Chemical Genetic Analysis of Echinochrome A Isolated from Evechinus chloroticus (Sea Urchin, Kina) in New Zealand

Abstract

Using unbiased genome-wide proteomic and genomic analyses available to Saccharomyces cerevisiae, this thesis aims to provide further insight into the mode of action of Echinochrome A (Ech A) extracted from the shells of New Zealand kina (Evechinus chloroticus). Abundance was measured for 4,100 proteins every 30 minutes for four hours using fluorescent microscopy, which resulted in the identification of 92 proteins with significant alterations in protein abundance caused by Ech A treatment that were overrepresented with specific changes in DNA replication, repair and RNA binding at 30 minutes,followed by metabolism of metal ions (specifically iron and copper) from 60-240 minutes. Further analysis indicated that Ech A chelated iron, and that iron supplementation negated the growth inhibition caused by Ech A. Likewise, copper supplementation negated the growth inhibition caused by Ech A. Via a genome-wide analysis of 4,800 gene deletion strains that measured sensitivity of growth of these deletion strains, it was identified that 20 gene deletion strains were sensitive to Ech A and these genes were over-represented in the cellular response to oxidative stress. To directly evaluate the potential antioxidant properties of Ech A suggested by the proteomic and genomic analyses, yeast growth was measured in the presence and absence of oxidative stress induced by hydrogen peroxide, whereby Ech A suppressed growth inhibition caused by the oxidative stress. Building off publications investigating Ech A in mammalian models, there were a number of mitochondrial gene deletions with significantly altered growth and mitochondrial proteins with significantly altered abundance. Overall, my thesis identified the iron chelation mechanism of Ech A as a key causative factor in Ech A bioactivity via the identification of novel genes and proteins that are essential to the iron chelation and antioxidant molecular mechanisms of Ech A.