Proteomic approach to the identification of novel delta-lactoferrin target genes: Characterization of DcpS, an mRNA scavenger decapping enzyme

作者：Mariller Christophe^*; Hardiville Stephan; Hoedt Esthelle; Benaissa Monique; Mazurier Joel; Pierce Annick

来源：Biochimie, 2009, 91(1): 109-122.

DOI：10.1016/j.biochi.2008.07.009

• Delta-lactoferrin
• Transcription factor
• Scavenger decapping enzyme DcpS
• mRNA turnover
• O-GlcNAc/P interplay
• Proteomics

The expression of the transcription factor Delta Lf is deregulated in cancer cells. Its overexpression provokes cell cycle arrest along with anti proliferative effects and we recently showed that the Skp1 gene promoter was a target of Delta Lf. Skp1 belongs to the Skp1/Cullin-1/F-box ubiquitin ligase complex responsible for the ubiquitination and the proteosomal degradation of numerous cellular regulators. The transcriptional activity of Delta Lf is highly controlled and negatively regulated by O-GlcNAc, a dynamic post-translational modification known to regulate the functions of many intracellular proteins. We, therefore, constructed a Delta Lf-M4 mutant corresponding to a constitutively active Delta Lf isoform in which all the glycosylation sites were mutated. In order to discover novel targets of Delta Lf transcriptional activity and to investigate the impact of the O-GlcNAc regulation on this activity in situ we compared the proteome profiles of Delta Lf and Delta Lf-M4-expressing HEK293 cells versus null plasmid transfected cells. A total of 14 differentially expressed proteins were visualized by 2D electrophoresis and silver staining and eight proteins were identified by mass spectrometry analyses (MALDI-TOF; LC-MS/MS), all of which were upregulated. The identified proteins are involved in several processes such as mRNA maturation and stability, cell viability, proteasomal degradation, protein and mRNA quality control. Among these proteins, only DcpS and TCPB were also upregulated at the mRNA level. Analysis of their respective promoters led to the detection of a cis-regulating element in the DcpS promoter. The S1(Dcps) is 80% identical to the S1 sequence previously described by He and Furmanski [Sequence specificity and transcriptional activation in the binding of lactoferrin to DNA, Nature 373 (1995) 721-724]. Reporter gene analyses and ChIP assays demonstrated that Delta Lf interacts specifically with the DcpS promoter in vivo. These data established that DcpS, a key enzyme in mRNA decay, is a new target of AU transcriptional activity.

• 出版日期2009-1