Post-translational modification of key proteins during exposure of intact prokaryotic and eukaryotic cells to organomercurials.
EMSL Project ID
34915
Abstract
Mercury (Hg) contaminates many DOE sites, notably Oak Ridge National Laboratory (ORNL) where its effects have been monitored for decades. Hg is also released from fossil fuels and minimization of this costs tens of millions of dollars annually. Surprisingly, the exact targets of Hg(II) toxicity are not known in any cellular system. In vitro, Hg(II) binds protein thiols, eliminating their functions. Paradoxically, Hg(II) is toxic at sub-micromolar levels despite most bacteria and eukaryotes having millimolar levels of small thiols such as glutathione to trap it. Summers' and Miller's groups study plasmid-encoded bacterial mercury resistance (mer), the only naturally occurring, dedicated biotic metal detoxification system. mer operon proteins convert organomercurials to Hg(II) which they reduce to volatile Hg(0) that diffuses away, thereby restoring thiol homeostasis. What is not known is which cellular proteins are most vulnerable to Hg(II). This question is central for understanding the ubiquity of the mer operon and its role in bioremediation, as well as understanding the actual risks to humans, animals and plants that Hg poses, since they lack the protection of a mer operon. In our current project we have found that stable post-translational modifications by Hg and RHg at protein cysteine residues are reproducibly observed by high resolution mass spectrometry in the most abundant proteins (e.g. those involved in glycoslysis, amino acid and protein synthesis) of E.coli cells exposed in vivo to toxic and subtoxic levels of Hg/RHg compounds. We propose to use diverse extraction techniques to enrich subsets of less abundant, but essential, proteins including transcription factors, respiratory proteins, and membrane transporters to test the hypothesis that many of them will also form stable, post-translational RHg/Hg adducts. After testing these techniques in E.coli, we will apply them to the model eukaryotic facultative anaerobe, Saccharomyces cerevisiae, to test the hypothesis that Hg-vulnerability of certain proteins is conserved across biological kingdoms.
Mass spectrometry-enabled global proteomics has dramatically enhanced discernment of the mechanisms of intoxication caused by this ubiquitous heavy metal. The techniques we devise for seeing low abundance Hg-vulnerable proteins will expose its damage at the subtlest levels of control and homeostasis. The impact of the proposed work will be the ability to identify key proteins as biomarkers for Hg/RHg exposure and cell damage in bacteria and eukaryotes. It will serve as a basis to extend this technique to fish and other endangered wildlife as well as humans.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2009-10-07
End Date
2012-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Benjamin J. Polacco, Samuel O. Purvine, Erika M. Zink, Stephen P. LaVoie, Mary S. Lipton, Anne O. Summers, Susan M. Miller (2011) Discovering mercury protein modifications in whole proteomes using natural isotope distributions observed in liquid chromatography-tandem mass spectrometry. Molecular & Cellular Proteomics, 30 April 2011 (online)
Benjamin J. Polacco, Samuel O. Purvine, Erika M. Zink, Stephen P. LaVoie, Mary S. Lipton, Anne O. Summers, Susan M. Miller (2011) Discovering mercury protein modifications in whole proteomes using natural isotope distributions observed in liquid chromatography-tandem mass spectrometry. Molecular & Cellular Proteomics, 30 April 2011 (online) - SUPPLEMENTAL MATERIAL ONLINE