Biology subjects

Bachhawat, A.

Publications and source records attributed to Bachhawat, A..

3 recordsLinked to original sources

bioRxiv2025

A yeast model of 5-oxoproline accumulation reveals a general toleration to 5-oxoproline

5-oxoproline (5-OP) or pyroglutamic acid is an intermediate of the degradation arc of the glutathione cycle. It is metabolized into glutamate through the action of the 5-oxoprolinase enzyme, the only enzyme known to act on this metabolite. 5-OP has long been known to be relatively inert with a proposed role as an osomoprotectant. Recent studies on the 5-oxoprolinase enzyme in mammalian cells have, however, shown that knockdown or deletions of the 5-oxoprolinase make mice (and humans) prone to heart failure. This was ascribed to oxidative stress due to a two-fold elevation in 5-OP. To examine the consequences of 5-oxoproline accumulation more rigorously, we have created a yeast model for 5-oxoproline accumulation. We observed retardation of growth only when intracellular levels of 5-OP were increased 12-20 fold over the normal levels. A transcriptomics study was carried out under these conditions. We observed that while a large number of genes were regulated upto 2-fold, there were no single prominent pathways amongst them. Among the key genes upregulated were different efflux pumps. Using knockouts and also overexpression of selected genes, we could observe that many of the upregulated genes were involved in the cellular response to 5-OP accumulation. However, it did not appear that there was any significant oxidative stress response. The study suggests a need to reevaluate previous suppositions of the 5-OP induced oxidative stress response. Instead, we propose an alternative model to explain the possible consequences of 5-oxoprolinase deficiency based on these findings made with the yeast model.

systems biology↗

bioRxiv2025

Plasma membrane folate transport in fungi and plants is mediated by members of the oligopeptide transporter (OPT) family

Folates are essential for all organisms. They are acquired either through de novo biosynthesis or from the diet. Yeast, fungi and plants make their own folates and it has not been clear if plasma membrane folate transporters exist in these organisms. Using a synthetic lethal screen in Saccharomyces cerevisiae we observed that deletions in a gene encoding the previously identified glutathione transporter, OPT1, was synthetically sick with a disruption in folate biosynthesis. Uptake experiments confirmed that Opt1p/Hgt1p can transport folinic acid and the naturally abundant methyl tetrahydrofolate. As S. cerevisiae Opt1p was able to transport both folate and glutathione, we used alanine-scanning mutants of the residues in the transmembrane domains of the channel to identify the residues required specifically for the uptake of folates and distinct from those required for glutathione. We further examined the oligopeptide transporter family of other organisms for the presence of folate transporters. In C. albicans, CaOPT1, the orthologue of S. cerevisiae OPT1 efficiently transported folate but not glutathione, while the previously characterized glutathione transporter, CaOPT7 could not transport folate. Aspergillus fumigatus has eight homologues of the oligopeptide transporter family, of which OptB and OptH could transport folates. In the plant Arabidopsis thaliana, the Opt1 homologs AtOpt2, AtOpt4, and AtOpt6 could transport folates. This discovery of folate transporters across fungi and plants fills a critical gap in our understanding of folate metabolism, and can benefit the exploitation of these pathways in pathogenic fungi, and in plants.

genetics↗

bioRxiv2024

Identification of Juglone, a "first-in-class" inhibitor of the human glutathione degrading enzyme, ChaC1, using yeast-based high throughput screens

The cytosolic glutathione-degrading enzyme, ChaC1, is highly upregulated in several cancers, with the upregulation correlating to poor prognosis. The ability to inhibit ChaC1 thus becomes important in pathophysiological situations where elevated glutathione levels would be beneficial. As no inhibitors of ChaC1 are known, in this study we have focussed on this goal. We have initially taken a computational approach where a systemic structure-based virtual screening was performed. However, none of the predicted hits proved to be effective inhibitors. We also evaluated synthetic substrate analogs, but these too were not inhibitory. As both these approaches targeted the active site, we shifted to developing two high-throughput, robust, yeast-based assays that were active site independent. A small molecule compound library was screened using an automated liquid handling system using these screens. The hits were further analyzed using in vitro assays. Among them, juglone, a naturally occurring naphthoquinone, completely inhibited ChaC1 activity with an IC50 of 8.7 {micro}M. It was also effective against the ChaC2 enzyme. Kinetic studies indicated that the inhibition was not competitive with the substrate. Juglone is known to form adducts with glutathione and is also known to selectively inhibit enzymes by covalently binding to their active site cysteine residues. However, juglone continued to inhibit a cysteine-free ChaC1 variant, indicating that it was acting through a novel mechanism. We evaluated different inhibitory mechanisms, and also analogues of juglone, and found plumbagin effective as an inhibitor. These compounds represent the first-in-class inhibitors of the ChaC enzymes discovered using a robust yeast screen.

biochemistry↗