High-throughput immunoblotting identifies biotin-dependent signaling proteins in HepG2 hepatocarcinoma cells.
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 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-mediated 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.
Department of Nutrition and Health Sciences, University of Nebraska at Lincoln, Lincoln, NE, USA.
Rocio
R
Rodriguez-Melendez
Jacob B
JB
Griffin
Gautam
G
Sarath
Janos
J
Zempleni
D001483:Base Sequence; D001710:Biotin / Q000378:metabolism*; D019921:Biotinylation; D043182:Carboxylesterase / Q000235:genetics / Q000378:metabolism; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D017931:DNA Primers; D006801:Humans; D015151:Immunoblotting; D008113:Liver Neoplasms; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D015345:Oligonucleotide Probes; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*
D017931:DNA Primers; D009363:Neoplasm Proteins; D015345:Oligonucleotide Probes; D001710:Biotin; D043182:Carboxylesterase