Transcript PSI

Prions as proteinaceous genetic
material
Michael D. Ter-Avanesyan
Michael D. Ter-Avanesyan
Amyloids
 Fibrils form via autocatalytic non-covalent protein polymerization, accompanied
by deep conformational rearrangement of polymerizing protein monomers
 Have a specific cross-β structure, in which the β strands are perpendicular to
the fibril axis, while β sheets formed by separate monomers are parallel to it
 Rigid, insoluble in detergents, resistant to proteases, bind Congo red and thioflavine T
2
Yeast
Sup35,
transmission
electron
microscopy
β-2 microglobulin,
Atomic force microscopy
Kajava et al., PNAS USA, 2004
Shewmaker et al., PNAS USA, 2006
2
Amyloid diseases
Transmissible (prion)
amyloidoses:
Non-transmissible (non-prion)
amyloidoses:
Creutzfeldt–Jakob disease
Alzheimer disease
Gerstmann– Straussler–Scheinker disease
Parkinson disease
Fatal familial insomnia
Huntington disease…
Kuru
________________
Sheep scrapie
~40 diseases
Bovine spongiform encephalopathy
Infectious amyloids of the PrP protein
Amyloid form of various
unrelated proteins
3
Proteins forming functional amyloid fibrils
Protein
Organism
Function of the amyloid fibrils
Curlin
Escherichia coli
(bacterium)
To colonize inert surfaces and mediate
binding to host proteins
Chaplins
Streptomyces
coelicolor
(bacterium)
To lower the water surface tension and
allow the development of aerial
hyphae
Hydrophobin EAS
Neurospora crassa
(fungus)
To lower the water surface tension and
allow the development of aerial
hyphae
Sup35 and other prions
fungi (mostly
Saccharomyces
cerevisiae)
To underlay protein-based inheritance
of traits
Spidroin
Nephila edulis (spider)
To form the silk fibers of the web
Proteins of the chorion of the
eggshell
Bombyx mori
(silkworm)
To protect the oocyte and the
developing embryo from environmental
hazard
Neuron-specific isoform of
CPEB
Aplisia californica
(marine snail)
To promote long-term maintenance of
synaptic changes associated with
memory storage
Pmel17
Homo sapiens
To form fibers upon which melanin
granules form in melanosomes
4
Emergence of mechanisms preventing
protein aggregation and amyloidogenesis
Primordial peptides prone
to form amyloids
(scaffold structures)
Polypeptides evolved to
acquire biologically
relevant globular
structure
Cells developed
aggregation-clearing
mechanisms
__________________________________________
Nowadays - amyloidogenic proteins can be both detrimental and beneficial
5
Amyloid-like polymers of ACS adaptor act as a platform
for caspase activation
LRR
NLPR3
NLRP3
NBD
PYD
ACS
PYD
CARD
ACS
CARD
Caspase-1
Caspase-1 filaments
Caspase
domain
Lu et al., Cell, 2014
6
Role of amyloid in melanin polymerization
(Pmel17 amyloid provides a scaffold for melanin synthesis)
From: Inge-Vechtomov et al., Prion, 2007
7
In mammals
prions are proteinaceous infectious agents
Bolton D., McKinley M., Prusiner S. 1982. Identification of protein that
purifies with the scrapie prion. Science, 218, 1309-1311
In lower eukaryotes
prions are proteinaceous genetic material
Wickner R, 1994. [URE3] as an altered URE2 protein: evidence for a
prion analog in S. cerevisiae. Science, 264, 566-569
8
Prions of lower eukaryotes
Function of nonprion form of the
protein
Manifestation
Sup35
Translation termination
factor
Read through of nonsense
codons
[URE3]
Ure2
Regulator of nitrogen
metabolism
Utilization of poorly assimilated
nitrogen sources
S. cerevisiae
[PIN+]
Rnq1
