The study aimed to investigate the effects of small ubiquitin-related modifier (SUMO) specific protease 1 (SENP1) on human PXR-mediated MDR1 transcriptional activity and mRNA expression. Empty vector and expression plasmids, including PXR, SENP1 and SENP1 mutant (SENP1m) were transiently transfected into HepG2 and LS174T cells using Lipo2000. Transcriptional activity was detected by dual luciferase reporter gene assay, and mRNA level was measured using real-time polymerase chain reaction. The results showed that SENP1 could remarkably reduce the rifampicin (RIF)-induced MDR1 reporter activity and mRNA level in hPXR over expressed HepG2 and LS174T cells (P < 0.05), whereas adding SENP1m restored the RIF-induced increases (P < 0.05). These results indicated that SENP1 could repress the RIF-induced hPXR-mediated MDR1 transcriptional activity and mRNA expression.
ATP Binding Cassette Transporter, Sub-Family B ; metabolism ; Cysteine Endopeptidases ; Endopeptidases ; metabolism ; Gene Expression ; Hep G2 Cells ; Humans ; Peroxisome-Targeting Signal 1 Receptor ; RNA, Messenger ; Receptors, Cytoplasmic and Nuclear ; metabolism ; Transcriptional Activation

The peroxisomal matrix proteins involved in many important biological metabolism pathways in eukaryotic cells are encoded by nucleal genes, synthesized in the cytoplasm and then transported into the organelles. Targeting and import of these proteins depend on their two peroxisomal targeting signals (PTS1 and PTS2) in sequence as we have known so far. The vectors of the fluorescent fusions with PTS, i.e., green fluorescence protein (GFP)-PTS1, GFP-PTS2 and red fluorescence protein (RFP)-PTS1, were constructed and introduced into Magnaporthe oryzae Guy11 cells. Transformants containing these fusions emitted fluorescence in a punctate pattern, and the locations of the red and green fluorescence overlapped exactly in RFP-PTS1 and GFP-PTS2 co-transformed strains. These data indicated that both PTS1 and PTS2 fusions were imported into peroxisomes. A probable higher efficiency of PTS1 machinery was revealed by comparing the fluorescence backgrounds in GFP-PTS1 and GFP-PTS2 transformants. By introducing both RFP-PTS1 and GFP-PTS2 into Deltamgpex6 mutants, the involvement of MGPEX6 gene in both PTS1 and PTS2 pathways was proved. In addition, using these transformants, the inducement of peroxisomes and the dynamic of peroxisomal number during the pre-penetration processes were investigated as well. In summary, by the localization and co-localization of PTS1 and PTS2, we provided a useful tool to evaluate the biological roles of the peroxisomes and the related genes.
Base Sequence ; DNA Primers ; genetics ; DNA, Fungal ; genetics ; Fungal Proteins ; genetics ; metabolism ; Genes, Fungal ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; metabolism ; Luminescent Proteins ; genetics ; metabolism ; Magnaporthe ; genetics ; metabolism ; Microscopy, Fluorescence ; Mutation ; Peroxisomal Targeting Signal 2 Receptor ; Peroxisome-Targeting Signal 1 Receptor ; Peroxisomes ; metabolism ; Receptors, Cytoplasmic and Nuclear ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transformation, Genetic