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Professor Mick Tuite

Professor of Molecular Biology

School of Biosciences

 

Professor Mick Tuite joined the School of Biosciences in 1983 after conducting postdoctoral research at the University of California (1978-81) and the University of Oxford (1981-83). He began his research studies on yeast whilst a PhD student under the supervision of Dr Brian Cox at the 'Botany School' in Oxford. His research interests have largely focused on the mechanism and control of translation in yeast but more recently his interests have moved to yeast prion proteins and molecular chaperones. The research in his group has been extensively funded by grants from the BBSRC, Wellcome Trust and the Leverhulme Trust.

Mick is currently the Director of Research in the school of Biosciences, and is a member of the Centre for Molecular Processing, the Yeast Molecular Biology Group and the Kent Fungal Group.

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Articles

    Tuite, Mick F. and Howard, Mark J. and Xue, Wei-Feng (2014) Dynamic Prions Revealed by Magic. Chemistry & Biology, 21 (2). pp. 295-305. ISSN 10745521.

    Abstract

    Prion proteins can be propagated as amyloid fibrils with several different conformational variants. By providing structural information at atomic level for two such variants of a yeast prion, Frederick and colleagues, in this issue of Chemistry & Biology, reveal how conformational flexibility can generate pheno- typic diversity.

    Mead, Emma J and Masterton, Rosalyn J. and von der Haar, Tobias et al. (2014) Control and regulation of mRNA translation. Biochemical Society Transactions, 42 (1). pp. 151-154. ISSN 0300-5127.

    Abstract

    Translational control is central to the gene expression pathway and was the focus of the 2013 annual Translation UK meeting held at the University of Kent. The meeting brought together scientists at all career stages to present and discuss research in the mRNA translation field, with an emphasis on the presentations on the research of early career scientists. The diverse nature of this field was represented by the broad range of papers presented at the meeting. The complexity of mRNA translation and its control is emphasized by the interdisciplinary research approaches required to address this area with speakers highlighting emerging systems biology techniques and their application to understanding mRNA translation and the network of pathways controlling it.

    Chu, Dominique and Kazana, Eleanna and Bellanger, Noémie et al. (2014) Translation elongation can control translation initiation on eukaryotic mRNAs. Embo Journal, 33 (9). pp. 937-1085. ISSN 0261-4189.

    Abstract

    Synonymous codons encode the same amino acid, but differ in other biophysical properties. The evolutionary selection of codons whose properties are optimal for a cell generates the phenomenon of codon bias. Although recent studies have shown strong effects of codon usage changes on protein expression levels and cellular physiology, no translational control mechanism is known that links codon usage to protein expression levels. Here, we demonstrate a novel translational control mechanism that responds to the speed of ribosome movement immediately after the start codon. High initiation rates are only possible if start codons are liberated sufficiently fast, thus accounting for the observation that fast codons are overrepresented in highly expressed proteins. In contrast, slow codons lead to slow liberation of the start codon by initiating ribosomes, thereby interfering with efficient translation initiation. Codon usage thus evolved as a means to optimise translation on individual mRNAs, as well as global optimisation of ribosome availability.

    Staniforth, Gemma L. and Tuite, Mick F. (2014) Monoculture Breeds Poor Social Skills. Cell, 158 (5). pp. 975-977. ISSN 00928674.

    Marchante, Ricardo and Rowe, Michelle L. and Zenthon, Jo et al. (2013) Structural Definition Is Important for the Propagation of the Yeast [PSI+] Prion. Molecular Cell, 50 (5). pp. 675-685. ISSN 10972765.

    Preiss, Thomas and Bauer, Johann W. and Brandl, Clemens et al. (2013) Specialized Yeast Ribosomes: A Customized Tool for Selective mRNA Translation. PLoS ONE, 8 (7). pp. e67609. ISSN 1932-6203.

    Abstract

    Evidence is now accumulating that sub-populations of ribosomes - so-called specialized ribosomes - can favour the translation of subsets of mRNAs. Here we use a large collection of diploid yeast strains, each deficient in one or other copy of the set of ribosomal protein (RP) genes, to generate eukaryotic cells carrying distinct populations of altered ‘specialized’ ribosomes. We show by comparative protein synthesis assays that different heterologous mRNA reporters based on luciferase are preferentially translated by distinct populations of specialized ribosomes. These mRNAs include reporters carrying premature termination codons (PTC) thus allowing us to identify specialized ribosomes that alter the efficiency of translation termination leading to enhanced synthesis of the wild-type protein. This finding suggests that these strains can be used to identify novel therapeutic targets in the ribosome. To explore this further we examined the translation of the mRNA encoding the extracellular matrix protein laminin ?3 (LAMB3) since a LAMB3-PTC mutant is implicated in the blistering skin disease Epidermolysis bullosa (EB). This screen identified specialized ribosomes with reduced levels of RP L35B as showing enhanced synthesis of full-length LAMB3 in cells expressing the LAMB3-PTC mutant. Importantly, the RP L35B sub-population of specialized ribosomes leave both translation of a reporter luciferase carrying a different PTC and bulk mRNA translation largely unaltered.

    Jossé, Lyne and Smales, C. Mark and Tuite, Mick F. (2012) Engineering the Chaperone Network of CHO Cells for Optimal Recombinant Protein Production and Authenticity. Methods in Molecular Biology, 824 (6). pp. 595-608. ISSN 1064-3745.

    Abstract

    All proteins fold into a defined three-dimensional shape that is compatible with the cellular role and/or biological activity of those proteins. Molecular chaperones are a family of proteins whose role is to assist the folding and targeting of proteins in both normal and stressed cells. The rational manipulation of chaperone levels in a cell line engineered to produce a defined recombinant protein (rP) can significantly improve both the achievable steady-state levels and authenticity of a wide range of recombinant proteins. Here, we describe the methodology associated with expressing a variety of molecular chaperones in Chinese hamster ovary (CHO) lines in order to improve their recombinant protein production capacity. These chaperones include both those that facilitate the folding of the polypeptide chain (i.e. Hsp70, Hsp40) and those that can re-fold proteins that have misfolded in the cell (i.e. ClpB/Hsp104). This latter property is particularly important given the propensity for highly expressed recombinant proteins to misfold in the “foreign” cellular environment.

    Jossé, Lyne and Marchante, Ricardo and Zenthon, Jo et al. (2012) Probing the role of structural features of mouse PrP in yeast by expression as Sup35-PrP fusions. Prion, 6 (3). pp. 201-210. ISSN 1933-6896.

    Abstract

    The yeast Saccharomyces cerevisiae is a tractable model organism in which both to explore the molecular mechanisms underlying the generation of disease-associated protein misfolding and to map the cellular responses to potentially toxic misfolded proteins. Specific targets have included proteins which in certain disease states form amyloids and lead to neurodegeneration. Such studies are greatly facilitated by the extensive ‘toolbox’ available to the yeast researcher that provides a range of cell engineering options. Consequently, a number of assays at the cell and molecular level have been set up to report on specific protein misfolding events associated with endogenous or heterologous proteins. One major target is the mammalian prion protein PrP because we know little about what specific sequence and/or structural feature(s) of PrP are important for its conversion to the infectious prion form, PrPSc. Here, using a study of the expression in yeast of fusion proteins comprising the yeast prion protein Sup35 fused to various regions of mouse PrP protein, we show how PrP sequences can direct the formation of non-transmissible amyloids and focus in particular on the role of the mouse octarepeat region. Through this study we illustrate the benefits and limitations of yeast-based models for protein misfolding disorders.

    Afanasieva, Evgenia G. and Kushnirov, Vitaly V. and Tuite, Mick F. et al. (2011) Molecular Basis for Transmission Barrier and Interference between Closely Related Prion Proteins in Yeast. Journal of Biological Chemistry, 286 (18). pp. 15773-15780. ISSN 0021-9258.

    Abstract

    Replicating amyloids, called prions, are responsible for transmissible neurodegenerative diseases in mammals and some heritable phenotypes in fungi. The transmission of prions between species is usually inhibited, being highly sensitive to small differences in amino acid sequence of the prion-forming proteins. To understand the molecular basis of this prion interspecies barrier, we studied the transmission of the [PSI+] prion state from Sup35 of Saccharomyces cerevisiae to hybrid Sup35 proteins with prion-forming domains from four other closely related Saccharomyces species. Whereas all the hybrid Sup35 proteins could adopt a prion form in S. cerevisiae, they could not readily acquire the prion form from the [PSI+] prion of S. cerevisiae. Expression of the hybrid Sup35 proteins in S. cerevisiae [PSI+] cells often resulted in frequent loss of the native [PSI+] prion. Furthermore, all hybrid Sup35 proteins showed different patterns of interaction with the native [PSI+] prion in terms of co-polymerization, acquisition of the prion state, and induced prion loss, all of which were also dependent on the [PSI+] variant. The observed loss of S. cerevisiae [PSI+] can be related to inhibition of prion polymerization of S. cerevisiae Sup35 and formation of a non-heritable form of amyloid. We have therefore identified two distinct molecular origins of prion transmission barriers between closely sequence-related prion proteins: first, the inability of heterologous proteins to co-aggregate with host prion polymers, and second, acquisition by these proteins of a non-heritable amyloid fold.

    Sideri, Theodora C. and Koloteva-Levine, Nadejda and Tuite, Mick F. et al. (2011) Methionine Oxidation of Sup35 Protein Induces Formation of the [PSI+] Prion in a Yeast Peroxiredoxin Mutant. Journal of Biological Chemistry, 286 (45). pp. 38924-38931. ISSN 0021-9258.

    Abstract

    The frequency with which the yeast [PSI+] prion form of Sup35 arises de novo is controlled by a number of genetic and environmental factors. We have previously shown that in cells lacking the antioxidant peroxiredoxin proteins Tsa1 and Tsa2, the frequency of de novo formation of [PSI+] is greatly elevated. We show here that Tsa1/Tsa2 also function to suppress the formation of the [PIN+] prion form of Rnq1. However, although oxidative stress increases the de novo formation of both [PIN+] and [PSI+], it does not overcome the requirement of cells being [PIN+] to form the [PSI+] prion. We use an anti-methionine sulfoxide antibody to show that methionine oxidation is elevated in Sup35 during oxidative stress conditions. Abrogating Sup35 methionine oxidation by overexpressing methionine sulfoxide reductase (MSRA) prevents [PSI+] formation, indicating that Sup35 oxidation may underlie the switch from a soluble to an aggregated form of Sup35. In contrast, we were unable to detect methionine oxidation of Rnq1, and MSRA overexpression did not affect [PIN+] formation in a tsa1 tsa2 mutant. The molecular basis of how yeast and mammalian prions form infectious amyloid-like structures de novo is poorly understood. Our data suggest a causal link between Sup35 protein oxidation and de novo [PSI+] prion formation.

