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Title: HIGH-THROUGHOUT IMMUNOBLOTTING IDENTIFIES BIOTIN-DEPENDENT SIGNALING PROTEINS IN HEPG2 HEPATOCARCINOMA CELLS

Author
item RODRIGUEZ-MELENDEZ, ROCIO - UNI OF NE, LINCOLN
item GRIFFIN, JACOB - UNI OF NE, LINCOLN
item Sarath, Gautam
item ZEMPLENI, JANOS - UNI OF NE, LINCOLN

Submitted to: Journal of Nutritional Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/14/2005
Publication Date: 7/1/2005
Citation: Rodriguez-Melendez, R., Griffin, J.B., Sarath, G., Zempleni, J. 2005. High-throughout immunoblotting identifies biotin-dependent signaling proteins in hepg2 hepatocarcinoma cells. Journal of Nutritional Biochemistry. vol 135:1659-1666.

Interpretive Summary: Biotin is an essential vitamin and affects different aspects of cellular functioning. Human-derived hepatocarcinoma (HepG2) cells were grown under deficient, physiological and high biotin levels and extracted cellular proteins were analyzed using a very large array of antibodies. Our data are consistent for a role of biotin in cell signaling and gene expression, and with the existence of clusters of biotin-responsive proteins that control distinct biological processes.

Technical Abstract: Biotin affects the abundance of mRNA coding for approximately 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 1,009 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-mediated signaling were identified as targets of biotin; the abundance of these proteins was greater in biotin-deficient cells compared with controls. This was associated with increased DNA-binding activities of transcription factors Fos and Jun, and increased expression of a reporter gene driven by AP1-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 posttranscriptional step. Additional clusters of biotin-responsive proteins were identified that play roles in cytoskeleton homeostasis, nuclear structure and transport, and neuroscience. Collectively, the studies presented here are consistent with roles of biotin in cell signaling and gene expression, and with the existence of clusters of biotin-responsive proteins in distinct biological processes.