Prions as proteinaceous genetic Prions as proteinaceous genetic materialmaterial

Michael D. Ter-Avanesyan

Michael D. Ter-Avanesyan

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

Amyloids

Yeast Sup35,

transmission electron

microscopy

β-2 microglobulin,Atomic force microscopy

Kajava et al., PNAS USA, 2004

Shewmaker et al., PNAS USA, 2006

2

2

Amyloid diseases

Transmissible (prion)amyloidoses:

Creutzfeldt–Jakob disease

Gerstmann– Straussler–Scheinker disease

Fatal familial insomnia

Kuru

Sheep scrapie

Bovine spongiform encephalopathy

Non-transmissible (non-prion)amyloidoses:

Alzheimer disease

Parkinson disease

Huntington disease…

________________

~40 diseases

Infectious amyloids of the PrP protein Amyloid form of various unrelated proteins

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Protein Organism Function of the amyloid fibrils

Curlin

Chaplins

Hydrophobin EAS

Sup35 and other prions

Spidroin

Proteins of the chorion of the eggshell

Neuron-specific isoform of CPEB

Pmel17

Escherichia coli (bacterium)

Streptomycescoelicolor

(bacterium)

Neurospora crassa (fungus)

fungi (mostly Saccharomyces

cerevisiae)

Nephila edulis (spider)

Bombyx mori (silkworm)

Aplisia californica (marine snail)

Homo sapiens

To colonize inert surfaces and mediate binding to host proteins

To lower the water surface tension and allow the development of aerial

hyphae

To lower the water surface tension and allow the development of aerial

hyphae

To underlay protein-based inheritance of traits

To form the silk fibers of the web

To protect the oocyte and the developing embryo from

environmental hazard

To promote long-term maintenance of synaptic changes associated with

memory storage

To form fibers upon which melanin granules form in melanosomes

Proteins forming functional amyloid fibrils

4

Primordial peptides prone to form amyloids

(scaffold structures)

Polypeptides evolved to acquire biologically

relevant globularstructure

Cells developed aggregation-clearing

mechanisms

5

Emergence of mechanisms preventing protein aggregation and amyloidogenesis

__________________________________________ Nowadays - amyloidogenic proteins can be both detrimental and beneficial

ACSACS

NLPR3 NLRP3

Caspase-1 filaments

Caspase-1

Lu et al., Cell, 2014

Amyloid-like polymers of ACS adaptor act as a platform for caspase activation

LRR

NBD

PYD

PYD

CARD

CARD

Caspasedomain

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

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

Bolton D., McKinley M., Prusiner S. 1982. Identification of protein that purifies with the scrapie prion. Science, 218, 1309-1311

8

Prions of lower eukaryotes

Organism Prion ProteinFunction of non-prion form of the

proteinManifestation

S. cerevisiae [PSI+] Sup35 Translation termination factor

Read through of nonsense codons

S. cerevisiae [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+] Mod5tRNA

isopentiniltransferaseRegulation of sterol synthesis,

resistance to antifungal agents, regulation of sterol biosynthesis

P. anserina [Het-s] HET-s Control of vegetative incompatibility

Death of heterokaryons

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Central dogma of molecular biology

F. Crick, Nature, 1970

(Information flow in biological systems)

DNA

RNA

Protein

FunctionFunction

Protein

Addition____________

[PSI+] determinant

[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

HypothesisHypothesis

The [PSI+] phenotype reflects conversion of the Sup35 protein into a prion form related to its aggregation and functional inactivation

Wickner, Science, 1994

Cox, Heredity, 1965(Mendel, 1865)

11

The system for [PSI+] detection(Suppression of ade2 nonsense mutations)

mRNA

Translation

mRNA

Translation

mRNA

Translation

Wild type

Nonsense mutant

Suppression

**

**12

Domain structure of the Sup35 protein

Only N domain is required for [PSI+]

