Biology subjects

Wasko, B. M.

Publications and source records attributed to Wasko, B. M..

4 recordsLinked to original sources

bioRxiv2026

BUDDY: An interactive web platform to accelerate the functional modeling of human missense variants using Saccharomyces cerevisiae orthologs

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).

bioinformatics↗

bioRxiv2025

An mTOR Inhibitor Discovery System Using Drug-Sensitized Yeast

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 {micro}M of Torin1 and 100 {micro}M 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 {micro}M, 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.

molecular biology↗

bioRxiv2020

Evolution of Natural Lifespan Variation and Molecular Strategies of Extended Lifespan

To understand the genetic basis and selective forces acting on longevity, it is useful to examine lifespan variation among closely related species, or ecologically diverse isolates of the same species, within a controlled environment. In particular, this approach may lead to understanding mechanisms underlying natural variation in lifespan. Here, we analyzed 76 ecologically diverse wild yeast isolates and discovered a wide diversity of replicative lifespan. Phylogenetic analyses pointed to genes and environmental factors that strongly interact to modulate the observed aging patterns. We then identified genetic networks causally associated with natural variation in replicative lifespan across wild yeast isolates, as well as genes, metabolites and pathways, many of which have never been associated with yeast lifespan in laboratory settings. In addition, a combined analysis of lifespan-associated metabolic and transcriptomic changes revealed unique adaptations to interconnected amino acid biosynthesis, glutamate metabolism and mitochondrial function in long-lived strains. Overall, our multi-omic and lifespan analyses across diverse isolates of the same species shows how gene-environment interactions shape cellular processes involved in phenotypic variation such as lifespan.

genetics↗

bioRxiv2020

Loss of vacuolar acidity results in iron sulfur cluster defects and divergent homeostatic responses during aging in Saccharomyces cerevisiae

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.

molecular biology↗