prion protein in sporadic Creutzfeldt-Jakob disease. N Engl J Med.2003; 349(19):1812-1820.
11. Gray BC, Skipp P, O'Connor VM, Perry VH. Increased expression of glial fibrillary acidic protein fragments and mu-calpain activation within the hippocampus of prion-infected mice. Biochem Soc Trans.2006; 34:51-54.
12. Vilette D, Madelaine MF, Laude H. Establishment of astrocyte cell lines from sheep genetically susceptible to scrapie. In Vitro Cell Dev Biol Anim.2000; 36: 45-49.
13. Stahl N, Borchelt DR, Hsiao K, Prusiner SB. Scrapie prion protein contains a phosphatidylinositol glycolipid. Cell.1987; 51:229-240,
14. Mironov A Jr, Latawiec D, Wille H, Bouzamondo-Bernstein E, Legname G, Williamson RA, Burton D, DeArmond SJ, Prusiner SB, Peters PJ. Cytosolic prion protein in neurons. J Neurosci.2003; 23:7183-7193.
15. Donne DG, Viles JH, Groth D, Mehlhorn I, James TL, Cohen FE, Prusiner SB, Wright PE, Dyson HJ. Structure of the recombinant full-length hamster prion protein PrP (29-231):the N terminus is highly flexible. Proc Natl Acad Sci U S A. 1997; 94(25):13452-13457.
16. Shyng SL, Moulder KL, Lesko A, Harris DA. The N-terminal domain of a glycolipid-anchored prion protein is essential for its endocytosis via clathrin-coated pits. J Biol Chem.1995; 270(24):14793-14800.
17. Walter ED, Stevens DJ, Visconte MP, Millhauser GL. The prion protein is a combined zinc and copper binding protein:Zn2+ alters the distribution of Cu2+ coordination modes. J Am Chem Soc.2007; 129(50):15440-15451.
18. Hachiya NS, Watanabe K, Sakasegawa Y, Kaneko K. Microtubules-associated intracellular localization of the NH2-terminal cellular prion protein fragment, Biochem Biophys Res Commun.2004; 313:818-823.
19. Borchelt DR, Koliatsos VE, Guarnieri M, Pardo CA, Sisodia SS. Rapid anterograde axonal transport of the cellular prion glycoprotein in the peripheral and central nervous systems. J Biol Chem.1994; 269:14711-14714.
20. Brown DR. Altered toxicity of the prion protein peptide PrP106-126 carrying the Ala (117)-Val mutation. Biochem J.2000; 346:785-791.
21. Kirschner MW, Mitchison T. Microtubule dynamics. Nature.1986; 324:621-631.
22. Su QN, Cai Q, Gerwin C, Smith CL, Sheng ZH. Syntabulin is a microtubule-associated protein implicated in syntaxin transport in neurons. Nature Cell Biol.2004; 6:941-953.
23. Jordan MA, Wilson L. Microtubules as a target for anticancer drugs. Nat Rev Cancer.2004; 4:253-265.
24. Mollinedo F, Gajate C. Microtubules, microtubule-interfering agents and apoptosis. Apoptosis.2003; 8:413-450.
25. Pribyl P, Cepa'k V and Zachleder V. Cytoskeletal alterations in interphase cells of the green alga Spirogyra decimina in response to heavy metals exposure:Ⅱ. The effect of aluminium, nickel and copper. Toxicol In Vitro 2008,22:1160-1168.
26. Nieznanski K, Nieznanska H, Skowronek K J. Direct interaction between prion protein and tubulin. Biochem Biophys Res Commun.2005; 334:403-411.
27. Nieznanski K, Podlubnaya ZA, Nieznanska H. Prion protein inhibits microtubule assembly by inducing tubulin oligomerization. Biochemical and biophysical research communications,2006; 349:11391-11399.
28. Dong CF, Shi S, Wang XF, An R, Li P, Chen JM, Wang X, Wang GR, Shan B, Zhang BY, Han J, Dong XP. The N-terminus of PrP is responsible for interacting with tubulin and fCJD related PrP mutants possess stronger inhibitive eVect on microtubule assembly in vitro. Arch Biochem Biophys.2008; 470:83-92.
29. Riek R, Hornemann S, Wider G, Glockshuber R, Wuthrich K. NMR characterization of the full-length recombinant murine prion protein, mPrP (23-231). FEBS Lett.1997; 413(2):282-288.