N M C

1 124 254 685

Structurally similar to translationelongation factor eEF1A

Essential for viability

Rich in: Gln, Asn Glu, Lys

Nonconservative

Nonessential for translation termination

and viability

mRNA

protein

ААА

Sup45/35

Sup45 (eRF1) andSup35 (eRF3) –translation termination factors

Ter-Avanesyan et al., Mol. Microbiol.,1993; Genetics,1994 13

Structural organization of the Sup35 fibril

Paushkin et al., Mol. Cell. Biol.,1997Baxa et al., Mol.Microbiol., 2011

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Sup35 forms prion aggregates in [PSI+] cells

Paushkin et al., EMBO J., 1996; Science, 1997

Prion inheritance in vitro

Sup35[PSI+] is aggregated

Centrifugation

1 2 3 4 5 6 7 8 9 10 11 12

soluble pellet

ribosomes

[PSI+]

[psi-]

Sup35

[PSI+] pellet pellet pellet

psi- psi- psi- psi-

Sup35 is 400-fold mutiplicated in 4 cycles

Serial propagation of the [PSI+] prion

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Prion transformation of yeast(Proof of the prion concept)

Tanaka, Weissman et al., Nature, 2004(Avery et al., J. Exptl. Med., 1944)

Sup35 polymers are infectious and underlie the [PSI+] determinant

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)

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Role of chaperones in [PSI+] maintenance

Control SSA1 YDJ1SSA1+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

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Hsp104 plays a key role in [PSI+] maintenance

Hsp104 [PSI+] cannot propagate in the absence of Hsp104

ParadoxParadox:: Overproduction of Hsp104 also can cause [PSI+] loss

To explain the role of Hsp104, we relied on two considerations:

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

Sup35 fibrils formedin vitro

Chernoff et. al., Science, 1995

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The model of prion replication

Polymerization(only Sup35 is required)

Fragmentation(Hsp104 is required)

ParadoxParadox:: Hsp104, which has evolved for destruction of protein aggregates is essential for maintenance and inheritance of prion aggregates

Hsp104Sup35:

Kushnirov and Ter-Avanesyan, Cell, 1998

Hsp104-mediated fragmentation of prion particles multiplicates them which is necessary for their stable inheritance

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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

Sup35NM GFPGFP

Patino, Lindquist, Science, 1996

[PSI+] [psi-]

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Electrophoretic analysis of prion polymers

MonomersSup35

StartPolymers

Sup35

0 25 50 70 10037 37 42SDS, % 2 2 2 5 2 2 2 2

Stability of Sup35 polymers in the presence of SDS

SDS disrupts aggregatesto polymers

Start

4200 (titin)

740 (nebulin)

205 (myosin)Sup35 monomers

kDa

[psi

-]

[PS

I+]

Kryndushkin et al., J. Biol. Chem., 2003

Polymer

Analysis of polymers in agarose gel with SDS

Hsp104 fragments prion polymers

Sup35мономер

Start

4200

740

205

kDa

0 1 2 3 Generations on GuHCl

1 2 3Hours after GuHCl

0 1 2 3 4 5 6

Cell generations

Start

4200

740

205Sup35monomer

kDa

210 100 55 30 17 11 8 % Hsp104

The size of Sup35 polymer is a characteristic traitof the [PSI+] prion

Decrease in the level of Hsp104 causes increase of the polymer size

Both deletion of HSP104 and incubation of cells on medium with GuHCl cause loss of [PSI+]; GuHCl inhibitsinhibits Hsp104 activity

Kryndushkin et al., J. Biol. Chem., 200322

[PSI+] variants (“alleles”) differ in suppressor phenotype and the size of Sup35 polymers

Sup35 polymers corresponding to different [PSI+] variants differ by susceptibility to fragmentation by Hsp104

Hypothesis:

weak [PSI+]

weak [PSI+]

strong [PSI+]

[psi -] weak [PSI+]

strong [PSI+]

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prionvariant: W S S S S W S S

[PSI+] [PSIPS+]

1 2 3 4 5 6 7 8

W - weak; S - strong

Insertion of tyrosine residues into the polyglutamine stretch enhances efficiency of polymer fragmentation

