4What 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
5The normal protein is called PrPC (for cellular) is a naturally occurring protein encoded by the Prnp geneis a transmembrane glycoprotein predominantly found on the surface of neuronsits secondary structure is dominated by 3 alpha helicesis solubleis easily digested by proteasesin a given cell type PrPC is necessary but not sufficient for the conversion of prions
6What is the physiological function of PrPC function is still elusivefunctions attributed so far:immunoregulationsignal transductioncopper bindingsynaptic transmissioninduction or protection against apoptosisA. Aguzzi, Cell 116, 313 (2004)
7The abnormal protein is called PrPSc (for scrapie) same primary structure as the PrPC 1its secondary structure is dominated by beta sheetsis insolubleis highly resistant to digestion by proteasesPrPSc molecules bind and form aggregates in the cytoplasmic vesicles of diseased individualsif in contact with PrPc it is capable of converting it into PrPSc1 Stanley B. Prusiner, Biochemistry, 32, 1991 (1993)
8The PRION diseases (animal) Mechanism of pathogenesisScrapie (sheep)Infection in genetically susceptible sheepBovine Spongiform Encephalopathy (BSE, cattle)Infection with prion-contaminated MBMTransmissible mink Encephalopathy (TME, mink)Infection with prions from sheep and cattleChronic wasting disease (CWD, mule deer, elk)UnknownFeline spongiform encephalopathy (FSE, cats)Exotic ungulate encephalopathy (EUE, greater kudu, nyalal, oryx)MBM = meat and bone mealHGH = human growth hormoneStanley B. Prusiner, Science, 278, 245 (1997)
9The PRION diseases (human) Mechanism of pathogenesisKuru (Fore people)Infection through ritualistic cannibalismVariant Creutzfeld-Jakob disease (vCJD)Infection from prion-contaminated HGH, dura mater grafts etc.Familial Creutzfeld-Jakob disease (fCJD)Germline mutation in PrP geneGerstmann-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)
11Biosafety Hamster recombinant protein CL1 requirements Bovine PrP seal joints in surfacesbag-in / bag-out HEPA BSC’sautoclave in laboratorydedicated laboratory & equipment
12Protein 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)
15Identification 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 peptideSRM (selective reaction monitoring) mode for ion current monitoring of the b2 product ion m/z = 216±0.3As expected, the high complexity of the sample precluded any peptide identification when theHPLC eluate was monitored by total ion current in the full mass mode (Fig. 2A). However, the hamsterprion peptide 107–110 could be detected using a SIM mode (Fig. 2B). The averaged mass spectrum acquiredin the narrow scan range of 491–495 amu for the intact ion and its fragmentation pattern are bothin perfect agreement with that calculated for the hamster prion reporter peptide (Figs. 2C and 2D). Onthe 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 PrPreporter peptide ion.Going one step further on this approach, we attempted to identify the PrP reporter peptide directlymonitoring for the b2 product ion (m/z of 216.0) according to the fragmentation pattern in Fig. 2D. Asknown, 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)
16Future 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?
18Structure of the PrPC flexible N-terminus 3 alpha helices 2 small beta strands2 N – glycosylations
19Structure of the PrPCFigure 1. Primary structure of the cellular PrP including post-translational modificationsGPI = glycosyl phosphatidyl inositolA. Aguzzi, M. Heikenwalder, Microbiology, 4, 765 (2006)
20Figure 2. Tertiary structure of the cellular PrP Structure of the PrPCFigure 2. Tertiary structure of the cellular PrPA. Aguzzi, M. Heikenwalder, Microbiology, 4, 765 (2006)
22ObjectiveUse Mass Spectrometry to characterize the aggregating and aggregated PrPSc.
23The SHPrPC 90 – 231 Sequence of the SHPrPC with the purification tag: MGSSHHHHHHSSGLVPRGSHMLEGQGGGTHNQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPMMHFGNDWEDRYYRENMNRYPNQVYYRPVDQYNNQNNFVHDCVNITIKQHTVTTTTKGENFTETDIKIMERVVEQMCTTQYQKESQAYYDGRRSSNumber of AA: 166Molecular weight: DaTheoretical pI: 8.85Instability index: 38.01GRAVY:
26Sequence coverage for the Tryptic digest Sequence identified: 126 AAMGSSHHHHHHSSGLVPRGSHMLEGQGGGTHNQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPMMHFGNDWEDRYYRENMNRYPNQVYYRPVDQYNNQNNFVHDCVNITIKQHTVTTTTKGENFTETDIKIMERVVEQMCTTQYQKESQAYYDGRRSSIdentified only by MSCoverage: 76%Sequence identified by MSMS: 97 AACoverage: 58%