Full humanization of the glycolytic pathway in Saccharomyces cerevisiae

Although transplantation of single genes in yeast plays a key role in elucidating gene functionality in metazoans, technical challenges hamper humanization of full pathways and processes. Empowered by advances in synthetic biology, this study demonstrates the feasibility and implementation of full humanization of glycolysis in yeast. Single gene and full pathway transplantation revealed the remarkable conservation of glycolytic and moonlighting functions and, combined with evolutionary strategies, brought to light context-dependent responses. Human hexokinase 1 and 2, but not 4, required mutations in their catalytic or allosteric sites for functionality in yeast, whereas hexokinase 3 was unable to complement its yeast ortholog. Comparison with human tissues cultures showed preservation of turnover numbers of human glycolytic enzymes in yeast and human cell cultures. This demonstration of transplantation of an entire essential pathway paves the way for establishment of species-, tissue-, and disease-specific metazoan models

Evolutionary rescue of phosphomannomutase deficiency in yeast models of human disease

The most common cause of human congenital disorders of glycosylation (CDG) are mutations in the phosphomannomutase gene PMM2, which affect protein N-linked glycosylation. The yeast gene SEC53 encodes a homolog of human PMM2. We evolved 384 populations of yeast harboring one of two human-disease-associated alleles, sec53-V238M and sec53-F126L, or wild-type SEC53. We find that after 1000 generations, most populations compensate for the slow-growth phenotype associated with the sec53 human-disease-associated alleles. Through whole-genome sequencing we identify compensatory mutations, including known SEC53 genetic interactors. We observe an enrichment of compensatory mutations in other genes whose human homologs are associated with Type 1 CDG, including PGM1, which encodes the minor isoform of phosphoglucomutase in yeast. By genetic reconstruction, we show that evolved pgm1 mutations are dominant and allele-specific genetic interactors that restore both protein glycosylation and growth of yeast harboring the sec53-V238M allele. Finally, we characterize the enzymatic activity of purified Pgm1 mutant proteins. We find that reduction, but not elimination, of Pgm1 activity best compensates for the deleterious phenotypes associated with the sec53-V238M allele. Broadly, our results demonstrate the power of experimental evolution as a tool for identifying genes and pathways that compensate for human-disease-associated alleles

Rapid selection of cyclic peptides that reduce alpha-synuclein toxicity in yeast and animal models

Phage display has demonstrated the utility of cyclic peptides as general protein ligands but cannot access proteins inside eukaryotic cells. Expanding a new chemical genetics tool, we describe the first expressed library of head-to-tail cyclic peptides in yeast (Saccharomyces cerevisiae). We applied the library to selections in a yeast model of alpha-synuclein toxicity that recapitulates much of the cellular pathology of Parkinson's disease. From a pool of 5 million transformants, we isolated two related cyclic peptide constructs that specifically reduced the toxicity of human alpha-synuclein. These expressed cyclic peptide constructs also prevented dopaminergic neuron loss in an established Caenorhabditis elegans Parkinson's model. This work highlights the speed and efficiency of using libraries of expressed cyclic peptides for forward chemical genetics in cellular models of human disease

Warburg effect and translocation-induced genomic instability: two yeast models for cancer cells

Yeast has been established as an efficient model system to study biological principles underpinning human health. In this review we focus on yeast models covering two aspects of cancer formation and progression (i) the activity of pyruvate kinase (PK), which recapitulates metabolic features of cancer cells, including the Warburg effect, and (ii) chromosome bridge-induced translocation (BIT) mimiking genome instability in cancer. Saccharomyces cerevisiae is an excellent model to study cancer cell metabolism, as exponentially growing yeast cells exhibit many metabolic similarities with rapidly proliferating cancer cells. The metabolic reconfiguration includes an increase in glucose uptake and fermentation, at the expense of respiration and oxidative phosphorylation (the Warburg effect), and involves a broad reconfiguration of nucleotide and amino acid metabolism. Both in yeast and humans, the regulation of this process seems to have a central player, PK, which is up-regulated in cancer, and to occur mostly on a post-transcriptional and post-translational basis. Furthermore, BIT allows to generate selectable translocation-derived recombinants ("translocants"), between any two desired chromosomal locations, in wild-type yeast strains transformed with a linear DNA cassette carrying a selectable marker flanked by two DNA sequences homologous to different chromosomes. Using the BIT system, targeted non-reciprocal translocations in mitosis are easily inducible. An extensive collection of different yeast translocants exhibiting genome instability and aberrant phenotypes similar to cancer cells has been produced and subjected to analysis. In this review, we hence provide an overview upon two yeast cancer models, and extrapolate general principles for mimicking human disease mechanisms in yeast

