Genetic Studies of Aldo–Keto Reductases

Abstract

Abstract: : Purpose: Aldo–keto reductases (AKR) comprise a family of enzymes that are evolutionarily conserved in all eukaryotes. Aldose reductase, a prototypic AKR, has long been recognized as a factor in diabetic cataract and retinopathy. Due to a multiplicity of aldose reductase–like genes in mammals, a targeted gene knock out approach in mice is unlikely to uncover physiological roles that relate to specific AKRs. Taking advantage of a more robust genetic system, we are utilizing the budding yeast S. cerevisiae as a model to probe the physiological roles of AKRs. Methods: Seed cultures of isogenic wild type and AKR deletion strains were grown to saturation in regular synthetic growth medium. Cultures were shifted to medium supplemented with/without inositol to resume growth. Relative transcript levels of selected genes among different strains were evaluated by real–time RT–PCR. For genetic analysis, a constitutively active mutant allele of Ras2^val19 gene was introduced into the AKR deletion mutant. For phenotype rescue experiment, the cDNA encoding inositol 3–phosphate synthase (INO1), whose function is in the rate–limiting step for the de novo synthesis of inositol, was amplified by PCR from genomic DNA isolated from wild type yeast, cloned into a low–copy centromeric expression vector, and transformed into the AKR deletion mutant. Results: Deletion of AKR genes leads to an inositol auxotrophic phenotype (Ino^–). This phenotype appears to be linked to a defect in expression of inositol synthase (INO1), one of many genes involved with phospholipid synthesis. Indeed, other genes that are co–regulated with INO1 in the pathway of phospholipid synthesis were also found to be relatively repressed under conditions that should induce transcriptional activation. Ectopic expression of INO1 restored inositol prototrophy in the AKR deletion strain. Genetic data suggest that the Ino^– phenotype of the AKR deletion mutant, which could be exacerbated by genetic enhancement of Ras signaling, was not mediated through increased PKA activity. Conclusions: The Ino^– phenotype of the AKR deletion mutant suggests that the AKR family genes are functionally essential to maintain the homeostatic state of inositol biosynthesis in yeast. The genetic interaction of AKR gene with Ras signaling suggests that the AKR genes may share overlapping functions with the Ras mediated signaling cascade. These results lay the foundation for future translational research to study the homologous mammalian aldo–keto reductases in more detail.

Keywords: cataract • diabetes • metabolism