Development of a fluorescent reporter to assess iron regulatory protein activity in living cells

Rebecca J. Henderson, Stephanie Patton, James Connor

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Through the insertion of an iron responsive element (IRE) into a pd2ECFP vector, we demonstrate a noninvasive method for determining alterations in iron regulatory protein (IRP) activity that results in changes in protein translation in living cells. This construct takes advantage of the specifically iron-dependent interaction between IRPs that bind IREs on mRNAs to posttranscriptionally regulate protein expression in a manner similar to ferritin production. In this report, we demonstrate, using HEK-293 cells, that an IRE-driven fluorescent reporter can be used to observe changes in cellular iron status that are sufficient to alter protein synthesis. When iron availability was decreased, there was less cyan fluorescent protein (CFP) expression, suggesting that IRPs bind to the IRE and block protein translation. Conversely, exposing the cells to iron increased CFP fluorescence. This construct has advantages over traditionally used dyes and existing IRE driven constructs because it can be used to repeatedly study iron-influenced protein production over extended periods of time. The future applications of this construct include investigation of how mutations in cells may impact cellular iron metabolism and how various types of exogenously applied trophic, stress, and therapeutic agents may impact cellular iron metabolism.

Original languageEnglish (US)
Pages (from-to)162-168
Number of pages7
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Volume1743
Issue number1-2
DOIs
StatePublished - Mar 22 2005

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Iron-Regulatory Proteins
Iron
Protein Biosynthesis
Proteins
HEK293 Cells
Ferritins

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

Cite this

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Development of a fluorescent reporter to assess iron regulatory protein activity in living cells. / Henderson, Rebecca J.; Patton, Stephanie; Connor, James.

In: Biochimica et Biophysica Acta - Molecular Cell Research, Vol. 1743, No. 1-2, 22.03.2005, p. 162-168.

Research output: Contribution to journalArticle

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