    Tuite, Mick F. and Marchante, Ricardo and Kushnirov, Vitaly V. (2011) Fungal Prions: Structure, Function and Propagation. Topics in Current Chemistry, 305. pp. 257-298. ISSN 0340-1022.

    Abstract

    Prions are not uniquely associated with rare fatal neurodegenerative diseases in the animal kingdom; prions are also found in fungi and in particular the yeast Saccharomyces cerevisiae. As with animal prions, fungal prions are proteins able to exist in one or more self-propagating alternative conformations, but show little primary sequence relationship with the mammalian prion protein PrP. Rather, fungal prions represent a relatively diverse collection of proteins that participate in key cellular processes such as transcription and translation. Upon switching to their prion form, these proteins can generate stable, sometimes beneficial, changes in the host cell phenotype. Much has already been learnt about prion structure, and propagation and de novo generation of the prion state through studies in yeast and these findings are reviewed here.

    Merritt, Gloria H. and Naemi, Wesley R. and Mugnier, Pierre M. et al. (2010) Decoding accuracy in eRF1 mutants and its correlation with pleiotropic quantitative traits in yeast. Nucleic acids research, 38 (16). pp. 5479-5492. ISSN 1362-4962.

    Abstract

    Translation termination in eukaryotes typically requires the decoding of one of three stop codons UAA, UAG or UGA by the eukaryotic release factor eRF1. The molecular mechanisms that allow eRF1 to decode either A or G in the second nucleotide, but to exclude UGG as a stop codon, are currently not well understood. Several models of stop codon recognition have been developed on the basis of evidence from mutagenesis studies, as well as studies on the evolutionary sequence conservation of eRF1. We show here that point mutants of Saccharomyces cerevisiae eRF1 display significant variability in their stop codon read-through phenotypes depending on the background genotype of the strain used, and that evolutionary conservation of amino acids in eRF1 is only a poor indicator of the functional importance of individual residues in translation termination. We further show that many phenotypes associated with eRF1 mutants are quantitatively unlinked with translation termination defects, suggesting that the evolutionary history of eRF1 was shaped by a complex set of molecular functions in addition to translation termination. We reassess current models of stop-codon recognition by eRF1 in the light of these new data.

    Tuite, Mick F. and Serio, Tricia R. (2010) The prion hypothesis: from biological anomaly to basic regulatory mechanism. Nature Reviews Molecular Cell Biology, 11 (12). pp. 823-833. ISSN 1471-0072.

    Abstract

    Prions are unusual proteinaceous infectious agents that are typically associated with a class of fatal degenerative diseases of the mammalian brain. However, the discovery of fungal prions, which are not associated with disease, suggests that we must now consider the effect of these factors on basic cellular physiology in a different light. Fungal prions are epigenetic determinants that can alter a range of cellular processes, including metabolism and gene expression pathways, and these changes can lead to a range of prion-associated phenotypes. The mechanistic similarities between prion propagation in mammals and fungi suggest that prions are not a biological anomaly but instead could be a newly appreciated and perhaps ubiquitous regulatory mechanism.

    Sideri, Theodora C. and Stojanovski, Klement and Tuite, Mick F. et al. (2010) Ribosome-associated peroxiredoxins suppress oxidative stress-induced de novo formation of the [PSI+] prion in yeast. Proceedings of the National Academy of Sciences, 107 (14). pp. 6394-6399. ISSN 0027-8424.

    Abstract

    Peroxiredoxins (Prxs) are ubiquitous antioxidants that protect cells against oxidative stress. We show that the yeast Tsa1/Tsa2 Prxs colocalize to ribosomes and function to protect the Sup35 translation termination factor against oxidative stress–induced formation of its heritable [PSI+] prion conformation. In a tsa1 tsa2 [psi-] [PIN+] strain, the frequency of [PSI+] de novo formation is significantly elevated. The Tsa1/Tsa2 Prxs, like other 2-Cys Prxs, have dual activities as peroxidases and chaperones, and we show that the peroxidase activity is required to suppress spontaneous de novo [PSI+] prion formation. Molecular oxygen is required for [PSI+] prion formation as growth under anaerobic conditions prevents prion formation in the tsa1 tsa2 mutant. Conversely, oxidative stress conditions induced by exposure to hydrogen peroxide elevates the rate of de novo [PSI+] prion formation leading to increased suppression of all three termination codons in the tsa1 tsa2 mutant. Altered translational fidelity in [PSI+] strains may provide a mechanism that promotes genetic variation and phenotypic diversity (True HL, Lindquist SL (2000) Nature 407:477–483). In agreement, we find that prion formation provides yeast cells with an adaptive advantage under oxidative stress conditions, as elimination of the [PSI+] prion from tsa1 tsa2 mutants renders the resulting [psi-] [pin-] cells hypersensitive to hydrogen peroxide. These data support a model in which Prxs function to protect the ribosomal machinery against oxidative damage, but when these systems become overwhelmed, [PSI+] prion formation provides a mechanism for uncovering genetic traits that aid survival during oxidative stress conditions.

    Moosavi, Behrooz and Wongwigkarn, Jintana and Tuite, Mick F. (2010) Hsp70/Hsp90 co-chaperones are required for efficient Hsp104-mediated elimination of the yeast [PSI+] prion but not for prion propagation. Yeast, 27 (3). pp. 167-179. ISSN 0749-503X.

    Abstract

    he continued propagation of the yeast [PSI+] prion requires the molecular chaperone Hsp104 yet in cells engineered to overexpress Hsp104; prion propagation is impaired leading to the rapid appearance of prion-free [psi?] cells. The underlying mechanism of prion loss in such cells is unknown but is assumed to be due to the complete dissolution of the prion aggregates by the ATP-dependent disaggregase activity of this chaperone. To further explore the mechanism, we have sought to identify cellular factors required for prion loss in such cells. Sti1p and Cpr7p are co-chaperones that modulate the activity of Hsp70/Ssa and Hsp90 chaperones and bind to the C-terminus of Hsp104. Neither Sti1p nor Cpr7p is necessary for prion propagation but we show that deletion of the STI1 and CPR7 genes leads to a significant reduction in the generation of [psi?] cells by Hsp104 overexpression. Deletion of the STI1 and CPR7 genes does not modify the elimination of [PSI+] by guanidine hydrochloride, which inhibits the ATPase activity of Hsp104 but does block elimination of [PSI+] by overexpression of either an ATPase-defective mutant of Hsp104 (hsp104K218T/K620T) or a ‘trap’ mutant Hsp104 (hsp104E285Q/E687Q) that can bind its substrate but can not release it. These results provide support for the hypothesis that [PSI+] elimination by Hsp104 overexpression is not simply a consequence of complete dissolution of the prion aggregates but rather is through a mechanism distinct from the remodelling activity of Hsp104.

    Jossé, Lyne and Smales, C. Mark and Tuite, Mick F. (2010) Transient expression of human TorsinA enhances secretion of two functionally distinct proteins in cultured Chinese hamster ovary (CHO) cells. Biotechnology and Bioengineering, 105 (3). pp. 556-566. ISSN 0006-3592.

    Abstract

    Cultured mammalian cells, particularly Chinese hamster ovary (CHO) cells, are widely exploited as hosts for the production of recombinant proteins, but often yields are limiting. Such limitations may be due in part to the misfolding and subsequent degradation of the heterologous proteins. Consequently we have determined whether transiently co-expressing yeast and/or mammalian chaperones that act to disaggregate proteins, in CHO cell lines, improve the levels of either a cytoplasmic (Fluc) or secreted (Gluc) form of luciferase or an immunoglobulin IgG4 molecule. Over-expression of the yeast ‘protein disaggregase’ Hsp104 in a CHO cell line increased the levels of Fluc more significantly than for Gluc although levels were not further elevated by over-expression of the yeast or mammalian Hsp70/40 chaperones. Over-expression of TorsinA, a mammalian protein related in sequence to yeast Hsp104, but located in the ER, significantly increased the level of secreted Gluc from CHO cells by 2.5-fold and to a lesser extent the secreted levels of a recombinant IgG4 molecule. These observations indicate that the over-expression of yeast Hsp104 in mammalian cells can improve recombinant protein yield and that over-expression of TorsinA in the ER can promote secretion of heterologous proteins from mammalian cells.

    Byrne, Lee J. and Cole, Diana J. and Cox, Brian S. et al. (2009) The Number and Transmission of [PSI+] Prion Seeds (Propagons) in the Yeast Saccharomyces Cerevisiae. PLoS ONE, Online. ISSN eISSN-1932-6203.

    Abstract

    Abstract Top Background Yeast (Saccharomyces cerevisiae) prions are efficiently propagated and the on-going generation and transmission of prion seeds (propagons) to daughter cells during cell division ensures a high degree of mitotic stability. The reversible inhibition of the molecular chaperone Hsp104p by guanidine hydrochloride (GdnHCl) results in cell division-dependent elimination of yeast prions due to a block in propagon generation and the subsequent dilution out of propagons by cell division. Principal Findings Analysing the kinetics of the GdnHCl-induced elimination of the yeast [PSI+] prion has allowed us to develop novel statistical models that aid our understanding of prion propagation in yeast cells. Here we describe the application of a new stochastic model that allows us to estimate more accurately the mean number of propagons in a [PSI+] cell. To achieve this accuracy we also experimentally determine key cell reproduction parameters and show that the presence of the [PSI+] prion has no impact on these key processes. Additionally, we experimentally determine the proportion of propagons transmitted to a daughter cell and show this reflects the relative cell volume of mother and daughter cells at cell division. Conclusions While propagon generation is an ATP-driven process, the partition of propagons to daughter cells occurs by passive transfer via the distribution of cytoplasm. Furthermore, our new estimates of n0, the number of propagons per cell (500–1000), are some five times higher than our previous estimates and this has important implications for our understanding of the inheritance of the [PSI+] and the spontaneous formation of prion-free cells.

