Lombari, Patrizia (2006) Modulazione dell'espressione della Serpina Proteasi Nexina 1 (PN1) in sistemi cellulari neurali e gliali. [Tesi di dottorato] (Unpublished)


Download (3MB) | Preview
[error in script] [error in script]
Item Type: Tesi di dottorato
Resource language: Italiano
Title: Modulazione dell'espressione della Serpina Proteasi Nexina 1 (PN1) in sistemi cellulari neurali e gliali
Lombari, PatriziaUNSPECIFIED
Date: 2006
Date type: Publication
Number of Pages: 96
Institution: Università degli Studi di Napoli Federico II
Department: Biochimica e biotecnologie mediche
Dottorato: Biochimica e biologia cellulare e molecolare
Ciclo di dottorato: 17
Coordinatore del Corso di dottorato:
D’Alessio, GiuseppeUNSPECIFIED
Pietropaolo, ConcettaUNSPECIFIED
Date: 2006
Number of Pages: 96
Keywords: Proteasi Nexina I, Sistema Tet-Off, RNA Interference
References: 1. Almeida, R. and Allshire, R.C. (2005) RNA silencing and genome regulation. Trends Cell Biol, 15, 251-258. 2. Aymerich, M.S., Alberdi, E.M., Martinez, A. and Becerra, S.P. (2001) Evidence for pigment epithelium-derived factor receptors in the neural retina. Invest Ophthalmol Vis Sci, 42, 3287-3293. 3. Baker, J.B. and Gronke, R.S. (1986) Protease nexins and cellular regulation. Semin Thromb Hemost, 12, 216-220. 4. Banda, M.J., Rice, A.G., Griffin, G.L. and Senior, R.M. (1988) The inhibitory complex of human alpha 1-proteinase inhibitor and human leukocyte elastase is a neutrophil chemoattractant. J Exp Med, 167, 1608-1615. 5. Bar-Shavit, R., Kahn, A., Mudd, M.S., Wilner, G.D., Mann, K.G. and Fenton, J.W., 2nd. (1984) Localization of a chemotactic domain in human thrombin. Biochemistry, 23, 397-400. 6. Bartel, D.P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 116, 281-297. 7. Baulcombe, D. (2004) RNA silencing in plants. Nature, 431, 356- 363. 8. Baulcombe, D. (2005) RNA silencing. Trends Biochem Sci, 30, 290-293. 9. Benda, P., Lightbody, J., Sato, G., Levine, L. and Sweet, W. (1968) Differentiated rat glial cell strain in tissue culture. Science, 161, 370-371. 10. Bernstein, E., Caudy, A.A., Hammond, S.M. and Hannon, G.J. (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, 409, 363-366. 11. Biales, B., Dichter, M. and Tischler, A. (1976) Electrical excitability of cultured adrenal chromaffin cells. J Physiol, 262, 743-753. 12. Blasi, F., Ciarrocchi, A., Luddi, A., Strazza, M., Riccio, M., Santi, S., Arcone, R., Pietropaolo, C., D'Angelo, R., Costantino- Ceccarini, E. et al. (2002) Stage-specific gene expression in early differentiating oligodendrocytes. Glia, 39, 114-123. 13. Bode, W. and Huber, R. (1992) Natural protein proteinase inhibitors and their interaction with proteinases. Eur J Biochem, 204, 433-451. 14. Brummelkamp, T.R., Bernards, R. and Agami, R. (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science, 296, 550-553. 15. Buchholz, M., Biebl, A., Neebetae, A., Wagner, M., Iwamura, T., Leder, G., Adler, G. and Gress, T.M. (2003) SERPINE2 (protease nexin I) promotes extracellular matrix production and local invasion of pancreatic tumors in vivo. Cancer Res, 63, 4945- 4951. 16. Carrell RW, P.P., Boswell DR. (1987) The serpins: evolution and adaptation in a family of protease inhibitors. The serpins: evolution and adaptation in a family of protease inhibitors., 52, 527-535. 17. Chen, X. (2004) A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science, 303, III 2022-2025. 18. Clark M, J.S., Leharach H, Lee R, Li F, Marra M, Eddy S, Hillier L, Kucaba T, Martin J, Beck C, Wylie T, Underwood K, Stepoe M, Theising B, Allen M, Bowers Y, Person B, Swaller T, Gibbons M, Pape D, Harvey N, Schurk R, Ritter E, Kohn S, Shin T, Jackson Y, Cardenas M, McCann R, Waterson R and Wilson R (1998) Genbank, entry 9821294. 