Brian M. Wasko

The advent of next-generation sequencing has transformed clinical genetics, accelerating the identification of human genetic variants. However, the labor-intensive process of functional validation of Variants of Uncertain Significance (VUS) remains a significant diagnostic barrier. While expressing humanized genes in Saccharomyces cerevisiae is a common approach for modeling missense variants with functional assays, using native yeast orthologs provides a more streamlined approach that preserves species-specific interactions within multisubunit complexes. However, taking this approach relies on a fragmented workflow that requires bioinformatic experience. To address this issue, we developed the Bioinformatic Utility for Diagnostic Discovery in Yeast (BUDDY), an integrated web platform designed to streamline variant analysis by connecting bioinformatic tools and databases including ClinVar. Features include amino acid alignments, a protein structure viewer that displays a superposition of yeast and human proteins, pathogenicity prediction data, CRISPR-Cas9 reagent design, and yeast phenotypes to provide potential functional assays. The platform also contains an artificial intelligence (AI)-assisted experimental design feature that evaluates the utility of modeling specific variant and gene combinations in yeast and can assist with phenotype selection. BUDDY significantly accelerated the bioinformatic preparation time needed for ortholog variant modeling by an experienced biologist. Additionally, without prior bioinformatics training, a first-year medical student used BUDDY to develop a self-directed project and proposed a study of five Lynch syndrome-associated VUS using a forward mutation rate assay. BUDDY is designed to accelerate the investigation of human variants in yeast and has a high potential utility for use in resource-constrained settings such as course-based undergraduate research experiences (CUREs).

Inhibition of the target of rapamycin (TOR/mTOR) protein kinase by the drug rapamycin extends lifespan and health span across diverse species. However, rapamycin has potential off-target and side effects that warrant the discovery of additional TOR inhibitors. TOR was initially discovered in Saccharomyces cerevisiae (yeast) which contains two TOR paralogs, TOR1 and TOR2. Yeast lacking functional Tor1 are viable but are hypersensitive to growth inhibition by TORC1 inhibitors, which is a property of yeast that can be exploited to identify TOR inhibitors. Additionally, yeast lacking FK506-sensitive proline rotamase (FPR1) or containing a tor1-1 allele (a mutation in the Fpr1-rapamycin binding domain of Tor1) are robustly and selectively resistant to rapamycin and analogs that allosterically inhibit TOR activity via an FPR1-dependent mechanism. To facilitate the identification of TOR inhibitors, we generated a panel of yeast strains with mutations in TOR pathway genes combined with the removal of 12 additional genes involved in drug efflux. This creates a drug-sensitive strain background that can sensitively and effectively identify TOR inhibitors. In a wild-type yeast strain background, 25 µM of Torin1 and 100 µM of GSK2126458 (omipalisib) are necessary to observe TOR1-dependent growth inhibition by these known TOR inhibitors. In contrast, 100 nM Torin1 and 500 nM GSK2126458 (omipalisib) are sufficient to identify TOR1-dependent growth inhibition in the drug-sensitized background. This represents a 200-fold and 250-fold increase in detection sensitivity for Torin1 and GSK2126458, respectively. Additionally, for the TOR inhibitor AZD8055, the drug-sensitive system resolves that the compound results in TOR1-dependent growth sensitivity at 100 µM, whereas no growth inhibition is observed in a wild-type yeast strain background. Our platform also identifies the caffeine analog aminophylline as a TOR1-dependent growth inhibitor via selective tor1 growth sensitivity. We also tested nebivolol, isoliquiritigenin, canagliflozin, withaferin A, ganoderic acid A, and taurine and found no evidence for TOR inhibition using our yeast growth-based model. Our results demonstrate that this system is highly effective at identifying compounds that inhibit the TOR pathway. It offers a rapid, cost-efficient, and sensitive tool for drug discovery, with the potential to expedite the identification of new TOR inhibitors that could serve as geroprotective and/or anti-cancer agents.