    Studte, Patrick and Zink, Sabrina and Jablonowski, Daniel et al. (2008) tRNA and protein methylase complexes mediate zymocin toxicity in yeast. Molecular Microbiology, 69 (5). pp. 1266-1277. ISSN 0950-382X.

    Abstract

    Modification of Saccharomyces cerevisiae tRNA anticodons at the wobble uridine (U34) position is required for tRNA cleavage by the zymocin tRNase killer toxin from Kluyveromyces lactis. Hence, U34 modification defects including lack of the U34 tRNA methyltransferase Trm9 protect against tRNA cleavage and zymocin. Using zymocin as a tool, we have identified toxin-resistant mutations in TRM9 that are likely to affect the U34 methylation reaction. Most strikingly, C-terminal truncations in Trm9 abolish interaction with Trm112, a protein shown to individually purify with Lys9 and two more methylases, Trm11 and Mtq2. Downregulation of a GAL1-TRM112 allele protects against zymocin whereas LYS9, TRM11 and MTQ2 are dosage suppressors of zymocin. Based on immune precipitation studies, the latter scenario correlates with competition for Trm112 and in excess, some of these Trm112 partners interfere with formation of the toxin-relevant Trm9.Trm112 complex. In contrast to trm11 Delta or lys9 Delta cells, trm112 Delta and mtq2 Delta null mutants are zymocin resistant. In line with the identified role that methylation of Sup45 by Mtq2 has for translation termination by the release factor dimer Sup45.Sup35, we observe that SUP45 overexpression and sup45 mutants suppress zymocin. Intriguingly, this suppression correlates with upregulated levels of tRNA species targeted by zymocin's tRNase activity.

    Tuite, Mick F. and Stojanovski, Klement and Ness, Frederique et al. (2008) Cellular factors important for the de novo formation of yeast prions. Biochemical Society Transactions, 36 (Part 5). pp. 1083-1087. ISSN 0300-5127.

    Abstract

    Prions represent an unusual structural form of a protein that is 'infectious'. in mammals, prions are associated with fatal neurodegenerative diseases such as CJD (Creutzfeldt-jakob disease), while in fungi they act as novel epigenetic regulators of phenotype. Even though most of the human prion diseases arise spontaneously, we still know remarkably little about how infectious prions form de novo. The [PSI+] prion of the yeast Saccharomyces cerevisiae provides a highly tractable model in which to explore the underlying mechanism of de novo prion formation, in particular identifying key cis- and trans-acting factors. Most significantly, the de novo formation of [PSI+] requires the presence of a second prion called [PIN+], which is typically the prion form of Rnq1p, a protein rich in glutamine and aspartic acid residues. The molecular mechanism by which the [PIN+] prion facilitates de novo [PSI+] formation is not fully established, but most probably involves some form of cross-seeding. A number of other cellular factors, in particular chaperones of the Hsp70 (heat-shock protein 70) family, are known to modify the frequency of de novo prion formation in yeast.

    Byrne, Lee J. and Cox, Brian S. and Cole, Diana J. et al. (2007) Cell division is essential for elimination of the yeast [PSI+] prion by guanidine hydrochloride. Proceedings of the National Academy of Sciences of the United States of America, 104 (28). pp. 11688-11693. ISSN 0027-8424.

    Abstract

    Guanidine hydrochloride (Gdn center dot HCl) blocks the propagation of yeast prions by inhibiting Hsp104, a molecular chaperone that is absolutely required for yeast prion propagation. We had previously proposed that ongoing cell division is required for Gdn center dot HCl-induced loss of the [PSI+] prion. Subsequently, Wu et al. [Wu Y, Greene LE, Masison DC, Eisenberg E (2005) Proc Nat] Acad Sci USA 102:1278912794] claimed to show that Gdn center dot HCl can eliminate the [PSI+] prion from alpha-factor-arrested cells leading them to propose that in Gdn center dot HCl center dot treated cells the prion aggregates are degraded by an Hsp104-independent mechanism. Here we demonstrate that the results of Wu et al can be explained by an unusually high rate of alpha-factor-induced cell death in the [PSI+] strain (780-1D) used in their studies. What appeared to be no growth in their experiments was actually no increase in total cell number in a dividing culture through a counterbalancing level of cell death. Using media-exchange experiments, we provide further support for our original proposal that elimination of the [PSI+] prion by Gdn center dot HCl requires ongoing cell division and that prions are not destroyed during or after the evident curing phase.

    von der Haar, Tobias and Jossé, Lyne and Wright, P. et al. (2007) Development of a novel yeast cell-based system for studying the aggregation of Alzheimer's disease-associated A beta peptides in vivo. Neurodegenerative Diseases, 4 (2-3). pp. 136-147. ISSN 1660-2854.

    Abstract

    Alzheimer's disease is the most common neurodegenerative disease, affecting -50% of humans by age 85. The disease process is associated with aggregation of the AP peptides, short 39-43 residue peptides generated through endoproteolytic cleavage of the Alzheimer's precursor protein. While the process of aggregation of purified AP peptides in vitro is beginning to be well understood, little is known about this process in vivo. In the present study, we use the yeast Saccharomyces cerevisiae as a model system for studying A beta-mediated aggregation in an organism in vivo. One of this yeast's endogenous prions, Sup35/[PSI+] loses the ability to aggregate when the prion-forming domain of this protein is deleted. We show that insertion of AP pepticle sequences in place of the original prion domain of this protein restores its ability to aggregate. However, the aggregates are qualitatively different from [PSI+] prions in their sensitivity to detergents and in their requirements on transacting factors that are normally needed for [PSI+] propagation. We conclude that we have established a useful new tool for studying the aggregation of AP peptides in an organism in vivo.

    von der Haar, Tobias and Tuite, Mick F. (2007) Regulated translational bypass of stop codons in yeast. Trends in Microbiology, 15 (2). pp. 78-86. ISSN 0966-842X.

    Abstract

    Stop codons are used to signal the ribosome to terminate the decoding of an mRNA template. Recent studies on translation termination in the yeast Saccharomyces cerevisiale have not only enabled the identification of the key components of the termination machinery, but have also revealed several regulatory mechanisms that might enable the controlled synthesis of C-terminally extended polypeptides via stop-codon readthrough. These include both genetic and epigenetic mechanisms. Rather than being a translation 'error', stop-codon readthrough can have important effects on other cellular processes such as mRNA degradation and, in some cases, can confer a beneficial phenotype to the cell

    Cole, Diana J. and Ridout, Martin S. and Morgan, Byron J. T. et al. (2007) Approximations for expected generation number. Biometrics, 63 (4). pp. 1023-1030. ISSN 0006-341X.

    Abstract

    A deterministic formula is commonly used to approximate the expected generation number of a population of growing cells. However, this can give misleading results because it does not allow for natural variation in the times that individual cells take to reproduce. Here we present more accurate approximations for both symmetric and asymmetric cell division. Based on the first two moments of the generation time distribution, these approximations are also robust. We illustrate the improved approximations using data that arise from monitoring individual yeast cells under a microscope and also demonstrate how the approximaitions can be used when such detailed data are not available.

    Ridout, Martin S. and Cole, Diana J. and Morgan, Byron J. T. et al. (2006) New approximations to the Malthusian parameter. Biometrics, 62 (4). pp. 1216-1223. ISSN 0006-341X.

    Abstract

    Approximations to the Malthusian parameter of an age-dependent branching process are obtained in terms of the moments of the lifetime distribution, by exploiting a link with renewal theory. In several examples, the new approximations are more accurate than those currently in use, even when based on only the first two moments. The new approximations are extended to include a form of asymmetric cell division that occurs in some species of yeast. When used for inference, the new approximations are shown to have high efficiency.

    Shkundina, Irina S. and Kushnirov, Vitaly V. and Tuite, Mick F. et al. (2006) The role of the N-terminal oligopeptide repeats of the yeast Sup35 prion protein in propagation and transmission of prion variants. Genetics, 172 (2). pp. 827-835. ISSN 0016-6731.

    Abstract

    The cytoplasmic [PSI+] determinant of Saccharomyces cerevisiae is the prion form of the Sup35 protein. Oligopeptide repeats within the Sup35 N-terminal domain (PrD) presumably are required for the stable [PSI+] inheritance that in turn involves fragmentation of Sup35 polymers by the chaperone Hsp104. The nonsense suppressor [PSI+] phenotype can vary in efficiency probably due to different inheritable Sup35 polymer structures. Here we study the ability of Sup35 mutants with various deletions of the oligopeptide repeats to support [PSI+] propagation. We define the minimal region of the Sup35-PrD necessary to support [PSI+] as amino acids 1-64, which include the first two repeats, although a longer fragment, 1-83, is required to maintain weak [PSI+] variants. Replacement of wild-type Sup35 with deletion mutants decreases the strength of the [PSI+] phenotype. However, with one exception, reintroducing the wild-type Sup35 restores the original phenotype. Thus, the specific prion fold defining the [PSI+] variant can be preserved by the mutant Sup35 protein despite the change of phenotype. Coexpression of wild-type and mutant Sup35 containing three, two, one, or no oligopeptide repeats causes variant-specific [PSI+] elimination. These data suggest that [PSI+] variability is primarily defined by differential folding of the Sup35-PrD oligopeptide-repeat region.

    Tuite, Mick F. and Cox, Brian S. (2006) The [PSI+] prion of yeast: a problem of inheritance. Methods, 39 (1). pp. 9-22. ISSN 1046-2023.

    Abstract

    The [PSI(+)] prion of the yeast Saccharomyces cerevisiae was first identified by Brian Cox some 40 years ago as a non-Mendelian genetic element that modulated the efficiency of nonsense suppression. Following the suggestion by Reed Wickner in 1994 that such elements might be accounted for by invoking a prion-based model, it was subsequently established that the [PSI(+)] determinant was the prion form of the Sup35p protein. In this article, we review how a combination of classical genetic approaches and modern molecular and biochemical methods has provided conclusive evidence of the prion basis of the [PSI(+)] determinant. In so doing we have tried to provide a historical context, but also describe the results of more recent experiments aimed at elucidating the mechanism by which the [PSI(+)] (and other yeast prions) are efficiently propagated in dividing cells. While understanding of the [PSI(+)] prion and its mode of propagation has, and will continue to have, an impact on mammalian prion biology nevertheless the very existence of a protein-based mechanism that can have a beneficial impact on a cell's fitness provides equally sound justification to fully explore yeast prions.