Unknown
Assistance in generation of other
prions
S. cerevisiae
[SWI+]
Swi1
Transcriptional factor
Sugar assimilation
S. cerevisiae
[ISP+]
Sfp1
Transcriptional factor
Decrease of nonsense codon
read through efficiency
S. cerevisiae
[NUP100+]
Nup100
Nucleoporin
Transport of mRNAs and proteins
across the nuclear membrane
S. cerevisiae
[OCT+]
Cyc8
Transcriptional factor
Utilization of lactate by cyc1Δ
cells
S. cerevisiae
[MOT3+]
Mot3
Transcriptional factor
Regulation of cell wall synthesis
S. cerevisiae
[MOD+]
Mod5
tRNA
isopentiniltransferase
Regulation of sterol synthesis,
resistance to antifungal agents,
regulation of sterol biosynthesis
P. anserina
[Het-s]
HET-s
Control of vegetative
incompatibility
Death of heterokaryons
Organism
Prion
Protein
S. cerevisiae
[PSI+]
S. cerevisiae
9
Central dogma of molecular biology
(Information flow in biological systems)
DNA
Addition
____________
RNA
Protein
Protein
Function
Function
F. Crick, Nature, 1970
10
[PSI+] determinant
Cox, Heredity, 1965
(Mendel, 1865)
 [PSI+] manifests a nonsense suppressor phenotype
 [PSI+] is inherited in a non-Mendelian fashion and can be
transmitted from one cell to another with cytoplasm
 No extrachromosomal DNA or RNA have been found to be
associated with [PSI+]
 [PSI+] can be efficiently eliminated by protein denaturing agent,
guanidine hydrochloride, or by exposure to stress-inducing
factors
 The curing of [PSI+] is reversible
Hypothesis
The [PSI+] phenotype reflects conversion of the Sup35 protein into
a prion form related to its aggregation and functional inactivation
Wickner, Science, 1994
11
The system for [PSI+] detection
(Suppression of ade2 nonsense mutations)
mRNA
Wild type
Translation
mRNA
Nonsense
mutant
*
Translation
mRNA
Suppression
*
Translation
12
Domain structure of the Sup35 protein
mRNA
ААА
Sup45 (eRF1) andSup35 (eRF3) –
translation termination factors
protein
Sup45/35
1
124
N
Rich in: Gln, Asn
254
M
685
C
Glu, Lys
Nonconservative
Structurally similar to translation
elongation factor eEF1A
Nonessential
for translation termination
and viability
Essential for viability
Only N domain is required for [PSI+]
Ter-Avanesyan et al., Mol. Microbiol.,1993; Genetics,1994 13
Structural organization of the Sup35 fibril
Baxa et al., Mol.Microbiol., 2011
Paushkin et al., Mol. Cell. Biol.,1997
14
Prion inheritance in vitro
Sup35 forms prion aggregates in [PSI+] cells
Serial propagation of the [PSI+] prion
Centrifugation
soluble
[PSI+] pellet
pellet
pellet pellet
ribosomes
1
2
3
4
5
6
7
8
9 10 11 12
[PSI+]
Sup35
psi-
psi-
psi-
psi-
[psi-]
Sup35[PSI+] is aggregated
Sup35 is 400-fold mutiplicated in 4 cycles
Paushkin et al., EMBO J., 1996; Science, 1997
15
Prion transformation of yeast
(Proof of the prion concept)
Sup35NM was produced in E. coli, purified and
polymerized in vitro
Yeast spheroplasts was co-transformed with
Sup35 fibrils and a plasmid carrying the URA3
selectable marker
Transformation resulted in the appearance of
clones with the [PSI+] phenotype (white color)
Sup35 polymers are infectious and underlie
the [PSI+] determinant
Tanaka, Weissman et al., Nature, 2004
(Avery et al., J. Exptl. Med., 1944)
16
Role of chaperones in [PSI+] maintenance
Control SSA1
SSA1+
YDJ1 YDJ1 SSB1 HSP104
[PSI+PS]
[PSI+]
Overproduction of Ssa1, Ssb1
(Hsp70/DnaK) and Ydj1(Hsp40/DnaJ)
chaperones can destabilize [PSI+]
Kryndushkin et al., J. Biol. Chem., 2002
17
Hsp104 plays a key role in [PSI+] maintenance