Inhibition of the target of rapamycin (TOR/mTOR) protein kinase by the drug rapamycin extends lifespan and healthspan across diverse species. However, rapamycin has potential off-target and side effects that warrant the discovery of additional TOR inhibitors. TOR was initially discovered in Saccharomyces cerevisiae (yeast) which contains two TOR paralogs, TOR1 and TOR2 . Yeast lacking functional Tor1 are viable but are hypersensitive to growth inhibition by TORC1 inhibitors, which is a property of yeast that can be exploited to identify TOR inhibitors. Additionally, yeast lacking FK506-sensitive proline rotamase ( FPR1 ) or containing a tor1-1 allele (a mutation in the Fpr1-rapamycin binding domain of Tor1) are robustly and selectively resistant to rapamycin and analogs that allosterically inhibit TOR activity via an FPR1 -dependent mechanism. To facilitate the identification of TOR inhibitors, we generated a panel of yeast strains with mutations in TOR pathway genes combined with the removal of 12 additional genes involved in drug efflux. This creates a drug sensitive strain background that can sensitively and effectively identify TOR inhibitors. In a wildtype yeast strain background, 25 µM of Torin1 and 100 µM of GSK2126458 (omipalisib) are necessary to observe TOR1 -dependent growth inhibition by these known TOR inhibitors. In contrast, 100 nM Torin1 and 500 nM GSK2126458 (omipalisib) are sufficient to identify TOR1- dependent growth inhibition in the drug sensitized background. This represents a 200-fold and 250-fold increase in detection sensitivity for Torin1 and GSK2126458, respectively. Additionally, for the TOR inhibitor AZD8055, the drug sensitive system resolves that the compound results in TOR1- dependent growth sensitivity at 100 µM, whereas no growth inhibition is observed in a wildtype yeast strain background. Our platform also identifies the caffeine analog aminophylline as a TOR1 -dependent growth inhibitor via selective tor1 growth sensitivity. We also tested nebivolol, isoliquiritigenin, canagliflozin, withaferin A, ganoderic acid A, and taurine, and found no evidence for TOR inhibition using our yeast growth-based model. Our results demonstrate that this system is highly effective at identifying compounds that inhibit the TOR pathway. It offers a rapid, cost-efficient, and sensitive tool for drug discovery, with the potential to expedite the identification of new TOR inhibitors that could serve as geroprotective and/or anti-cancer agents.

The Ku complex performs multiple functions inside eukaryotic cells, including protection of chromosomal DNA ends from degradation and fusion events, recruitment of telomerase, and repair of double-strand breaks (DSBs). Inactivation of Ku complex genes YKU70 or YKU80 in cells of the yeast Saccharomyces cerevisiae gives rise to mutants that exhibit shortened telomeres and temperature-sensitive growth. In this study, we have investigated the mechanism by which overexpression of telomerase suppresses the temperature sensitivity of yku mutants. Viability of yku cells was restored by overexpression of the Est2 reverse transcriptase and TLC1 RNA template subunits of telomerase, but not the Est1 or Est3 proteins. Overexpression of other telomerase- and telomere-associated proteins (Cdc13, Stn1, Ten1, Rif1, Rif2, Sir3, and Sir4) did not suppress the growth defects of yku70 cells. Mechanistic features of suppression were assessed using several TLC1 RNA deletion derivatives and Est2 enzyme mutants. Supraphysiological levels of three catalytically inactive reverse transcriptase mutants (Est2-D530A, Est2-D670A, and Est2-D671A) suppressed the loss of viability as efficiently as the wild-type Est2 protein, without inducing cell senescence. Roles of proteins regulating telomere length were also determined. The results support a model in which chromosomes in yku mutants are stabilized via a replication-independent mechanism involving structural reinforcement of protective telomere cap structures.

Histone acetylations are important epigenetic markers for transcriptional activation in response to metabolic changes and various stresses. Using the high-throughput SEquencing-Based Yeast replicative Lifespan screen method and the yeast knockout collection, we demonstrate that the HDA complex, a class-II histone deacetylase (HDAC), regulates aging through its target of acetylated H3K18 at storage carbohydrate genes. We find that, in addition to longer lifespan, disruption of HDA results in resistance to DNA damage and osmotic stresses. We show that these effects are due to increased promoter H3K18 acetylation and transcriptional activation in the trehalose metabolic pathway in the absence of HDA. Furthermore, we determine that the longevity effect of HDA is independent of the Cyc8-Tup1 repressor complex known to interact with HDA and coordinate transcriptional repression. Silencing the HDA homologs in C. elegans and Drosophila increases their lifespan and delays aging-associated physical declines in adult flies. Hence, we demonstrate that this HDAC controls an evolutionarily conserved longevity pathway.

The question of why and how some species or individuals within a population live longer than others is among the most important questions in the biology of aging. A particularly useful model to understand the genetic basis and selective forces acting on the plasticity of lifespan are closely related species or ecologically diverse individuals of the same species widely different in lifespan. Here, we analyzed 76 diverse wild isolates of two closely related budding yeast species Saccharomyces cerevisiae and Saccharomyces paradoxus and discovered a diversity of natural intra-species lifespan variation. We sequenced the genomes of these organisms and analyzed how their replicative lifespan is shaped by nutrients and transcriptional and metabolite patterns. We identified sets of genes and metabolites to regulate aging pathways, many of which have not been previously associated with lifespan regulation. We also identified and characterized long-lived strains with elevated intermediary metabolites and differentially regulated genes for NAD metabolism and the control of epigenetic landscape through chromatin silencing. Our data further offer insights into the evolution and mechanisms by which caloric restriction regulates lifespan by modulating the availability of nutrients without decreasing fitness. Overall, our study shows how the environment and natural selection interact to shape diversity of lifespan.

