11/29/16
SS Prions Course, 2014. Lecture 1-2: Addi onal Reading
1. "Deadly Feasts," by Richard Rhodes. Mee ng 1: Infec ous prion disease and basics of protein structure. 2. Basic concepts in Protein structure: h p://www.proteinstructures.com 3. h p://www.scien ficamerican.com/ar cle/the-prion-diseases/ Mee ng 2: Structural mechanisms of prion transmission. Gene cs of prions in yeast. 4. Nobel lectures by Gajdusek and Prusiner. 5. Movie (available on YouTube). “Science and Sorcery” Mee ng 3. Prion mechanisms in neurodegenera ve disease. Proposed physiological mechanisms for prion-like conforma onal transi ons.
Mee ng 4. Intrinsically disordered protein domains and their cellular func ons.
Mani Ramaswami (X8400) [email protected]
2
Daniel CARLETON GAJDUSEK(1923-2008) Physiology and Medicine Nobel Prize in 1976
John Enders Hungarian Rabies and plague Linus Pauling Walter Reed (Joe Smadel) Kuru and related diseases
Frank Burnet Viruses Hemorrhagic fever Vincent Zigas and Kuru h ps://www.theguardian.com/science/2009/feb/25/ carleton-gajdusek-obituary
1 11/29/16
Kuru h p://www.youtube.com/watch?v=vw_tClcS6To
Kuru: Science and Sorcery
1500 miles
Mins 3:07 – 5:30.
Cannibalism: 26:45 - 29:00 ; 1:00- 1:10 6
Vincent Zigas, Jack Baker en Carleton Gajdusek onderzoeken kuru-hersenen (in de pan op de ee afel) in Okapa, Nieuw Guinea,1957 Pathology: including lack of inflamma on
- Mechanism of infec on and iden ty of the pathogen
(Contact, food, environment, genes?) Kuru
Creutzfeldt-JaKob Disease. Occurs in elderly male or female pa ents in all communi es (20 cases were known) - Igor Klatzo, NIH. CJD
7
2 11/29/16
1880s German Physician Robert Koch’s Postulates.
1. The agent must be present in every case of the disease.
2. Inocula on of pure cultures of the disease should produce disease in animals
3. Cultures of disease agent repurified from infected animals must produce disease again
“We tried everything from snakes to bears” - Carleton Gajdusek
University of Minnesota and then the Rocky Mountain Labs in Montana.
9 10
Transmission? Disease transmission tests: Kuru to Chimps
(Daisy and George e)
Kigea, an 11-year-old girl from Waisa village who died of kuru.
(CJD was also transmi ed)
(Michael Alpers) Movie: 29:14-30:17 11 12
3 11/29/16
Scrapie or “tremblante,” the trembling disease -Kuru , Scrapie and CJD are similar. -All three are transmissable.- -Kuru may be transmi ed by endocannibalism. 1700s - 1950s
Similar symptoms; Epidemics; Transmissable to other sheep and goats (incuba on me of 3-4 years in sheep and one year in goats) – Bill Hadlow.
William Gordon, a UK Veterinarian: vaccine for a virus (formaldehyde fixed homogenate of infected sheep ssue.).
Can be transmi ed to mice, hamsters and many other species.
13 14
Animal prion diseases can be transmi ed by feeding An anthropologist, who traced Kuru back to a single individual in the early 1900s
Kuru = CJD = BSE = Scrapie - Downer ca le. -Why no inflamma on -Why a long incuba on period
15 -Why and how do different strains arise? 16
4 11/29/16
High Tech Endocannibalism Bovine Spongiform Encephali s
The Guardian Observer, Sunday 22 October 2000 BSE vic ms to get millions: Labour accepts moral case for compensa on 1. Tissue transplanta ons - corneal dura membranes Kamal Ahmed and Antony Barne The Government has agreed a mul -million-pound compensa on package for 2. Hospital equipment sufferers of the human form of BSE, or mad cow disease, a er agreeing it would be 'morally impossible' to turn them down, The deal, which will delight the 84 families whose lives have been blighted by the disease, is set to be announced on Thursday, 3. Human growth hormone along with the publica on of Lord Phillips's long-awaited report on the BSE crisis that has stalked Britain since the Eigh es. Senior Whitehall sources said that, although there were s ll prac cal and legal difficul es that needed to be overcome, the Cabinet commi ee set up to deal with the Phillips report has agreed a no-fault scheme that From ea ng or contac ng infected meat? will pay families hundreds of thousands of pounds.