Hsp104 [PSI+] cannot propagate in the absence of Hsp104
Paradox:
Overproduction of Hsp104 also can cause [PSI+] loss
Chernoff et. al., Science, 1995
To explain the role of Hsp104, we relied on two considerations:
Sup35 fibrils formed
in vitro
 Sup35 prion particles have fibrillar shape
 Hsp104 act on fibrils in the same way as on
aggregates of thermally denatured proteins,
i.e. disrupt them
18
The model of prion replication
Polymerization
(only Sup35 is required)
Sup35:
Fragmentation
(Hsp104 is required)
Hsp104
Hsp104-mediated fragmentation of prion particles multiplicates them
which is necessary for their stable inheritance
Kushnirov and Ter-Avanesyan, Cell, 1998
Paradox:
Hsp104, which has evolved for destruction of protein aggregates
is essential for maintenance and inheritance of prion aggregates
19
Methods of prion particles analysis
 Isolation of aggregates by centrifugation
 Microscopic observation of aggregated GFP hybrid proteins
Both methods do not allow to analyze the size of polymers
[PSI+]
[psi-]
Sup35NM GFP
Patino, Lindquist, Science, 1996
20
Electrophoretic analysis of prion polymers
Start
SDS, %
Start
Polymers
Sup35
2
0
2
2
25 37
5
2
2
2
kDa
2
4200 (titin)
37 42 50 70 100

740 (nebulin)
Monomers
Sup35
205 (myosin)
Sup35 monomers
[psi-]
Polymer
SDS disrupts aggregates
to polymers
[PSI+]
Stability of Sup35 polymers
in the presence of SDS
Analysis of polymers in
agarose gel with SDS
Kryndushkin et al., J. Biol. Chem., 2003
21
Hsp104 fragments prion polymers
Both deletion of HSP104 and incubation of cells on medium with GuHCl cause loss of [PSI+];
GuHCl inhibits Hsp104 activity
Start
Start
kDa
kDa
4200
4200
740
740
205
Sup35
monomer
210 100 55 30 17 11 8
0 1 2 3 4 5 6
% Hsp104
Cell generations
Decrease in the level of
Hsp104 causes increase of
the polymer size
205
Sup35
мономер
0 1 2 3 1 2 3
Generations Hours after
on GuHCl
GuHCl
The size of Sup35 polymer is a characteristic trait
of the [PSI+] prion
Kryndushkin et al., J. Biol. Chem., 2003
22
[PSI+] variants (“alleles”) differ in suppressor
phenotype and the size of Sup35 polymers
1
2
3
prion
variant: W
S
S
4
5
6
7
8
weak [PSI+]
weak
[PSI+]
[psi -]
weak
[PSI+]
[PSI+]
S S W S
S
[PSIPS+]
W - weak; S - strong
strong
[PSI+]
strong [PSI+]
Hypothesis:
Sup35 polymers corresponding to different
[PSI+] variants differ by susceptibility to
fragmentation by Hsp104
23
Insertion of tyrosine residues into the polyglutamine stretch
enhances efficiency of polymer fragmentation
(125)
polyQ/QY
(254)
(685)
M
C
Sup35MC
polyQ: MSG-(QQQQQ)m-QSQGA
polyQY: MSG-(QQQYQ)m-QSQGA
polyQ/QY proteins
KDa
4000
30QY
46QY
50QY
76QY
120QY
25Q
45Q
51Q
56Q
65Q
70Q
131Q
730
Tyrosine residues stimulate
fragmentation
Alexandrov et al., J. Biol. Chem., 2008
Polymers in SDS-agarose gel
24
Inhibit
polymerization
Fragmentation efficiency
Non-Q/N residues in yeast prion domains
a.c./Protein
Y Tyr
W Trp
F Phe
A Ala
H His
S Ser
T Thr
C Cys
M Met
I Ile
V Val
N Asn
Q Gln
G Gly
K Lys*
R Arg
E Glu
D Asp*
P Pro
L Leu
Sup35
16,13
0
3,23
4,84
0
3,23
0
0
0,81
0
0
16,13
28,23
16,13
1,61
1,61
0
Rnq1
5,93
0
3,56
5,14
1,58
15,42
0,79
0
1,98
0
0
16,21
26,88
16,6
0
1,19
1,19
Ure3
0
0
2,35
1,18
1,18
11,76
5,88
0
2,35
3,53
4,71
38,82
7,06
5,88
1,18
4,71
3,53
Cyc8
0,57
0
0,57
20,45
2,27
2,84
1,7
0
1,14
1,14
2,27
0,57
51,7
1,7
0,57
0,57
0,57
Sfp1
1,27
0
0
8,86
7,59
13,9
8,86
0
5,06
5,06
0
24,1
13,9
1,27
2,53
1,27
0
Swi1
1,72
0,19
4,39
6,11
0,76
10,69
7,63
0,19
1,72
4,01
2,86
