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

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Presentation on theme: "Prion Project Presented by Béla Reiz Supervisor: Dr. Liang Li."— Presentation transcript:

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

2 Outline 1.Introduction 2.Structure 3.Experimental 4.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 PrP C (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 PrP C is necessary but not sufficient for the conversion of prions

6 What is the physiological function of PrP C 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 PrP Sc (for scrapie) same primary structure as the PrP C 1 its secondary structure is dominated by beta sheets is insoluble is highly resistant to digestion by proteases PrP Sc molecules bind and form aggregates in the cytoplasmic vesicles of diseased individuals if in contact with PrP c it is capable of converting it into PrP Sc 1 Stanley B. Prusiner, Biochemistry, 32, 1991 (1993)

8 The PRION diseases (animal) DiseaseMechanism of pathogenesis Scrapie (sheep)Infection in genetically susceptible sheep Bovine Spongiform Encephalopathy (BSE, cattle) Infection with prion-contaminated MBM Transmissible mink Encephalopathy (TME, mink) Infection with prions from sheep and cattle Chronic wasting disease (CWD, mule deer, elk) Unknown Feline spongiform encephalopathy (FSE, cats) Infection with prion-contaminated MBM Exotic ungulate encephalopathy (EUE, greater kudu, nyalal, oryx) Infection with prion-contaminated MBM Stanley B. Prusiner, Science, 278, 245 (1997)

9 The PRION diseases (human) DiseaseMechanism of pathogenesis Kuru (Fore people)Infection through ritualistic cannibalism Variant Creutzfeld-Jakob disease (vCJD) Infection from prion-contaminated HGH, dura mater grafts etc. Familial Creutzfeld-Jakob disease (fCJD) Germline mutation in PrP gene Gerstmann-Sträussler-Scheinker disease (GSS) Germline mutation in PrP gene Fatal familial insomnia (FFI)Germline mutation in PrP gene (D178N and M129) Sporadic Creutzfeld-Jakob disease (sCJD) Somatic mutation of spontaneuous conversion of PrP C into PrP Sc ? 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

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

16 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?

17 2.Structure

18 Structure of the PrP C flexible N-terminus 3 alpha helices 2 small beta strands 2 N – glycosylations

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

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

21 3.Experimental

22 Objective Use Mass Spectrometry to characterize the aggregating and aggregated PrP Sc.

23 The SHPrP C 90 – 231 Sequence of the SHPrP C with the purification tag: MGSSHHHHHHSSGLVPRGSHMLEGQGGGTHNQWNKPSKPKTNMK HMAGAAAAGAVVGGLGGYMLGSAMSRPMMHFGNDWEDRYYRENM NRYPNQVYYRPVDQYNNQNNFVHDCVNITIKQHTVTTTTKGENFTET DIKIMERVVEQMCTTQYQKESQAYYDGRRSS Number of AA: 166 Molecular weight: Da Theoretical pI: 8.85 Instability index: GRAVY:

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

25 HPLC spectrum of the SHPrP C tryptic digest

26 Sequence coverage for the Tryptic digest Sequence identified: 126 AA MGSSHHHHHHSSGLVPRGSHMLEGQGGGTHNQWNKPSKPKTN MKHMAGAAAAGAVVGGLGGYMLGSAMSRPMMHFGNDWEDRYY RENMNRYPNQVYYRPVDQYNNQNNFVHDCVNITIKQHTVTTTTKG ENFTETDIKIMERVVEQMCTTQYQKESQAYYDGRRSS Identified only by MS Coverage: 76% Sequence identified by MSMS: 97 AA Coverage: 58%

27 Sequence coverage for the Chymotryptic digest

28 4.Future Work

29 Use MS to determine: 1.surface residue location 2.cysteine placement 3.residue-residue proximity 4.residue-specific hydrogen exchange 5.secondary structure Future Work

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

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

32 Thanks! Dr. Liang Li All Li group members


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