JoVE Journal > Immunology and Infection
Summary
Abstract
Protocol
Discussion
Divulgazioni
Acknowledgements
Materials
Riferimenti
Traduzione automatica
Please note that all translations are automatically generated. Click here for the English version.
Immunologia e infezioni

Yüzey Plasmon Rezonans Host Cytosol Toksin Translokasyon Tespiti

Published: January 03, 2012
doi:
10.3791/3686
, , ,
1Department of Molecular Biology and Microbiology,University of Central Florida

Summary

Bu raporda, yüzey plazmon rezonans ev sahibi sitoplazmada içine toksin girişini tespit etmek için nasıl kullanıldığını açıklar. Bu son derece duyarlı bir yöntem sitozolik toksin miktarı nicel veri sağlayabilir ve toksinlerin bir dizi yapılabilir.

Abstract

AB toksinler A alt biriminin bir enzimatik ve hücre bağlayıcı B alt ünite 1 oluşur. Bu toksinler ekstraselüler ortam içine salgılanan, ancak ökaryotik sitoplazmada içinde hedefleri doğrultusunda hareket. Bazı AB toksinler hücre yüzeyinde sitoplazmada 2-4 girmeden önce endoplazmik retikulum (ER) vezikül taşıyıcılar tarafından seyahat ER, toksin ve hamle geri kalanı protein iletken bir kanal aracılığıyla katalitik Bir zincir ayrışmaktadır onun sitozolik hedef 5 ulaşmak için . ER toksin ticareti son derece verimsiz bir süreç olmasından dolayı, transloke, sitozolik zinciri tespit etmek zordur: en içselleştirilmiş toksin golgi ve ER 6 ulaşır yüzey bağlı toksin yüzden sadece küçük bir bölümünü, bozulması için lizozomlar yönlendirilir -12.

ER kültürlü hücrelerde sitoplazmada toksin translokasyon izlemek için highl Subselüler fraksiyonasyon protokolü kombineyüzey plazmon rezonans (SPR) 13-15 y hassas algılama yöntemi. Toksin-tedavili hücrelerde plazma zarı seçici, daha sonra bir anti-toksin A zinciri antikor ile kaplı bir SPR sensör üzerinden perfüze bir sitozolik fraksiyonu toplanması için izin verirler digitonin ile permeabilize. Antikor kaplı sensör sitozolik toksin pg / mL miktarlarda yakalamak ve tespit edebilir. Bu protokol ile, sitoplazmada toksin giriş kinetiği takip etmek ve translokasyon olayı üzerine inhibitör etkileri karakterize etmek mümkündür. Sitozolik toksin konsantrasyonu sensör üzerindeki perfüze edilmiştir zinciri standartları bilinen miktarlarda üretilen standart bir eğri de hesaplanabilir. Bizim yöntem, hedef toksin Radyoaktif veya diğer modifikasyonlar gerektirmeyen bir, hızlı, duyarlı, ve kantitatif algılama sistemi temsil eder.

Protocol

1. Digitonin, hazırlanması Bir mikrosantrifüj tüp 500% 100 etanol mcL ekleyin ve 80 ° C de 10 dakika süreyle bir ısı bloğu yerleştirin. Digitonin% 1 hisse çözüm üretmek için ısıtmalı etanol 250 mcL digitonin 2.5 mg eritin. % 0.04 digitonin çalışan bir çözüm üretmek için, 960 mcL HCN tampon (50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM CaCl 2, 10 mM N-ethylmaleimide, ve proteaz inhibitörü digitonin stok solüsyonu 40 mcL ekleyin kokteyl). <p class="…

Discussion

Mevcut metodoloji ile Karşılaştırma

Bizim SPR tabanlı translokasyon tahlil ev sahibi sitoplazmada içine toksin teslim algılamak için hızlı, duyarlı ve kantitatif bir yöntem temsil eder. Teknik Radyoaktif veya toksin diğer modifikasyonlar gerektirebilir ve bu zinciri antikor mevcut olan bir anti-toksin herhangi bir toksin uygulanabilir. Sitoplazmada içine toksin geçişini izlemek için varolan yöntemleri de ayrı sitoplazmada ve membran fraksiyonları 11, 23, 27, 28 b…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

Bu çalışma K. Teter NIH hibe R01 AI073783 tarafından finanse edildi. Biz yazının eleştirel okuma Subselüler fraksiyonasyon protokolü ve Helen Burress gelişimi Dr. Shane Massey yardım için teşekkür ederim.

