Biology subjects

Armstrong, J. O.

Publications and source records attributed to Armstrong, J. O..

3 recordsLinked to original sources

bioRxiv2024

URA6 mutations provide an alternative mechanism for 5-FOA resistance in Saccharomyces cerevisiae

The URA3 gene is frequently used in the budding yeast community as the mutation target for 5-fluoroorotic acid (5-FOA) resistance. We identified a class of ura6 mutants that can grow in the presence of 5-FOA. Unlike ura3 mutants, ura6 mutants remain prototrophic and are able to grow in the absence of uracil. In addition to 5-FOA resistance, we found that ura6 mutants are also resistant to 5-fluorocytosine (5-FC) and 5-fluorouracil (5-FU). In total, we identified 50 unique missense mutations across 32 unique amino acid positions of Ura6 which confer resistance to 5-FOA. We found that 28 out of the 32 affected positions are located in regions conserved between Saccharomyces cerevisiae and three clinically relevant pathogenic fungi. Metabolic analysis revealed a build-up of uridine monophosphate (UMP) and 5-fluorouridine monophosphate (5-FUMP) in ura6 mutants, indicating a reduction in Ura6 activity. Despite the accumulation of UMP in ura6 mutants, uridine diphosphate and triphosphate (UDP, UTP) levels were similar across mutants and wild type. These findings suggest that missense mutations to URA6, can result in reduction of uridylate kinase activity and lead to cross resistance to fluorinated prodrugs that incorporate into both the de novo pyrimidine synthesis and the pyrimidine salvage pathway.

genetics↗

bioRxiv2023

Systematic Profiling of Ale Yeast Protein Dynamics across Fermentation and Repitching

Studying the genetic and molecular characteristics of brewing yeast strains is crucial for understanding their domestication history and adaptations accumulated over time in fermentation environments, and for guiding optimizations to the brewing process itself. Saccharomyces cerevisiae (brewing yeast) is amongst the most profiled organisms on the planet, yet the temporal molecular changes that underlie industrial fermentation and beer brewing remain understudied. Here, we characterized the genomic makeup of a Saccharomyces cerevisiae ale yeast widely used in the production of Hefeweizen beers, and applied shotgun mass spectrometry to systematically measure the proteomic changes throughout two fermentation cycles which were separated by 14 rounds of serial repitching. The resulting brewing yeast proteomics resource includes 64,740 protein abundance measurements. We found that this strain possesses typical genetic characteristics of Saccharomyces cerevisiae ale strains and displayed progressive shifts in molecular processes during fermentation based on protein abundance changes. We observed protein abundance differences between early fermentation batches compared to those separated by 14 rounds of serial repitching. The observed abundance differences occurred mainly in proteins involved in the metabolism of ergosterol and isobutyraldehyde. Our systematic profiling serves as a starting point for deeper characterization of how the yeast proteome changes during commercial fermentations and additionally serves as a resource to guide fermentation protocols, strain handling, and engineering practices in commercial brewing and fermentation environments. Finally, we created a web interface (https://brewing-yeast-proteomics.ccbb.utexas.edu/) to serve as a valuable resource for yeast geneticists, brewers, and biochemists to provide insights into the global trends underlying commercial beer production.

systems biology↗

bioRxiv2023

Multidimensional proteomics identifies molecular trajectories of cellular aging and rejuvenation

The declining capacity of cells to maintain a functional proteome is a major driver of cellular dysfunction and decreased fitness in aging. Here we assess the impact of aging on multiple proteome dimensions, which are reflective of function, across the replicative lifespan of Saccharomyces cerevisiae. We quantified protein abundance, protein turnover, protein thermal stability, and protein phosphorylation in mother yeast cells and their derived progeny at different ages. We find progressive and cumulative proteomic alterations that are reflective of dysregulation of complex assemblies, mitochondrial remodeling, post-translational activation of the AMPK/Snf1 energy sensor in mother cells, and an overall shift from biosynthetic to energy-metabolic processes. Our multidimensional proteomic study systematically corroborates previous findings of asymmetric segregation and daughter cell rejuvenation, and extends these concepts to protein complexes, protein phosphorylation, and activation of signaling pathways. Lastly, profiling age-dependent proteome changes in a caloric restriction model of yeast provided mechanistic insights into longevity, revealing minimal remodeling of energy-metabolic pathways, improved mitochondrial maintenance, ameliorated protein biogenesis, and decreased stress responses. Taken together, our study provides thousands of age-dependent molecular events that can be used to gain a holistic understanding of mechanisms of aging.

systems biology↗