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search.filters.applied.f.advisor: Chernoff, Yury O. × End date: 2016 × Has files: Yes × search.filters.applied.f.isOrgUnitOfPublicationTitle: College of Sciences ×

Publication Search Results

Now showing 1 - 10 of 13
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Roles of protein sequence and cell environment in cross-species prion transmission and amyloid interference

2014-07-08 , Bruce, Kathryn Lyn

Proteinaceous infectious particles, termed 'prions' are self-perpetuating protein isoforms that transmit neurodegenerative diseases in mammals and phenotypic traits in yeast. Each conformational variant of a prion protein is faithfully propagated to a homologous protein in the same cell environment. However, a reduction in the efficiency of prion transmission between different species is often observed and is termed "species barrier". Prion transmission to a heterologous protein may, in some cases, permanently change the structure of the prion variant, and divergent proteins may interfere with prion propagation in a species-specific manner. To identify the importance of both protein sequence and the cell environment on prion interference and cross-species transmission, we employed heterologous Sup35 proteins from three Saccharomyces sensu stricto species: Saccharomyces cerevisiae (Sc), Saccharomyces paradoxus (Sp), and Saccharomyces bayanus (Sb). We performed our experiments in two different cell environments (Sc and Sp). Our data show that Sup35 from one species can form a prion in another, and we employed a transfection procedure to perform cross-species transfer of the prion. Using a shuffle procedure, we demonstrate that the specificity of prion transmission is determined by the protein itself rather than the cell environment. Interestingly, we noted that variant-specific prion patterns can be altered irreversibly during cross-species transmission through S. bayanus module II. We further show that prion interference does not always correlate with cross-species prion transmission, and the identity of particular regions or even a specific amino acid, rather than the overall level of PrD homology is crucial for determining cross-species transmission and interference. Lastly we provide evidence to suggest that prion interference is specific to the cell environment.

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Genetic and physical interaction of Sgt2 protein with prion-chaperone machinery

2011-08-10 , Pan, Tao

The word "Prion" refers to self-perpetuating protein aggregates that cause neurodegenerative diseases in mammals. It is a protein isoform that has undergone a conformational change which converts the normal form of the protein into the infectious form with the same amino acid sequence. Yeast [PSI+] prion is the prion isoform of Sup35 protein, a translation termination factor eRF3. It has been suggested that prion [PSI+] is controlled by the ensemble of chaperones with Hsp104 playing the major role. The previous work performed in the Chernoffs lab showed that the defective GET pathway caused by get led to the defect in [PSI+] curing by excess Hsp104. The GET pathway is a system responsible for transporting newly synthesized TA-protein to the ER membrane, and the components which have been proven to be involved in this pathway include: Get1, Get2, Get3, Get4, Get5 and Sgt2. In this study we describe the mechanism underlying the effect of the defective GET pathway on [PSI+]. We demonstrate that Sgt2, one of the components of GET pathway, interacts with Sup35 in both [PSI+] and [psi-] strains through its prion domain. Overproduction of Sgt2 and Hsp70-Ssa is triggered by the defective GET pathway and leads to the protection of [PSI+] aggregates from curing by excess Hsp104. We show that the direct interaction between Sgt2 and Hsp70-Ssa is not required for this protective effect.

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Effects of the components of the Get pathway on prion propagation

