Transcript Mass Spectrometry of Prions

Prion Project
Presented by Béla Reiz
Supervisor: Dr. Liang Li
Outline
1.
Introduction
2.
Structure
3.
Experimental
4.
Future Work
1.Introduction
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
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
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)
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)
The PRION diseases (animal)
Disease
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 Infection with prion-contaminated
(FSE, cats)
MBM
Exotic ungulate encephalopathy
(EUE, greater kudu, nyalal, oryx)
Infection with prion-contaminated
MBM
Stanley B. Prusiner, Science, 278, 245 (1997)
The PRION diseases (human)
Disease
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)
Germline mutation in PrP gene
Fatal familial insomnia (FFI)
Germline mutation in PrP gene
(D178N and M129)
Sporadic Creutzfeld-Jakob disease Somatic mutation of spontaneuous
(sCJD)
conversion of PrPC into PrPSc?
Stanley B. Prusiner, Science, 278, 245 (1997)
Prion infection mechanism
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
Protein only model of infection
T. Alper, W.A. Cramp, D.A. Haig, M.C. Clarke, Nature, 214, 764 (1967).
“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)
Identification of PrP using HPLC-MS
Schinina et al., Pure Appl. Chem., 75, 2-3 (2003)
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?
2.Structure
Structure of the PrPC
• flexible N-terminus
• 3 alpha helices
• 2 small beta strands
• 2 N – glycosylations
Structure of the PrPC
Figure 1. Primary structure of the cellular PrP including post-translational
modifications
A. Aguzzi, M. Heikenwalder, Microbiology, 4, 765 (2006)
Structure of the PrPC
Figure 2. Tertiary structure of the cellular PrP
A. Aguzzi, M. Heikenwalder, Microbiology, 4, 765 (2006)
3.Experimental
Objective
Use Mass Spectrometry to characterize the aggregating and aggregated PrPSc.
The SHPrPC 90 – 231
• Sequence of the SHPrPC with the purification tag:
MGSSHHHHHHSSGLVPRGSHMLEGQGGGTHNQWNKPSKPKTNMK
HMAGAAAAGAVVGGLGGYMLGSAMSRPMMHFGNDWEDRYYRENM
NRYPNQVYYRPVDQYNNQNNFVHDCVNITIKQHTVTTTTKGENFTET
DIKIMERVVEQMCTTQYQKESQAYYDGRRSS
• Number of AA: 166
• Molecular weight: 18866.9 Da
• Theoretical pI: 8.85
• Instability index: 38.01
• GRAVY: -0.989
Experimental Procedure
Protein
MS
Digestion (Peptides)
MS
LC-MALDI
MS
MSMS
Database Search
HPLC spectrum of the SHPrPC tryptic digest
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%
Sequence coverage for the Chymotryptic digest
4.Future Work
Future Work
Use MS to determine:
1.
surface residue location
2.
cysteine placement
3.
residue-residue proximity
4.
residue-specific hydrogen exchange
5.
secondary structure
Chemical modification of surface exposed
residues using N-bromosuccinimide (NBS)
Yash P. Myer, Biochemistry, 11, 23 (1972)
Nitrosylation of surface exposed Tyrosine
residues using tetranitromethane (TNM)
J.F. Leite, M. Cascio, Biochemistry, 41, 19 (2002)
Thanks!
• Dr. Liang Li
• All Li group members