    Zenthon, Joanna F. and Ness, Frederique and Cox, Brian S. et al. (2006) The [PSI+] prion of Saccharomyces cerevisiae can be propagated by an Hsp104 orthologue from Candida albicans. Eukaryotic Cell, 5 (2). pp. 217-225. ISSN 1535-9778.

    Abstract

    The molecular chaperone Hsp104 is not only a key component of the cellular machinery induced to disassemble aggregated proteins in stressed cells of Saccharomyces cerevisiae but also plays an essential role in the propagation of the [PSI+], [URE3], and [RNQ/PIN+] prions in this organism. Here we demonstrate that the fungal pathogen Candida albicans carries an 899-residue stress-inducible orthologue of Hsp104 (CaHsp104) that shows a high degree of amino acid identity to S. cerevisiae Hsp104 (ScHsp104). This identity is significantly lower in the N- and C-terminal regions implicated in substrate recognition and cofactor binding, respectively. CaHsp104 is able to provide all known functions of ScHsp104 in an S. cerevisiae hsp104 null mutant, i.e., tolerance to high-temperature stress, reactivation of heat-denatured proteins, and propagation of the [PSI+] prion. As also observed for ScHsp104, overexpression of CaHsp104 leads to a loss of the [PSI+] prion. However, unlike that of ScHsp104, CaHsp104 function is resistant to guanidine hydrochloride (GdnHCl), an inhibitor of the ATPase activity of this chaperone. These findings have implications both in terms of the mechanism of inhibition of Hsp104 by GdnHCl and in the evolution of the ability of fungal species to propagate prions.

    Alderton, Alex J. and Burr, Ian and Mühlschlegel, Fritz A. et al. (2006) Zeocin resistance as a dominant selective marker for transformation and targeted gene deletions in Candida glabrata. Mycoses, 49 (6). pp. 445-451. ISSN 0933-7407.

    Abstract

    Many of the genetic tools used to generate gene knockouts in Candida glabrata exploit auxotrophic markers but this is not suitable for use with clinical strains. Antibiotic resistance markers, however, allow one to target genes to be deleted without any prior genetic manipulation of clinical isolates. Such antibiotic selection markers have been widely reported for the manipulation of Saccharomyces cerevisiae. However, very few antibiotic resistance markers have been shown to be useful in C. glabrata. Here, we report the use of Zeocin resistance (ZeoR), encoded by the ble gene from Streptoalloteichus hindustanus, as a new positive selection marker for the genetic manipulation of C. glabrata including clinical strains that we show are significantly more sensitive to Zeocin than to G418. The potential of the ZeoR marker for targeted gene disruption in C. glabrata was confirmed by constructing deletions of the ADE2 in both a laboratory and a clinical strain of C. glabrata, using both short (90 bp) and long (400 bp) homology cassettes.

    Klengel, Torsten and Liang, Wei-Jun and Chaloupka, James et al. (2005) Fungal adenylyl cyclase integrates CO2 sensing with cAMP signaling and virulence. Current Biology, 15 (22). pp. 2021-2026. ISSN 0960-9822.

    Abstract

    The ascomycete Candida albicans is the most common fungal pathogen in immunocompromised patients . Its ability to change morphology, from yeast to filamentous forms, in response to host environmental cues is important for virulence . Filamentation is mediated by second messengers such as cyclic adenosine 3',5'-monophosphate (cAMP) synthesized by adenylyl cyclase . The distantly related basidiomycete Cryptococcus neoformans is an encapsulated yeast that predominantly infects the central nervous system in immunocompromised patients . Similar to the morphological change in C. albicans, capsule biosynthesis in C. neoformans, a major virulence attribute, is also dependent upon adenylyl cyclase activity . Here we demonstrate that physiological concentrations of CO2/HCO3- induce filamentation in C. albicans by direct stimulation of cyclase activity. Furthermore, we show that CO2/HCO3- equilibration by carbonic anhydrase is essential for pathogenesis of C. albicans in niches where the available CO2 is limited. We also demonstrate that adenylyl cyclase from C. neoformans is sensitive to physiological concentrations of CO2/HCO3-. These data demonstrate that the link between cAMP signaling and CO2/HCO3- sensing is conserved in fungi and reveal CO2 sensing to be an important mediator of fungal pathogenesis. Novel therapeutic agents could target this pathway at several levels to control fungal infections.

    Jones, Gareth J.F. and Tuite, Mick F. (2005) Chaperoning prions: the cellular machinery for propagating an infectious protein? Bioessays, 27 (8). pp. 823-832. ISSN 0265-9247.

    Abstract

    Newly made polypeptide chains require the help of molecular chaperones not only to rapidly reach their final three-dimensional forms, but also to unfold and then correctly refold them back to their biologically active form should they misfold. Most prions are an unusual type of protein that can exist in one of two stable conformations, one of which leads to formation of an infectious alternatively folded form. Studies in Baker's yeast (Saccharomyces cerevisiae) have revealed that prions can exploit the molecular chaperone machinery in the cell in order to ensure stable propagation of the infectious, aggregation-prone form. The disaggregation of yeast prion aggregates by molecular chaperones generates forms of the prion protein that can seed the protein polymerisation that underlies the prion propagation cycle. In this article, we review what we have learnt about the role of molecular chaperones in yeast prion propagation, describe a model that can explain the role of various classes of molecular chaperones and their co-chaperones, and speculate on the possible involvement of chaperones in the propagation of mammalian prions.

    Lund, Peter A. and Tuite, Mick F. (2005) Preventing illicit liaisons in Poland. EMBO Reports, 6 (12). pp. 1126-1130. ISSN 1469-221X.

    O'Callaghan, K.J. and Byrne, Lee J. and Tuite, Mick F. (2005) Extraction and denaturing gel electrophoretic methodology for the analysis of yeast proteins. Methods in Molecular Biology, 308. pp. 357-373. ISSN 1064-3745.

    Byrne, Lee J. and O'Callaghan, K.J. and Tuite, Mick F. (2005) Heterologous gene expression in yeast. Methods in Molecular Biology, 308. pp. 51-64. ISSN 1064-3745.

    Klengel, Torsten and Liang, Wei-Jun and Chaloupka, James et al. (2005) Fungal adenylyl cyclase integrates CO2 sensing with cAMP signaling and virulence. Current Biology, 15 (23). pp. 2177-2177. ISSN 0960-9822.

    Osherovich, Lev Z. and Cox, Brian S. and Tuite, Mick F. et al. (2004) Dissection and design of yeast prions. PLoS Biology, 2 (4). pp. 442-451. ISSN 1544-9173.

    Abstract

    Many proteins can misfold into beta-sheet-rich, self-seeding polymers (amyloids). Prions are exceptional among such aggregates in that they are also infectious. In fungi, prions are not pathogenic but rather act as epigenetic regulators of cell physiology, providing a powerful model for studying the mechanism of prion replication. We used prion-forming domains from two budding yeast proteins (Sup35p and New1p) to examine the requirements for prion formation and inheritance. In both proteins, a glutamine/asparagine-rich (Q/N-rich) tract mediates sequence-specific aggregation, while an adjacent motif, the oligopeptide repeat, is required for the replication and stable inheritance of these aggregates. Our findings help to explain why although Q/N-rich proteins are relatively common, few form heritable aggregates: prion inheritance requires both an aggregation sequence responsible for self-seeded growth and an element that permits chaperone-dependent replication of the aggregate. Using this knowledge, we have designed novel artificial prions by fusing the replication element of Sup35p to aggregation-prone sequences from other proteins, including pathogenically expanded polyglutamine.

    Cole, Diana J. and Morgan, Byron J. T. and Ridout, Martin S. et al. (2004) Estimating the number of prions in yeast cells. Mathematical Medicine and Biology, 21 (4). pp. 369-395. ISSN 1477-8599.

    Abstract

    Certain yeast cells contain proteins that behave like the mammalian prion PrP and are called yeast prions. The yeast prion protein Sup35p can exist in one of two stable forms, giving rise to phenotypes [PSI+] and [psi(-)]. If the chemical guanidine hydrochloride (GdnHCl) is added to a culture of growing [PSI+] cells, the proportion of [PSI+] cells decreases overtime. This process is called curing and is due to a failure to propagate the prion form of Sup35p. We describe how curing can be modelled, and improve upon previous models for the underlying processes of cell division and prion segregation; the new model allows for asymmetric cell division and unequal prion segregation. We conclude by outlining plans for future experimentation and modelling.

    Santos, Manuel and Moura, Gabriela and Massey, Steven E. et al. (2004) Driving change: the evolution of alternative genetic codes. Trends in Genetics, 20 (2). pp. 95-102. ISSN 0168-9525.

    Abstract

    Pioneering studies in the 1960s that elucidated the genetic code suggested that all extant forms of life use the same genetic code. This early presumption has subsequently been challenged by the discovery of deviations of the universal genetic code in prokaryotes, eukaryotic nuclear genomes and mitochondrial genomes. These studies have revealed that the genetic code is still evolving despite strong negative forces working against the fixation of mutations that result in codon reassignment. Recent data from in vitro, in vivo and in silico comparative genomics studies are revealing significant, previously overlooked links between modified nucleosides in tRNAs, genetic code ambiguity, genome base composition, codon usage and codon reassignment.

    Tuite, Mick F. (2004) Cell biology: the strain of being a prion. Nature, 428 (6980). pp. 265-267. ISSN 0028-0836.

    Tuite, Mick F. and Koloteva-Levine, Nadejda (2004) Propagating prions in fungi and mammals. Molecular Cell, 14 (5). pp. 541-552. ISSN 1097-2765.