Materials

Name of the reagent Company Catalogue number
Digitonin Sigma D141
Ethanol Acros 61509-0010
DMEM Invitrogen 11995065
Fetal Bovine Serum Atlanta Biologicals S11550
Ganglioside GM1 Sigma G7641
CTA Sigma C2398
PTS1 List 182
NHS (N-Hydroxysuccinimide) Pierce 24500
EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) Thermo Scientific 22981
Ethanolamine Sigma E0135
PBST Medicago 09-8903-100
Anti-CTA antibody Santa Cruz Biotech sc-80747
Anti-CTB antibody Calbiochem 227040
Anti-PTS1 antibody Santa Cruz Biotech sc-57639
Refractometer Reichert SR7000, SR7000DC
SPR sensor slides Reichert 13206060
Syringe pump Cole Palmer 780200C
DOWNLOAD MATERIALS LIST

Riferimenti

  1. Sandvig, K., van Deurs, B. Membrane traffic exploited by protein toxins. Annu. Rev. Cell Dev. Biol. 18, 1-24 (2002).
  2. Watson, P., Spooner, R. A. Toxin entry and trafficking in mammalian cells. Adv. Drug Deliv. Rev. 58, 1581-1596 (2006).
  3. Carbonetti, N. H. Pertussis toxin and adenylate cyclase toxin: key virulence factors of Bordetella pertussis and cell biology tools. Future Microbiol. 5, 455-469 (2010).
  4. Wernick, N. L. B., Chinnapen, D. J. -. F., Cho, J. A., Lencer, W. I. Cholera toxin: an intracellular journey into the cytosol by way of the endoplasmic reticulum. Toxins. 2, 310-325 (2010).
  5. Lord, J. M., Roberts, L. M., Lencer, W. I. Entry of protein toxins into mammalian cells by crossing the endoplasmic reticulum membrane: co-opting basic mechanisms of endoplasmic reticulum-associated degradation. Curr. Top. Microbiol. Immunol. 300, 149-168 (2005).
  6. Lencer, W. I. Entry of cholera toxin into polarized human intestinal epithelial cells. Identification of an early brefeldin A sensitive event required for A1-peptide generation. J. Clin. Invest. 92, 2941-2951 (1993).
  7. Orlandi, P. A., Curran, P. K., Fishman, P. H. Brefeldin A blocks the response of cultured cells to cholera toxin. Implications for intracellular trafficking in toxin action. J. Biol. Chem. 268, 12010-12016 (1993).
  8. Sandvig, K., Prydz, K., Hansen, S. H., van Deurs, B. Ricin transport in brefeldin A-treated cells: correlation between Golgi structure and toxic effect. J. Cell. Biol. 115, 971-981 (1991).
  9. Sandvig, K., Prydz, K., Ryd, M., van Deurs, B. Endocytosis and intracellular transport of the glycolipid-binding ligand Shiga toxin in polarized MDCK cells. J. Cell Biol. 113, 553-562 (1991).
  10. van Deurs, B. Estimation of the amount of internalized ricin that reaches the trans-Golgi network. J. Cell Biol. 106, 253-267 (1988).
  11. Tam, P. J., Lingwood, C. A. Membrane cytosolic translocation of verotoxin A1 subunit in target cells. Microbiology. 153, 2700-2710 (2007).
  12. Plaut, R. D., Carbonetti, N. H. Retrograde transport of pertussis toxin in the mammalian cell. Cell. Microbiol. 10, 1130-1139 (2008).
  13. Willander, M., Al-Hilli, S. Analysis of biomolecules using surface plasmons. Methods Mol. Biol. 544, 201-229 (2009).
  14. Homola, J. Present and future of surface plasmon resonance biosensors. Anal. Bioanal. Chem. 377, 528-539 (2003).
  15. Medaglia, M. V., Fisher, R. J., Golemis, E. . Protein-Protein Interactions. , 255-272 (2002).
  16. Banerjee, T. Contribution of subdomain structure to the thermal stability of the cholera toxin A1 subunit. Biochimica. 49, 8839-8846 (2010).
  17. Massey, S. Stabilization of the tertiary structure of the cholera toxin A1 subunit inhibits toxin dislocation and cellular intoxication. J. Mol. Biol. 393, 1083-1096 (2009).