22,71
13,55
2,48
3,24
2,48
2,29
1,61
4,84
1,61
0
0,79
2,77
2,35
0
3,53
0
6,25
5,11
1,27
1,27
3,8
2,67
4,2
6,11
Average in
Mot3
yeast
3,7
3,4
0
1
1,23
4,5
0
5,6
0
2,1
3,7
8,9
0
5,9
0
1,3
11,11
2,1
1,23
6,5
1,23
5,6
29,63
6,1
29,63
3,9
7,41
5,1
0
7,3
4,94
4,4
1,23
6,5
1,23
2,47
1,23
5,8
4,4
9,5
25
Role of the exposed region of the
Sup35 prion domain in fragmentation of polymers
Hypothesis:
Difference in [PSI+] phenotypes is
related to variation in exposure of
certain non-Q/N amino acids in prion
domains
Alexandrov et al., PLoS One, 2012
26
Role of chaperones in fragmentation of Sup35
prion polymers
27
Origin of prion proteins in yeast: hypothesis
Background:
 PolyQ domains can expand and contract
 PolyQ often serve to mediate interaction between proteins
 Expansion of polyQ stretches can result in toxicity of corresponding proteins
while their contraction may inhibit biological function of these proteins
 Insertion of non-Q/N amino acid residues into polyQ should stabilize their length
and may decrease toxicity
Hypothesis:
Yeast prion domains may have derived from polyQ tracts via accumulation
and amplification of mutations
Alexandrov, Ter-Avanesyan , Prion, 2013
28
Main contributors:
V. Kushnirov
S. Paushkin (now in USA)
A. Alexandrov
D. Kryndushkin (now in USA)
I. Alexandrov (now in USA)
Thank you for your attention
29
Pellet
Soluble
fraction
Pellet
Soluble
fraction
Excess of Hsp104 dissolves prion aggregates of
Sup35 and decreases their size
[PSI+PS]
Sup35PS
Sup35
[PSI+]
Hsp104:
Wild type level
Enhanced level
Kushnirov et al., EMBO J., 2000
17
Sup35 forms prion aggregates in [PSI+]
cells
Centrifugation
soluble
pellet
Proteinase K
(mkg/ml)
Sup35
Sup35
ribosomes
1
2
3
4
5
6
7
8
8.0 4.0 2.0 1.0 0.4 0.2
[PSI+]
9 10 11 12
[PSI+]
[psi-]
[psi-]
Sup35[PSI+] is aggregated
Sup35[PSI+] is persistent to
proteinase К
Paushkin et al., EMBO J., 1996
13
Prion inheritance in vitro
Serial propagation of the [PSI+]
prion
[PSI+] pellet
psi-
psi-
pellet pellet
psi-
psi-
Sup35 is 400-fold mutiplicated in 4 cycles
Paushkin et al., Science, 1997
14
Origin of prion proteins in yeast: hypothesis
Background:
 PolyQ domains can expand and contract
 PolyQ often serve to mediate interaction between proteins
 Expansion of polyQ stretches can result in toxicity of corresponding proteins
while their contraction may inhibit biological function of these proteins
 Insertion of non-Q/N amino acid residues into polyQ should stabilize their length
and may decrease toxicity
Hypothesis:
Yeast prion domains could derive and evolve due to mutational insertion some
amino acid residues into polyQ and their subsequent amplification
Alexandrov, Ter-Avanesyan , Prion, 2013
38
Prions in lower eukaryotes can be considered as:
 Molecular basis of inheritance of acquired traits
 Proteinaceous genes
 An addition to the central dogma of molecular biology
5
Prions as proteinaceous genes
 How do proteinaceous genes determine phenotypic traits?
 What kind of information they encode?
 How do proteinaceous genes replicate?
 What is the nature of their “alleles”?
 What is the biological significance of prions
(are they a bate or benefit)?
7
[PSI+] variants (“alleles”) differ in suppressor
phenotype and the size of Sup35 polymers
weak [PSI+]
weak
[PSI+]
[psi -]
weak
[PSI+]
strong
[PSI+]
strong [PSI+]
Hypothesis:
Sup35 polymers corresponding to different [PSI+] variants differ
by susceptibility to fragmentation by Hsp104
22