    Abstract

    Prions constitute a rare class of protein, which can switch to a robust amyloid form and then propagate that form in the absence of a nucleic acid determinant, thereby creating a unique, protein-only infectious agent. Details of the mechanism that drives conversion to the prion form and then subsequent propagation of that form are beginning to emerge using a range of in vivo and in vitro approaches. Recent studies on both mammalian and fungal prions are providing a greater understanding of the structural features that distinguish prions from non-transmissible amyloids.

    Resende, Catarina and Outeiro, Tiago F. and Sands, Laina et al. (2003) Prion protein gene polymorphisms in Saccharomyces cerevisiae. Molecular Microbiology, 49 (4). pp. 1005-1018. ISSN 0950-382X.

    Abstract

    The yeast Saccharomyces cerevisiae genome encodes several proteins that, in laboratory strains, can take up a stable, transmissible prion form. In each case, this requires the Asn/Gln-rich prion-forming domain (PrD) of the protein to be intact. In order to further understand the evolutionary significance of this unusual property, we have examined four different prion genes and their corresponding PrDs, from a number of naturally occurring strains of S. cerevisiae. In 4 of the 16 strains studied we identified a new allele of the SUP35 gene (SUP35delta19) that contains a 19-amino-acid deletion within the N-terminal PrD, a deletion that eliminates the prion property of Sup35p. In these strains a second prion gene, RNQ1, was found to be highly polymorphic, with eight different RNQ1 alleles detected in the six diploid strains studied. In contrast, for one other prion gene (URE2) and the sequence of the NEW1 gene encoding a PrD, no significant degree of DNA polymorphism was detected. Analysis of the naturally occurring alleles of RNQ1 and SUP35 indicated that the various polymorphisms identified were associated with DNA tandem repeats (6, 12, 33, 42 or 57 bp) within the coding sequences. The expansion and contraction of DNA repeats within the RNQ1 gene may provide an evolutionary mechanism that can ensure rapid change between the [PRION+] and [prion-] states.

    Cox, Brian S. and Ness, Frederique and Tuite, Mick F. (2003) Analysis of the generation and segregation of propagons: entities that propagate the [PSI+] prion in yeast. Genetics, 165 (1). pp. 23-33. ISSN 0016-6731.

    Abstract

    The propagation of the prion form of the yeast Sup35p protein, the so-called [PSI(+)] determinant, involves the generation and partition of a small number of particulate determinants that we propose calling "propagons." The numbers of propagons in [PSI(+)] cells can be inferred from the kinetics of elimination of [PSI(+)] during growth in the presence of a low concentration of guanidine hydrochloride (GdnHCl). Using this and an alternative method of counting the numbers of propagons, we demonstrate considerable clonal variation in the apparent numbers of propagons between different [PSI(+)] yeast strains, between different cultures of the same [PSI(+)] yeast strain, and between different cells of the same [PSI(+)] culture. We provide further evidence that propagon generation is blocked by growth in GdnHCl and that it is largely confined to the S phase of the cell cycle. In addition, we show that at low propagon number there is a bias toward retention of propagons in mother cells and that production of new propagons is very rapid when cells with depleted numbers of propagons are rescued into normal growth medium. The implications of our findings with respect to yeast prion propagation mechanisms are discussed.

    Tuite, Mick F. and Cox, Brian S. (2003) Propagation of yeast prions. Nature Reviews Molecular Cell Biology, 4 (11). pp. 878-889. ISSN 1471-0072.

    Abstract

    Discusses the mechanism by which certain yeast proteins can take on and propagate a transmissible prion form. Information on the prion-associated phenotypes in yeast; Criteria that establish a prion; Details on the key sequence features for prion conversion and propagation.

    Massey, Steven E. and Moura, Gabriela and Beltrao, Pedro et al. (2003) Comparative evolutionary genomics unveils the molecular mechanism of reassignment of the CTG codon in Candida spp. Genome Research, 13 (4). pp. 544-557. ISSN 1088-9051.

    Abstract

    Using the (near) complete genome sequences of the yeasts Candida albicans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, we address the evolution of a unique genetic code change, which involves decoding of the standard leucine-CTG codon as serine in Candida spp. By using two complementary comparative genomics approaches, we have been able to shed new light on both the origin of the novel Candida spp. Ser-tRNA(CAG), which has mediated CTG reassignment, and on the evolution of the CTG codon in the genomes of C. albicans, S. cerevisiae, and S. pombe. Sequence analyses of newly identified tRNAs from the C. albicans genome demonstrate that the Ser-tRNA(CAG) is derived from a serine and not a leucine tRNA in the ancestor yeast species and that this codon reassignment occurred approximately 170 million years ago, but the origin of the Ser-tRNA(CAG) is more ancient, implying that the ancestral Leu-tRNA that decoded the CTG codon was lost after the appearance of the Ser-tRNA(CAG). Ambiguous CTG decoding by the Ser-tRNA(CAG) combined with biased AT pressure forced the evolution of CTG into TTR codons and have been major forces driving evolution of the CTN codon family in C. albicans. Remarkably, most of the CTG codons present in extant C. albicans genes are encoded by serine and not leucine codons in homologous S. cerevisiae and S. pombe genes, indicating that a significant number of serine TCN and AGY codons evolved into CTG codons either directly by simultaneous double mutations or indirectly through an intermediary codon. In either case, CTG reassignment had a major impact on the evolution of the coding component of the Candida spp. genome.

    Ness, Frederique and Ferreira, Paulo C. and Cox, Brian S. et al. (2002) Guanidine hydrochloride inhibits the generation of prion "seeds" but not prion protein aggregation in yeast. Molecular and Cellular Biology, 22 (15). pp. 5593-5605. ISSN 0270-7306.

    Abstract

    [PSI(+)] strains of the yeast Saccharomyces cerevisiae replicate and transmit the prion form of the Sup35p protein but can be permanently cured of this property when grown in millimolar concentrations of guanidine hydrochloride (GdnHCl). GdnHCl treatment leads to the inhibition of the replication of the [PSI(+)] seeds necessary for continued [PSI(+)] propagation. Here we demonstrate that the rate of incorporation of newly synthesized Sup35p into the high-molecular-weight aggregates, diagnostic of [PSI(+)] strains, is proportional to the number of seeds in the cell, with seed number declining (and the levels of soluble Sup35p increasing) in the presence of GdnHCl. GdnHCl does not cause breakdown of preexisting Sup35p aggregates in [PSI(+)] cells. Transfer of GdnHCl-treated cells to GdnHCl-free medium reverses GdnHCl inhibition of [PSI(+)] seed replication and allows new prion seeds to be generated exponentially in the absence of ongoing protein synthesis. Following such release the [PSI(+)] seed numbers double every 20 to 22 min. Recent evidence (P. C. Ferreira, F. Ness, S. R. Edwards, B. S. Cox, and M. F. Tuite, Mol. Microbiol. 40:1357-1369, 2001; G. Jung and D. C. Masison, Curr. Microbiol. 43:7-10, 2001), together with data presented here, suggests that curing yeast prions by GdnHCl is a consequence of GdnHCl inhibition of the activity of molecular chaperone Hsp104, which in turn is essential for [PSI(+)] propagation. The kinetics of elimination of [PSI(+)] by coexpression of a dominant, ATPase-negative allele of HSP104 were similar to those observed for GdnHCl-induced elimination. Based on these and other data, we propose a two-cycle model for "prionization" of Sup35p in [PSI(+)] cells: cycle A is the GdnHCl-sensitive (Hsp104-dependent) replication of the prion seeds, while cycle B is a GdnHCl-insensitive (Hsp104-independent) process that converts these seeds to pelletable aggregates.

    Fernandez-Bellot, Eric and Guillemet, Elisabeth and Ness, Frederique et al. (2002) The [URE3] phenotype: evidence for a soluble prion in yeast. EMBO Reports, 3 (1). pp. 76-81. ISSN 1469-221X.

    Abstract

    The aggregation of the two yeast proteins Sup35p and Ure2p is widely accepted as a model for explaining the prion propagation of the phenotypes [PSI+] and [URE3], respectively. Here, we demonstrate that the propagation of [URE3] cannot simply be the consequence of generating large aggregates of Ure2p, because such aggregation can be found in some conditions that are not related to the prion state of Ure2p. A comparison of [PSI+] and [URE3] aggregation demonstrates differences between these two prion mechanisms. Our findings lead us to propose a new unifying model for yeast prion propagation.

    Moura, Gabriela and Miranda, Isabel and Cheesman, Caroline et al. (2002) Stop codon decoding in Candida albicans: from non-standard back to standard. Yeast, 19 (9). pp. 727-733. ISSN 0749-503X.

    Abstract

    The human pathogen Candida albicans translates the standard leucine-CUG codon as serine. This genetic code change is mediated by a novel ser-tRNA(CAG), which induces aberrant mRNA decoding in vitro, resulting in retardation of the electrophoretic mobility of the polypeptides synthesized in its presence. These non-standard decoding events have been attributed to readthrough of the UAG and UGA stop codons encoded by the Brome Mosaic Virus RNA 4, which codes for the virion coat protein, and the rabbit globin mRNAs, respectively. In order to fully elucidate the behaviour of the C. albicans ser-tRNA(CAG) towards stop codons, we have used other cell-free translation systems and reporter genes. However, the reporter systems used encode several CUG codons, making it impossible to distinguish whether the slow migration of the polypeptides is caused by the replacement of leucines by serines at the CUG codons, readthrough, or a combination of both. Therefore, we have constructed new reporter systems lacking CUG codons and have used them to demonstrate that aberrant mRNA decoding in vitro is not a result from stop codon readthrough or any other non-standard translational event. Our data show that a single leucine to serine replacement at only one of the four CUG codons encoded by the BMV RNA-4 gene is responsible for the aberrant migration of the BMV coat protein on SDS-PAGE, suggesting that this amino acid substitution (ser for leu) significantly alters the structure of the virion coat protein. The data therefore show that the only aberrant event mediated by the ser-tRNA(CAG) is decoding of the leu-CUG codon as serine.

    Resende, Catarina and Parham, Steven N. and Tinsley, Caroline et al. (2002) The Candida albicans Sup35p protein (CaSup35p): function, prion-like behaviour and an associated polyglutamine length polymorphism. Microbiology, 148 (Pt 4). pp. 1049-1060. ISSN 0002-4564.

