High-throughput immunoblotting identifies biotin-dependent signaling proteins in HepG2 hepatocarcinoma cells

Rocio Rodriguez-Melendez, Jacob B. Griffin, Gautam Sarath, Janos Zempleni

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

Biotin affects the abundance of mRNA coding for ∼10% of genes expressed in human-derived hepatocarcinoma (HepG2) cells. Here, we determined whether effects of biotin on gene expression are associated with changes in the abundance of distinct proteins in cell signaling and structure. HepG2 cells were cultured in media containing the following concentrations of biotin: 0.025 nmol/L (denoted "deficient"), 0.25 nmol/L ("physiological" = control), and 10 nmol/L ("pharmacological") for 10 d before harvesting. The abundance of 1009 proteins from whole-cell extracts was quantified by using high-throughput immunoblots. The abundance of 44 proteins changed by at least 25% in biotin-deficient and biotin-supplemented cells compared with physiological controls. One third of these proteins participate in cell signaling. Specifically, proteins associated with receptor tyrosine kinase-rnediated signaling were identified as targets of biotin; the abundance of these proteins was greater in biotin-deficient cells than in controls. This was associated with increased DNA-binding activities of the transcription factors Fos and Jun, and increased expression of a reporter gene driven by activator protein (AP)1-binding elements in biotin-deficient cells compared with physiological controls. The abundance of selected signaling proteins was not paralleled by the abundance of mRNA, suggesting that biotin affects expression of these genes at a post-transcriptional step. Additional clusters of biotin-responsive proteins were identified that play roles in cytoskeleton homeostasis, nuclear structure and transport, and neuroscience. This study is consistent with the existence of clusters of biotin-responsive proteins in distinct biological processes, including signaling by Fos/Jun; the latter might mediate the proinflammatory and antiapoptotic effects of biotin deficiency.

Original languageEnglish (US)
Pages (from-to)1659-1666
Number of pages8
JournalJournal of Nutrition
Volume135
Issue number7
DOIs
StatePublished - Jul 2005

Keywords

  • Biotin
  • Cell signaling
  • HepG2 cells
  • Human
  • Tyrosine kinase

ASJC Scopus subject areas

  • Medicine (miscellaneous)
  • Nutrition and Dietetics

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