19. Cogoni, C., Irelan, J.T., Schumacher, M., Schmidhauser, T.J., Selker, E.U. and Macino, G. (1996) Transgene silencing of the al- 1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does not depend on DNA-DNA interactions or DNA methylation. Embo J, 15, 3153-3163. 20. Coughlin, S.R. (1999) How the protease thrombin talks to cells. Proc Natl Acad Sci U S A, 96, 11023-11027. 21. Cox, D.W. and Billingsley, G.D. (1989) Rare deficiency types of alpha 1-antitrypsin: electrophoretic variation and DNA haplotypes. Am J Hum Genet, 44, 844-854. 22. Crisp, R.J., Knauer, D.J. and Knauer, M.F. (2000) Roles of the heparin and low density lipid receptor-related protein-binding sites of protease nexin 1 (PN1) in urokinase-PN1 complex catabolism. The PN1 heparin-binding site mediates complex retention and degradation but not cell surface binding or internalization. J Biol Chem, 275, 19628-19637. 23. Cucina, A., Borrelli, V., Di Carlo, A., Pagliei, S., Corvino, V., Santoro-D'Angelo, L., Cavallaro, A. and Sterpetti, A.V. (1999) Thrombin induces production of growth factors from aortic smooth muscle cells. J Surg Res, 82, 61-66. 24. Cullen, B.R. (2003) Nuclear mRNA export: insights from virology. Trends Biochem Sci, 28, 419-424. 25. Cunningham, D.D. (1992) Regulation of neuronal cells and astrocytes by protease nexin-1 and thrombin. Ann N Y Acad Sci, 674, 228-236. 26. Cunningham, D.D. and Gurwitz, D. (1989) Proteolytic regulation of neurite outgrowth from neuroblastoma cells by thrombin and protease nexin-1. J Cell Biochem, 39, 55-64. 27. Cunningham, D.D., Pulliam, L. and Vaughan, P.J. (1993) Protease nexin-1 and thrombin: injury-related processes in the brain. Thromb Haemost, 70, 168-171. 28. Davis, R.L., Shrimpton, A.E., Holohan, P.D., Bradshaw, C., Feiglin, D., Collins, G.H., Sonderegger, P., Kinter, J., Becker, L.M., Lacbawan, F. et al. (1999) Familial dementia caused by polymerization of mutant neuroserpin. Nature, 401, 376-379. 29. Dawson, D.W., Volpert, O.V., Gillis, P., Crawford, S.E., Xu, H., Benedict, W. and Bouck, N.P. (1999) Pigment epithelium-derived factor: a potent inhibitor of angiogenesis. Science, 285, 245-248. 30. Debeir, T., Benavides, J. and Vige, X. (1998) Involvement of protease-activated receptor-1 in the in vitro development of mesencephalic dopaminergic neurons. Neuroscience, 82, 739- 752. 31. Detwiler, T.C., Chang, A.C., Speziale, M.V., Browne, P.C., IV Miller, J.J. and Chen, K. (1992) Complexes of thrombin with proteins secreted by activated platelets. Semin Thromb Hemost, 18, 60-66. 32. Dichter, M.A., Tischler, A.S. and Greene, L.A. (1977) Nerve growth factor-induced increase in electrical excitability and acetylcholine sensitivity of a rat pheochromocytoma cell line. Nature, 268, 501-504. 33. Donovan, F.M., Vaughan, P.J. and Cunningham, D.D. (1994) Regulation of protease nexin-1 target protease specificity by collagen type IV. J Biol Chem, 269, 17199-17205. 34. Drapkin, P.T., Monard, D. and Silverman, A.J. (2002) The role of serine proteases and serine protease inhibitors in the migration of gonadotropin-releasing hormone neurons. BMC Dev Biol, 2, 1. 35. Dykxhoorn, D.M., Novina, C.D. and Sharp, P.A. (2003) Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol, 4, 457-467. 36. Eaton, D.L. and Baker, J.B. (1983) Evidence that a variety of cultured cells secrete protease nexin and produce a distinct cytoplasmic serine protease-binding factor. J Cell Physiol, 117, 175-182. 37. Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 411, 494-498. 38. Elliott, P.R., Abrahams, J.P. and Lomas, D.A. (1998) Wild-type alpha 1-antitrypsin is in the canonical inhibitory conformation. J Mol Biol, 275, 419-425. 39. Evans, D.L., McGrogan, M., Scott, R.W. and Carrell, R.W. (1991) Protease specificity and heparin binding and activation of recombinant protease nexin I. J Biol Chem, 266, 22307-22312. 