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1.  A protein polymerization cascade mediates toxicity of non-pathological human huntingtin in yeast 
Scientific Reports  2015;5:18407.
Several neurodegenerative amyloidoses, including Huntington disease, are caused by expansion of polyglutamine (polyQ) stretches in otherwise unrelated proteins. In a yeast model, an N-terminal fragment of mutant huntingtin with a stretch of 103 glutamine residues aggregates and causes toxicity, while its non-toxic wild type variant with a sequence of 25 glutamines (Htt25Q) does not aggregate. Here, we observed that non-toxic polymers of various proteins with glutamine-rich domains could seed polymerization of Htt25Q, which caused toxicity by seeding polymerization of the glutamine/asparagine-rich Sup35 protein thus depleting the soluble pools of this protein and its interacting partner, Sup45. Importantly, only polymers of Htt25Q, but not of the initial benign polymers, induced Sup35 polymerization, indicating an intermediary role of Htt25Q in cross-seeding Sup35 polymerization. These data provide a novel insight into interactions between amyloidogenic proteins and suggest a possible role for these interactions in the pathogenesis of Huntington and other polyQ diseases.
PMCID: PMC4682096  PMID: 26673834
2.  Could yeast prion domains originate from polyQ/N tracts? 
Prion  2013;7(3):209-214.
A significant body of evidence shows that polyglutamine (polyQ) tracts are important for various biological functions. The characteristic polymorphism of polyQ length is thought to play an important role in the adaptation of organisms to their environment. However, proteins with expanded polyQ are prone to form amyloids, which cause diseases in humans and animals and toxicity in yeast. Saccharomyces cerevisiae contain at least 8 proteins which can form heritable amyloids, called prions, and most of them are proteins with glutamine- and asparagine-enriched domains. Yeast prion amyloids are susceptible to fragmentation by the protein disaggregase Hsp104, which allows them to propagate and be transmitted to daughter cells during cell divisions. We have previously shown that interspersion of polyQ domains with some non-glutamine residues stimulates fragmentation of polyQ amyloids in yeast and that yeast prion domains are often enriched in one of these residues. These findings indicate that yeast prion domains may have derived from polyQ tracts via accumulation and amplification of mutations. The same hypothesis may be applied to polyasparagine (polyN) tracts, since they display similar properties to polyQ, such as length polymorphism, amyloid formation and toxicity. We propose that mutations in polyQ/N may be favored by natural selection thus making prion domains likely by-products of the evolution of polyQ/N.
PMCID: PMC3783105  PMID: 23764835
amyloid; Hsp104; polyglutamine; polyasparagine; polyQ; polyN; prion; yeast
3.  Proteomic Screening for Amyloid Proteins 
PLoS ONE  2014;9(12):e116003.
Despite extensive study, progress in elucidation of biological functions of amyloids and their role in pathology is largely restrained due to the lack of universal and reliable biochemical methods for their discovery. All biochemical methods developed so far allowed only identification of glutamine/asparagine-rich amyloid-forming proteins or proteins comprising amyloids that form large deposits. In this article we present a proteomic approach which may enable identification of a broad range of amyloid-forming proteins independently of specific features of their sequences or levels of expression. This approach is based on the isolation of protein fractions enriched with amyloid aggregates via sedimentation by ultracentrifugation in the presence of strong ionic detergents, such as sarkosyl or SDS. Sedimented proteins are then separated either by 2D difference gel electrophoresis or by SDS-PAGE, if they are insoluble in the buffer used for 2D difference gel electrophoresis, after which they are identified by mass-spectrometry. We validated this approach by detection of known yeast prions and mammalian proteins with established capacity for amyloid formation and also revealed yeast proteins forming detergent-insoluble aggregates in the presence of human huntingtin with expanded polyglutamine domain. Notably, with one exception, all these proteins contained glutamine/asparagine-rich stretches suggesting that their aggregates arose due to polymerization cross-seeding by human huntingtin. Importantly, though the approach was developed in a yeast model, it can easily be applied to any organism thus representing an efficient and universal tool for screening for amyloid proteins.
PMCID: PMC4280166  PMID: 25549323
4.  The Effects of Amino Acid Composition of Glutamine-Rich Domains on Amyloid Formation and Fragmentation 
PLoS ONE  2012;7(10):e46458.
Fragmentation of amyloid polymers by the chaperone Hsp104 allows them to propagate as prions in yeast. The factors which determine the frequency of fragmentation are unclear, though it is often presumed to depend on the physical strength of prion polymers. Proteins with long polyglutamine stretches represent a tractable model for revealing sequence elements required for polymer fragmentation in yeast, since they form poorly fragmented amyloids. Here we show that interspersion of polyglutamine stretches with various amino acid residues differentially affects the in vivo formation and fragmentation of the respective amyloids. Aromatic residues tyrosine, tryptophan and phenylalanine strongly stimulated polymer fragmentation, leading to the appearance of oligomers as small as dimers. Alanine, methionine, cysteine, serine, threonine and histidine also enhanced fragmentation, while charged residues, proline, glycine and leucine inhibited polymerization. Our data indicate that fragmentation frequency primarily depends on the recognition of fragmentation-promoting residues by Hsp104 and/or its co-chaperones, rather than on the physical stability of polymers. This suggests that differential exposure of such residues to chaperones defines prion variant-specific differences in polymer fragmentation efficiency.
PMCID: PMC3468588  PMID: 23071575
5.  Amyloid-Mediated Sequestration of Essential Proteins Contributes to Mutant Huntingtin Toxicity in Yeast 
PLoS ONE  2012;7(1):e29832.
Polyglutamine expansion is responsible for several neurodegenerative disorders, among which Huntington disease is the most well-known. Studies in the yeast model demonstrated that both aggregation and toxicity of a huntingtin (htt) protein with an expanded polyglutamine region strictly depend on the presence of the prion form of Rnq1 protein ([PIN+]), which has a glutamine/asparagine-rich domain.
Principal Findings
Here, we showed that aggregation and toxicity of mutant htt depended on [PIN+] only quantitatively: the presence of [PIN+] elevated the toxicity and the levels of htt detergent-insoluble polymers. In cells lacking [PIN+], toxicity of mutant htt was due to the polymerization and inactivation of the essential glutamine/asparagine-rich Sup35 protein and related inactivation of another essential protein, Sup45, most probably via its sequestration into Sup35 aggregates. However, inhibition of growth of [PIN+] cells depended on Sup35/Sup45 depletion only partially, suggesting that there are other sources of mutant htt toxicity in yeast.
The obtained data suggest that induced polymerization of essential glutamine/asparagine-rich proteins and related sequestration of other proteins which interact with these polymers represent an essential source of htt toxicity.
PMCID: PMC3256205  PMID: 22253794

Results 1-5 (5)