1. Presented by Béla Reiz Supervisor: Dr. Liang Li
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)
Mechanism 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)
Exotic ungulate encephalopathy (EUE, greater kudu, nyalal, oryx)
MBM = meat and bone meal
HGH = human growth hormone
Stanley B. Prusiner, Science, 278, 245 (1997)

9. The PRION diseases (human)
Mechanism 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)
Fatal familial insomnia (FFI)
Germline mutation in PrP gene (D178N and M129)
Sporadic Creutzfeld-Jakob disease (sCJD)
Somatic mutation of spontaneuous conversion of PrPC into PrPSc?
Stanley B. Prusiner, Science, 278, 245 (1997)

10. Prion infection mechanism

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

12. Protein only model of infection
This is a famous paper (379 citations) commonly said to show that nucleic acids were not part of the scrapie agent.
They irradiated with UV light at two wavelengths, using the highly resistant organism Micorcoccus radiodurans as control. UV light forms cyclobutadiene thymine dimers in DNA whenever two T's are adjacent; these prevent the DNA from being replicated. Earlier studies had found the same result with ionizing radiation. Scrapie resistance was complete at dosages that knocked the control viability down 3 logs in activity, giving rise to the oft-cited Fig1 and 2 of this paper. Residual scrapie titer was measured by serial dilution, using intra-cranial injection in 7-8 mice at every dilution.
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)

15. Identification of PrP using HPLC-MS
RP-HPLC C18 column, 2.1x220, 5 μm particle size, 300 Å pore size, Vydac.
Linear gradient of 1-60% ACN containing 0.05% TFA in 60 min.
ES-IT, mod. LCQ, ThermoFinnigan; pozitive mode, on-line;
SIM (selected ion monitoring) mode was used for ion current monitoring of the reporter peptide
SRM (selective reaction monitoring) mode for ion current monitoring of the b2 product ion m/z = 216±0.3
As expected, the high complexity of the sample precluded any peptide identification when the
HPLC eluate was monitored by total ion current in the full mass mode (Fig. 2A). However, the hamster
prion peptide 107–110 could be detected using a SIM mode (Fig. 2B). The averaged mass spectrum acquired
in the narrow scan range of 491–495 amu for the intact ion and its fragmentation pattern are both
in perfect agreement with that calculated for the hamster prion reporter peptide (Figs. 2C and 2D). On
the contrary, peptide 186–194 gave only a weak signal, probably due to its chemicophysical properties
(data not shown). For this reason, in the following analyses we considered only peptide 107–110 as PrP
reporter peptide ion.
Going one step further on this approach, we attempted to identify the PrP reporter peptide directly
monitoring for the b2 product ion (m/z of 216.0) according to the fragmentation pattern in Fig. 2D. As
known, this latter mode of MS analysis is generally referred to as SRM mode. As indicated in Fig. 3,
unequivocal detection of the hamster prion in the complex matrix was obtained.
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 PrPC flexible N-terminus 3 alpha helices
2 small beta strands
2 N – glycosylations

19. Structure of the PrPC
Figure 1. Primary structure of the cellular PrP including post-translational modifications
GPI = glycosyl phosphatidyl inositol
A. Aguzzi, M. Heikenwalder, Microbiology, 4, 765 (2006)

20. Figure 2. Tertiary structure of the cellular PrP
Structure of the PrPC
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 PrPSc.

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

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

25. HPLC spectrum of the SHPrPC tryptic digest

26. 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%

27. Sequence coverage for the Chymotryptic digest

28. 4.Future Work

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

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