Lyne Jossé

Development of a Novel Yeast Cell-Based System for Studying the Aggregation of Alzheimer’s Disease-Associated Aβ Peptides in vivo

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 Aβ peptides, short 39–43 residue peptides generated through endoproteolytic cleavage of the Alzheimer’s precursor protein. While the process of aggregation of purified Aβ 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 Aβ-mediated aggregation in an organism in vivo. One ofthis 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 Aβ peptide 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 trans-acting factors that are normally needed for [PSI+] propagation. We conclude that we have established a useful new tool for studying the aggregation of Aβ peptides in an organism in vivo.

Transient expression of human TorsinA enhances secretion of two functionally distinct proteins in cultured Chinese hamster ovary (CHO) cells.

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. Biotechnol. Bioeng. 2010; 105: 556–566.

Transcriptomic and phenotypic analysis of the effects of T-2 toxin on Saccharomyces cerevisiae: evidence of mitochondrial involvement

At 5 μg mL(-1) , T-2 toxin significantly upregulated the transcription of 281 genes and downregulated 86. Strongly upregulated genes included those involved in redox activity, mitochondrial functions, the response to oxidative stress, and cytoplasmic rRNA transcription and processing. Highly repressed genes have roles in mitochondrial biogenesis, and the expression and stability of cytoplasmic rRNAs. T-2 toxin inhibition of growth was greater in a medium requiring respiration, and was antagonized by antioxidants. T-2 toxin treatment induced reactive oxygen species, caused nucleolytic damage to DNA, probably mitochondrial, and externalization of phosphatidylserine. Deletion mutations causing respiratory deficiency substantially increased toxin tolerance, and deletion of some TOR (target of rapamycin) pathway genes altered T-2 toxin sensitivity. Deletion of FMS1, which plays an indirect role in cytoplasmic protein synthesis, markedly increased toxin tolerance. Overall, the findings suggest that T-2 toxin targets mitochondria, generating oxy-radicals and repressing mitochondrial biogenesis genes, thus inducing oxidative stress and redox enzyme genes, and triggering changes associated with apoptosis. The large transcriptional changes in genes needed for rRNA transcription and expression and the effects of deletion of FMS1 are also consistent with T-2 toxin damage to the cytoplasmic translational mechanism, although it is unclear how this relates to the mitochondrial effects.

mTORC1 signalling and eIF4E/4E-BP1 translation initiation factor stoichiometry influence recombinant protein productivity from GS-CHOK1 cells

Many protein-based biotherapeutics are produced in cultured Chinese hamster ovary (CHO) cell lines. Recent reports have demonstrated that translation of recombinant mRNAs and global control of the translation machinery via mammalian target of rapamycin (mTOR) signalling are important determinants of the amount and quality of recombinant protein such cells can produce. mTOR complex 1 (mTORC1) is a master regulator of cell growth/division, ribosome biogenesis and protein synthesis, but the relationship between mTORC1 signalling, cell growth and proliferation and recombinant protein yields from mammalian cells, and whether this master regulating signalling pathway can be manipulated to enhance cell biomass and recombinant protein production (rPP) are not well explored. We have investigated mTORC1 signalling and activity throughout batch culture of a panel of sister recombinant glutamine synthetase-CHO cell lines expressing different amounts of a model monoclonal IgG4, to evaluate the links between mTORC1 signalling and cell proliferation, autophagy, recombinant protein expression, global protein synthesis and mRNA translation initiation. We find that the expression of the mTORC1 substrate 4E-binding protein 1 (4E-BP1) fluctuates throughout the course of cell culture and, as expected, that the 4E-BP1 phosphorylation profiles change across the culture. Importantly, we find that the eIF4E/4E-BP1 stoichiometry positively correlates with cell productivity. Furthermore, eIF4E amounts appear to be co-regulated with 4E-BP1 amounts. This may reflect a sensing of either change at the mRNA level as opposed to the protein level or the fact that the phosphorylation status, as well as the amount of 4E-BP1 present, is important in the co-regulation of eIF4E and 4E-BP1.

Probing the role of structural features of mouse PrP in yeast by expression as Sup35-PrP fusions

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.

Modulation of phosducin-like protein 3 (PhLP3) levels promotes cytoskeletal remodelling in a MAPK and RhoA-dependent manner.

