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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