40. Fagard, M., Boutet, S., Morel, J.B., Bellini, C. and Vaucheret, H. (2000) AGO1, QDE-2, and RDE-1 are related proteins required for post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference in animals. Proc Natl Acad Sci U S A, 97, 11650-11654. 41. Fenton, J.W., 2nd. (1988) Regulation of thrombin generation and functions. Semin Thromb Hemost, 14, 234-240. 42. Festoff, B.W., Rao, J.S. and Hantai, D. (1991) Plasminogen activators and inhibitors in the neuromuscular system: III. The serpin protease nexin I is synthesized by muscle and localized at neuromuscular synapses. J Cell Physiol, 147, 76-86. 43. Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E. and Mello, C.C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391, 806-811. 44. Gettins, P.G. (2002) Serpin structure, mechanism, and function. Chem Rev, 102, 4751-4804. 45. Gil, J. and Esteban, M. (2000) Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): mechanism of action. Apoptosis, 5, 107-114. V 46. Gingrich, M.B. and Traynelis, S.F. (2000) Serine proteases and brain damage - is there a link? Trends Neurosci, 23, 399-407. 47. Gloor, S., Odink, K., Guenther, J., Nick, H. and Monard, D. (1986) A glia-derived neurite promoting factor with protease inhibitory activity belongs to the protease nexins. Cell, 47, 687- 693. 48. Gossen, M. and Bujard, H. (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A, 89, 5547-5551. 49. Grabham, P. and Cunningham, D.D. (1995) Thrombin receptor activation stimulates astrocyte proliferation and reversal of stellation by distinct pathways: involvement of tyrosine phosphorylation. J Neurochem, 64, 583-591. 50. Greene, L.A. and Rein, G. (1977) Release, storage and uptake of catecholamines by a clonal cell line of nerve growth factor (NGF) responsive pheo-chromocytoma cells. Brain Res, 129, 247-263. 51. Greene, L.A. and Rukenstein, A. (1981) Regulation of acetylcholinesterase activity by nerve growth factor. Role of transcription and dissociation from effects on proliferation and neurite outgrowth. J Biol Chem, 256, 6363-6367. 52. Greene, L.A. and Tischler, A.S. (1976) Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A, 73, 2424-2428. 53. Grimmond, S., Van Hateren, N., Siggers, P., Arkell, R., Larder, R., Soares, M.B., de Fatima Bonaldo, M., Smith, L., Tymowska- Lalanne, Z., Wells, C. et al. (2000) Sexually dimorphic expression of protease nexin-1 and vanin-1 in the developing mouse gonad prior to overt differentiation suggests a role in mammalian sexual development. Hum Mol Genet, 9, 1553-1560. 54. Gurwitz, D. and Cunningham, D.D. (1990) Neurite outgrowth activity of protease nexin-1 on neuroblastoma cells requires thrombin inhibition. J Cell Physiol, 142, 155-162. 55. Hagglund, A.C., Ny, A., Liu, K. and Ny, T. (1996) Coordinated and cell-specific induction of both physiological plasminogen activators creates functionally redundant mechanisms for plasmin formation during ovulation. Endocrinology, 137, 5671- 5677. 56. Hammond, S.M., Bernstein, E., Beach, D. and Hannon, G.J. (2000) An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature, 404, 293-296. 57. Hillen, W., Unger, B. and Klock, G. (1982) Analysis of tet operator-TET repressor complexes by thermal denaturation studies. Nucleic Acids Res, 10, 6085-6097. 58. Hopkins, P.C. and Whisstock, J. (1994) Function of maspin. Science, 265, 1893-1894. 59. Huntington, J.A. (2003) Mechanisms of glycosaminoglycan activation of the serpins in hemostasis. J Thromb Haemost, 1, 1535-1549. 60. Huntington, J.A., Read, R.J. and Carrell, R.W. (2000) Structure VI of a serpin-protease complex shows inhibition by deformation. Nature, 407, 923-926. 