30. Pauly PC, Harris DA. Copper stimulates endocytosis of the prion protein. J Biol Chem 1998,273:33107-33110.
31. Bounhar Y, Zhang Y, Goodyer CG, LeBlanc A. Prion protein protects human neurons against Bax-mediated apoptosis. J Biol Chem 2001,276:39145-39149.
32. Gunawardena S, Goldstein LB. Cargo-carrying motor vehicles on the neuronal highway:transport pathways and neurodegenerative disease. J Neurobiol.2003; 58:258-271.
33. Mandelkow E, Mandelkow EM. Kinesin motors and disease. Trends Cell Biol. 2002; 12:585-591.
34. Pazour GJ, Rosenbaum JL. Intraflagellar transport and cilia-dependent diseases. Trends Cell Biol.2002; 12:551-555.
35. Moya KL, Hassig R, Creminon C, Laffont I, Di Giamberardino L. Enhanced detection and retrograde axonal transport of PrPc in peripheral nerve. J Neurochem.2004; 88:155-160.
36. Butowt R, Abdelraheim S, Brown DR, von Bartheld CS. Anterograde axonal transport of the exogenous cellular isoform of prion protein in the chick visual system. Mol Cell Neurosci.2006,31:97-108.
37. Kelkar S, Pfister KK, Crystal RG, Leopold PL. Cytoplasmic dynein mediates adenovirus binding to microtubules. J Virol.2004; 78:10122-10132.
38. Han J, Zhang J, Yao H, Wang X, Li F, Chen L and Gao C, et al. Study on interaction between microtubule associated protein tau and prion protein. Sci China C Life Sci 2006; 49:473-479.
39. Lippard SJ. Free copper ions in the cell? Science 1999; 284:748-749.
40. Liliom K, Wa'gner G, Pa'cz A, Cascante M, Kova'cs J, Ova'di J. Organization-dependent effects of toxic bivalent ions microtubule assembly and glycolysis. Eur J Biochem.2000; 267:4731-4739.
41. Stockel J, Safar J, Wallace AC, Cohen FE, Prusiner SB. Prion protein selectively binds copper (Ⅱ) ions. Biochemistry.1998; 37:7185-7193.
42. Steinebach OM, Wolterbeek HT. Role of cytosolic copper, metallothionein and glutathione in copper toxicity in rat hepatoma tissue culture cells. Toxicology. 1994; 92:75-90.
43. McKenzie D, Bartz J, Mirwald J, Olander D, Marsh R, Aiken J. Reversibility of scrapie inactivation is enhanced by copper. J Biol Chem.1998; 273: 25545-25547.
44. Quaglio E, Chiesa R, Harris DA. Copper converts the cellular prion protein into a protease-resistant species that is distinct from the scrapie isoform. J Biol. Chem. 2001; 276:11432-11438.
45. Qin K, Yang DS, Yang Y, Chishti MA, Meng L, Kretzschmar HA, Yip CM, Fraser PE, Westaway D. Copper(Ⅱ)-induced conformational changes and protease resistance in recombinant and cellular PrP. Effect of protein age and deamidation. J Biol. Chem.2000; 275:19121-19131.
46. Miura T, Hori-i A, Mototani H, Takeuchi H. Raman spectroscopic study on the copper (Ⅱ) binding mode of prion octapeptide and its pH dependence. Biochemistry.1999; 38:11560-11569.
47. Wadsworth JD, Hill AF, Joiner S, Jackson GS, Clarke AR, Collinge J. Strain-specific prion-protein conformation determined by metal ions. Nat Cell Biol.1999; 1:55-59.
48. Deignan ME, Prior M, Stuart LE, Comerford EJ, McMahon HE. The structure function relationship for the Prion protein. J Alzheimers Dis.2004; 6:283-289.
49. Blagosklonny MV, Giannakakou P, El-Deiry WS, Kingston DG, Higgs PI, Neckers L, Fojo T. Raf-1/Bcl-2 phosphorylation:a step from microtubule damage to cell death. Cancer Res.1997; 57:130-135.
50. Wang XF, Dong CF, Zhang J, Wan YZ, Li F, Huang YX, Han L, Dong XP. Human tau protein forms complex with PrP and some GSS-and fCJD-related PrP mutants possess stronger binding activities with tau in vitro. Mol Cell Biochem; 2008; 310:49-55.
