Faculty Opinions recommendation of Cross-Species Complementation of Nonessential Yeast Genes Establishes Platforms for Testing Inhibitors of Human Proteins.

AbstractPathway transplantation from one organism to another represents a means to a more complete understanding of a biochemical or regulatory process. The purine biosynthesis pathway, a core metabolic function, was transplanted from human to yeast. We replaced the entireSaccharomyces cerevisiaeadenine de novo pathway with the cognate human pathway components. A yeast strain was “humanized” for the full pathway by deleting all relevant yeast genes completely and then providing the human pathway in trans using a neochromosome expressing the human protein coding regions under the transcriptional control of their cognate yeast promoters and terminators. The “humanized” yeast strain grows in the absence of adenine, indicating complementation of the yeast pathway by the full set of human proteins. While the strain with the neochromosome is indeed prototrophic, it grows slowly in the absence of adenine. Dissection of the phenotype revealed that the human ortholog ofADE4, PPAT, shows only partial complementation. We have used several strategies to understand this phenotype, that point toPPAT/ADE4as the central regulatory node. Pathway metabolites are responsible for regulatingPPAT’sprotein abundance through transcription and proteolysis as well as its enzymatic activity by allosteric regulation in these yeast cells. Extensive phylogenetic analysis of PPATs from diverse organisms hints at adaptations of the enzyme-level regulation to the metabolite levels in the organism. Finally, we isolated specific mutations in PPAT as well as in other genes involved in the purine metabolic network that alleviate incomplete complementation byPPATand provide further insight into the complex regulation of this critical metabolic pathway.

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Humanization of yeast genes with multiple human orthologs reveals principles of functional divergence between paralogs

10.1101/668335 ◽

2019 ◽

Cited By ~ 5

Author(s):

Jon M. Laurent ◽

Riddhiman K. Garge ◽

Ashley I. Teufel ◽

Claus O. Wilke ◽

Aashiq H. Kachroo ◽

...

Keyword(s):

Functional Divergence ◽

Gene Families ◽

Evolutionary Divergence ◽

Orthologous Genes ◽

Specific Expression ◽

Human Genes ◽

Human Orthologs ◽

Human Proteins ◽

Yeast Genes ◽

Opening Up

AbstractDespite over a billion years of evolutionary divergence, several thousand human genes possess clearly identifiable orthologs in yeast, and many have undergone lineage-specific duplications in one or both lineages. The ortholog conjecture postulates that orthologous genes between species retain ancestral functions despite divergence over vast timescales, but duplicated genes will be free to diverge in function. However, the retention of ancestral functions among co-orthologs between species and within gene families has been difficult to test experimentally at scale. In order to investigate how ancestral functions are retained or lost post-duplication, we systematically replaced hundreds of essential yeast genes with their human orthologs from gene families that have undergone lineage-specific duplications, including those with single duplications (one yeast gene to two human genes, 1:2) or higher-order expansions (1:>2) in the human lineage. We observe a variable pattern of replaceability across different ortholog classes, with an obvious trend towards differential replaceability inside gene families, rarely observing replaceability by all members of a family. We quantify the ability of various properties of the orthologs to predict replaceability, showing that in the case of 1:2 orthologs, replaceability is predicted largely by the divergence and tissue-specific expression of the human co-orthologs, i.e. the human proteins that are less diverged from their yeast counterpart and more ubiquitously expressed across human tissues more often replace their single yeast ortholog. These trends were consistent with in silico simulations demonstrating that when only one ortholog is replaceable, it tends to be the least diverged of the pair. Replaceability of yeast genes having more than two human co-orthologs was marked by retention of orthologous interactions in functional or protein networks as well as by more ancestral subcellular localization. Overall, we performed >400 human gene replaceability assays revealing 56 new human-yeast complementation pairs, thus opening up avenues to further functionally characterize these human genes in a simplified organismal context.

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Role of the Tsc1-Tsc2 Complex in Signaling and Transport Across the Cell Membrane in the Fission Yeast Schizosaccharomyces pombe

Genetics ◽

10.1093/genetics/161.3.1053 ◽

2002 ◽

Vol 161 (3) ◽

pp. 1053-1063 ◽

Cited By ~ 10

Author(s):

Sanae Matsumoto ◽

Amitabha Bandyopadhyay ◽

David J Kwiatkowski ◽

Umadas Maitra ◽

Tomohiro Matsumoto

Keyword(s):

Fission Yeast ◽

Protein Trafficking ◽

Similar Process ◽

Benign Tumors ◽

Amino Acid Permease ◽

Human Proteins ◽

P Factor ◽

Yeast Genes ◽

Uptake Of Nutrients

Abstract Heterozygous inactivation of either human TSC1 or TSC2 causes tuberous sclerosis (TSC), in which development of benign tumors, hamartomas, occurs via a two-hit mechanism. In this study, fission yeast genes homologous to TSC1 and TSC2 were identified, and their protein products were shown to physically interact like the human gene products. Strains lacking tsc1+ or tsc2+ were defective in uptake of nutrients from the environment. An amino acid permease, which is normally positioned on the plasma membrane, aggregated in the cytoplasm or was confined in vacuole-like structures in Δtsc1 and Δtsc2 strains. Deletion of tsc1+ or tsc2+ also caused a defect in conjugation. When a limited number of the cells were mixed, they conjugated poorly. The conjugation efficiency was improved by increased cell density. Δtsc1 cells were not responsive to a mating pheromone, P-factor, suggesting that Tsc1 has an important role in the signal cascade for conjugation. These results indicate that the fission yeast Tsc1-Tsc2 complex plays a role in the regulation of protein trafficking and suggest a similar function for the human proteins. We also show that fission yeast Int6 is involved in a similar process, but functions in an independent genetic pathway.