61. Irving, J.A., Pike, R.N., Lesk, A.M. and Whisstock, J.C. (2000) Phylogeny of the serpin superfamily: implications of patterns of amino acid conservation for structure and function. Genome Res, 10, 1845-1864. 62. Irving, J.A., Steenbakkers, P.J., Lesk, A.M., Op den Camp, H.J., Pike, R.N. and Whisstock, J.C. (2002) Serpins in prokaryotes. Mol Biol Evol, 19, 1881-1890. 63. Jalink, K., van Corven, E.J., Hengeveld, T., Morii, N., Narumiya, S. and Moolenaar, W.H. (1994) Inhibition of lysophosphatidateand thrombin-induced neurite retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding protein Rho. J Cell Biol, 126, 801-810. 64. Janciauskiene, S. (2001) Conformational properties of serine proteinase inhibitors (serpins) confer multiple pathophysiological roles. Biochim Biophys Acta, 1535, 221-235. 65. Jesty, J. (1979) Dissociation of complexes and their derivatives formed during inhibition of bovine thrombin and activated factor X by antithrombin III. J Biol Chem, 254, 1044-1049. 66. Ketting, R.F. and Plasterk, R.H. (2000) A genetic link between co-suppression and RNA interference in C. elegans. Nature, 404, 296-298. 67. Kim, S., Buonanno, A. and Nelson, P.G. (1998) Regulation of prothrombin, thrombin receptor, and protease nexin-1 expression during development and after denervation in muscle. J Neurosci Res, 53, 304-311. 68. Knauer, D.J., Majumdar, D., Fong, P.C. and Knauer, M.F. (2000) SERPIN regulation of factor XIa. The novel observation that protease nexin 1 in the presence of heparin is a more potent inhibitor of factor XIa than C1 inhibitor. J Biol Chem, 275, 37340-37346. 69. Knauer, M.F., Hawley, S.B. and Knauer, D.J. (1997) Identification of a binding site in protease nexin I (PN1) required for the receptor mediated internalization of PN1-thrombin complexes. J Biol Chem, 272, 12261-12264. 70. Knauer, M.F., Kridel, S.J., Hawley, S.B. and Knauer, D.J. (1997) The efficient catabolism of thrombin-protease nexin 1 complexes is a synergistic mechanism that requires both the LDL receptorrelated protein and cell surface heparins. J Biol Chem, 272, 29039-29045. 71. Kretschmer-Kazemi Far, R. and Sczakiel, G. (2003) The activity of siRNA in mammalian cells is related to structural target accessibility: a comparison with antisense oligonucleotides. Nucleic Acids Res, 31, 4417-4424. 72. Kurdowska, A. and Travis, J. (1990) Acute phase protein stimulation by alpha 1-antichymotrypsin-cathepsin G complexes. Evidence for the involvement of interleukin-6. J Biol Chem, 265, 21023-21026. 73. Kury, P., Schaeren-Wiemers, N. and Monard, D. (1997) Protease VII nexin-1 is expressed at the mouse met-/mesencephalic junction and FGF signaling regulates its promoter activity in primary met- /mesencephalic cells. Development, 124, 1251-1262. 74. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680- 685. 75. Ley, T.J., Anagnou, N.P., Pepe, G. and Nienhuis, A.W. (1982) RNA processing errors in patients with beta-thalassemia. Proc Natl Acad Sci U S A, 79, 4775-4779. 76. Ligoxygakis, P., Roth, S. and Reichhart, J.M. (2003) A serpin regulates dorsal-ventral axis formation in the Drosophila embryo. Curr Biol, 13, 2097-2102. 77. Liu, Z., Zhou, X., Shapiro, S.D., Shipley, J.M., Twining, S.S., Diaz, L.A., Senior, R.M. and Werb, Z. (2000) The serpin alpha1- proteinase inhibitor is a critical substrate for gelatinase B/MMP-9 in vivo. Cell, 102, 647-655. 78. Luthi, A., Van der Putten, H., Botteri, F.M., Mansuy, I.M., Meins, M., Frey, U., Sansig, G., Portet, C., Schmutz, M., Schroder, M. et al. (1997) Endogenous serine protease inhibitor modulates epileptic activity and hippocampal long-term potentiation. J Neurosci, 17, 4688-4699. 79. Mangoura, D., Sakellaridis, N., Jones, J. and Vernadakis, A. (1989) Early and late passage C-6 glial cell growth: similarities with primary glial cells in culture. Neurochem Res, 14, 941-947. 