Springer Protocols: Abstract: 2. Investigation of PrPC Metabolism and Function in Live Cells: Methods for Studying Individual Cells and Cell Populations

2. Investigation of PrPC Metabolism and Function in Live Cells: Methods for Studying Individual Cells and Cell Populations

By: Cathryn L. Haigh³, David R. Brown⁴

Abstract

Full Text

Download PDF (602K)

Prion protein (PrP)^C expression levels and protein localization are known to be affected by factors such as metal ions and oxidative stress. By the development of a green fluorescent protein (GFP)-PrP^C fusion protein, the movement of PrP can be followed in real time. Furthermore, alterations in cellular metabolism can be detected while cells are still viable. The internalization response of PrP to 20 μM manganese (Mn) in divalent metal ion-depleted media is used to demonstrate the movement of GFP-tagged proteins in live cells and real tim0e. A live cell microtiter plate assay shows that PrP null cells are less capable of dealing with Mn-induced oxidative stress. In addition, this chapter outlines several complementary techniques for studying live cells and GFP fusion proteins.

Affiliation(s): (3) Department of Pathology and Mental Health Research Institute of Victoria, University of Melbourne, Melbourne, Australia
(4) Department of Biochemistry, Bath University, Bath, UK

Book Title: Prion Protein Protocols

Series: Methods in Molecular Biology | Volume: 459 | Pub. Date: Jun-04-2008 | Page Range: 21-34 | DOI: 10.1007/978-1-59745-234-2_2

Subject: Protein Science

Key Words: Confocal imaging - colocalization - dichlorodihydrofluorescein diace-tate (DCFDA) - native polyacrylamide gel electrophoresis (PAGE) - reactive oxygen species (ROS) - trafficking.

References

Download References

1.	Nunziante, M. Gilch, S. Schatzl, HM. (2003) Essential role of the prion protein N-terminus in subcellular trafficking and half-life of PrPc. Journal of Biological Chemistry 278(6), 3726–3734.

2.	Winklhofer, KF., Heske, J., Heller, U., Reintjes, A., Muranyi, W., Moarefi, I., Tatzelt, J. (2003) Determinants of the in vivo folding of the prion protein: a bipartate function of helix 1 in folding and aggregation. Journal of Biological Chemistry 278(17), 14961–14970.

3.	Hornshaw, MP., McDermott, JR., Candy, JM., Lakey, JH. (1995) Copper binding to the N-ter-minal tandem repeat region of mammalian and avian prion protein: structural studies using synthetic peptides. Biochemical and Biophysical Research Communications 214(3), 993–999.

4.	Brown, DR., Qin, K., Herms, JW., Madlung, A., Manson, J., Strome, R., Fraser, PE., Kruck, T., Bohlen, A., Schulz-Schaeffer, W., Giese, A., Westaway, D., Kretzschmar, H. (1997) The cellular prion protein binds copper in vivo. Nature 390, 684–687.

5.	Hasnain, SS., Murphy, LM., Strange, RW., Grossmann, JG., Clarke, AR., Jackson, GS., Collinge, J. (2001) XAFS study of the high-affinity copper-binding site of human PrP^91–231 and its low-resolution structure in solution. Journal of Molecular Biology 311, 467–473.

6.	Jackson, GS., Murray, I., Hosszu, LLP., Gibbs, N., Waltho, JP., Clarke, AR., Collinge, J. (2001) Location and properties of metal-binding sites on the human prion protein. Proceedings of the National Academy of Sciences USA. 98(15), 8531–8535.

7.	Pauly, PC., Harris, DA. (1998) Copper stimulates endocytosis of the prion protein. Journal of Biological Chemistry 273(50), 33107–33110.

8.	Lee, KS., Magalhães, AC., Zanata, SM., Brentani, RR., Martins, VR. Prado, MA. (2001) Internalisation of mammalian fluorescent cellular prion protein and N-terminal deletion mutants in living cells. Journal of Neurochemistry 79(1), 79–87.

9.	Sunyach, C., Jen, A., Deng, J., Fitzgerald, KT., Frobert, Y. , Grassi, J., McCaffrey, MW., Morris, R. (2003) The mechanism of internalisation of glycosylphosphatidylinositol-anchored prion protein. EMBO Journal 22(14), 3591–3601.

10.	Haigh, CL., Edwards, KE., Brown, DR. (2005) Copper binding is the governing determinant of prion protein turnover. Molecular and Cellular Neuroscience 30, 186–96.

11.	Brown, DR., Schmidt, B., Kretzschmar, HA. (1998) Effects of copper on survival of prion protein knockout neurons and glia. Journal of Neurochemistry 70, 1686–93.

12.	Wong, BS., Brown, DR., Pan, T., Whitemann, M., Liu, T., Bu, X., Li, R., Gambetti, P., Olesik, J., Rubenstein, R., Sy, MS. (2001) Oxidative impairment in scrapie-infected mice is associated with brain metal perturbations and altered anti-oxidant activities. Journal of Neurochemistry 79, 689–698.

13.	Thackray, AM., Knight, R., Haswell, SJ., Bujdoso, R., Brown, DR. (2002) Metal imbalance and compromised anti-oxidant function are early changes in prion disease. Biochemical Journal 362, 253–258.

14.	Brown, DR., Besinger, A. (1998) Prion protein expression and superoxide dismutase activity. Biochemical Journal 334, 423–429.

15.	Brown, DR., Clive, C., Haswell, SJ. (2001) Antioxidant activity related to copper binding of native prion protein. Journal of Neurochemistry 76, 69–76.

16.	Brown, DR., Hafiz, F., Glasssmith, LL., Wong, BS., Jones, IM., Clive, C., Haswell, SJ. (2000) Consequences of manganese replacement of copper for prion protein function and proteinase resistance. EMBO Journal 19(6), 1180–1186.

17.	Holme, A., Daniels, M., Sassoon, J., Brown, DR. A novel method of generating neuronal cell lines from gene-knockout mice to study prion protein membrane orientation. European Journal of Neuroscience 18, 571–579.

18.	Dawson, RM., Elliott, DC., Elliott, WH., Jones, KM. (1994) Data for Biochemical Research (third edition). Oxford Scientific Publications, Oxford, UK.

19.	Martin, BD., Schoenhard, JA., Sugden, KD. (1998) Hypervalent chromium mimics reactive oxygen species as measured by the oxidant-sensitive dyes 2′,7′-dichlorofluorescin and dihydrorhodamine. Chemical Research and Toxicology 11(12), 1402–10.

Comments (Loading...)

Post comment/View all comments