Misfolding Diseases and Protein
Folding by Ribosome
(lecture 2)
Suparna Sanyal
ICM
Uppsala University
Misfolding Diseases in Human
Disease
Protein or precursor involved
in disease
Cystic fibrosis
CFTR
Creutzfeldt-Jakob disease
Prion protein
Alzheimer´s disease
ß-amyloid protein
Tay-Sachs disease
ß-hexosaminidase
Huntington‘s disease
Huntingtin (CAG repeats)
Osteogenesis imperfecta
Collagen (type I procollagen)
Parkinson`s disease
Alpha-Synuclein
Cataracts
Crystalllins
Retinitis pigmentosa
Rhodopsin
Protein folding and misfolding diseases
Huntington’s Disease
•Neurodegenerative disorder where part of the
brain becomes malfunctional.
•Caused by a genetic defect on chromosome 4 in
Huntingtin gene.
•Multiple occurance of CAG repeats (40 – 120 times
instead of 10 – 28 times which is normal).
•Inserts poly glutamine stretch.
•Folding disorder.
•Inherited trait.
Protein folding and misfolding diseases
Parkinson’s Disease
•a degenerative disorder of the central nervous system.
•caused by the accumulation of a protein called alphasynuclein into inclusions called Lewy bodies in neurons
•Lewy bodies are abnormal aggregates of protein
• Insufficient formation and activity of dopamine
produced in certain neurons at the midbrain.
• Lewy bodies are the pathological hallmark of the
idiopathic disorder, and the distribution of the Lewy
bodies throughout the Parkinsonian brain varies from one
individual to another.
Protein folding and misfolding diseases
Parkinson’s Disease: Lewy Bodies
•Lewy bodies are abnormal aggregates of protein alphasynuclein.
60X magnification
20X magnification
Protein folding and misfolding diseases
Alzheimer’s Disease
•A progressive neurologic disease of the brain leading to
the irreversible loss of neurons
•The loss of intellectual abilities, including memory and
reasoning.
•Caused by the accumulation of a beta-amyloid (βA)
deposits
•The amyloid precursor protein (APP) is located on
chromosome 21  irregular expression with age.
•Another theory: DR6 receptors, which help in self
degradation of the N-terminus of APP (adjacent to Aβ
peptide) is overexpressed in Alz brain. This indicates
that the DR6 mediated degradation pathway is
accelerated  more APP is degraded  Aβ amyloids.
Protein folding and misfolding diseases
Alzheimer’s Disease: Amyloid Plaques
•Brain section shows visual loss or reduction + holes
•Neurons become irregular in shape and show plaques..
Protein folding and misfolding diseases
Alzheimer’s Disease: Amyloid Plaques
•Brain section shows visual loss or reduction + holes
•Neurons become irregular in shape and show plaques..
Protein folding and misfolding diseases
Prion Disease (Transmissible Spongiform
Encephalopathy or TSE)
Sectional View of Brain in Normal and Infected Animals –
Holes, Spongiform appearance, Astrocytes
Protein folding and misfolding diseases
Prion Diseases in Human and Animals
Creutzfeld Jakob
Disease ( CJD)
human
Inherited
or
Sporadic
Mutation in Prnp gene
Gerstmann Staussler
Scheinker (GSS)
human
Inherited
Mutation in Prnp gene
Fatal Familial Insomnia
(FFI)
human
Inherited
Mutation in Prnp gene
Scrapie
Sheep
Infectious
???
Mad Cow Disease
(BSE)
Cow
Infectious
Acquired from diet
containing infected
sheep tissues
Variant CJD
human
Infectious
Acquired from cattle
with BSE
Kuru
human
Infectious
Acquired from cannibolism of infected
brain components
Other animal TSE
Cat, mink,
elk, mule,
deer etc.
Infectious
Infected by food ?