80. Mansuy, I.M., van der Putten, H., Schmid, P., Meins, M., Botteri, F.M. and Monard, D. (1993) Variable and multiple expression of Protease Nexin-1 during mouse organogenesis and nervous system development. Development, 119, 1119-1134. 81. Matzke, M.A. and Matzke, A.J. (1991) Differential inactivation and methylation of a transgene in plants by two suppressor loci containing homologous sequences. Plant Mol Biol, 16, 821-830. 82. Mbebi, C., Hantai, D., Jandrot-Perrus, M., Doyennette, M.A. and Verdiere-Sahuque, M. (1999) Protease nexin I expression is upregulated in human skeletal muscle by injury-related factors. J Cell Physiol, 179, 305-314. 83. Meins, M., Piosik, P., Schaeren-Wiemers, N., Franzoni, S., Troncoso, E., Kiss, J.Z., Brosamle, C., Schwab, M.E., Molnar, Z. and Monard, D. (2001) Progressive neuronal and motor dysfunction in mice overexpressing the serine protease inhibitor protease nexin-1 in postmitotic neurons. J Neurosci, 21, 8830- 8841. 84. Mendes de Aguiar, C.B., Garcez, R.C., Alvarez-Silva, M. and Trentin, A.G. (2002) Undersulfation of proteoglycans and proteins alter C6 glioma cells proliferation, adhesion and extracellular matrix organization. Int J Dev Neurosci, 20, 563- 571. 85. Molinari, F., Meskanaite, V., Munnich, A., Sonderegger, P. and Colleaux, L. (2003) Extracellular proteases and their inhibitors in genetic diseases of the central nervous system. Hum Mol Genet, 12 Spec No 2, R195-200. VIII 86. Moll, S., Schaeren-Wiemers, N., Wohlwend, A., Pastore, Y., Fulpius, T., Monard, D., Sappino, A.P., Schifferli, J.A., Vassalli, J.D. and Izui, S. (1996) Protease nexin 1 in the murine kidney: glomerular localization and up-regulation in glomerulopathies. Kidney Int, 50, 1936-1945. 87. Monard, D. (1988) Cell-derived proteases and protease inhibitors as regulators of neurite outgrowth. Trends Neurosci, 11, 541- 544. 88. Murer, V., Spetz, J.F., Hengst, U., Altrogge, L.M., de Agostini, A. and Monard, D. (2001) Male fertility defects in mice lacking the serine protease inhibitor protease nexin-1. Proc Natl Acad Sci U S A, 98, 3029-3033. 89. Napoli, C., Lemieux, C. and Jorgensen, R. (1990) Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. Plant Cell, 2, 279-289. 90. Neumann, S., Petfalski, E., Brugger, B., Grosshans, H., Wieland, F., Tollervey, D. and Hurt, E. (2003) Formation and nuclear export of tRNA, rRNA and mRNA is regulated by the ubiquitin ligase Rsp5p. EMBO Rep, 4, 1156-1162. 91. Niclou, S.P., Suidan, H.S., Pavlik, A., Vejsada, R. and Monard, D. (1998) Changes in the expression of protease-activated receptor 1 and protease nexin-1 mRNA during rat nervous system development and after nerve lesion. Eur J Neurosci, 10, 1590- 1607. 92. Noda-Heiny, H., Fujii, S. and Sobel, B.E. (1993) Induction of vascular smooth muscle cell expression of plasminogen activator inhibitor-1 by thrombin. Circ Res, 72, 36-43. 93. Ohlsson, K. and Laurell, C.B. (1976) The disappearance of enzyme-inhibitor complexes from the circulation of man. Clin Sci Mol Med, 51, 87-92. 94. Paddison, P.J., Caudy, A.A. and Hannon, G.J. (2002) Stable suppression of gene expression by RNAi in mammalian cells. Proc Natl Acad Sci U S A, 99, 1443-1448. 95. Palauqui, J.C., Elmayan, T., Pollien, J.M. and Vaucheret, H. (1997) Systemic acquired silencing: transgene-specific posttranscriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions. Embo J, 16, 4738-4745. 96. Parker, K.K., Norenberg, M.D. and Vernadakis, A. (1980) "Transdifferentiation" of C6 glial cells in culture. Science, 208, 179-181. 97. Patston, P.A. (2000) Serpins and other serine protease inhibitors. Immunol Today, 21, 354. 98. Perez Canadillas, J.M. and Varani, G. (2003) Recognition of GUrich polyadenylation regulatory elements by human CstF-64 protein. Embo J, 22, 2821-2830. 