Background Phosducin-like protein 3 (PhLP3) forms a ternary complex with the ATP-dependent molecular chaperone CCT and its folding client tubulin. In vitro studies suggest PhLP3 plays an inhibitory role in β-tubulin folding while conversely in vivo genetic studies suggest PhLP3 is required for the correct folding of β-tubulin. We have a particular interest in the cytoskeleton, its chaperones and their role in determining cellular phenotypes associated with high level recombinant protein expression from mammalian cell expression systems. Methodology/Principal Findings As studies into PhLP3 function have been largely carried out in non mammalian systems, we examined the effect of human PhLP3 over-expression and siRNA silencing using a single murine siRNA on both tubulin and actin systems in mammalian Chinese hamster ovary (CHO) cell lines. We show that over-expression of PhLP3 promotes an imbalance of α and β tubulin subunits, microtubule disassembly and cell death. In contrast, β-actin levels are not obviously perturbed. On-the-other-hand, RNA silencing of PhLP3 increases RhoA-dependent actin filament formation and focal adhesion formation and promotes a dramatic elongated fibroblast-like change in morphology. This was accompanied by an increase in phosphorylated MAPK which has been associated with promoting focal adhesion assembly and maturation. Transient overexpression of PhLP3 in knockdown experiments rescues cells from the morphological change observed during PhLP3 silencing but mitosis is perturbed, probably reflecting a tipping back of the balance of PhLP3 levels towards the overexpression state. Conclusions Our results support the hypothesis that PhLP3 is important for the maintenance of β-tubulin levels in mammalian cells but also that its modulation can promote actin-based cytoskeletal remodelling by a mechanism linked with MAPK phosphorylation and RhoA-dependent changes. PhLP3 levels in mammalian cells are thus finely poised and represents a novel target for engineering industrially relevant cell lines to evolve lines more suited to suspension or adherent cell growth.

20 Yeast Prions and Their Analysis InVivo

Publisher Summary This chapter discusses the three yeast (Saccharomyces cerevisiae) prions—Ure2p, Sup35p, and , and Rnq1p—in terms of their associated phenotypes and their cell biological and biochemical properties and reviews the experimental approaches that can be taken to study the three native prions in vivo. The chapter also discusses the way a researcher establishes whether a new or novel phenotype is associated with the prion-like behavior of a cellular protein. Yeast prions are transmitted efficiently from cell-to-cell during mitosis and meiosis, indicating that a very effective mechanism must exist that ensures their continued propagation even in rapidly dividing cells. Two different, but not mutually exclusive, models have been put forward to explain the self-propagation of prions: template-directed refolding and seeded polymerization. The more widely accepted seeded polymerization model is based on the prion protein existing in an altered conformational state, which is in a reversible dynamic equilibrium with a soluble form of that protein. The generation and transmission of propagons in yeast appears to be dependent upon a number of different molecular chaperones with one particular chaperone, the heat-shock-inducible protein Hsp104p, being essential for the propagation of all three native prions.

A cell culture platform for Cryptosporidium that enables long-term cultivation and new tools for the systematic investigation of its biology

Graphical abstract

20 Yeast Prions and Their Analysis

Publisher Summary This chapter discusses the three yeast (Saccharomyces cerevisiae) prions—Ure2p, Sup35p, and , and Rnq1p—in terms of their associated phenotypes and their cell biological and biochemical properties and reviews the experimental approaches that can be taken to study the three native prions in vivo. The chapter also discusses the way a researcher establishes whether a new or novel phenotype is associated with the prion-like behavior of a cellular protein. Yeast prions are transmitted efficiently from cell-to-cell during mitosis and meiosis, indicating that a very effective mechanism must exist that ensures their continued propagation even in rapidly dividing cells. Two different, but not mutually exclusive, models have been put forward to explain the self-propagation of prions: template-directed refolding and seeded polymerization. The more widely accepted seeded polymerization model is based on the prion protein existing in an altered conformational state, which is in a reversible dynamic equilibrium with a soluble form of that protein. The generation and transmission of propagons in yeast appears to be dependent upon a number of different molecular chaperones with one particular chaperone, the heat-shock-inducible protein Hsp104p, being essential for the propagation of all three native prions.

8 Reporter Genes and Their Uses in Studying Yeast Gene Expression

Publisher Summary Reporter genes are the tools of ever-increasing importance in the study of yeast gene expression. Their versatility and the ease of use have made them widely available and ongoing developments will undoubtedly continue to increase their usefulness and their potential applications. A few examples of studies on transcriptional, translational, and post-translational regulation of gene expression are used in this chapter to illustrate some general principles of the use of reporter genes. Many of the reporters developed for use in yeast were originally designed to investigate promoter activity. A good example for this is the original description of the chloramphenicol acetyltransferase (CAT) assay in yeast. The authors of this study demonstrated the validity of CAT as a yeast reporter gene by constructing vectors that contained the coding region of the bacterial cat gene, but no promoter. Any essential gene can be used as a reporter in the corresponding knockout yeast strains because in these strains the ability to grow depends on the expression of the respective gene. The growth rate, thus, provides an easy readout for the expression of the gene.