2007-11-15 , Bariar, Bhawana

Yeast prions e.g. [PSI+], [PIN+] and [URE3] are similar to mammalian amyloids that cause neurodegenerative diseases. [PSI+] is the aggregated self-perpetuating (prion) isoform of Sup35, a translation termination factor. The molecular chaperone Hsp104 plays a crucial role in the maintenance and propagation of [PSI+]. Deletion of the GET2 gene has been shown to cause a [PSI+] curing defect by excess Hsp104 and [PSI+] instability on synthetic medium (S. Muller, J. Patterson and Y. Chernoff, unpublished data; and J. Patterson Honors Thesis). Get2 is a membrane protein working in a complex with Get1 and Get3 proteins. This complex, called GET (Golgi-to-ER Traffic), is known to retrieve resident ER proteins from Golgi. In this particular study we provide further evidence for the connection between the GET pathway and yeast prions. The get2 deletion also leads to a detectable loss of [PIN+] prion on synthetic medium. The role of the other two members of the Get complex in prion propagation is also explored. The levels and the activity of Hsp104 in the get2 mutants is analyzed. The size of [PSI+] aggregates in the get2Δ strain is compared to that found in wild type. Finally, other possible mechanisms for the effect of get2 on prion maintenance and propagation are addressed.

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An investigation of the genetic control of protein mutability : the role of the ubiquitin system in protein based inheritance in Saccharomyces Cerevisiae

2003-05 , Allen, Kim D.

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Studies of genetic factors modulating polyglutamine toxicity in the yeast model

2011-09-28 , Gong, He

Polyglutamine-expanded fragments, derived from the human huntingtin protein, are aggregation-prone and toxic in yeast cells, bearing endogenous QN-rich proteins in the aggregated (prion) form. Attachment of the proline-rich region targets polyglutamine aggregates to the large perinuclear deposit (aggresome). Aggresome targeting ameliorates polyglutamine cytotoxicity in the presence of the prion form of Rnq1 protein, however, aggresome-forming construct remains toxic in the presence of the prion form of translation termination (release) factor Sup35 (eRF3). Disomy by chromosome II partly ameliorates polyglutamine toxicity in the strains containing Sup35 prion. The chromosome II gene, coding for another release factor, and interaction partner of Sup35, named Sup45 (eRF1), is responsible for amelioration of toxicity. Plasmid-mediated overproduction of Sup45, or expression of the Sup35 derivative that lacks the QN-rich domain and is unable to be incorporated into prion aggregates, also ameliorate polyglutamine toxicity. Protein analysis indicates that polyglutamines alter aggregation patterns of the Sup35 prion and promote aggregation of Sup45, while excess Sup45 counteracts these effects. In the absence of Sup35 prion, disomy by chromosome II is still able to decrease polyglutamine toxicity. However, SUP45 is no longer the gene responsible for such an effect. Taken together with the finding that the presence of both the Rnq1 prion and the Sup35 prion has an additive effect on polyQ toxicity, one gene or few genes on chromosome II are able to ameliorate polyQ toxicity through a SUP45-independent pathway. The identification of such a gene is currently ongoing. Monosomy by chromosome VIII in diploid heterozygous by AQT (Anti-polyQ Toxicity mutants that are disomic by chromosome II) counteracted the effect of AQT. Similarly, deletion of the arg4 gene in chromosome VIII in AQT haploid was able to eliminate the AQT effect. Moreover, analysis of genes involved in the arginine and polyamine synthesis indicated that loss of genes in later stages of arginine biosynthesis causes increase of polyglutamine toxicity. Deletion of genes arg1, arg4, arg8 (arginine pathway) and spe1 (polyamine pathway) all suppressed the Sup35 prion phenotype expression in the nonsense suppression system. Further analysis regarding the mechanisms behind those effects is needed. Our data uncover the mechanisms by which genetic and epigenetic factors may influence polyglutamine toxicity, and demonstrate that one and the same type of polyglutamine deposits could be cytoprotective or cytotoxic, depending on the prion composition of a eukaryotic cell.