    Abstract

    The Sup35p protein of Saccharomyces cerevisiae is an essential translation factor whose prion-like properties give rise to the non-Mendelian genetic element [PSI(+)]. In this study the SUP35 gene from the related yeast species Candida albicans has been characterized. The CaSUP35 gene encodes a protein (CaSup35p) of 729 aa which shows 65% amino acid identity to the S. cerevisiae Sup35p protein (ScSup35p), with the C-terminal region showing greater identity (79%) than the N-terminal region. The full-length CaSup35p can functionally replace ScSup35p in S. cerevisiae although complementation is only complete when CaSup35p is overexpressed. Complementation only requires expression of the CaSup35p C domain. In S. cerevisiae the full-length CaSup35p is unable to establish a prion-like aggregated state even in the presence of endogenous ScSup35p prion 'seeds', thus confirming the existence of a species barrier in fungal prion propagation. Subcellular localization studies in C. albicans show that although CaSup35p is normally ribosome-associated, when not ribosome-associated, it does not form pelletable high-molecular-mass aggregates characteristic of the ScSup35p in [PSI(+)] strains. Unlike the ScSup35p, the CaSup35p N domain contains a number of polyglutamine repeats although it does contain seven copies of the peptide GGYQQ that is repeated in the ScSup35p N domain. Analysis of the CaSUP35 gene from 14 different strains of C. albicans identified four naturally occurring polymorphisms associated with changes in the length of the largest of the polyglutamine repeats. These findings have important implications for the evolution of fungal prion genes.

    Parham, Steven N. and Resende, Catarina and Tuite, Mick F. (2001) Oligopeptide repeats in the yeast protein Sup35p stabilize intermolecular prion interactions. EMBO Journal, 20 (9). pp. 2111-2119. ISSN 0261-4189.

    Abstract

    The nuclear-encoded Sup35p protein is responsible for the prion-like [PSI(+)] determinant of yeast, with Sup35p existing largely as a high molecular weight aggregate in [PSI(+)] strains. Here we show that the five oligopeptide repeats present at the N-terminus of Sup35p are responsible for stabilizing aggregation of Sup35p in vivo. Sequential deletion of the oligopeptide repeats prevented the maintenance of [PSI(+)] by the truncated Sup35p, although deletants containing only two repeats could be incorporated into pre-existing aggregates of wild-type Sup35p. The mammalian prion protein PrP also contains similar oligopeptide repeats and we show here that a human PrP repeat (PHGGGWGQ) is able functionally to replace a Sup35p oligopeptide repeat to allow stable [PSI(+)] propagation in vivo. Our data suggest a model in which the oligopeptide repeats in Sup35p stabilize intermolecular interactions between Sup35p proteins that initiate establishment of the aggregated state. Modulating repeat number therefore alters the rate of yeast prion conversion in vivo. Furthermore, there appears to be evolutionary conservation of function of the N-terminally located oligopeptide repeats in prion propagation.

    Ferreira, Paulo C. and Ness, Frederique and Edwards, Suzanne R. et al. (2001) The Elimination of the Yeast [PSI+] Prion by Guanidine Hydrochloride is the result of Hsp104 Inactivation. Molecular Microbiology, 40 (6). pp. 1357-1369. ISSN 0950-382X.

    Abstract

    In the yeast Saccharomyces cerevisiae, Sup35p (eRF3), a subunit of the translation termination complex, can take up a prion-like, self-propagating conformation giving rise to the non-Mendelian [PSI+] determinant. The replication of [PSI+] prion seeds can be readily blocked by growth in the presence of low concentrations of guanidine hydrochloride (GdnHCl), leading to the generation of prion-free [psi-] cells. Here, we provide evidence that GdnHCl blocks seed replication in vivo by inactivation of the molecular chaperone Hsp104. Although growth in the presence of GdnHCl causes a modest increase in HSP104 expression (20-90%), this is not sufficient to explain prion curing. Rather, we show that GdnHCl inhibits two different Hsp104-dependent cellular processes, namely the acquisition of thermotolerance and the refolding of thermally denatured luciferase. The inhibitory effects of GdnHCl protein refolding are partially suppressed by elevating the endogenous cellular levels of Hsp104 using a constitutive promoter. The kinetics of GdnHCl-induced [PSI+] curing could be mimicked by co-expression of an ATPase-negative dominant HSP104 mutant in an otherwise wild-type [PSI+] strain. We suggest that GdnHCl inactivates the ATPase activity of Hsp104, leading to a block in the replication of [PSI+] seeds.

    O'Sullivan, Justin M. and Davenport, J. Bernard and Tuite, Mick F. (2001) Codon Reassignment and the Evolving Genetic Code: Problems and Pitfalls in Post-genome Analysis. Trends in Genetics, 17 (1). pp. 20-22. ISSN 0168-9525.

    Abstract

    The in silico translation of open reading frames, using the 'universal genetic code', must be approached with caution. The uncovering of a number of codon reassignments in nuclear and organellar genomes highlights the importance of experimentally confirming the assignments of all 64 codons for the species whose genome is under investigation. Such alterations to codon meaning also suggest that the genetic code is not 'frozen' and continues to evolve.

    O'Sullivan, Justin M. and Mihr, Marian J. and Santos, Manuel et al. (2001) The Candida Albicans Gene Encoding the Cytoplasmic Leucyl-tRNA Synthetase: Implications for the Evolution of CUG Codon Reassignment. Gene, 275 (1). pp. 133-140. ISSN 0378-1119.

    Abstract

    In a number of Candida species the 'universal' leucine codon CUG is decoded as serine. To help understand the evolution of such a codon reassignment we have analyzed the Candida albicans leucyl-tRNA synthetase (CaLeuRS) gene (CaCDC60). The predicted CaLeuRS sequence shows a significant level of amino acid identity to LeuRS from other organisms. A mitochondrial LeuRS (ScNAM2) homologue, which shared low identity with the CaLeuRS, was also identified in C. albicans. Antigenically-related LeuRSs were identified in a range of Candida species decoding the CUG codon as both serine and leucine, using an antibody raised against the N-terminal 15 amino acids of the CaLeuRS. Complementation experiments demonstrated that the CaLeuRS was able to functionally complement a Saccharomyces cerevisiae cdc60:kanMX null mutation. We conclude that there is no alteration in tRNA recognition and aminoacylation by the C. albicans LeuRS, which argues against it having a role in codon reassignment. The nucleotide sequences of the CaCDC60 and CaNAM2 genes were deposited at GenBank under Accession numbers AF293346 and AF352020, respectively.

    O'Sullivan, Justin M. and Mihr, Marian J. and Santos, Manuel et al. (2001) Seryl-tRNA Synthetase is not responsible for the Evolution of CUG Codon Reassignment in Candida Albicans. Yeast, 18 (4). pp. 313-322. ISSN 0749-503X.

    Abstract

    A number of Candida species translate the standard leucine-CUG codon as serine using a novel ser-tRNA(CAG). This tRNA, which has an unusual anticodon stem-loop structure, has been implicated in the evolution of this codon reassignment. However, such a sense codon reassignment might also require a change in the specificity of the cognate aminoacyl tRNA-synthetase, in this case the ser-tRNA synthetase. Here we describe the cloning and sequence analysis of the C. albicans seryl aminoacyl-tRNA synthetase (CaSerRS) gene (CaSES1). The predicted CaSerRS sequence shows a significant level of amino acid identity to SerRs from other organisms and fully complements a S. cerevisiae SerRS null strain without any apparent defect in growth rate. This suggests that the SerRS recognizes and charges S. cerevisiae ser-tRNAs with similar efficiency to that of the S. cerevisiae SerRS. Using an antibody raised against CaSerRS, we also demonstrate the presence of SerRS in a range of Candida spp. showing CUG codon reassignment. We conclude that the key element in CUG reassigment in Candida spp. is the tRNA that decodes the CUG codon rather than a SerRS structural change. The nucleotide sequence of the CaSES1 gene has been deposited at GenBank under Accession No. AF290915.

    Tuite, Mick F. (2000) Cell biology. Sowing the protein seeds of prion propagation. Science, 289 (5479). pp. 556-557. ISSN 0036-8075.

    Abstract

    Ever since Prusiner first proposed his radical "protein-only" hypothesis to explain how certain infectious proteins (prions) are transmitted from one mammal to another in the absence of DNA or RNA, scientists have been trying to prove him right (or wrong). The study of mammalian prions, such as those causing Creutzfeldt-Jakob disease in humans, scrapie in sheep and mad cow disease in cattle, has been slow to yield answers. However, as Tuite discusses in his Perspective, the Sup35p and Ure2p proteins of yeast that exist in both normal and infectious forms are providing evidence that the "protein-only" hypothesis may be right (Sparrer et al.).

    Eaglestone, Simon S. and Ruddock, Lloyd W. and Cox, Brian S. et al. (2000) Guanidine hydrochloride blocks a critical step in the propagation of the prion-like determinant [PSI+] of Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences of the United States of America, 97 (1). pp. 240-244. ISSN 0027-8424.

    Abstract

    The cytoplasmic heritable determinant [PSI+] of the yeast Saccharomyces cerevisiae reflects the prion-like properties of the chromosome-encoded protein Sup35p. This protein is known to be an essential eukaryote polypeptide release factor, namely eRF3. In a [PSl(+)] background, the prion conformer of Sup35p forms large oligomers, which results in the intracellular depletion of functional release factor and hence inefficient translation termination. We have investigated the process by which the [PSI+] determinant can be efficiently eliminated from strains, by growth in the presence of the protein denaturant guanidine hydrochloride (GuHCl). Strains are "cured" of [PSI+] by millimolar concentrations of GuHCl, well below that normally required for protein denaturation. Here we provide evidence indicating that the elimination of the [PSI+] determinant is not derived from the direct dissolution of self-replicating [PSI+] seeds by GuHCl. Although GuHCl does elicit a moderate stress response, the elimination of [PSI+] is not enhanced by stress, and furthermore, exhibits an absolute requirement for continued cell division. We propose that GuHCl inhibits a critical event in the propagation of the prion conformer and demonstrate that the kinetics of curing by GuHCl fit a random segregation model whereby the heritable [PSI+] element is diluted from a culture. after the total inhibition of prion replication by GuHCl.

