May 2006
Volume 47, Issue 13
ARVO Annual Meeting Abstract  |   May 2006
Genetic Studies of Human and Yeast Aldo–Keto Reductases
Author Affiliations & Notes
  • J. Petrash
    Ophthalmology, Washington University, Saint Louis, MO
  • Q. Chang
    Ophthalmology, Washington University, Saint Louis, MO
  • Footnotes
    Commercial Relationships  J. Petrash, None; Q. Chang, None.
  • Footnotes
    Support  NIH Grants EY05856 and EY02687 and Research to Prevent Blindness, Inc.
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 4125. doi:
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      J. Petrash, Q. Chang; Genetic Studies of Human and Yeast Aldo–Keto Reductases . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4125.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Aldo–keto reductases (AKRs) are NADPH–dependent oxidoreductases that reduce a wide range of substrates including aldo–sugars, lipid–derived aldehydes, ketones, steroid hormones, and xenobiotics. Human aldose reductase (HAR), which encodes a prototypical AKR, is thought to be involved in the pathogenesis of diabetic cataract and retinopathy. However, the physiological roles of AKRs are largely unknown due to the complexity of the AKR gene family in humans and mammalian model systems. We have used a targeted gene knockout and replacement approach in the model organism Saccharomyces cerevisiae to genetically dissect the AKR gene family.

Methods: : Five open reading frames (ORFs) that encode AKRs were identified in the yeast genome by a BLAST query against the sequence for HAR. Kinetic analysis of recombinant proteins produced from each of the five ORFs verified the enzymatic nature of each putative AKR. Targeted disruptions were introduced into each of the five yeast AKR genes, producing a permuted collection of mutant strains that were null for one or more AKR genes. Deletion mutants were subjected to a cassette of phenotypic screenings to measure alternations in growth rate, nutritional auxotrophies, morphology and drug sensitivity. Site directed mutagenesis was used to catalytically inactivate selected AKRs by substituting an active site tyrosine side chain with phenylalanine.

Results: : Enhanced sensitivity to heat shock stress and inositol auxotrophy are the most prominent phenotypic features of AKR depleted strains. These phenotypes emerged when at least three of the five AKR genes were disrupted, and were enhanced when we disrupted the remaining two AKR genes. Transformation of AKR–depleted mutant strains with expression vectors encoding either human or yeast AKRs rescued these mutant phenotypes. However, phenotypic rescue was not observed if mutant strains were transformed with catalytically inactive mutant AKRs.

Conclusions: : Rescue of the heat shock and inositol phenotypes by ectopic expression of HAR indicates that these phenotypes result from one or more unmetabolized AKR substrates or a deficiency of AKR products normally generated in response to stress or culture conditions. Further studies are ongoing to identify analogous pathways and processes that are influenced by the action of AKRs in mammalian target tissues of diabetes mellitus.

Keywords: cataract • diabetes • genetics 

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