• BMB Reports
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• 제29권5호
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• pp.411-416
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• 1996

In order to study the role of the protein shell in both iron uptake and iron core formation of ferritin, we constructed a deletion mutant of the ferritin gene and expressed the mutant gene in Escherichia coli, This mutant was obtained by introducing an amber mutation at position Pro-157 and a deletion of the 19 amino acid residues at the carboxy-terminus of the recombinant tadpole H-chain ferritin. The deleted amino acids correspond to E-helix forming the hydrophobic channel in the protein. E. coli harboring the plasmid pTHP157, which contains the deleted gene, was grown at $23^{\circ}C$ in the presence of 0.1 mM IPTG, and the induced protein appeared to be partly soluble. Nondenaturing polyacrylamide gel electrophoresis showed that the expressed mutant H-chains coassemble into holoprotein, suggesting that E-helix is not necessary for assembly of the subunits as reported for human H-chain ferritin. Its ability in iron core formation was proven in an Fe staining gel, the result disagreeing with the observation that the hydrophobic channel is necessary for iron core formation in human H-chain ferritin.

• Journal of Microbiology
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• 제37권4호
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• pp.256-262
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• 1999

Latent membrane protein 1 (LMP1) of the Epstein-Barr virus (EBV) is an integral membrane protein with six transmembrane domains, which is essential for EBV-induced B cell transformation. LMP1 functions as a constitutively active tumor necrosis factor receptor (TNFR) like membrane receptor, whose signaling requires recruitment of TNFR-associated factors (TRAFs) and leads to NF-${\kappa}B$ activation. NF-${\kappa}B$ activation by LMP1 is critical for B cell transformation and has been linked to many phenotypic changes associated with EBV-induced B cell transformation. Deletion analysis has identified two NF-${\kappa}B$ activation regions in the carboxy terminal cytoplasmic domains of LMP1, termed CTAR1 (residues 194-232) and CTAR2 (351-386). The membrane proximal C-terminal domain was precisely mapped to a PXQXT motif (residues 204-208) involved in TRAF binding as well as NF-${\kappa}B$ activation. In this study, we dissected the CTAR2 region, which is the major NF-${\kappa}B$ signaling effector of LMP1, to determine a minimal functional sequence. A series of LMP1 mutant constructs systematically deleted for the CTAR2 region were prepared, and NF-${\kappa}B$ activation activity of these mutants were assessed by transiently expressing them in 293 cells and Jurkat T cells. The NF-${\kappa}B$ activation domain of CTAR2 appears to reside in a stretch of 6 amino acids (residues 379-384) at the end of the carboxy terminus.

• International Journal of Oral Biology
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• 제32권4호
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• pp.119-125
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• 2007

Dlx3 is a homeodomain protein and is known to play a role in development and differentiation of many tissues. Deletion of four base pairs in DLX3 (NT3198) is causally related to tricho-dento-osseous (TDO) syndrome (OMIM #190320), a genetic disorder manifested by taurodontism, hair abnormalities, and increased bone density in the cranium. The molecular mechanisms that explain the phenotypic characteristics of TDO syndrome have not been clearly determined. In this study, we examined phenotypic characteristics of wild type DLX3(wtDlx3) and 4-BP DEL DLX3 (TDO mtDlx3) in C2C12 cells. To investigate how wtDlx3 and TDO mtDlx3 differentially regulate osteoblastic differentiation, reporter assays were performed by using luciferase reporters containing the promoters of alkaline phosphatase, bone sialoprotein or osteocalcin. Both wtDlx3 and TDO mtDlx3 enhanced significantly all the reporter activities but the effect of mtDlx3 was much weaker than that of wtDlx3. In spite of these differences in reporter activity, electrophoretic mobility shift assay showed that both wtDlx3 and TDO mtDlx3 formed similar amounts of DNA binding complexes with Dlx3 binding consensus sequence or with ALP promoter oligonucleotide bearing the Dlx3 binding core sequence. TDO mtDlx3 exhibits a longer half-life than wtDlx3 and it corresponds to PESTfind analysis result showing that potential PEST sequence was missed in carboxy terminal of TDO mtDlx3. In addition, co-immunoprecipitation demonstrated that TDO mtDlx3 binds to Msx2 more strongly than wtDlx3. Taken together, though TDO mtDlx3 acted as a weaker transcriptional activator than wtDlx3 in osteoblastic cells, there is possibility that during in vivo osteoblast differentiation TDO mtDlx3 may antagonize transcriptional repressor activity of Msx2 more effectively and for longer period than wtDlx3, resulting in enhancement of osteoblast differentiation.