    Song, Haiwei and Mugnier, Pierre M. and Das, Amit K. et al. (2000) The crystal structure of human eukaryotic release factor eRF1 - Mechanism of stop codon recognition and peptidyl-tRNA hydrolysis. Cell, 100 (3). pp. 311-321. ISSN 0092-8674.

    Abstract

    The release factor eRF1 terminates protein biosynthesis by recognizing stop codons at the A site of the ribosome and stimulating peptidyl-tRNA bond hydrolysis at the peptidyl transferase center. The crystal structure of human eRF1 to 2.8 Angstrom resolution, combined with mutagenesis analyses of the universal GGQ motif, reveals the molecular mechanism of release factor activity. The overall shape and dimensions of eRF1 resemble a tRNA molecule with domains 1, 2, and 3 of eRF1 corresponding to the anticodon loop, aminoacyl acceptor stem, and T stem of a tRNA molecule, respectively. The position of the essential GGQ motif at an exposed tip of domain 2 suggests that the Gin residue coordinates a water molecule to mediate the hydrolytic activity at the peptidyl transferase center. A conserved groove on domain 1, 80 Angstrom from the GGQ motif, is proposed to form the codon recognition site.

Book Sections

    Tuite, Mick F. (2013) The Natural History of Yeast Prions. In: Sariaslani, Sima and Gadd, Geoffrey M. Advances in Applied Microbiology. Elsevier Inc., pp. 85-137. ISBN 9780124076730.

    Abstract

    Although prions were first discovered through their link to severe brain degenerative diseases in animals, the emergence of prions as regulators of the phenotype of the yeast Saccharomyces cerevisiae and the filamentous fungus Podospora anserina has revealed a new facet of prion biology. In most cases, fungal prions are carried without apparent detriment to the host cell, representing a novel form of epigenetic inheritance. This raises the question of whether or not yeast prions are beneficial survival factors or actually gives rise to a “disease state” that is selected against in nature. To date, most studies on the impact of fungal prions have focused on laboratory-cultivated “domesticated” strains of S. cerevisiae. At least eight prions have now been described in this species, each with the potential to impact on a wide range of cellular processes. The discovery of prions in nondomesticated strains of S. cerevisiae and P. anserina has confirmed that prions are not simply an artifact of “domestication” of this species. In this review, I describe what we currently know about the phenotypic impact of fungal prions. I then describe how the interplay between host genotype and the prion-mediated changes can generate a wide array of phenotypic diversity. How such prion-generated diversity may be of benefit to the host in survival in a fluctuating, often hazardous environment is then outlined. Prion research has now entered a new phase in which we must now consider their biological function and evolutionary significance in the natural world.

    Staniforth, Gemma L. and Tuite, Mick F. (2012) Fungal Prions. In: UNSPECIFIED Molecular Biology of Neurodegenerative Diseases. Progress in Molecular Biology and Translational Science, 107. Elsevier, pp. 417-456. ISBN 978012385883-2.

    Abstract

    For both mammalian and fungal prion proteins, conformational templating drives the phenomenon of protein-only infectivity. The conformational conversion of a protein to its transmissible prion state is associated with changes to host cellular physiology. In mammals, this change is synonymous with disease, whereas in fungi no notable detrimental effect on the host is typically observed. Instead, fungal prions can serve as epigenetic regulators of inheritance in the form of partial loss-of-function phenotypes. In the presence of environmental challenges, the prion state [PRION+], with its resource for phenotypic plasticity, can be associated with a growth advantage. The growing number of yeast proteins that can switch to a heritable [PRION+] form represents diverse and metabolically penetrating cellular functions, suggesting that the [PRION+] state in yeast is a functional one, albeit rarely found in nature. In this chapter, we introduce the biochemical and genetic properties of fungal prions, many of which are shared by the mammalian prion protein PrP, and then outline the major contributions that studies on fungal prions have made to prion biology.

    Jossé, Lyne and Smales, C. Mark and Tuite, Mick F. (2012) Engineering the Chaperone Network of CHO Cells for Optimal Recombinant Protein Production and Authenticity. In: UNSPECIFIED Recombinant Gene Expression. Springer New York, pp. 595-608. ISBN 9781617794339.

    Abstract

    All proteins fold into a defined three-dimensional shape that is compatible with the cellular role and/or biological activity of those proteins. Molecular chaperones are a family of proteins whose role is to assist the folding and targeting of proteins in both normal and stressed cells. The rational manipulation of chaperone levels in a cell line engineered to produce a defined recombinant protein (rP) can significantly improve both the achievable steady-state levels and authenticity of a wide range of recombinant proteins. Here, we describe the methodology associated with expressing a variety of molecular chaperones in Chinese hamster ovary (CHO) lines in order to improve their recombinant protein production capacity. These chaperones include both those that facilitate the folding of the polypeptide chain (i.e. Hsp70, Hsp40) and those that can re-fold proteins that have misfolded in the cell (i.e. ClpB/Hsp104). This latter property is particularly important given the propensity for highly expressed recombinant proteins to misfold in the “foreign” cellular environment.

    Tuite, Mick F. and Byrne, Lee J. and Jossé, Lyne et al. (2007) Yeast prions and their analysis in vivo. In: UNSPECIFIED Yeast gene analysis, second edition. Methods in Microbiology, 36. Elsevier Academic Press, San Diego, US, pp. 491-526. ISBN 0123694795.

    Cox, Brian S. and Byrne, Lee J. and Tuite, Mick F. (2007) Prion stability. In: Chernoff, Yury O. Protein-based inheritance. Landes Biosciences, pp. 56-72. ISBN 978-1-58706-138-7.

    Tuite, Mick F. and Cox, Brian S. (2007) The genetic control of the formation and propagation of the [PSI+] prion. In: Chernoff, Yury O. Protein-based inheritance. Landes Biosciences, pp. 14-29. ISBN 978-1-58706-138-7.

Reviews
Conference Items

    Whalley, Jacqueline L. and Tuite, Mick F. and Johnson, Colin G. (2002) A virtual lab for exploring the [PSI]+ yeast prion. In: Valafar, Faramarz CSERA Press, USA pp. 583-589. ISBN 1-892512-32-7.

    Abstract

    This paper discusses on going work on simulating the propogation within the cell of a prion protein in yeast. The biological background to the project is outlined, and a number of questions about this system are posed. The paper then discusses how computer simulation is being used to provide a ''virtual laboratory'' for this system, which will be employed to support and understand real experiments. Futhermore, the development of the application is detailed with emphasis on the challenges encountered to date.

Total publications in KAR: 139 [See all in KAR]

 

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Protein misfolding, prions and molecular chaperones

Creutzfeldt-Jakob Disease (CJD) is an unusual infectious disease of the human brain that leads to dementia and death. The most baffling fact about this and related diseases such as sheep scrapie and bovine spongiform encephalopathy (BSE, Mad Cow Disease) is that they appear to be triggered by a unique class of infectious agent known as a prion (protein-only infectious agent). Although CJD is a comparatively rare disease in humans (approximately 1 in a million people will contract the disease in any one year), sufferers show many of the pathological features seen in individuals suffering from other more common, non-infectious diseases of the brain such as Alzheimer's Disease and Parkinson's Disease. In spite of its infectious nature, the majority of cases of human CJD (~80%) are known to occur 'sporadically' i.e. appear spontaneously, without any evidence of the diseased individual acquiring an infectious prion from a third party, or through a mutation in the individual's genes. Prions are highly unusual infectious agents because they consist only of a single protein that has become structurally altered. In humans the prion protein responsible for CJD is called PrP, a protein normally found in the brain. Yet we know very little about how the PrP-based prions take up their altered structure spontaneously to cause sporadic CJD, what will trigger their formation or how prions are propagated and transmitted once formed in the cell.

To help us address these important questions we study prions that are found in Baker's yeast (Saccharomyces cerevisiae). Prions were first described in this fungus some 15 years ago by Reed Wickner and the many subsequent studies on yeast prions have revealed fascinating often surprising new aspects of prion biology not least the fact that their presence can be of benefit to the host they infect rather than detrimental.

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The yeast Saccharomyces cerevisiae in its many guises

Recent reviews published by the Tuite Laboratory

  • Tuite, M.F. and Serio, T.R. (2010). The prion hypothesis: from biological anomaly to basic regulatory mechanism. Nature Reviews in Molecular Cell Biology 11, 823-833.
  • Naeimi, W.R. and Tuite, M.F. (2010) Fungal prions as epigenetic determinants. The Biochemist 32: 30-33.
  • Tuite, M.F., Marchante, R. and Kushnirov, V.V. (2011) Fungal prions: structure, function and propagation. In: Prion Proteins (Topics in Current Chemistry, volume 305), Ed. Jorg Tatzelt, Springer, pp 257-98.
  • Staniforth, G.L and Tuite, M.F. (2012) Fungal prions. Progress in Molecular Biology and Translational Science 107: 417–456.
  • Naeimi, W.R. and Tuite, M.F. (2012) Prions as epigenetic regulators of phenotype in fungi. In Encyclopaedia of Molecular Cell Biology and Molecular Medicine: Epigenetic Regulation and Epigenomics (Ed. R A Meyers), Wiley-VCH Verlag GmbH & Co. pp741-770.

....and on-line seminar

  • Tuite, M.F.. (2008), "Mechanisms of yeast prion propagation", in Wickner, R. (ed.), Prions and Amyloids: Self-propagating protein structures in mammals, yeast and fungi, The Biomedical & Life Sciences Collection, Henry Stewart Talks Ltd, London (online at http://www.hstalks.com/?t=BL0521794-Tuite

Current projects

Defining the sequence and structural features important for amyloidogenesis and prion propagation in yeast

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The main objective of this project is to identify the role of a particular region of the yeast prion protein in question, namely the protein Sup35p, in the continued replication of the transmissible prion form. The region of the Sup35p protein molecule that will be investigated lies at the extreme amino terminus of the protein, a region known as the prion-forming domain (PrD). This region in turn has two functionally distinct elements: a 40 residue ‘aggregation domain’ and a 56 residue ‘propagation domain’. The important feature of the aggregation domain is its ability to drive the formation of amyloid-like fibres of the protein in the test tube. This property is due in part to its high content of amino acid residues glutamine (Q) and asparagine (N). This region, which is referred to as the QN-rich (QNR) region, will be the focus of the project. The major objectives of this project are:

  1. to define the minimal region of the QNR region with respect to prion propagation;
  2. to study the structure of the QNR region using NMR;
  3. to identify the important sequence and/or structural features of the oligopeptide repeat region (OPR) of the Sup35p-PrD necessary for stable propagation of the prion form of Sup35p.