1.Prusiner SB.1998. Prions. Proc Natl Acad Sci U S A,95:13363-13383.
2. Prusiner SB, Groth D, Serban A, Koehler R, Foster D, Torchia M, Burton D, Yang SL, DeArmond SJ. Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies. Proc Natl Acad Sci USA. 1993; 90:10608-10612.
3. Aguzzi A, Polymenidou M. Mammalian prion biology:one century of evolving concepts. Cell.2004; 116(2):313-327.
4. Bueler H, Aguzzi A, Sailer A, Greiner RA, Autenried P, Aguet M, Weissmann C. Mice devoid of PrP are resistant to scrapie. Cell.1993; 73:1339-1347.
5. Ma J, Lindquist S. Wild-type PrP and a mutant associated with prion disease are subject to retrograde transport and proteasome degradation. Proc Natl Acad Sci USA.2001; 98:14955-14960.
6. Ma J, Wollmann R, Lindquist S. Neurotoxicity and neurodegeneration when PrP accumulates in the cytosol. Science.2002; 298:1781-1785.
7. Angelika S, Rambold, Margit M, Doron R, Till B, Michael B, Konstanze F, Winklhofer, Jorg, T. Association of bcl-2 with misfolded prion protein is linked to the toxic potential of cytosolic PrP. Molecular Biology of the Cell.2006; 17: 3356-3368
8. Yedidia Y, Horonchik L, Tzaban S, Yanai A, Taraboulos A. Proteasomes and ubiquitin are involved in the turnover of the wild-type prion protein. EMBO J.2001; 20:5383-5391.
9. Lorenz H, Windl O, Kretzschmar HA. Cellular phenotyping of secretory and nuclear prion proteins associated with inherited prion diseases. J Biol Chem.2002; 277:8508-8516.
10. Bueler H, Aguzzi A, Sailer A, Greiner RA, Autenried P, Aguet M, Weissmann C. Mice devoid of PrP are resistant to scrapie. Cell.1993; 73:1339-1347.
11. Roucou X, Guo G, Zhang Y, Goodyer CG, LeBlanc AC. Cytosolic prion protein is not toxic and protects against Baxmediated cell death in human primary neurons. J Biol Chem.2003; 278:40877-40881.
12. Gunawardena S, Goldstein LB. Cargo-carrying motor vehicles on the neuronal highway:transport pathways and neurodegenerative disease. J Neurobiol.2003; 58:258-271.
13.Mandelkow E, Mandelkow EM. Kinesin motors and disease. Trends Cell Biol. 2002; 12:585-591.
14. Pazour GJ, Rosenbaum JL. Intraflagellar transport and cilia-dependent diseases. Trends Cell Biol.2002; 12:551-555.
15. Badano JL, Teslovich TM, Katsanis N. The centrosome in human genetic disease. Nature Rev Genet.2005; 6:194-205.
16. Gerdes JM, Katsanis N. Microtubule transport defects in neurological and ciliary disease. Cell Mol Life Sci.2005; 62:1556-1570.
17. Gao JM, Gao C, Han J, Zhou XB, Xiao XL, Zhang J, Chen L, Zhang BY, Hong T, Dong XP. Dynamic analyses of PrP and PrPSc in brain tissues of golden hamsters infected with scrapie strain 263K revealed various PrP forms. Biomed Environ Sci. 2004; 17:8-20.
18. Jordan MA, Wilson L. Microtubules as a target for anticancer drugs. Nat Rev Cancer.2005; 4:253-265.
19. Brandt R. The tau proteins in neuronal growth and development. Front Biosci. 1996; 1:118-130.
20. D' Andrea MR, Ilyin S, Plata-Salaman CR. Abnormal patterns of microtubule-associated protein-2 (MAP-2) immunolabeling in neuronal nuclei and Lewy bodies in Parkinson's disease substantia nigra brain tissues. Neurosci Lett. 2001;306(3):137-140.
21. Jellinger KA. Cell death mechanisms in Parkinson's disease. J Neural Transm. 2000; 107(1):1-29.
22. Saha AR, Hill J, Utton MA, Asuni AA, Ackerley S, Grierson AJ, Miller CC, Davies AM, Buchman VL, Anderton BH, Hanger DP. Parkinson's disease alpha-synuclein mutations exhibit defective axonal transport in cultured neurons. J Cell Sci.2004; 117(Pt 7):1017-1024.