  18. Taylor, M. A therapeutic chemical chaperone inhibits cholera intoxication and unfolding/translocation of the cholera toxin A1 subunit. PLoS ONE. 6, e18825-e18825 (2011).
  19. Taylor, M. Hsp90 is required for transfer of the cholera toxin A1 subunit from the endoplasmic reticulum to the cytosol. J. Biol. Chem. 285, 31261-31267 (2010).
  20. Donta, S. T., Beristain, S., Tomicic, T. K. Inhibition of heat-labile cholera and Escherichia coli enterotoxins by brefeldin A. Infect. Immun. 61, 3282-3286 (1993).
  21. Donta, S. T., Tomicic, T. K., Donohue-Rolfe, A. Inhibition of Shiga-like toxins by brefeldin. A. J. Infect. Dis. 171, 721-724 (1995).
  22. Nambiar, M. P., Oda, T., Chen, C., Kuwazuru, Y., Wu, H. C. Involvement of the Golgi region in the intracellular trafficking of cholera toxin. J. Cell. Physiol. 154, 222-228 (1993).
  23. Rapak, A., Falnes, P. O., Olsnes, S. Retrograde transport of mutant ricin to the endoplasmic reticulum with subsequent translocation to cytosol. Proc. Natl. Acad. Sci. U. S. A. 94, 3783-3788 (1997).
  24. Xu, Y., Barbieri, J. T. Pertussis toxin-mediated ADP-ribosylation of target proteins in Chinese hamster ovary cells involves a vesicle trafficking mechanism. Infect. Immun. 63, 825-832 (1995).
  25. Yoshida, T., Chen, C. C., Zhang, M. S., Wu, H. C. Disruption of the Golgi apparatus by brefeldin A inhibits the cytotoxicity of ricin, modeccin, and Pseudomonas toxin. Exp. Cell Res. 192, 389-395 (1991).
  26. Godber, B. Direct quantification of analyte concentration by resonant acoustic profiling. Clin. Chem. 51, 1962-1972 (2005).
  27. Bernardi, K. M., Forster, M. L., Lencer, W. I., Tsai, B. Derlin-1 facilitates the retro-translocation of cholera toxin. Mol. Biol. Cell. 19, 877-884 (2008).
  28. Wernick, N. L., De Luca, H., Kam, W. R., Lencer, W. I. N-terminal Extension of the Cholera Toxin A1-chain Causes Rapid Degradation after Retrotranslocation from Endoplasmic Reticulum to Cytosol. J. Biol. Chem. 285, 6145-6152 (2010).
  29. Simpson, J. C. Ricin A chain utilises the endoplasmic reticulum-associated protein degradation pathway to enter the cytosol of yeast. FEBS. Lett. 459, 80-84 (1999).
  30. Veithen, A., Raze, D., Locht, C. Intracellular trafficking and membrane translocation of pertussis toxin into host cells. Int. J. Med. Microbiol. 290, 409-413 (2000).
  31. Castro, M. G., McNamara, U., Carbonetti, N. H. Expression, activity and cytotoxicity of pertussis toxin S1 subunit in transfected mammalian cells. Cell. Microbiol. 3, 45-54 (2001).
  32. Schmitz, A., Herrgen, H., Winkeler, A., Herzog, V. Cholera toxin is exported from microsomes by the Sec61p complex. J. Cell Biol. 148, 1203-1212 (2000).
  33. Teter, K., Allyn, R. L., Jobling, M. G., Holmes, R. K. Transfer of the cholera toxin A1 polypeptide from the endoplasmic reticulum to the cytosol is a rapid process facilitated by the endoplasmic reticulum-associated degradation pathway. Infect. Immun. 70, 6166-6171 (2002).
  34. Winkeler, A., Godderz, D., Herzog, V., Schmitz, A. BiP-dependent export of cholera toxin from endoplasmic reticulum-derived microsomes. FEBS Lett. 554, 439-442 (2003).
  35. Yu, M., Haslam, D. B. Shiga toxin is transported from the endoplasmic reticulum following interaction with the luminal chaperone HEDJ/ERdj3. Infect. Immun. 73, 2524-2532 (2005).
  36. LaPointe, P., Wei, X., Gariepy, J. A role for the protease-sensitive loop region of Shiga-like toxin 1 in the retrotranslocation of its A1 domain from the endoplasmic reticulum lumen. J. Biol. Chem. 280, 23310-23318 (2005).
