Screening of proteins interacting with HSPC016 by yeast two-hybridization technique / 中华皮肤科杂志

ABSTRACT

Objective To screen and identify proteins interacting with hematopoietic stem/progenitor cell differentiation-related gene HSPC016, and to explore the molecular mechanism involved in the regulation by HSPC016 on the aggregative behavior of dermal papilla cells. Methods By using yeast two-hybridization,HSPC016 gene was sub-cloned into pGBKT7 to construct the bait plasmid (named as pGBKT7-HSPC016) in yeast AH109. The cDNA yeast expression library of human dermal papillae cells in yeast Y187 was screened with the bait plasmid and the proteins interacting with HSPC016 were identified. Yeast two-hybridization retransformation experiment was conducted to exclude the false positive clones and verify the interactions, then,the positive clones were sequenced and analyzed by using bioinformatic methods. Results The bait plasmid pGBKT7-HSPC016 was constructed successfully and there was no self-activation in or toxicity against yeast AH 109. Four proteins,including forkhead family of transcription factors (FOXO 1 ), mitogen-activated protein kinase 11 (MAPK 11 ), phosphoinositide-3-kinase (PIK3R3) and liver X receptor were screened and identified. Bioinformatic analysis revealed that these proteins had close relationship with intracellular energy metabolism and translational regulation. Conclusions HSPC016 may regulate the aggregative behavior of DPCs by regulating the levels of intracellular reactive oxygen species (ROS) and interacting with signaling molecules involved in intracellular energy metabolism and translational regulation.