730

4000

KDa

25

Q4

5Q

51

Q5

6Q

65

Q7

0Q

13

1Q

30

QY

46

QY

50

QY

76

QY

12

0Q

Y

M CpolyQ/QY

(125) (254) (685)

polyQ: MSG-(QQQQQ)m-QSQGApolyQY: MSG-(QQQYQ)m-QSQGA

Sup35MC

polyQ/QY proteins

Polymers in SDS-agarose gel

Tyrosine residues stimulate fragmentation

Alexandrov et al., J. Biol. Chem., 2008

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a.c./Protein Sup35 Rnq1 Ure3 Cyc8 Sfp1 Swi1 Mot3Average in

yeastY Tyr 16,13 5,93 0 0,57 1,27 1,72 3,7 3,4

W Trp 0 0 0 0 0 0,19 0 1

F Phe 3,23 3,56 2,35 0,57 0 4,39 1,23 4,5

A Ala 4,84 5,14 1,18 20,45 8,86 6,11 0 5,6

H His 0 1,58 1,18 2,27 7,59 0,76 0 2,1

S Ser 3,23 15,42 11,76 2,84 13,9 10,69 3,7 8,9

T Thr 0 0,79 5,88 1,7 8,86 7,63 0 5,9

C Cys 0 0 0 0 0 0,19 0 1,3

M Met 0,81 1,98 2,35 1,14 5,06 1,72 11,11 2,1

I Ile 0 0 3,53 1,14 5,06 4,01 1,23 6,5

V Val 0 0 4,71 2,27 0 2,86 1,23 5,6

N Asn 16,13 16,21 38,82 0,57 24,1 22,71 29,63 6,1

Q Gln 28,23 26,88 7,06 51,7 13,9 13,55 29,63 3,9

G Gly 16,13 16,6 5,88 1,7 1,27 2,48 7,41 5,1

K Lys* 1,61 0 1,18 0,57 2,53 3,24 0 7,3

R Arg 1,61 1,19 4,71 0,57 1,27 2,48 4,94 4,4

E Glu 0 1,19 3,53 0,57 0 2,29 1,23 6,5

D Asp* 1,61 0 2,35 0 1,27 2,67 1,23 5,8

P Pro 4,84 0,79 0 6,25 1,27 4,2 2,47 4,4

L Leu 1,61 2,77 3,53 5,11 3,8 6,11 1,23 9,5

Fra

gmen

tatio

n ef

ficie

ncy

Inhi

bit

poly

mer

izat

ion

Non-Q/N residues in yeast prion domains

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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

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Role of chaperones in fragmentation of Sup35 prion polymers

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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

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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

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So

lub

le

frac

tio

n

So

lub

le

frac

tio

n

Pel

let

Pel

let

Sup35PS

Sup35

[PSI+PS]

[PSI+]

Hsp104: Wild type level Enhanced level

Excess of Hsp104 dissolves prion aggregates of Sup35 and decreases their size

Kushnirov et al., EMBO J., 2000

17

Sup35 forms prion aggregates in [PSI+] cells

Sup35[PSI+] is persistent to proteinase КSup35[PSI+] is aggregated

Centrifugation

8.0 4.0 2.0 1.0 0.4 0.2Proteinase K(mkg/ml)

Sup35 [PSI+]

[psi-]

1 2 3 4 5 6 7 8 9 10 11 12

soluble pellet

ribosomes

[PSI+]

[psi-]

Sup35

Paushkin et al., EMBO J., 1996

13

Prion inheritance in vitro

[PSI+] pellet pellet pellet

psi- psi- psi- psi-

Sup35 is 400-fold mutiplicated in 4 cycles

Paushkin et al., Science, 1997

Serial propagation of the [PSI+]prion

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

Molecular basis of inheritance of acquired traits

Proteinaceous genes

An addition to the central dogma of molecular biology

Prions in lower eukaryotes can be considered as:

5

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)?

Prions as proteinaceous genes

7

[PSI+] variants (“alleles”) differ in suppressor phenotype and the size of Sup35 polymers

Sup35 polymers corresponding to different [PSI+] variants differ by susceptibility to fragmentation by Hsp104

Hypothesis:

weak [PSI+]

weak [PSI+]

strong [PSI+]

[psi -] weak [PSI+]

strong [PSI+]

22