Protein folding and misfolding diseases
Prion = Proteinaceous Infectious Particle
1982 It is a protein !!!
Science. 1982 Apr 9;216(4542):136-44.
Novel proteinaceous infectious
particles cause scrapie.
Prusiner SB et al..
Stanley Prusiner
Nobel Prize in medicine
1997
”PRION" (PROteinaceous
INfectious particle).
The protein was called PrP.
Protein folding and misfolding diseases
Prions are product of PrnP gene
1985
It’s in our gene !!!
Cell, 1985 Apr;40(4):735-46.
A cellular gene encodes scrapie PrP 27-30 protein.
Oesch B, Westaway D, Walchli M, McKinley MP, Kent SB,
Aebersold R, Barry RA, Tempst P, Teplow DB, Hood LE, et al.
•PrP = Prion Protein
•The gene is called PrnP gene.
•Prion diseases can only be transmitted to animals
that already carry PrnP gene or its homologue.
•We all have the cellular form of PrP in our brain.
Protein folding and misfolding diseases
History of Prions
1996 Variant CJD !!!
Mad Cow beef is fatal to people !!!
Symptoms: Similar to classic CJD.
Occurrence: By April 2005, 155 U.K. residents died in vCJD.
Protein folding and misfolding diseases
Two Forms of Prion-Protein (Prp)
Cellular form
PrPC
Soluble
-helix
Infectious form
PrPSc
Aggregation
-sheet
Protein folding and misfolding diseases
Two Forms of Prion Protein (Prp)
• Sensitivity to
PK
Proteinase-K (PK)
PrPC
- +
PrPSc
- +
• Larger aggregates
• Amyloid fibrils
• Brain cell death
Protein folding and misfolding diseases
Screening for Drug in Mammalian Cells
from Korth C. et al 2001
Disadvantages :
1. Health Risk for the researchers
2. Time and money consuming
3. High-throughput screening difficult
Protein folding and misfolding diseases
Alternative Yeast System for
Screening of Anti-Prion Drugs
Discovery of yeast prions. Wickner (1994) Science
[URE3] (Ure2P) and [Psi](Sup35)
Yeast prions have good reporter system.
 Hypothesis : Prion controlling mechanisms are
conserved from yeast to human.
Bach, S., Talarek, N., ………. and Blondel, M. (2003)
Isolation of drugs active against mammalian prions
using a yeast-based screening assay. Nature
Biotechnology 21, 1075-1081.
Protein folding and misfolding diseases
Yeast as An Alternative System for
Screening Anti-Prion Drugs
white
colonies
[PSI+] strain: Contains Prion
forms of Ure2p and Sup35p
The strain is Ade+
[PSI-] strain: Contains normal
forms of Ure2p and Sup35p
The strain is Ade-
Drugs
red
colonies
Advantages of yeast prion system:
 Easy screening of drug libraries
 High-throughput
Protein folding and misfolding diseases
Rationale for Screening Anti-Prion Drugs
based on Sup35
•Sup35 is a translation termination factor (functions as eRF3)
•Sup 35 is active in complex with Sup45 (equivalent to eRF1).
Reporter system:
ade 1-14
(additional STOP
codon in the
ORF)
Outcome
Adenine
synthesized
No Adenine
synthesis
Metabolic byproduct
accumulates
red
white
colonies
colonies
No metabolic
byproduct
YPD media
Protein folding and misfolding diseases
Why Psi+ strain forms white colonies?
[PSI+] strain:
•Contains prion form of Sup35
•Sup35 aggregated
•Can not form complex with Sup45
•Read-through of first stop
•Adenine synthesised (Ade+)
•No metabolic byproduct
•White colonies on rich YPD media
white
colonies
Protein folding and misfolding diseases
Why Psi- strain forms red colonies?