23. Ferrer I. Synaptic pathology and cell death in the cerebellum in Creutzfeldt-Jakob disease. Cerebellum.2002; 1(3):213-222.
24. Uppuluri S, Knipling L, Sackett DL, Wolff J. Localization of the colchicine-binding site of tubulin. Proc Natl Acad Sci USA,1993; 90 (24): 11598-11602.
25. Zuliani T, Denis V, Noblesse E, Schnebert S, Andre P, Dumas M, Dumas M, Ratinaud MH. Hydrogen peroxide-induced cell death in normal human keratinocytes is differentiation dependent. Free Radical Bio Med 2005; 38: 307-316.
26. Wang X, Dong CF, Shi Q, Shi S, Wang GR, Lei YJ, Xu K, An R, Chen JM, Jiang HY, Tian C, Gao C, Zhao YJ, Han J, Dong XP. Cytosolic prion protein induces apoptosis in human neuronal cell SH-SY5Y via mitochondrial disruption pathway. BMB Rep.2009; 42(7):444-449.
27. Chen L, Yang Y, Han J, Zhang BY, Zhao L, Nie K, Wang XF, Li F, Gao C, Dong XP, Xu CM. Removal of the Glycosylation of Prion Protein Provokes Apoptosis in SF126. J Biochem Mol Biol.2007; 41:662-669.
28. Kopito RR. ER quality control:the cytoplasmic connection. Cell.1997; 88: 427-430.
29. Fewell SW, Travers KJ, Weissman JS. The action of molecular chaperones in the early secretory pathway. Annu. Rev. Genet.2001; 35:149-191.
30. Holscher UC, Bach B, Dobberstein. Prion protein contains a second endoplasmic reticulum targeting signal sequence located at its C-terminus. J. Biol. Chem.2003; 276:13388-13394.
31. Nieznanski K, Nieznanska H, Skowronek KJ. Direct interaction between prion protein and tubulin. Biochem Biophys Res Commun.2005; 334:403-411.
32. Nieznanski K, Podlubnaya ZA, Nieznanska H. Prion protein inhibits microtubule assembly by inducing tubulin oligomerization. Biochemical and biophysical research communications,2006; 349:11,391-399.
33. Hachiya NS, Watanabe K, Sakasegawa Y, Kaneko K. Microtubules-associated intracellular localization of the NH2-terminal cellular prion protein fragment, Biochem Biophys Res Commun.2004; 313:818-823.
34. Dong CF, Shi S, Wang XF, An R, Li P, Chen JM, Wang X, Wang GR, Shan B, Zhang BY, Han J, Dong XP. The N-terminus of PrP is responsible for interacting with tubulin and fCJD related PrP mutants possess stronger inhibitive eVect on microtubule assembly in vitro. Arch Biochem Biophys.2008; 470:83-92.
35. Gourlay CW, Carpp LN, Timpson P, Winder SJ, Ayscough FR. A role for the actin cytoskeleton in cell death and aging in yeast. J Cell Biol.2004; 164(6): 803-809.
36. Olivia NT, Jocelyne H, Jacqueline B. Cytoskeleton and apoptosis. Biochem Pharmacol.2008; 76(1):11-18.
1. Prusiner SB. Novel proteinaceous infectious particles cause scrapie. Science. 1982; 216:136-144.
2. Horiuchi M, Yamazaki N, Ikeda T, Ishiguro N, Shinagawa M. A cellular form of prion protein exists in many non-neuronal tissues of sheep. J Gen Virol.1995; 76: 2583-2587.
3. Mironov AJ, Latawiec D, Wille H, Bernstein EB, Legname G, Williamson RA, Burton D, Dearmond SJ, Prusiner SB, Peters PJ. Cytosolic prion protein in neurons. J. Neurosci.2003; 23:7183-7193.
4. Linden R, Martins VR, Prado MA, Cammarota M, Izquierdo I, Brentani RR. Physiology of the Prion Protein. Physiol Rev.2008; 88:673-728.
5. Kasper M, Barth K. Bleomycin and its role in inducing apoptosis and senescence in lung cells-modulating effects of caveolin-1. Curr Cancer Drug Targets.2009; 9: 341-353.
6. Bu J, Bruckner SR, Sengoku T, Geddes JW, Estus S. Glutamate regulates caveolin expression in rat hippocampal neurons. J Neurosci Res.2003; 72: 185-190.