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

Genetics ◽

10.1534/genetics.119.302971 ◽

2020 ◽

Vol 214 (3) ◽

pp. 735-747 ◽

Cited By ~ 4

Author(s):

Akil Hamza ◽

Maureen R. M. Driessen ◽

Erik Tammpere ◽

Nigel J. O’Neil ◽

Philip Hieter

Keyword(s):

Drug Targets ◽

Chromosome Instability ◽

Yeast Complementation ◽

Synthetic Lethal ◽

Link Type ◽

Human Proteins ◽

Species Specific ◽

Yeast Genes

Cross-species complementation can be used to generate humanized yeast, which is a valuable resource with which to model and study human biology. Humanized yeast can be used as an in vivo platform to screen for chemical inhibition of human protein drug targets. To this end, we report the systematic complementation of nonessential yeast genes implicated in chromosome instability (CIN) with their human homologs. We identified 20 human–yeast complementation pairs that are replaceable in 44 assays that test rescue of chemical sensitivity and/or CIN defects. We selected a human–yeast pair (hFEN1/yRAD27), which is frequently overexpressed in cancer and is an anticancer therapeutic target, to perform in vivo inhibitor assays using a humanized yeast cell-based platform. In agreement with published in vitro assays, we demonstrate that HU-based PTPD is a species-specific hFEN1 inhibitor. In contrast, another reported hFEN1 inhibitor, the arylstibonic acid derivative NSC-13755, was determined to have off-target effects resulting in a synthetic lethal phenotype with yRAD27-deficient strains. Our study expands the list of human–yeast complementation pairs to nonessential genes by defining novel cell-based assays that can be utilized as a broad resource to study human drug targets.

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Rice with human proteins to take root in Kansas

Nature ◽

10.1038/news070514-17 ◽

2007 ◽

Author(s):

Emma Marris

Keyword(s):

Human Proteins

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The Effects of Amines on the Esterase Activities of Thrombin and Thrombokinase and on Several Clotting Tests

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649165 ◽

1974 ◽

Vol 31 (02) ◽

pp. 309-318

Author(s):

Phyllis S Roberts ◽

Raphael M Ottenbrite ◽

Patricia B Fleming ◽

James Wigand

Keyword(s):

Choline Chloride ◽

Polymerization Process ◽

Ammonium Bromide ◽

Bovine Thrombin ◽

One Stage ◽

Human Proteins ◽

Rate Of Hydrolysis ◽

The One ◽

Hydrolysis Of Esters ◽

Hydrolysis Of

Summary1. Choline chloride, 0.1 M (in 0.25 M Tris. HCl buffer, pH 7.4 or 8.0, 37°), doubles the rate of hydrolysis of TAME by bovine thrombokinase but has no effect on the hydrolysis of this ester by either human or bovine thrombin. Only when 1.0 M or more choline chloride is present is the hydrolysis of BAME by thrombokinase or thrombin weakly inhibited. Evidence is presented that shows that these effects are due to the quaternary amine group.2. Tetramethyl ammonium bromide or chloride has about the same effects on the hydrolysis of esters by these enzymes as does choline chloride but tetra-ethyl, -n.propyl and -n.butyl ammonium bromides (0.1 M) are stronger accelerators of the thrombokinase-TAME reaction and they also accelerate, but to a lesser degree, the thrombin-TAME reaction. In addition, they inhibit the hydrolysis of BAME by both enzymes. Their effects on these reactions, however, do not follow any regular order. The tetraethyl compound is the strongest accelerator of the thrombokinase-TAME reaction but the tetra-ethyl and -butyl compounds are the strongest accelerators of the thrombin-TAME reaction. The ethyl and propyl compounds are the best (although weak) inhibitors of the thrombokinase-BAME and the propyl compound of the thrombin-BAME reactions.3. Tetra-methyl, -ethyl, -n.propyl and -n.butyl ammonium bromides (0.01 M) inhibit the clotting of fibrinogen by thrombin (bovine and human proteins) at pH 7.4, imidazole or pH 6.1, phosphate buffers and they also inhibit, but to a lesser degree, a modified one-stage prothrombin test. In all cases the inhibition increases regularly as the size of the alkyl group increases from methyl to butyl. Only the ethyl com pound (0.025 M but not 0.01 M), however, significantly inhibits the polymerization of bovine fibrin monomers. It was concluded that inhibition of the fibrinogen-thrombin and the one-stage tests by the quaternary amines is not due to any effect of the com pounds on the polymerization process but probably due to inhibition of thrombin’s action on fibrinogen by the quaternary amines.

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