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Cytosolic and nuclear protein targets of thiol-reactive electrophiles

Michelle K Dennehy1, Karolyn A M Richards, Gregory R Wernke

  • 1Department of Biochemistry and Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.

Chemical Research in Toxicology
|January 18, 2006
PubMed

Insights

Researchers identified specific protein targets modified by reactive electrophiles, revealing key cysteine residues susceptible to damage from toxic substances and oxidative stress, advancing understanding of cellular responses to chemical exposure.

Area of Science:

  • Biochemistry
  • Proteomics
  • Toxicology

Background:

  • Reactive electrophiles from drugs, chemicals, and oxidative stress can covalently bind to proteins.
  • Understanding protein targets and susceptibility factors is crucial for deciphering adaptive and toxic cellular responses.
  • Current knowledge of protein targets for electrophilic modification remains limited.

Purpose of the Study:

  • To identify and characterize protein targets modified by thiol-reactive electrophiles.
  • To investigate protein structural features that confer susceptibility to electrophilic modification.
  • To establish a methodology for analyzing protein adducts in response to chemical exposure.

Main Methods:

  • Utilized two biotin-tagged electrophiles, PEO-IAB and BMCC, to modify proteins in HEK293 cell proteomes.
  • Employed affinity enrichment with neutravidin beads to capture biotinylated peptides.
  • Analyzed adducted peptides using multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Main Results:

  • Identified 897 adducts on cysteine residues across 539 proteins.
  • Demonstrated selective and reproducible protein adduction, with most proteins modified at one or two sites.
  • Discovered a core set of 125 cysteines consistently modified by both electrophiles, indicating specific susceptibility.

Conclusions:

  • Developed a robust method for identifying protein targets of electrophilic modification.
  • Revealed that certain protein domains and motifs are preferentially targeted, suggesting specific susceptibility factors.
  • This approach provides a foundation for studying protein damage in toxicology and disease.

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