[PSI-] strain:
•Contains native form of Sup35
•Sup35 not aggregated
•Forms complex with Sup45
•Stops at the first stop
•Adenine synthesis stopped
• (Ade-) phenotype
•Metabolic byproduct accumulated
•Red colonies on rich YPD media
red
colonies
Protein folding and misfolding diseases
How to Screen for Anti-Prion Drugs
(1) [psi+] strain grown in YPD media
(2) The culture spread on a petriplate containing same media
(3) Different compounds added in circular papers
(4) Incubation
(5) Scanning the plate  result
(6) Secondary Screening for URE3.
Protein folding and misfolding diseases
Anti-prion Drugs Identified and
Validated in Yeast Psi+ System
10mM
DMSO
10mM
Chlorpromazine
20mM
40mM
Quinacrine
10mM
6-aminophenanthridine
300mM
GuHCl
Protein folding and misfolding diseases
6AP (6-Aminophenanthridine) is an
antiprion compound
1. Active against both [PSI+] and [URE3]
yeast prions and mamalian prions.
2. 6APi is the inactive analogue of 6AP.
Protein folding and misfolding diseases
References
Isolation of drugs active against mammalian prions using a yeast-based screening
assay.Bach S, Talarek N, Andrieu T, Vierfond JM, Mettey Y, Galons H, Dormont D,
Meijer L, Cullin C, Blondel M.
Nat Biotechnol. 2003 Sep;21(9):1075-81. Epub 2003 Aug 10.
Antiprion drugs as chemical tools to uncover mechanisms of prion
propagation.Tribouillard D, Gug F, Galons H, Bach S, Saupe SJ, Blondel M.
Prion. 2007 Jan;1(1):48-52. Epub 2007 Jan 20. Review.
Protein folding and misfolding diseases
Identification of Cellular Targets for 6AP
(attach a linker and pull down assay)
1. Synthesis of the drug with a
linker attached to a resin
2. Activity test of the drug (white
to red) with the linker
3. Immobilise the
drug with the
resin in a column
Protein folding and misfolding diseases
Identification of Drug Target by
Pull-Down Assay
Cell-extract
Drug
immobilised
with the
resin in the
column
Free- drug
Target
bound with
the drug
immobilised
with the
resin in the
column
Target components
Unbound Components
Protein folding and misfolding diseases
What is bound to the drug-beads ?
tive
6A
P
+i
na
c
+f
ree
6A
P
em
pty
be
ad
s
- fr
ee
6A
P
6AP beads
Ribosomal
Proteins
Bound Nonspecifically
bound
Components
componenets
Protein folding and misfolding diseases
6AP and GA Target rRNA as the
protein bands disappear when trested
with RNAse
Protein folding and misfolding diseases
Mechanism of action of 6AP and GA
• Trans mechanism of action, do not act directly on
the prion protein but through RNA interactions.
• 6AP and GA bind to rRNA
 cure prion features.
 alters folding of prion protein.
• Question: If 6AP (and GA) binds to rRNA can the
ribosomes function properly?
Which function can be affected?
Protein folding and misfolding diseases
Ribosomal Components and Functions
Proteins (orange)
RNA (grey)
Primary Function: Protein Synthesis.
But, Protein Folding???
Protein folding and misfolding diseases
Protein folding activity of the ribosome
(PFAR) * Pioneering idea from Chanchal DasGupta, University of Calcutta, India
1992 It was first shown ribosome can act as folding modulators.
Das and Gupta
1992
1994 The protein-folding activity of E. coli ribosome was found to be
Chattopadhyay,
Das et al.
1994
This activity of the ribosome was demonstrated with 70S ribosome from E. coli, which could
refold bacterial alkaline phosphatase in vitro .
present in 50S particles and in 23S rRNA.
The 30S particle on 16S rRNA did not show any protein-folding activity.
1996 Domain V of 23S rRNA appears to play a crucial role in
Chattopadhyay,
Das et al.