  37. Teter, K., Jobling, M. G., Sentz, D., Holmes, R. K. The cholera toxin A13 subdomain is essential for interaction with ADP-ribosylation factor 6 and full toxic activity but is not required for translocation from the endoplasmic reticulum to the cytosol. Infect. Immun. 74, 2259-2267 (2006).
  38. Redmann, V. Dislocation of ricin toxin a chains in human cells utilizes selective cellular factors. J. Biol. Chem. 286, 21231-21238 (2011).
  39. Yamaizumi, M., Mekada, E., Uchida, T., Okada, Y. One molecule of diphtheria toxin fragment A introduced into a cell can kill the cell. Cell. 15, 245-250 (1978).
  40. Bellisola, G. Reductive activation of ricin and ricin A-chain immunotoxins by protein disulfide isomerase and thioredoxin reductase. Biochem. Pharmacol. 67, 1721-1731 (2004).
  41. McKee, M. L., FitzGerald, D. J. Reduction of furin-nicked Pseudomonas exotoxin A: an unfolding story. Biochimica. 38, 16507-16513 (1999).
  42. Orlandi, P. A. Protein-disulfide isomerase-mediated reduction of the A subunit of cholera toxin in a human intestinal cell line. J. Biol. Chem. 272, 4591-4599 (1997).
  43. Spooner, R. A. Protein disulphide-isomerase reduces ricin to its A and B chains in the endoplasmic reticulum. Biochem. J. 383, 285-293 (2004).
  44. Fujinaga, Y. Gangliosides that associate with lipid rafts mediate transport of cholera and related toxins from the plasma membrane to endoplasmic reticulm. Mol. Biol. Cell. 14, 4783-4793 (2003).
  45. Guerra, L. Cellular internalization of cytolethal distending toxin: a new end to a known pathway. Cell. Microbiol. 7, 921-934 (2005).
  46. Johannes, L., Tenza, D., Antony, C., Goud, B. Retrograde transport of KDEL-bearing B-fragment of Shiga toxin. J. Biol. Chem. 272, 19554-19561 (1997).
  47. Deeks, E. D. The low lysine content of ricin A chain reduces the risk of proteolytic degradation after translocation from the endoplasmic reticulum to the cytosol. Biochimica. 41, 3405-3413 (2002).
  48. Hazes, B., Read, R. J. Accumulating evidence suggests that several AB-toxins subvert the endoplasmic reticulum-associated protein degradation pathway to enter target cells. Biochimica. 36, 11051-11054 (1997).
  49. Rodighiero, C., Tsai, B., Rapoport, T. A., Lencer, W. I. Role of ubiquitination in retro-translocation of cholera toxin and escape of cytosolic degradation. EMBO Rep. 3, 1222-1227 (2002).
  50. Worthington, Z. E., Carbonetti, N. H. Evading the proteasome: absence of lysine residues contributes to pertussis toxin activity by evasion of proteasome degradation. Infect. Immun. 75, 2946-2953 (2007).
  51. Pande, A. H., Moe, D., Jamnadas, M., Tatulian, S. A., Teter, K. The pertussis toxin S1 subunit is a thermally unstable protein susceptible to degradation by the 20S proteasome. Biochimica. 45, 13734-13740 (2006).
  52. Pande, A. H. Conformational instability of the cholera toxin A1 polypeptide. J. Mol. Biol. 374, 1114-1128 (2007).

Tags

Toxin TranslocationSurface Plasmon ResonanceAB ToxinsCell-binding B SubunitVesicle CarriersEndoplasmic Reticulum (ER)Catalytic A ChainProtein-conducting ChannelCytosolic TargetToxin TraffickingLysosomesGolgi ApparatusSubcellular Fractionation ProtocolDigitonin PermeabilizationAnti-toxin A Chain AntibodyDetection Method
check_url/it/3686?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citazione di questo articolo
Taylor, M., Banerjee, T., VanBennekom, N., Teter, K. Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance. J. Vis. Exp. (59), e3686, doi:10.3791/3686 (2012).
Scarica citazione
Ristampe e autorizzazioni

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image