99. Pires Neto, M.A., Braga-de-Souza, S. and Lent, R. (1999) Extracellular matrix molecules play diverse roles in the growth and guidance of central nervous system axons. Braz J Med Biol Res, 32, 633-638. IX 100. Poulin, G., Nandakumar, R. and Ahringer, J. (2004) Genomewide RNAi screens in Caenorhabditis elegans: impact on cancer research. Oncogene, 23, 8340-8345. 101. Ragg, H., Lokot, T., Kamp, P.B., Atchley, W.R. and Dress, A. (2001) Vertebrate serpins: construction of a conflict-free phylogeny by combining exon-intron and diagnostic site analyses. Mol Biol Evol, 18, 577-584. 102. Reinhard, E., Suidan, H.S., Pavlik, A. and Monard, D. (1994) Gliaderived nexin/protease nexin-1 is expressed by a subset of neurons in the rat brain. J Neurosci Res, 37, 256-270. 103. Richard, B., Arocas, V., Guillin, M.C., Michel, J.B., Jandrot- Perrus, M. and Bouton, M.C. (2004) Protease nexin-1: a cellular serpin down-regulated by thrombin in rat aortic smooth muscle cells. J Cell Physiol, 201, 138-145. 104. Rovelli, G., Stone, S.R., Guidolin, A., Sommer, J. and Monard, D. (1992) Characterization of the heparin-binding site of gliaderived nexin/protease nexin-1. Biochemistry, 31, 3542-3549. 105. Rushlow, C. (2004) Dorsoventral patterning: a serpin pinned down at last. Curr Biol, 14, R16-18. 106. Saksela, O. and Rifkin, D.B. (1988) Cell-associated plasminogen activation: regulation and physiological functions. Annu Rev Cell Biol, 4, 93-126. 107. Sambrook, J. Fritsch, E.F. Maniatis, T. (1989) Molecular cloning a laboratory manual. Second edition, Cold Spring Harbour Laboratory Press. 108. Schechter, I. and Berger, A. (1967) On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun, 27, 157- 162. 109. Schuster, M.G., Enriquez, P.M., Curran, P., Cooperman, B.S. and Rubin, H. (1992) Regulation of neutrophil superoxide by antichymotrypsin-chymotrypsin complexes. J Biol Chem, 267, 5056-5059. 110. Scott, R.W., Bergman, B.L., Bajpai, A., Hersh, R.T., Rodriguez, H., Jones, B.N., Barreda, C., Watts, S. and Baker, J.B. (1985) Protease nexin. Properties and a modified purification procedure. J Biol Chem, 260, 7029-7034. 111. Seegers, W.H. (1986) Postclotting fates of thrombin. Semin Thromb Hemost, 12, 181-183. 112. Shea, T.B. (1995) Role of glial-derived nexin in neuronal differentiation and in acute brain injury and potential involvement in exacerbation of neurodegeneration in Alzheimer's disease. Brain Res Brain Res Rev, 20, 171-184. 113. Shikamoto, Y. and Morita, T. (1999) Expression of factor X in both the rat brain and cells of the central nervous system. FEBS Lett, 463, 387-389. 114. Silverman, G.A., Bird, P.I., Carrell, R.W., Church, F.C., Coughlin, P.B., Gettins, P.G., Irving, J.A., Lomas, D.A., Luke, C.J., Moyer, R.W. et al. (2001) The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, X and a revised nomenclature. J Biol Chem, 276, 33293-33296. 115. Smith-Swintosky, V.L., Zimmer, S., Fenton, J.W., 2nd and Mattson, M.P. (1995) Opposing actions of thrombin and protease nexin-1 on amyloid beta-peptide toxicity and on accumulation of peroxides and calcium in hippocampal neurons. J Neurochem, 65, 1415-1418. 116. Sommer, J., Gloor, S.M., Rovelli, G.F., Hofsteenge, J., Nick, H., Meier, R. and Monard, D. (1987) cDNA sequence coding for a rat glia-derived nexin and its homology to members of the serpin superfamily. Biochemistry, 26, 6407-6410. 117. Steele, F.R., Chader, G.J., Johnson, L.V. and Tombran-Tink, J. (1993) Pigment epithelium-derived factor: neurotrophic activity and identification as a member of the serine protease inhibitor gene family. Proc Natl Acad Sci U S A, 90, 1526-1530. 118. Stiernberg, J., Redin, W.R., Warner, W.S. and Carney, D.H. (1993) The role of thrombin and thrombin receptor activating peptide (TRAP-508) in initiation of tissue repair. Thromb Haemost, 70, 158-162. 