1994
1996 The ribosomes from various prokaryotic and eukaryotic sources
Das,Chattopadh
yay et al. 1996
1999 The ribosome from E. coli possess a general activity in protein
Chattopadhyay,
Pal et al. 1999
reactivation of denatured proteins.
were shown to be able to refold a number of proteins in vitro.
folding even in vivo.
Ribosome in Protein Folding:How to Study?
or its
components
Protein folding and misfolding diseases
Ribosome Refolds Proteins in vitro
80S W.germ
Proper folding = Enzyme
Activity
Denatured  Folded
80S rat liver
70S E.coli
Ribosomes purified from
different sources show
protein folding activity
in vitro.
Self folding
(Das B. et al., EJB, 1996)
Protein folding and misfolding diseases
% Re a ctiva tion
Ribosomal Protein Folding Modulators
80
50S
70
70S
60
23S rRNA
50
40
30
30S
20
10
0
0
25
50
75
100
125
Folding Modulators ( nM )
150
The active folding module of ribosome (70S) is the
RNA (23S rRNA) of the large subunit (50S).
(Chattopadhyay S. et al., BJ, 1994; Das B. et. al. EJB 1996))
Protein folding and misfolding diseases
Ribosomal RNA is active in protein folding
% Reactivation
70
60
Domain V of 23S rRNA
50
transcribed in vitro
40
30
Smaller RNA
fragments of domain V
20
10
0
0
30
60
90
120
150
180
RNA ( nM )
The domain V of 23S rRNA transcribed in vitro
can refold protein. Note that no report of
protein synthesis is available with that RNA
fragment
Protein folding and misfolding diseases
Location and Secondary Structure
of Domain V rRNA
Crystal structure of
Haloarcula ribosome 50S
subunit
Domain V of 23S rRNA from
E. coli ribosome
Secondary Structures of the Domain V
of 23S and 25S rRNA are Highly Similar
Domain V of 25S rRNA from
S. cerevisiae is 682
nucleotides
Domain V of 23S rRNA from
E. coli is 660 nucleotides
The location of critical folding domain
• The critical
site is located
in the subunit
interface.
50S viewed from the 30S side
• Exposed.
• Free from
ribosomal
proteins.
• Highly
conserved.
Domain V
23S rRNA
Protein folding and misfolding diseases
Ribosome is the Target of Anti-Prion
Drugs 6AP and GA
Two possibilities………
•Protein synthesis will be inhibited
•Ribosomal protein folding activity
will be inhibited
Protein folding and misfolding diseases
Qu
ina
crin
e
Ch
lor
pro
ma
zin
Bre
e
feld
in A
PS
I-1
14
Cy
clo
he
xim
GA
6-A
P
DM
SO
ide
Effect of 6AP on Translation in General
Autoradiogram
of total protein
SDS-PAGE using
35S Met.
No effect on protein synthesis.
Also, checked in 2D gel.
Protein folding and misfolding diseases
6AP and GA:No Effect on Protein Synthesis
Protein folding and misfolding diseases
6AP and GA Show No Effect on Peptide
Bond Formation in vitro
Tribouillard-Tanvier D et al., PLOS ONE, 2008
Protein folding and misfolding diseases
6AP and GA inhibit ribosome
assisted protein folding
Tribouillard-Tanvier D et al., PLOS ONE, 2008Protein folding and misfolding diseases
6AP Specifically Inhibits Sup35 Expression
Western-blot with anti-Sup35 antibody
DMSO
T0
T30
T150
6-AP 100M
T240
T0
T30
T150
T240
Western-blot with anti-Actin antibody
Protein folding and misfolding diseases
6AP & GA compete with the substrate
protein for its binding site on 23S rRNA
Black box – 6AP and GA binding site
Green box – Protein folding intermediate
binding site
Circles – Sites we mutated
Red circles – Inhibition in folding
Black circles – No effect in folding
Protein folding and misfolding diseases
6AP follows the kinetic model of
competitive inhibition
6AP
• 6AP inhibits protein folding by the
ribosome in a competitive manner.
