Investigation of Functional Roles for Thioredoxins in Protecting against Reversible Electrophile Induced Protein Damage in Saccharomyces cerevisiae
Thioredoxins directly repair oxidative protein damage by reducing functionally disruptive protein disulfide bonds. Recent work has demonstrated that thioredoxins also recognize and repair additional thiol modifications including cysteine S-sulfation, S-nitrosation, and S-glutathionylation. Given that substrates of Saccharomyces cerevisiae thioredoxins like the 2-cysteine peroxiredoxin Tsa1 are also targeted by organic electrophiles and that several classes of electrophiles react reversibly with thiols, the roles thioredoxins play in protecting against reversible electrophilic damage were explored in yeast. To this end, the acute toxicity of the reversible electrophiles n-butyl isothiocyanate (BITC) and 2-nitrofuran (NF) was determined using a simple cytotoxicity assay in the thioredoxin deficient yeast strains trx1Î” and trx2Î” and wild-type yeast. Single thioredoxin deletion strains trx1Î” and trx2Î” showed a similarly small amount of increased sensitivity to both BITC and NF, indicating that thioredoxins do confer some protection against reversible electrophiles. Furthermore, the mechanisms of toxicity of BITC and NF are clearly different from that of H2O2, which was much more toxic to trx2Î” than to trx1Î”. The lack of greatly increased sensitivity of both trx1Î” and trx2Î” to NF and BITC was attributed to a compensatory mechanism such as increased expression of the remaining cytosolic thioredoxin in these strains. Gene deletion constructs for trx2 and trr1 were successfully made. Future work is required to make the trx1Î”trx2Î” and trr1Î” strains necessary to fully evaluate the importance of thioredoxins in conferring protection against reversible electrophiles. GST fusion proteins GST-Tsa1 and GST-Trx2 were successfully constructed and expressed. Future work with proteins in GST pull-down experiments is necessary to determine whether Trx2 physically interacts with and repair Tsa1 modified by reversible electrophiles.
© Copyright 2013 Wesley J. Murphy