The loss of vacuolar/lysosomal acidity is an early event during aging that has been linked to mitochondrial dysfunction. However, it is unclear how loss of vacuolar acidity results in age-related dysfunction. Through unbiased genetic screens, we determined that increased iron uptake can suppress the mitochondrial respiratory deficiency phenotype of yeast vma mutants, which have lost vacuolar acidity due to genetic disruption of the vacuolar ATPase proton pump. Yeast vma mutants exhibited nuclear localization of Aft1, which turns on the iron regulon in response to iron-sulfur cluster (ISC) deficiency. This led us to find that loss of vacuolar acidity with age in wild-type yeast causes ISC defects and a DNA damage response. Using microfluidics to investigate aging at the single-cell level, we observe grossly divergent trajectories of iron homeostasis within an isogenic and environmentally homogeneous population. One subpopulation of cells fails to mount the expected compensatory iron regulon gene expression program, and suffers progressively severe ISC deficiency with little to no activation of the iron regulon. In contrast, other cells show robust iron regulon activity with limited ISC deficiency, which allows extended passage and survival through a period of genomic instability during aging. These divergent trajectories suggest that iron regulation and ISC homeostasis represent a possible target for aging interventions.

The loss of vacuolar/lysosomal acidity is an early event during aging that has been linked to mitochondrial dysfunction. However, it is unclear how loss of vacuolar acidity results in age-related dysfunction. Through unbiased genetic screens, we determined that increased iron uptake can suppress the mitochondrial respiratory deficiency phenotype of yeast vma mutants, which have lost vacuolar acidity due to genetic disruption of the vacuolar ATPase proton pump. Yeast vma mutants exhibited nuclear localization of Aft1, which turns on the iron regulon in response to iron sulfur cluster (ISC) deficiency. This led us to find that loss of vacuolar acidity with age in wildtype yeast causes ISC defects and a DNA damage response. Using microfluidics to investigate aging at the single cell level, we observe grossly divergent trajectories of iron homeostasis within an isogenic and environmentally homogeneous population. One subpopulation of cells fails to mount the expected compensatory iron regulon gene expression program, and suffers progressively severe ISC deficiency with little to no activation of the iron regulon. In contrast, other cells show robust iron regulon activity with limited ISC deficiency, which allows extended passage and survival through a period of genomic instability during aging. These divergent trajectories suggest that iron regulation and ISC homeostasis represent a possible target for aging interventions.

Significance: Mutations accumulate throughout life in every cell of the body. A long-standing question is to what extent these mutations contribute to aging. Age is the single greatest risk factor for cancer, a disease driven by random mutagenesis and selection for malignant phenotypes. We wondered whether cancer cells, being the most heavily mutagenized human cell, are short-lived due to mutagenesis. Single-cell measurements of replicative lifespan are not established for human cells. So, instead, we engineered diploid yeast to have cancer-associated defects that increase mutagenesis and then measured lifespan and mutation accumulation. Yeast cellular lifespan decreased as the total number of mutations increased to levels commonly found in human cancers, suggesting that the longevity of cancer cells may be inherently limited.

The yeast genome becomes unstable during stress, which often results in adaptive aneuploidy, allowing rapid activation of protective mechanisms that restore cellular homeostasis. In this study, we performed a genetic screen in Saccharomyces cerevisiae to identify genome adaptations that confer resistance to tunicamycin-induced endoplasmic reticulum (ER) stress. Whole-genome sequencing of tunicamycin-resistant mutants revealed that ER stress resistance correlated significantly with gains of chromosomes II and XIII. We found that chromosome duplications allow adaptation of yeast cells to ER stress independently of the unfolded protein response, and that the gain of an extra copy of chromosome II alone is sufficient to induce protection from tunicamycin. Moreover, the protective effect of disomic chromosomes can be recapitulated by overexpression of several genes located on chromosome II. Among these genes, overexpression of UDP-N-acetylglucosamine-1-P transferase (ALG7), a subunit of the 20S proteasome (PRE7), and YBR085C-A induced tunicamycin resistance in wild-type cells, whereas deletion of all three genes completely reversed the tunicamycin-resistance phenotype. Together, our data demonstrate that aneuploidy plays a critical role in adaptation to ER stress by increasing the copy number of ER stress protective genes. While aneuploidy itself leads to proteotoxic stress, the gene-specific effects of chromosome II aneuploidy counteract the negative effect resulting in improved protein folding. Significance In eukaryotes, about one-third of all proteins are folded in the endoplasmic reticulum (ER). When protein folding demand exceeds capacity of the ER, the unfolded protein response (UPR) is activated to counteract ER stress. However, the mechanisms of adaptation to ER stress are not restricted to the UPR. Here, we report that gain of chromosome II in yeast allows adaptation of cells to protein misfolding. We narrowed down the effect of chromosome II duplication to three genes, which promote ER stress resistance independently of the UPR. Overexpression of these genes was sufficient to protect cells from ER stress and led to the elimination of aneuploidy. Thus, aneuploidy can serve as an important genome adaptation protecting cells against ER stress.