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Inverted repeats as a source of eukaryotic genome instability

2008-07-08 , Narayanan, Vidhya

Chromosomal rearrangements play a major role in the evolution of eukaryotic genomes. Genomic aberrations are also a hallmark of many tumors and are associated with a number of hereditary diseases in humans. The presence of repetitive sequences that can adopt non-canonical DNA structures is one of the factors which can predispose chromosomal regions where they reside to instability. Palindromic sequences (inverted repeats with or without a unique sequence between them) that can adopt hairpin or cruciform structures are frequently found in regions that are prone for gross chromosomal rearrangements (GCRs) in somatic and germ cells in different organisms. Direct physical evidence was obtained that double-strand breaks (DSBs) occur at the location of long inverted repeats, a triggering event for the genomic instability. However, the mechanisms by which palindromic sequences lead to chromosomal fragility are largely unknown. The overall goal of this research is to elucidate the mechanisms of DSB and GCR generation by palindromic sequences in yeast, Saccharomyces cerevisiae.

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Interactions of the chaperones and components of UB system in the formation and propagation of the yeast prion [PSI+]

2005-06-28 , Tennant, Esther Paula

Three of the best-characterized prions of Saccharomyces cerevisiae are [PSI+], [URE3], and [PIN+]. This study focuses on the prions [PSI+] and [PIN+]. [PSI+] is the prion isoforms of the protein Sup35 that functions as a eRF3 translational termination factor. The presence of [PSI+] is detected by the partial loss of function of Sup35. The prion [PIN+] is the isoform of the protein Rnq1, and this proteins function is unknown. The presence of the prion [PIN+] is necessary for the de novo formation of the prion [PSI+] (Derkatch et al., 1997). The chaperone, Hsp104, belongs to an evolutionary conserved Hsp100 family of proteins that participate in a various number of cellular processes (Schirmer et al., 1996). Hsp104, in particular, is responsible for the cells adaptation to heat shock, it controls spore viability and the long-term viability of starving vegetative cells. (Sanchez and Linquist, 1990; Sanchez et al.,1992) It is an ATPase that has been shown to promote solubilization of aggregated protein (Parsel et al., 1991). A unique relationship exists between Hsp104 levels within the cell and the maintenance of the prion [PSI+]. The over production of Hsp104 eliminates [PSI+] (Chernoff 1995). This seems logical considering Hsp104 is a disaggregase, and it is reasonable to assume that the over production provides sufficient resources to break the aggregates into portions that are accessible to either other chaperones which would facilitate the proper folding or perhaps the system responsible for the elimination of unusable proteins, such as the ubiquitin-proteasome system. This study examines the role of the ubiquitin-proteasome system in curing of [PSI+] by Hsp104. The role of alternate pathways, in which the prion isoform is refolded into it correct, functional conformation by the action of the chaperones Ssb1 and Ssb2 is examined. These results suggest that the combination of both the degradation pathway and the refolding of proteins are involved in curing of [PSI+] by Hsp104 over production.

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Development of the new yeast-based assays for prion properties

2011-08-29 , Sun, Meng

Prion is an infectious isoform of a normal cellular protein which is capable of converting the non-prion form of the same protein into the alternative prion form. Mammalian prion protein PrP is responsible for prion formation in mammals, causing a series of fatal and incurable prion diseases. (1) We constructed, for the first time, a two-component system to phenotypically monitor the conformational status of PrP in the yeast cells. In this system, the prion domain of Sup35 (Sup35N) was fused to PrP90-230, and the initial formation of the PrPSc-like conformation stimulated prion formation of Sup35N, which in turn converted soluble Sup35 into the prion isoform, leading to a detectable phenotype. Prion-like properties of PrP were studied in this novel yeast model system. Additionally, we employed this system to study amyloidogenic protein Aβ42 aggregation in the yeast model. It has been suggested that the ability to form transmissible amyloids (prions) is widespread among yeast proteins and is likely intrinsic to proteins from other organisms. However, the distribution of yeast prions in natural conditions is not yet clear, which prevents us from understanding the relationship between prions and their adaptive roles in various environmental conditions. (2) We modified and developed sequence and phenotype-independent approaches for prion detection and monitoring. We employed these approaches for prion-profiling among yeast strains of various origins. (3) Lastly, we found a prion-like state [MCS+] causing nonsense suppression in the absence of the Sup35 prion domain. Our results suggested that [MCS+] is determined by both a prion factor and a nuclear factor. The prion-related properties of [MCS+] were studied by genetic and biochemical approaches.

