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Prion Project

Prion Project. Presented by B é la Reiz Supervisor: Dr. Liang Li. Outline. Introduction Structure Experimental Future Work. 1.Introduction. What are prions?.

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Prion Project

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  1. Prion Project Presented by Béla Reiz Supervisor: Dr. Liang Li

  2. Outline • Introduction • Structure • Experimental • Future Work

  3. 1.Introduction

  4. What are prions? • Prions are a type of infectious particles that turn out to be molecules of a normal body protein that have changed their three-dimensional configuration. • “Prion” is derived from small proteinaceous infectious particle which resists procedures that modify nucleic acids. • PrP = prion-related protein or protease-resistant protein

  5. The normal protein • is called PrPC (for cellular) • is a naturally occurring protein encoded by the Prnp gene • is a transmembrane glycoprotein predominantly found on the surface of neurons • its secondary structure is dominated by 3 alpha helices • is soluble • is easily digested by proteases • in a given cell type PrPC is necessary but not sufficient for the conversion of prions

  6. What is the physiological function of PrPC • function is still elusive • functions attributed so far: • immunoregulation • signal transduction • copper binding • synaptic transmission • induction or protection against apoptosis A. Aguzzi, Cell 116, 313 (2004)

  7. The abnormal protein • is called PrPSc (for scrapie) • same primary structure as the PrPC 1 • its secondary structure is dominated by beta sheets • is insoluble • is highly resistant to digestion by proteases • PrPSc molecules bind and form aggregates in the cytoplasmic vesicles of diseased individuals • if in contact with PrPc it is capable of converting it into PrPSc 1 Stanley B. Prusiner, Biochemistry, 32, 1991 (1993)

  8. The PRION diseases (animal) Stanley B. Prusiner, Science, 278, 245 (1997)

  9. The PRION diseases (human) Stanley B. Prusiner, Science, 278, 245 (1997)

  10. Prion infection mechanism

  11. Biosafety Hamster recombinant protein • CL1 requirements Bovine PrP • CL2 requirements • seal joints in surfaces • bag-in / bag-out HEPA BSC’s • autoclave in laboratory • dedicated laboratory & equipment

  12. Protein only model of infection T. Alper, W.A. Cramp, D.A. Haig, M.C. Clarke, Nature, 214, 764 (1967).

  13. “PRION” • After infection and a prolonged incubation period, the scrapie agent causes a degenerative disease of the central nervous system in sheep and goats. Six lines of evidence including sensitivity to proteases demonstrate that this agent contains a protein that is required for infectivity. Although the scrapie agent is irreversibly inactivated by alkali, five procedures with more specificity for modifying nucleic acids failed to cause inactivation. The agent shows heterogeneity with respect to size, apparently a result of its hydrophobicity; the smallest form may have a molecular weight of 50,000 or less. Because the novel properties of the scrapie agent distinguish it from viruses, plasmids, and viroids, a new term "prion" is proposed to denote a small proteinaceous infectious particle which is resistant to inactivation by most procedures that modify nucleic acids. Knowledge of the scrapie agent structure may have significance for understanding the causes of several degenerative diseases. Stanley B. Prusiner, Science, 216, 136 (1982)

  14. Identification of PrP using HPLC-MS Schinina et al., Pure Appl. Chem., 75, 2-3 (2003)

  15. Future of PRION science • What is the precise physical structure of the protein? • What is the biochemical basis of the prion strain? • Is there a species barrier? • What factors determine the species barrier in prion infections? • What are the host susceptibility factors that promote prion infection? • What are the molecular mechanisms that will underpin an efficacious therapy?

  16. 2.Structure

  17. Structure of the PrPC • flexible N-terminus • 3 alpha helices • 2 small beta strands • 2 N – glycosylations

  18. Structure of the PrPC Figure 1. Primary structure of the cellular PrP including post-translational modifications A. Aguzzi, M. Heikenwalder, Microbiology, 4, 765 (2006)

  19. Structure of the PrPC Figure 2. Tertiary structure of the cellular PrP A. Aguzzi, M. Heikenwalder, Microbiology, 4, 765 (2006)

  20. 3.Experimental

  21. Objective Use Mass Spectrometry to characterize the aggregating and aggregated PrPSc.

  22. The SHPrPC 90 – 231 • Sequence of the SHPrPC with the purification tag: MGSSHHHHHHSSGLVPRGSHMLEGQGGGTHNQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPMMHFGNDWEDRYYRENMNRYPNQVYYRPVDQYNNQNNFVHDCVNITIKQHTVTTTTKGENFTETDIKIMERVVEQMCTTQYQKESQAYYDGRRSS • Number of AA: 166 • Molecular weight: 18866.9 Da • Theoretical pI: 8.85 • Instability index: 38.01 • GRAVY: -0.989

  23. Protein MS Digestion (Peptides) MS LC-MALDI MS MSMS Database Search Experimental Procedure

  24. HPLC spectrum of the SHPrPC tryptic digest

  25. Sequence coverage for the Tryptic digest • Sequence identified: 126 AA MGSSHHHHHHSSGLVPRGSHMLEGQGGGTHNQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPMMHFGNDWEDRYYRENMNRYPNQVYYRPVDQYNNQNNFVHDCVNITIKQHTVTTTTKGENFTETDIKIMERVVEQMCTTQYQKESQAYYDGRRSS Identified only by MS Coverage: 76% • Sequence identified by MSMS: 97 AA • Coverage: 58%

  26. Sequence coverage for the Chymotryptic digest

  27. 4.Future Work

  28. Future Work Use MS to determine: • surface residue location • cysteine placement • residue-residue proximity • residue-specific hydrogen exchange • secondary structure

  29. Chemical modification of surface exposed residues using N-bromosuccinimide (NBS) Yash P. Myer, Biochemistry, 11, 23 (1972)

  30. Nitrosylation of surface exposed Tyrosine residues using tetranitromethane (TNM) J.F. Leite, M. Cascio, Biochemistry, 41, 19 (2002)

  31. Thanks! • Dr. Liang Li • All Li group members

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