A yeast phenomic model for the gene interaction network modulating CFTR-ΔF508 protein biogenesis

BackgroundThe overall influence of gene interaction in human disease is unknown. In cystic fibrosis (CF) a single allele of the cystic fibrosis transmembrane conductance regulator (CFTR-ΔF508) accounts for most of the disease. In cell models, CFTR-ΔF508 exhibits defective protein biogenesis and degradation rather than proper trafficking to the plasma membrane where CFTR normally functions. Numerous genes function in the biogenesis of CFTR and influence the fate of CFTR-ΔF508. However it is not known whether genetic variation in such genes contributes to disease severity in patients. Nor is there an easy way to study how numerous gene interactions involving CFTR-ΔF would manifest phenotypically.MethodsTo gain insight into the function and evolutionary conservation of a gene interaction network that regulates biogenesis of a misfolded ABC transporter, we employed yeast genetics to develop a 'phenomic' model, in which the CFTR-ΔF508-equivalent residue of a yeast homolog is mutated (Yor1-ΔF670), and where the genome is scanned quantitatively for interaction. We first confirmed that Yor1-ΔF undergoes protein misfolding and has reduced half-life, analogous to CFTR-ΔF. Gene interaction was then assessed quantitatively by growth curves for approximately 5,000 double mutants, based on alteration in the dose response to growth inhibition by oligomycin, a toxin extruded from the cell at the plasma membrane by Yor1.ResultsFrom a comparative genomic perspective, yeast gene interactions influencing Yor1-ΔF biogenesis were representative of human homologs previously found to modulate processing of CFTR-ΔF in mammalian cells. Additional evolutionarily conserved pathways were implicated by the study, and a ΔF-specific pro-biogenesis function of the recently discovered ER membrane complex (EMC) was evident from the yeast screen. This novel function was validated biochemically by siRNA of an EMC ortholog in a human cell line expressing CFTR-ΔF508. The precision and accuracy of quantitative high throughput cell array phenotyping (Q-HTCP), which captures tens of thousands of growth curves simultaneously, provided powerful resolution to measure gene interaction on a phenomic scale, based on discrete cell proliferation parameters.ConclusionWe propose phenomic analysis of Yor1-ΔF as a model for investigating gene interaction networks that can modulate cystic fibrosis disease severity. Although the clinical relevance of the Yor1-ΔF gene interaction network for cystic fibrosis remains to be defined, the model appears to be informative with respect to human cell models of CFTR-ΔF. Moreover, the general strategy of yeast phenomics can be employed in a systematic manner to model gene interaction for other diseases relating to pathologies that result from protein misfolding or potentially any disease involving evolutionarily conserved genetic pathways

Contribution to drug discovery and development for tauopathies using yeast as a model

