ABSTRACT
Squamous cell carcinomas (SCCs) are one of the most frequent solid cancer types in humans and are derived from stratified epithelial cells found in various organs. SCCs derived from various organs share common important properties, including genomic abnormalities in the tumor suppressor gene p53. There is a carcinogen-induced mouse model of SCC that produces benign papilloma, some of which progress to advanced carcinoma and metastatic SCCs. These SCCs undergo key genetic alterations that are conserved between humans and mice, including alterations in the genomic p53 sequence, and are therefore an ideal system to study the mechanisms of SCC tumorigenesis. Using this SCC model, we show that the PHLDA3 gene, a p53-target gene encoding a protein kinase B repressor, is involved in the suppression of benign and metastatic tumor development. Loss of PHLDA3 induces an epithelialâmesenchymal transition and can complement p53 loss in the formation of metastatic tumors. We also show that in human patients with SCC, low PHLDA3 expression is associated with a poorer prognosis. Collectively, this study identifies PHLDA3 as an important downstream molecule of p53 involved in SCC development and progression.
Subject(s)
Carcinoma, Squamous Cell , Papilloma , Skin Neoplasms , Animals , Carcinogenesis/genetics , Carcinoma, Squamous Cell/pathology , Epithelial Cells/metabolism , Humans , Mice , Nuclear Proteins , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
Protein-protein interactions (PPIs) are often mediated by helical, strand and/or coil secondary structures at the interface regions. We previously showed that non-naturally occurring, stable helical trimers of bicyclic ß-amino acids (Abh) with all-trans amide bonds can block the p53-MDM2/MDMX α-helix-helix interaction, which plays a role in regulating p53 function. Here, we conducted docking and molecular dynamics calculations to guide the structural optimization of our reported compounds, focusing on modifications of the C-terminal/N-terminal residues. We confirmed that the modified peptides directly bind to MDM2 by means of thermal shift assay, isothermal titration calorimetry, and enzyme-linked immunosorbent assay (ELISA) experiments. Biological activity assay in human osteosarcoma cell line SJSA-1, which has wild-type p53 and amplification of the Mdm2 gene, indicated that these peptides are membrane-permeable p53-MDM2/MDMX interaction antagonists that can rescue p53 function in the cells.
Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Oligopeptides/pharmacology , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Tumor Suppressor Protein p53/antagonists & inhibitors , Carbohydrate Conformation , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Humans , Molecular Docking Simulation , Oligopeptides/chemistry , Protein Binding/drug effects , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-mdm2/chemistry , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/metabolismABSTRACT
We developed bioluminescence probes to detect quantitative interaction of GPCRs with arrestin isoforms ß-arrestin1 and ß-arrestin2 based on split luciferase complementation. Time-dependent GPCR-ß-arrestin interactions showed two-types of remarkable variations that were consistent with a classification of GPCR classes. Positive charge residues in serine clusters located at the C-terminal region of GPCRs were necessary for binding to ß-arrestin. This quantitative method enables elucidation of the mechanisms of different classes of GPCRs that regulate ß-arrestin isoforms.