• It blocks the sites on rRNA which are
also involved in binding or recognition
of the protein (folding substrate).
• 6AP does not affect the rate of
folding of the proteins suggesting the
following model.
Protein folding and misfolding diseases
The Working Principle or Some Questions
Nascent Prion
Protein
Expressed in
Excess
Folded Prion
Protein
Chaperone
Activity of the
Ribosome
Amyloid Form
Antiprion Drugs
(Storage and
other function?)
6AP and GA
Degraded by
Protease
Prion Symptoms
Reversed
Protein folding and misfolding diseases
Summary
 Ribosome refolds various proteins in vitro and in vivo.
 This is primarily a function of 23S (or 28S) rRNA
from the large subunit of ribosome.
 Ribosome assisted protein folding has strong
implications in prion propagation.
 Antiprion drug 6AP targets ribosomal RNA in the cell
but do not inhibit protein synthesis by ribosome.
The binding sites for 6AP are overlapping with that of
the proteins which are substrates for folding.
 6AP competitively inhibits binding of the substrate
proteins to rRNA, thereby inhibiting their folding.
Protein folding and misfolding diseases
References
Protein folding activity of ribosomal RNA is a selective target of two unrelated
antiprion drugs.
Tribouillard-Tanvier D, Dos Reis S, Gug F, Voisset C, Béringue V, Sabate R, Kikovska E,
Talarek N, Bach S, Huang C, Desban N, Saupe SJ, Supattapone S, Thuret JY, Chédin S,
Vilette D, Galons H, Sanyal S, Blondel M.
PLoS One. 2008 May 14;3(5):e2174.
Role of the ribosome in protein folding. Das D, Das A, Samanta D, Ghosh J,
Dasgupta S, Bhattacharya A, Basu A, Sanyal S, Das Gupta C.
Biotechnol J. 2008 Aug;3(8):999-1009.
The antiprion compound 6-aminophenanthridine inhibits the protein folding activity of the
ribosome by direct competition.
Pang Y, Kurella S, Voisset C, Samanta D, Banerjee D, Schabe A, Das Gupta C, Galons
H, Blondel M, Sanyal S. J Biol Chem. 2013 Jun 28;288(26):19081-9. doi:
10.1074/jbc.M113.466748. Epub 2013 May 14.
Protein folding and misfolding diseases
What’s next?
 Evidence for protein folding activity of the ribosome
in vivo  Express folding deficient ribosomes in the cell
 Complement with in vitro experiments.
 Develop a kinetic assay to follow the conversion from
soluble prion (-helical) to insoluble fibrils (-sheet) 
Sup35 or Het-s prion system.
 Folding of nascent protein chains after release from
the ribosome  bottleneck is RNC (ribosome nascent
chain complex) production. We have developed a
reconstituted transcription translation folding system
(RTTF) for that. We can now produce RNC by excluding
RFs from RTTF mix.
Protein folding and misfolding diseases
Seminar Questions
1. Name some misfolding diseases in human. Discuss the
symptoms and causes for three common misfolding
diseases.
2. What is a prion? Name two common prion diseases.
Discuss the symptoms and appearence of brian in prion
infected animals.
3. Prion is an example of one protein attending two
completely different fold. Discuss.
4. What make the PrpSc proteins different from PrpC. Why the
prion proteins can not be easily digested?
5. Why yeast prions provide a better model than mammalian
prions for antiprion drug discovery and characterization?
6. Discuss why the Psi+ forms white colonies and Psi- forms
red colonies.
7. Describe the procedure for identification of the cellular
target of a drug molecule using 6AP as an example.
8. Discuss how 6AP can possibly inhibit prion propagation
pathway by targetting the ribosomal RNA.
9. Explain the proteostatis network and discuss the amyloid
fibrils.
Protein folding and misfolding diseases