7. Trushina E, Du CJ, Parisi J, McMurray CT. Neurological abnormalities in caveolin-1 knocked out mice. Behav Brain Res; 2006:172:24-32.
8. Mouillet R, Ermonval M, Chebassier C, Laplanche JL, Lehmann S, Launay JM, Kellermann O. Signal Transduction through prion protein. Science.2000; 289: 1925-1928.
9. Pantera B, Bini C, Cirri P, Paoli P, Camici G, Manao G, Caselli A. PrPc activation induces neurite outgrowth and differentiation in PC12 cells:role for caveolin-1 in the signal transduction pathway. J Neurochem.2009; 110:194-207.
10. Chiarini LB, Freitas AR, Zanata SM, Brentani RR, Martins VR, Linden R. Cellular prion protein transduces neuroprotective signals. Embo J.2002; 21: 3317-3326.
11. Zanata SM, Lopes MH, Mercadante AF, Hajj GN, Chiarini LB, Nomizo R, Freitas AR, Cabral AL, Lee KS, Juliano MA, Oliveira E, Jachieri SG,Burlingame A, Huang L, Linden R, Brentani RR, Martins VR. Stress-inducible protein 1 is a cell surface ligand for cellular prion that triggers neuroprotection. Embo J.2002; 21: 3307-3316.
12. Lopes MH, Hajj G, Muras AG, Mancini GL, Castro R, Ribeiro K, Brentani RR, Linden R, Martins VR. Interaction of cellular prion and stress-inducible protein 1 promotes neuritogenesis and neuroprotection by distinct signaling pathways. J Neurosci.2005;25:11330-11339.
13. Schneider B, Mutel V, Pietri M, Ermonval M, Richard SM, Kellermann O. NADPH oxidase and extracellular regulated kinases 1/2 are targets of prion protein signaling in neuronal and nonneuronal cells. Proc Natl Acad Sci USA. 2003; 100:13326-13331.
14. Uppington KM, Brown DR. Resistance of cell lines to prion toxicity aided by phospho-ERK expression. J Neurochem.2008; 105(3):842-852.
15. Kanaani J, Prusiner SB, Diacovo J, Beakkeskov S, Legname G. Recombinant prion protein induces rapid polarization and development of synapses in embryonic rat hippocampal neurons in vitro. J Neurochem.2005; 95:1373-1386.
16. Krebs B, Dorner CC, Schmalzbauer R, Vassallo N, Herms J, Kretzschmar HA. Prion protein induced signaling cascades in monocytes. Biochem Biophys Res Commun.2006; 340:13-22.
17. Malaga-Trillo E, Solis GP, Schrock Y, Geiss C, Luncz L, Thomanetz V, Stuermer C. Regulation of embryonic cell adhesion by the prion protein. PLOS Biol.2009; 7,576-590.
18. Fuhrmann M, Bittner T, Mitteregger G, Haider N, Moosmang S, Kretzschmar H, Herms J. Loss of the cellular prion protein affects the Ca2+homeostasis in hippocampal CA1 neurons. J Neurochem.2006; 98:1876-1885.
19. Brini M, Miuzzo M, Pierobon N, Nergo A, Sorgato MC. The prion protein and its paralogue Doppel affect calcium signaling in Chinese hamster ovary cells. Mol Biol Cell.2005; 16:2799-2808.
20. Stuermer CA, Langhorst MF, Wiechers MF, Legler DF, Hanwehr SH, Guse AH, Plattner H. PrPc capping in T cells promotes its association with the lipid raft proteins reggie-1 and reggie-2 and leads to signal transduction. FASEB J,2004, 18:1731-1733.
21. Botto L, Masserini M, Cassetti A, Palestini P. Immunoseparation of Prion protein-enriched domains from other detergent-resistant membrane fractions, isolated from neuronal cells. FEBS Lett.2004; 557:143-147.
22. Mazzoni IE, Ledebur HC Jr, Paramithiotis E, Cashman N. Lymphoid signal transduction mechanism linked to cellular prion protein. Biochem Cell Biol.2005; 83:644-653.
23. Janes KA, Yaffe MB. Data-driven modeling of signal-transduction networks. Nat Rev Mol Cell Biol.2006; 7:820-828.
24. Brodsky IE, Medzhitov R. Targeting of immune signaling networks by bacterial pathogens. Nat Cell Biol.2009; 11:521-526.