119. Stone, S.R., Brown-Luedi, M.L., Rovelli, G., Guidolin, A., McGlynn, E. and Monard, D. (1994) Localization of the heparinbinding site of glia-derived nexin/protease nexin-1 by sitedirected mutagenesis. Biochemistry, 33, 7731-7735. 120. Strehlow, D., Jelaska, A., Strehlow, K. and Korn, J.H. (1999) A potential role for protease nexin 1 overexpression in the pathogenesis of scleroderma. J Clin Invest, 103, 1179-1190. 121. Sui, G., Soohoo, C., Affar el, B., Gay, F., Shi, Y., Forrester, W.C. and Shi, Y. (2002) A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci U S A, 99, 5515-5520. 122. Tang, G. (2005) siRNA and miRNA: an insight into RISCs. Trends Biochem Sci, 30, 106-114. 123. Tischler, A.S. and Greene, L.A. (1978) Morphologic and cytochemical properties of a clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Lab Invest, 39, 77-89. 124. Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A, 76, 4350-4354. 125. Triezenberg, S.J., Kingsbury, R.C. and McKnight, S.L. (1988) Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression. Genes Dev, 2, 718-729. 126. Triezenberg, S.J., LaMarco, K.L. and McKnight, S.L. (1988) Evidence of DNA: protein interactions that mediate HSV-1 immediate early gene activation by VP16. Genes Dev, 2, 730- 742. 127. Turgeon, V.L. and Houenou, L.J. (1997) The role of thrombin-like (serine) proteases in the development, plasticity and pathology of the nervous system. Brain Res Brain Res Rev, 25, 85-95. XI 128. Turgeon, V.L., Lloyd, E.D., Wang, S., Festoff, B.W. and Houenou, L.J. (1998) Thrombin perturbs neurite outgrowth and induces apoptotic cell death in enriched chick spinal motoneuron cultures through caspase activation. J Neurosci, 18, 6882-6891. 129. Vaistij, F.E., Jones, L. and Baulcombe, D.C. (2002) Spreading of RNA targeting and DNA methylation in RNA silencing requires transcription of the target gene and a putative RNA-dependent RNA polymerase. Plant Cell, 14, 857-867. 130. van der Krol, A.R., Mol, J.N. and Stuitje, A.R. (1988) Antisense genes in plants: an overview. Gene, 72, 45-50. 131. van Gent, D., Sharp, P., Morgan, K. and Kalsheker, N. (2003) Serpins: structure, function and molecular evolution. Int J Biochem Cell Biol, 35, 1536-1547. 132. Vernadakis, A., Kentroti, S., Brodie, C., Mangoura, D. and Sakellaridis, N. (1991) C-6 glioma cells of early passage have progenitor properties in culture. Adv Exp Med Biol, 296, 181- 195. 133. Vouret-Craviari, V., Van Obberghen-Schilling, E., Rasmussen, U.B., Pavirani, A., Lecocq, J.P. and Pouyssegur, J. (1992) Synthetic alpha-thrombin receptor peptides activate G proteincoupled signaling pathways but are unable to induce mitogenesis. Mol Biol Cell, 3, 95-102. 134. Wilmouth, R.C., Edman, K., Neutze, R., Wright, P.A., Clifton, I.J., Schneider, T.R., Schofield, C.J. and Hajdu, J. (2001) X-ray snapshots of serine protease catalysis reveal a tetrahedral intermediate. Nat Struct Biol, 8, 689-694. 135. Wolfer, D.P., Lang, R., Cinelli, P., Madani, R. and Sonderegger, P. (2001) Multiple roles of neurotrypsin in tissue morphogenesis and nervous system development suggested by the mRNA expression pattern. Mol Cell Neurosci, 18, 407-433. 136. Woodcock, D.M., Crowther, P.J., Doherty, J., Jefferson, S., DeCruz, E., Noyer-Weidner, M., Smith, S.S., Michael, M.Z. and Graham, M.W. (1989) Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res, 17, 3469-3478. 137. Ye, S., Cech, A.L., Belmares, R., Bergstrom, R.C., Tong, Y., Corey, D.R., Kanost, M.R. and Goldsmith, E.J. (2001) The structure of a Michaelis serpin-protease complex. Nat Struct Biol, 8, 979-983. 138. Yoshida, S. and Shiosaka, S. (1999) Plasticity-related serine proteases in the brain (review). Int J Mol Med, 3, 405-409.