Variant CJD can occur in the very young 13 years +
18
What is the transmissable agent?
The Scrapie agent survives: Kuru Infec on through mortuary feasts (ritual endocannibalism) 1. 30 minutes of boiling. CJD Unknown Mechanism. 2. 60 days of freezing. V-CJD Ea ng contaminated meat or bloodborne transmission 3. Strong formaldehyde. 4. Dessica on for 2 years. Iatrogenic CJD Infec on from surgical ra s, equipment or human 5. Intense UV exposure. pituitary derived hormones. 6. Survives electron bombardment that should destroy a genome 1000 mes smaller than lambda.
19 20
5 11/29/16
What is the transmissable agent? EM images of scrapie-associated fibrils Patricia Merz, 1981 CJD Hamster CJD Human Scrapie The Assay:
Inoculum - Inject host – assay disease (short-incuba on model in strain 263K of golden hamsters). Purifica on: Either (a) inhibit necessary components and then add them back (or) (b) separate individual components and test them.
22
Animal prion diseases can be transmi ed by feeding What is the transmissable agent?
The Assay:
Inoculum - Inject host – assay disease
Purifica on: Either (a) inhibit necessary components and then add them back (or) (b) separate individual components and test them.
(Inoculum concentra on determines incuba on period)
- Downer ca le.
23 24
6 11/29/16
Problems using mice Prusiner’s fast hamster assay.
1. Mouse: assays take 12 months. Hamsters show disease in 70-90 days. 60m mice per sample About 300 samples Phenotypes so clear that 4 animals per sample suffice. Inject every 3 months 72000 mice per year; $2 million per year in late 1970s.
80-fold increase in the rate at which frac ons could be studied! 2. Different mouse strains are different. (Could do in one year – what would have taken a 80 years)
25 26
Prion: Protein Infection (named by Stan Prusiner)
Red: Acidic Violet Basic; Green: nonpolar; Blue Polar
22-aa signal pep de cleaved during transla on 15-aa C-terminal cleaved and glycophospho dylinositol (GPI) anchored (230). 181, 196 N-linked glycosyla on. 5 Octapep de repeats ((PH/QGGG(G)WGQ)
27 28
7 11/29/16
h p://www.federa onofscien sts.org/pmpanels/tse/priprogene.asp Crystalliza on models for prion disease.
JS Griffith 1967 and later Carleton Gajdusek
Examples: Salts Ethylene diamine tartrate factories Ice 9 (Kurt Vonnegut)
29 30
Prion: Protein Infection (named by Stan Prusiner)
31 32
8 11/29/16
Protein folding and structure
(GN Ramachandran’s Phi-Psi plot) How Proteins Fold?
– Consider a protein with 100 a.a.
• 10100 possible conforma ons (avg. of 10 conforma ons/a.a.) • If it converts from one conforma on to another in ~10-13 sec then the avg. me to sample all conforma ons would be 1077 years.
Cosmic Term: longer than the life of earth/universe
33 34
Anfinson, 1961
Levinthal’s Paradox
• However, in vivo, proteins fold in 10-1- 103 seconds, a mismatch of >98 orders of magnitude
• Conclusion: Folding is not random. It is determinis c (directed)
35 36
9 11/29/16
Hydrophobic groups are usually buried
37 38
Secondary structure Key concepts in protein structure Alpha helices Beta sheets
Sequence
Secondary structures (helices and beta sheets)
Motifs (specific functional organization of secondary structure units)
Domains (autonomous folded unit)
Full protein (can be multi domain and/or multisubunit) (local structures) (between sequences that are separated)
39 40
10 11/29/16
Structural motifs and Protein Folds Ribbon representations Helix Bundle TIM Barrel
Different beta sheet motifs
β-hairpin
41 42
No new folds have been discovered since 2008! Domains and multidomain proteins
Red bars – show total number of known folds in each year (2012 to the le ) - Maxes out at about 1300.
44
11 11/29/16
45 46
The precision and reproducibility of protein-folding processes within cells is such that protein molecules of the same amino acid sequence have 3-D conforma ons sufficiently homogenous to form macroscopic crystals.
This property is absent from most other polymers:
• polyesters,
• celluloses,
• polysccharides,
• fa y acids,
• long DNA
47
12