Cross-Species Complementation of Nonessential Yeast Genes Establishes Platforms for Testing Inhibitors of Human Proteins

Hamza, Akil; Driessen, Maureen R M; Tammpere, Erik; O’Neil, Nigel J.; Hieter, Philip

doi:10.1534/genetics.119.302971

Cited by 15 publications

(17 citation statements)

References 106 publications

(152 reference statements)

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“…The yeast S. cerevisiae has been used as an important tool to understand the function of heterologous enzymes and for the search and study of small molecule inhibitors [76][77][78][79][80][81]. Functional replacement of essential genes in yeast by their counterparts from the target pathogenic organism has been successfully employed to support target-based drug discovery programs for proteins recalcitrant to recombinant production, such as LmDHSp/DHSc here.…”

Section: Development Of a S Cerevisiae-based Assay For Dhs Activitymentioning

confidence: 99%

Structural features and development of an assay platform of the parasite target deoxyhypusine synthase of Brugia malayi and Leishmania major

et al. 2020

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Deoxyhypusine synthase (DHS) catalyzes the first step of the post-translational modification of eukaryotic translation factor 5A (eIF5A), which is the only known protein containing the amino acid hypusine. Both proteins are essential for eukaryotic cell viability, and DHS has been suggested as a good candidate target for small molecule-based therapies against eukaryotic pathogens. In this work, we focused on the DHS enzymes from Brugia malayi and Leishmania major, the causative agents of lymphatic filariasis and cutaneous leishmaniasis, respectively. To enable B. malayi (Bm)DHS for future target-based drug discovery programs, we determined its crystal structure bound to cofactor NAD +. We also reported an in vitro biochemical assay for this enzyme that is amenable to a high-throughput screening format. The L. major genome encodes two DHS paralogs, and attempts to produce them recombinantly in bacterial cells were not successful. Nevertheless, we showed that ectopic expression of both LmDHS paralogs can rescue yeast cells lacking the endogenous DHSencoding gene (dys1). Thus, functionally complemented dys1Δ yeast mutants can be used to screen for new inhibitors of the L. major enzyme. We used the known human DHS inhibitor GC7 to validate both in vitro and yeast-based DHS assays. Our results show that BmDHS is a homotetrameric enzyme that shares many features with its human homologue, whereas LmDHS paralogs are likely to form a heterotetrameric complex and have a distinct regulatory mechanism. We expect our work to facilitate the identification and development of new DHS inhibitors that can be used to validate these enzymes as vulnerable targets for therapeutic interventions against B. malayi and L. major infections.

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Section: Development Of a S Cerevisiae-based Assay For Dhs Activitymentioning

confidence: 99%

Structural features and development of an assay platform of the parasite target deoxyhypusine synthase of Brugia malayi and Leishmania major

et al. 2020

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“…By exploring phenotypes ranging from minimal complementation to stress resistance, we were able to characterize the levels of functional conservation of human splicing isoforms and paralogs. In well-characterized cases of complementation, humanized yeast may be a useful platform to investigate the functional effects of human mutations, isoforms and splicing variants (Mayfield et al 2012;Hamza et al 2015), or the efficacy of drug treatments (Hamza et al 2020).…”

Section: We Can Learn From Humanized Yeastmentioning

confidence: 99%

Precise Replacement of Saccharomyces cerevisiae Proteasome Genes with Human Orthologs by an Integrative Targeting Method

Yellman

2020

G3 Genes|Genomes|Genetics

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Artificial induction of a chromosomal double-strand break in Saccharomyces cerevisiae enhances the frequency of integration of homologous DNA fragments into the broken region by up to several orders of magnitude. The process of homologous repair can be exploited to integrate, in principle, any foreign DNA into a target site, provided the introduced DNA is flanked at both the 5' and 3' ends by sequences homologous to the region surrounding the double-strand break. I have developed tools to precisely direct double-strand breaks to chromosomal target sites with the meganuclease I-SceI and select integration events at those sites. The method is validated in two different applications. First, the introduction of site-specific single-nucleotide phosphorylation site mutations into the S. cerevisiae gene SPO12. Second, the precise chromosomal replacement of eleven S. cerevisiae proteasome genes with their human orthologs. Placing the human genes under S. cerevisiae transcriptional control allowed us to update our understanding of cross-species functional gene replacement. This experience suggests that using native promoters may be a useful general strategy for the coordinated expression of foreign genes in S. cerevisiae. I provide an integrative targeting tool set that will facilitate a variety of precision genome engineering applications.

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“…The Embden-Meyerhof-Parnas (EMP) pathway of glycolysis, which is near ubiquitous to eukaryotes, has a central role in carbon metabolism and is involved in a wide range of diseases in mammals, including cancer with the well-known Warburg effect [20]. So far, few single human glycolytic enzymes have been transplanted into yeast, mostly in large-scale complementation studies [6, 8, 9, 22–24]. Whether all human glycolytic enzymes can complement their yeast orthologs is however unknown.…”

Section: Introductionmentioning

confidence: 99%

A yeast with muscle doesn’t run faster: full humanization of the glycolytic pathway in Saccharomyces cerevisiae

Boonekamp

Knibbe

Vieira-Lara

et al. 2021

Preprint

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While transplantation of single genes in yeast plays a key role in elucidating gene functionality in metazoans, technical challenges hamper the 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 both glycolytic and moonlighting functions and, combined with evolutionary strategies, brought to light novel, context-dependent responses. Remarkably, human hexokinase 1 and 2, but not 4, required mutations in their catalytic or allosteric sites for functionality in yeast, while hexokinase 3 was unable to complement its yeast ortholog. Comparison with human tissues cultures showed the 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 to the establishment of species, tissue and disease-specific metazoan models.

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Cross-Species Complementation of Nonessential Yeast Genes Establishes Platforms for Testing Inhibitors of Human Proteins

Cited by 15 publications

References 106 publications

Structural features and development of an assay platform of the parasite target deoxyhypusine synthase of Brugia malayi and Leishmania major

Structural features and development of an assay platform of the parasite target deoxyhypusine synthase of Brugia malayi and Leishmania major

Precise Replacement of Saccharomyces cerevisiae Proteasome Genes with Human Orthologs by an Integrative Targeting Method

A yeast with muscle doesn’t run faster: full humanization of the glycolytic pathway in Saccharomyces cerevisiae

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