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Prion species barrier at the short phylogenetic distances in the yeast model

2008-07-07 , Chen, Buxin

Prions are self-perpetuating and, in most cases, aggregation-prone protein isoforms that transmit neurodegenerative diseases in mammals and control heritable traits in yeast. Prion conversion requires a very high level of identity of the interacting protein sequences. Decreased transmission of the prion state between divergent proteins is termed "species barrier" and was thought to occur due to the inability of divergent prion proteins to co-aggregate. Species barrier can be overcome in cross-species infections, for example from "mad cows" to humans. We studied the counterparts of yeast prion protein Sup35, originated from three different species of the Saccharomyces sensu stricto group and exhibiting the range of prion domain divergence that overlaps with the range of divergence observed among distant mammalian species. Heterologous Sup35 proteins co-aggregated in S. cerevisiae cells. However, in vivo cross-species prion conversion was decreased and in vitro polymerization was cross-inhibited in at least some heterologous combinations, thus demonstrating the existence of prion species barrier. Our data suggests that species-specificity of prion transmission is controlled at the level of conformational transition rather than co-aggregation. We have shown the Sup35 prion domain is sufficient for the species barrier among the S. sensu stricto species, and constructed SUP35 chimeric prion domains, combining the subregions of various origins Our data demonstrated in different cross-species combinations, different modules of prion domain play a crucial role in the controlling of species-specificity of prion transmission. One essential amino acid position has been identified in S. cerevisiae and S. paradoxus system. Our data support a model suggesting that identity of the short amyloidogenic sequences is crucial for the species barrier. Sup35 originated from three different species of the S. sensu stricto group were capable of forming a prion in S. cerevisiae. However, it was not known whether they are capable of generating and maintaining the prion state in the homologous cell environment. We have constructed the S. paradoxus and S. bayanus strains with appropriate markers, and we were able to demonstrate de novo [PSI+] formation in S. paradoxus but not in S. bayanus. Our data show that [PSI+] formation is not a unique property of S. cerevisiae.

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Interactions between endogenous prions, chaperones and polyglutamine proteins in the yeast model

2005-03-16 , Gokhale, Kavita Chandan

Poly-Q expanded exon 1 of huntingtin (Q103) fused to GFP is toxic to yeast cells containing endogenous yeast prions, [PIN+] ([RNQ+]) and/or [PSI+], which presumably serve as aggregation nuclei. Propagation of yeast prions is modulated by the chaperones of Hsp100/70/40 complex. While some chaperones were reported to influence poly-Q aggregation in yeast, it was not clear whether they do it directly or via affecting yeast prions. Our data show that while dominant negative Hsp104 mutants antagonize poly-Q aggregation and toxicity by eliminating endogenous yeast prions, some mutant alleles of Hsp104 decreases size and ameliorate toxicity of poly-Q aggregates without affecting prion propagation. Elevated levels of the yeast Hsp40 proteins, Ydj1 and Sis1, exhibit opposite effects on poly-Q aggregation and toxicity without influencing prion propagation. Among the yeast Hsp70s, only overproduction of Ssa4 antagonized poly-Q toxicity. We have also isolated dominant Anti-poly-Q-toxicity (AQT) mutants counteracting poly-Q toxicity only in the absence of the major ubiquitin-conjugating enzyme Ubc4. Prion forming potential of other Q-rich proteins and influence of Q and P-rich regions on prion propagation were also studied. Our data connects poly-Q aggregation and toxicity to the stress defense pathway in yeast. As many stress-defense proteins are conserved between yeast and mammals, our data shed light on possible mechanisms modulating poly-Q aggregation and toxicity in mammalian cells.

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