Abstract: CCAAT/Enhancer Binding Protein d (C/EBPd) plays a key role in mammary epithelial cell G0 growth arrest and "loss of function" alterations in C/EBPd have been reported in breast cancer and acute myeloid leukemia (AML). C/EBPd is regulated at the transcriptional, post-transcriptional and post-translational levels, suggesting tight control of C/EBPd content and function. Protein inhibitors of activated STATs (PIASs) regulate a growing number of transcription factors, including C/EBPs. HC11 nontransformed mammary epithelial cells express PIAS3, PIASxss and PIASy and all three PIAS family members repress C/EBPd transcriptional activity. PIASy is the most potent however, repressing C/EBPd transcriptional activity by >80%. PIASy repression of C/EBPd transcriptional activity is dependent upon interaction between the highly conserved PIASy N-terminal nuclear matrix binding domain (SAPD) and the C/EBPd transactivation domain (TAD). PIASy repression of C/EBPd transcriptional activity is independent of histone deacetylase activity, PIASy E3 SUMO ligase activity and C/EBPd sumoylation status. PIASy expression is associated with C/EBPd translocation from nuclear foci, where C/EBPd co-localizes with p300, to the nuclear periphery. PIASy-mediated translocation of C/EBPd is dependent upon the PIASy SAPD and C/EBPd TAD. PIASy reduces the expression of C/EBPd adhesion-related target genes and enhances repopulation of open areas within a cell monolayer in the in vitro "scratch" assay. These results demonstrate that PIASy represses C/EBPd by a mechanism that requires interaction between the PIASy SAPD and C/EBPd TAD and does not require PIASy SUMO ligase activity or C/EBPd sumoylation. PIASy alters C/EBPd nuclear localization, reduces C/EBPd transcriptional activity and enhances cell proliferation/migration.
Abstract: Cyclic peptides provide attractive lead compounds for drug discovery and excellent molecular probes in biomedical research. In this work, a novel method has been developed for the high-throughput synthesis, screening, and identification of cyclic peptidyl ligands against macromolecular targets. Support-bound cyclic phosphotyrosyl peptide libraries containing randomized amino acid sequences and different ring sizes (theoretical diversity of 3.2 x 10 (6)) were synthesized and screened against the SH2 domains of Grb2 and tensin. Potent, selective inhibitors were identified from the libraries and were generally more effective than the corresponding linear peptides. One of the inhibitors selected against the Grb2 SH2 domain inhibited human breast cancer cell growth and disrupted actin filaments. This method should be applicable to the development of cyclic peptidyl inhibitors against other protein domains, enzymes, and receptors.
Abstract: CCAAT/enhancer binding protein δ (C/EBPδ) is a member of the C/EBP family of nuclear proteins that function in the control of cell growth, survival, differentiation and apoptosis. We previously demonstrated that C/EBPδ gene transcription is highly induced in G0 growth arrested mammary epithelial cells but the C/EBPδ protein exhibits a t1/2 of only ~ 120 minutes. The goal of this study was to investigate the role of C/EBPδ modification by ubiquitin and C-EBPδ proteasome-mediated degradation. Structural and mutational analyses demonstrate that an intact leucine zipper is required for C/EBPδ ubiquitination; however, the leucine zipper does not provide lysine residues for ubiquitin conjugation. C/EBPδ ubiquitination is not required for proteasome mediated C/EBPδ degradation and the presence of ubiquitin does not increase C/EBPδ degradation by the proteasome. Instead, the leucine zipper stabilizes the C/EBPδ protein by forming homodimers that are poor substrates for proteasome degradation. To investigate the cellular conditions associated with C/EBPδ ubiquitination we treated G0 growth arrested mammary epithelial cells with DNA damage and oxidative stress inducing agents and found that C/EBPδ ubiquitination is induced in response to hydrogen peroxide (H2O2). However, C/EBPδ protein stability is not influenced by H2O2 treatment. In conclusion, our results demonstrate that proteasome-mediated protein degradation of C/EBPδ is ubiquitin-independent.
Abstract: Chondroitin sulfate (CS) has a variety of biological activities, most of them due to chondroitin sulfate's sulfonic acid groups and carboxyl groups as well. To gain insight into the mechanism of interaction between the spectroscopic probe and chondroitin sulfate, we have used Methylene Blue (MB), one cationic dye, by a spectrophotometric method. This paper developed a new experimental method for determining the maximum binding number Ne, which expresses the binding ability of this dye with CS. Meanwhile, By using an interaction theory model we have established, the maximum binding number Nc can be calculated from the linear regression equation. The research results show that the Ne value is in agreement with that of Nc.
Abstract: The interaction of hyaluronic aicd (HA) and azur A(AA) was studied by absorption spectra. The influence of pH and the molar ratio of AA/HA on the spectra was investigated. The critical molar ratio (1.28Ã10^3) necessary fro the hypsochromism of the absorption spectra of AA-HA complex and the maximum binding number (3.22Ã10^3) were obtained by spectrometry. The maximum binding number is consistent with the theoretical value.