This work aimed to contribute to drug discovery and development (DDD) for tauopathies, while expanding our knowledge on this group of neurodegenerative disorders, including Alzheimer’s disease (AD). Using yeast, a recognized model for neurodegeneration studies, useful models were produced for the study of tau interaction with beta-amyloid (Aβ), both AD hallmark proteins. The characterization of these models suggests that these proteins co-localize and that Aβ1-42, which is toxic to yeast, is involved in tau40 phosphorylation (Ser396/404) via the GSK-3β yeast orthologue, whereas tau seems to facilitate Aβ1-42 oligomerization. The mapping of tau’s interactome in yeast, achieved with a tau toxicity enhancer screen using the yeast deletion collection, provided a novel framework, composed of 31 genes, to identify new mechanisms associated with tau pathology, as well as to identify new drug targets or biomarkers. This genomic screen also allowed to select the yeast strain mir1Δ-tau40 for development of a new GPSD2TM drug discovery screening system. A library of unique 138 marine bacteria extracts, obtained from the Mid-Atlantic Ridge hydrothermal vents, was screened with mir1Δ-tau40. Three extracts were identified as suppressors of tau toxicity and constitute good starting points for DDD programs. mir1Δ strain was sensitive to tau toxicity, relating tau pathology with mitochondrial function. SLC25A3, the human homologue of MIR1, codes for the mitochondrial phosphate carrier protein (PiC). Resorting to iRNA, SLC25A3 expression was silenced in human neuroglioma cells, as a first step towards the engineering of a neural model for replicating the results obtained in yeast. This model is essential to understand the mechanisms of tau toxicity at the mitochondrial level and to validate PiC as a relevant drug target. The set of DDD tools here presented will foster the development of innovative and efficacious therapies, urgently needed to cope with tau-related disorders of high human and social-economic impact

Dihydropyrimidine-thiones and clioquinol synergize to target beta-amyloid cellular pathologies through a metal-dependent mechanism

The lack of therapies for neurodegenerative diseases arises from our incomplete understanding of their underlying cellular toxicities and the limited number of predictive model systems. It is critical that we develop approaches to identify novel targets and lead compounds. Here, a phenotypic screen of yeast proteinopathy models identified dihydropyrimidine-thiones (DHPM-thiones) that selectively rescued the toxicity caused by β-amyloid (Aβ), the peptide implicated in Alzheimer’s disease. Rescue of Aβ toxicity by DHPM-thiones occurred through a metal-dependent mechanism of action. The bioactivity was distinct, however, from that of the 8-hydroxyquinoline clioquinol (CQ). These structurally dissimilar compounds strongly synergized at concentrations otherwise not competent to reduce toxicity. Cotreatment ameliorated Aβ toxicity by reducing Aβ levels and restoring functional vesicle trafficking. Notably, these low doses significantly reduced deleterious off-target effects caused by CQ on mitochondria at higher concentrations. Both single and combinatorial treatments also reduced death of neurons expressing Aβ in a nematode, indicating that DHPM-thiones target a conserved protective mechanism. Furthermore, this conserved activity suggests that expression of the Aβ peptide causes similar cellular pathologies from yeast to neurons. Our identification of a new cytoprotective scaffold that requires metal-binding underscores the critical role of metal phenomenology in mediating Aβ toxicity. Additionally, our findings demonstrate the valuable potential of synergistic compounds to enhance on-target activities, while mitigating deleterious off-target effects. The identification and prosecution of synergistic compounds could prove useful for developing AD therapeutics where combination therapies may be required to antagonize diverse pathologies.D.F.T was funded by NRSA Fellowship NIH 5F32NS061419. D.F.T. and S.L. were supported by WIBR funds in support of research on Regenerative Disease, the Picower/JPB Foundation, and the Edward N. and Della L. Thome Foundation. G.A.C. and S.L. were funded by a Howard Hughes Medical Institute (HHMI) Collaborative Innovation Award. L.E.B., R.T., and S.E.S. were funded by NIH GM086180, NIH GM067041, and NIH GM111625. (5F32NS061419 - NRSA Fellowship NIH; WIBR funds in support of research on Regenerative Disease; Picower/JPB Foundation; Edward N. and Della L. Thome Foundation; Howard Hughes Medical Institute (HHMI) Collaborative Innovation Award; GM086180 - NIH; NIH GM067041 - NIH; NIH GM111625 - NIH)https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5705239/Accepted manuscrip

A fruitful fly forward : the role of the fly in drug discovery for neurodegeneration

AD, Alzheimer’s disease; APP, amyloid precursor protein; BBB, blood brain barrier; GFP, green fluorescent protein; HTS, high-throughput screening; HD, Huntington’s disease; LB, Lewy bodies; PD, Parkinson’s disease; PolyQ, Polyglutamine; RNAi, RNA interference; SNCA, α-synuclein gene; UAS, Upstream Activating Sequence.peer-reviewe

Fungal model systems and the elucidation of pathogenicity determinants

Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.Peer reviewedPublisher PD