Researchers: Dr Ricardo Marchante (postdoc), Faye Russell (MSc student)

Collaborators: Dr Mark Howard (Kent), Dr Vitaly Kushnirov (Cardiology Research Centre, Moscow),Dr Wei-Feng Xue Biosciences (Kent)

Funded by: The Wellcome Trust.

Modelling prion dynamics in the living yeast cell

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Two models have been used to explain the self-propagation of prions in yeast and mammals; the template-directed refolding model ('seeded polymerisation') and the inhibitor titration model. The former model has gained general acceptance and invokes the formation of oligomeric seeds that drive the structural alteration and polymerisation of soluble molecules of the prion protein thus forming the characteristic high molecular weight amyloid fibrils. Stable propagation of yeast prions thus requires continued generation of new prion seeding molecular entities (which we call propagons) and their efficient transmission to daughter cells at cell division. In this project we are exploiting a novel, stochastic modelling approach to establish the key molecular events in the propagation and transmission of the [PSI+] prion.

Our current models provide an important phenomenological description of how the [PSI+] prion is eliminated in the absence of a mechanism to generate new propagons. In this new project we are developing new mechanistic models that not only take into account the kinetics of prion protein polymerisation, but also polymer fragmentation in the growing yeast cell. To achieve this we are generating stochastic simulation models for yeast prion polymer kinetics and supporting this model building by experimentally establishing the values of the important model factors including the cellular levels of Sup35p and the key chaperone proteins Hsp104 and Sis1p, the number and sizes of Sup35p prion polymers and the rate of synthesis and turnover of Sup35p in its various cellular forms.

To evaluate the emerging simulation models we are also assessing the in vivo consequences of modulating levels of Sup35p, Hsp104 and Sis1p all of which are essential for [PSI+] propagation. One crucial parameter in our models is pi, the proportion of propagons transmitted to the daughter cell. We wish to identify factors that lead to a change in pi, particularly in relation to the number and length of Sup35p polymers in the cell. We also carry out model sensitivity and validation studies in the light of experimental data obtained in order to generate a definitive stochastic simulation model for yeast prion polymer kinetics in the dividing cell. This project involves a close interplay between bioscience-led research and integrated mathematical modelling with the aim of shedding new light on how infectious amyloids are generated and transmitted in vivo.

Researchers: Dr Wesley Naeimi (postdoc), Dr Vasileios Giagos (postdoc), Jintana Wongwigkarn (PhD student)


Collaborators: Professors Byron Morgan and Martin Ridout (School of Mathematics, Statistics and Actuarial Science, University of Kent)

Funded by: BBSRC

Induction of yeast prions by reactive oxygen species (ROS)

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How yeast and mammalian prions form spontaneously into infectious amyloid-like structures is poorly understood at present yet the majority of cases of the human prion disease Creutzfeldt Jakob Disease (CJD) are sporadic i.e. without underlying infection or genetic change. The overall objective of this project is to establish the molecular mechanism by which 'sporadic' prion formation is triggered by oxidative stress and other environmental triggers using the well established and widely exploited yeast prion model.

We are using two different yeast prions in this study, namely [PSI+]/Sup35 and [PIN+]/Rnq1, and are focussing on the role of the Tsa1/Tsa2 peroxiredoxins (Prxs). Prxs are antioxidant enzymes that have multiple functions in stress protection and, in collaboration with the laboratory of Professor Chris Grant at the University of Manchester, we have recently shown that the two major cytoplasmic Prxs (that both are ribosome-associated) suppress oxidative stress-induced de novo formation of the[PSI+] prion. Using high throughput assays for de novo formation of the [PSI+] and [PIN+] prions, we are therefore defining the role of Tsa1/Tsa2 in prion formation.

These important new findings strongly implicated oxidative damage of Sup35 as an important trigger of the formation of the heritable prion conformation in yeast. Oxidative damage has also been implicated in the de novo formation of mammalian prions and a trigger for other protein misfolding diseases of man. We have recently established that direct oxidation of the Sup35 polypeptides lead to structural transitions favouring conversion to the transmissible amyloid-like form and hence plays a role in the mechanism of induction of de novo prion formation. These studies are also being extended to other disease-associated, amyloid-forming proteins expressed in yeast including alpha-synuclein. The overall aim of this project is to define the mechanism by which oxidative stress induces yeast prion formation de novo and also the misfolding of human amyloidogenic proteins.

Researchers: Dr Gemma Staniforth (postdoc)

Collaborator: Professor Chris Grant, Dr Vicki Doronina, University of Manchester

Funded by: BBSRC

The natural history of yeast prions

The [PSI+] and [URE3] prions modify the yeast cell phenotype without apparent detriment, suggesting that prions could also represent a novel form of epigenetic inheritance. What we must now establish is whether prions exist in S. cerevisiae strains isolated from a variety of ecosystems and natural environments and are not simply an artifact of 'domestication' of this species.

The main aim of this project is to establish whether [PSI+] and [PIN+], two different and well characterised prions, are present in a wide range of wild strains of S. cerevisiae isolated from 'natural' environments. We will also establish whether the [PSI+] and [PIN+] prions from laboratory strains can replicate in such wild strains and vice versa. The hypothesis that underpins this project is that the [PSI+] and [PIN+] prions can exist in wild strains of S. cerevisiae and that apparently 'prion-free' wild strains have the molecular machinery to replicate one or both prions. Our laboratory was the first to report that a yeast prion (in this case [PIN+]) is present in some wild strains of S. cerevisiae, but in that study we only looked at relatively atypical pathogenic strains isolated from immunocompromised human patients. Although widely viewed as a domesticated organism, nevertheless S. cerevisiae has been found in a variety of ecological niches including tree bark and the surface of fruits. Each niche represents a unique environment in which the organism has to adapt and retain viability and fertility. As first suggested by Susan Lindquist and her colleagues at MIT, the ability to modulate protein function in a reversible manner without the immediate need to fix changes in DNA sequence could be of benefit to the host both in short-term adaptation and in the longer term, for the evolution of new traits.

Researchers: Professor Mick Tuite


Collaborators: Professor Susan Lindquist (MIT-WIBR)


Funded by: The Leverhulme Trust

Yeast models of protein misfolding diseases

To facilitate the analysis of the cause and consequences of disease-associated protein misfolding, a number of 'neurodegenerative disease models' have been established in the budding yeast Saccharomyces cerevisiae. S. cerevisiae is a highly tractable but simple eukaryote that nevertheless has many of the protein misfolding and quality control mechanisms that exist in higher eukaryotes. A number of the high profile human neurodegenerative diseases described to date, including Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD) are being successfully studied in yeast models. The main objective of using a yeast-based model approach to further our understanding of these diseases is to establish the molecular basis of the causation and pathology of what are a globally important group of diseases. For example, by genetically manipulating S. cerevisiae to accumulate misfolded proteins allows the molecular mechanisms important in the cellular folding machinery and the response to misfolded proteins, to be identified. The factors which trigger misfolding and the generation of the toxic entity associated with this misfolding can also be established in order to identify potential intervention strategies.
In this project we are looking at two different disease models to try and establish the associated mechanism(s) of proteotoxicity:


Amyotrophic Lateral Sclerosis (ALS), a non-transmissible neurodegenerative disease affecting the upper and lower motor neurons of the brain and spinal cord. The most common fALS-associated mutation occurs in the gene encoding superoxide dismutase 1 (SOD1), an enzyme that converts superoxide anions to hydrogen peroxide and oxygen thus protecting cells against oxidative damage. We have developed a model in which we have introduced disease-associated mutations into the yeast SOD1 protein and are using a range of cell biological and biochemical approaches to pinpoint the associated toxicity that we see in yeast.


Amyloid toxicity. Two 'model' amyloids are being used one of which (Rnq1p) is a native yeast prion protein. The other 'model' is a protein carrying a polyglutamine expansion and based on the Huntington Disease-associated huntingtin protein. By carrying out genetic and cell biological screens in yeast expressing the disease-associated forms of these proteins we are identifying how the cell responds to amyloid-induced toxicity and the cellular factors that can be manipulated to abrogate the toxicity.


Researchers: Emma Bastow (PhD student), Selena Li (PhD student)


Collaborators: Dr Campbell Gourlay (University of Kent)

Funded by: BBSRC

Heterozygous inhibition of the amyloid state

A number of single-site mutations have been identified in the yeast Sup35p prion protein that lead to a failure to propagate the [PSI+] prion. One of the interesting properties of these so-called 'PNM – [PSI+] no more - mutations is that the prion form cannot propagate in the heterozygous configuration i.e. in diploid cells expressing equal levels of both normal and mutant Sup35p. This state mimics what has been observed with CJD and other prion diseases when there is heterozygosity at the PrP-encoding locus. This is very easy to show in yeast by mating two strains with different SUP35 alleles and showing that the diploid produced is unable to propagate the [PSI+] prion state.

The amyloids formed by Aβ, tau, huntingtin, α-synuclein, serum amyloid A and numerous other proteins responsible for amyloidoses in humans may or may not be self-propagating and none has been found to have resistance polymorphisms in populations. There are good reasons for this lack which have nothing to do with whether 'resistance' mutations analogous to the SUP35-based PNM mutations, may occur. In this project we are exploring the hypothesis that resistance mutants can be found such that, as in prions, heterozygosity at certain sites may prevent the formation of a non-self-propagating amyloid state.


Researchers: Dr Brian Cox


Collaborators: Professor Mike Resnick


Funded by: The Leverhulme Trust

 

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This page is currently being updated with new information

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Year 2

  • BI501 Gene Expression and Its Control (Module convenor)

Final Year

  • BI601 Skills for Biochemists
  • BI651 Skills for Biologists III
  • BI631 Skills for Biomedical Scientists II
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Director of Research, Biosciences

Member of Editorial Boards of

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Enquiries: Phone: +44 (0)1227 823743

School of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ

Last Updated: 28/08/2013