Lead poisoning is the most common environmentally-caused disease in the United States and is threat to human health worldwide. Although efforts to prevent lead exposure have increased throughout the years, lead poisoning still remains a common problem. Lead toxicity is more prevalent in children, who suffer from permanent neurodevelopment and cognitive symptom even in case of low blood lead levels. In addition, lead poisoning in both children and adults is often observed with iron insufficiency demonstrating interplay between lead toxicity and iron homeostasis. Here, this work describes the molecular mechanism for lead's toxicity in S. cerevisiae, a model organism to investigate heavy metal homeostasis. S. cerevisiae presents a good model organism for the investigation of metal ion homeostasis and transport due to the conspicuous parallels found in higher eukaryotes. Interrogation of the molecular consequences propagated by lead provides a better understanding of not only how to improve human health by avoiding the consequences following heavy metal exposure, but also how natural and beneficial metals are handled. Since the mechanism for lead poisoning is largely unknown, molecular approaches that aimed at revealing global changes upon lead exposure were utilized. To determine the effects of lead on gene expression in the model organism, genechip microarrays and quantitative real-time RT-PCR were performed. These studies revealed that lead represses several genes that are typically upregulated by the iron responsive transcription factor Aft1 in absence of iron. Moreover, transcription-based assays using a reporter system with the iron responsive promoter for fet3 fused to the E. coli lacZ gene demonstrate that Aft1 activity is reduced in presence of lead even under iron-deficient conditions. The activity of the dominant positive mutation of Aft1, Aft1up1, is also suppressed upon lead treatment suggesting that lead specifically disrupts either Aft1 or proteins that directly interact with it. Localization of Aft1-GFP changes in response to lead treatment suggesting that lead interferes with the proper localization of Aft1 by decreasing the extent of nuclear localization under iron deprivation. The recent discovery of the regulation of Aft1 indicates that the monothiol glutaredoxins, Grx3 and Grx4, act as repressors of Aft1. Our research demonstrates that Grx3 is also implicated in the mechanism for lead specific repression of Aft1. Using the transcriptional assay described above, we show that the lead specific inhibition of Aft1 is absent in strains lacking grx3, Δgrx3 and Δgrx3Δgrx4. The same effect was observed regardless of the intracellular iron status. Complementation in trans, using a plasmid-encoding grx3, on a background that lacks grx3 restores the lead induced inhibition of Aft1. Collectively, these findings ascribe a role of Grx3 to the lead-specific Aft1 repression.

Last modified

• 08/31/2018

Creator

• Dimitar Ivanov

DOI

• https://doi.org/10.21985/N2KX6C

Subject

• Interdepartmental Biological Sciences Program

Keyword

• Lead
• Toxicity
• Grx3
• S. cerevisiae
• Aft1

Date created

• 2008-05-06

Resource type

• Dissertation

Rights statement

• In Copyright