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/10 - Biochimica
Date Deposited: 04 Aug 2008
Last Modified: 29 Oct 2014 12:11
URI: http://www.fedoa.unina.it/id/eprint/626
DOI: 10.6092/UNINA/FEDOA/626

Collection description

Le serpine (serine proteinase inhibitors) sono una superfamiglia di proteine caratterizzate da un comune ripiegamento e dal meccanismo d'inibizione che è del tipo substrato suicida. Il meccanismo di inibizione consiste nella formazione di un complesso irreversibile tra serpina e proteasi che una volta internalizzato viene degradato. Tra le serpine la Proteasi Nexina-1 (PN1), uno tra i maggiori inibitori della trombina, è presente principalmente nella matrice del sistema nervoso centrale. La sua funzione in questo distretto si pensa sia legato all’inibizione della trombina, che su cellule nervose differenziate causa retrazione dei neuriti. Inoltre, la PN1 è espressa con un particolare pattern spaziale e temporale durante lo sviluppo e l’organogenesi del sistema nervoso centrale, che fa supporre un suo coinvolgimento in questi processi. Per chiarire le funzioni della PN1 sono stati messi a punto un sistema per l’over-espressione inducibile e un sistema per il silenziamento genico di questa proteina. Nel primo caso è stato utilizzato come modello sperimentale il sistema tet-off in cellule PC12, cellule di feocromocitoma di ratto. Le cellule PC12 sono in grado di differenziarsi in neuroni mediante aggiunta di Nerve Growth Factor (NGF) o mediante deprivazione di siero. Il sistema tet-off permette di indurre l’over-espressione della proteina di interesse e studiarne gli effetti sulle cellule. Il plasmide per l’over-espressione inducibile contenente il cDNA della PN1 è stato cotransfettato insieme al plasmide contenente il gene per la resistenza all’igromicina nelle cellule PC12 tet-off. I cloni stabili sono stati isolati ed amplificati. Dall’analisi delle proteine, da lisati totali mediante western blot, e dall’analisi dell’mRNA, mediante RT-PCR semiquantitativa, alcuni dei cloni analizzati sono risultati inducibili. I cloni che hanno mostrato una maggiore produzione di PN1, successivamente all’induzione, sono stati analizzati morfologicamente durante il differenziamento, indotto sia dall’aggiunta di NGF al mezzo, sia da deprivazione di siero. Gli esperimenti condotti in presenza di NGF hanno mostrato un ruolo protettivo, nei confronti delle cellule differenziate, dell’over-espressione di PN1 in caso di aggiunta di trombina al mezzo di coltura. Gli esperimenti condotti in assenza di siero mostrano una diminuzione del numero di cellule differenziate in caso di over-espressione di PN1. Dai campioni analizzati morfologicamente durante il differenziamento sono stati preparati i lisati totali per osservare la produzione di PN1 mediante western blot. Da tale analisi è risultato un aumento di espressione della serpina indipendente dall’induzione del sistema tet-off. Tale aumento, da successivi esperimenti su cellule PC12 wild-type, è risultato dipendente sia dal tempo, sia dalla concentrazione di trombina. Per ottenere il silenziamento genico della PN1, è stato utilizzato l’RNA interference in cellule C6. A questo scopo sono stati clonati nel plasmide pAVU6+27 vari tipi di oligonucleotidi, in grado di attivare il processo di RNA interference, a valle di un promotore per piccoli RNA. I plasmidi ottenuti sono stati transfettati nelle cellule C6. A 48 ore dalla transfezione le cellule sono state raccolte per estrarre RNA totale e lisati totali. Dall’analisi dell’RNA mediante northern blot è risultata una diminuzione di mRNA di PN1 variabile a seconda dei costrutti utilizzati, comunque mai superiore al 60%. Dall’analisi dei lisati, mediante western blot, è risultata una diminuzione della proteina di circa 80% in tre dei campioni analizzati. Sebbene il processo di RNA interference non provochi un’efficiente degradazione del mRNA di PN1, c’è un efficiente blocco della traduzione


Downloads per month over past year

Actions (login required)

View Item View Item