ABSTRACT
Reactivation of the p53 pathway by a potential therapeutic antagonist, which inhibits HDM2 and HDMX, is an attractive strategy for drug development in oncology. Developing blockers towards conserved hydrophobic pockets of both HDMs has mainly focused on small synthetic compounds; however, this approach has proved challenging. Here we describe an approach to generate a potent HDM dual inhibitor, p53LZ2, by rational protein grafting of the p53 transactivation domain onto a homodimeric leucine zipper. p53LZ2 shows tight binding affinity to both HDMs compared with wild-type p53 in vitro. X-ray crystallographic, comparative modelling and small-angle X-ray scattering studies of p53LZ2-HDM complexes show butterfly-shaped structures. A cell-permeable TAT-p53LZ2 effectively inhibits the cancer cell growth in wild-type but not mutant p53 by arresting cell cycle and inducing apoptosis in vitro. Thus, p53LZ2, designed by rational grafting, shows a potential therapeutic approach against cancer.
Subject(s)
Leucine Zippers/genetics , Nuclear Proteins/antagonists & inhibitors , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Recombinant Proteins/pharmacology , Tumor Suppressor Protein p53/genetics , Amino Acid Sequence , Animals , Apoptosis/genetics , Cell Cycle Checkpoints/genetics , Cell Cycle Proteins , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Crystallography, X-Ray , Female , HCT116 Cells , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Molecular Dynamics Simulation , Multiprotein Complexes/ultrastructure , Neoplasm Transplantation , Neoplasms/drug therapy , Protein Engineering , Protein Structure, Tertiary , Recombinant Proteins/ultrastructure , Sequence Alignment , Transplantation, Heterologous , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/ultrastructureABSTRACT
A new visualizing method was developed for monitoring protein-protein (P-P) interactions in live mammalian cells. P-P interactions are visualized by directing localization of a bait protein to endosomes. This method is sufficiently robust to analyze signal-dependent P-P interactions such as calcium-dependent protein interactions. We visualized interactions between activated calmodulin and calmodulin-binding proteins, and observed oscillatory interactions via time-lapse imaging. In addition, this new method can simultaneously monitor multiple P-P interactions in a single live cell, which allows comparison of interactions between several prey proteins and a single bait protein. We observed that CaMKK1 and CaMKIIalpha bind calmodulin with distinct binding affinities in live cell, which indicates that calcium signaling is fine-tuned by distinct activation patterns of CaM kinases. This method will enable investigation of cellular processes based on dynamic P-P interactions.
Subject(s)
Calcium Signaling , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Calmodulin/metabolism , Image Processing, Computer-Assisted/methods , Two-Hybrid System Techniques , Calcium/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/chemistry , Calmodulin/chemistry , HeLa Cells , Humans , Microscopy, ConfocalABSTRACT
Interferon regulatory factor 3 (IRF3) is activated in response to various environmental stresses including viral infection and DNA-damaging agents. However, the biological function of IRF3 in cell growth is not well understood. We demonstrated that IRF3 markedly inhibited growth and colony formation of cells. IRF3 blocked DNA synthesis and induced apoptosis. Based on this negative control of cell growth by IRF3, we examined whether functional loss of IRF3 may contribute to oncogenic transformation. IRF3 activity was specifically inhibited by expression of its dominant negative mutant. This mutant lacks a portion of the DNA binding domain like IRF3a, an alternative splice form of IRF3 in the cells. This dominant negative inhibition blocked expression of specific IRF3 target genes. Mutant IRF3 efficiently transformed NIH3T3 cells, as demonstrated by anchorage-independent growth in soft agar and tumorigenicity in nude mice. These results imply that IRF3 may function as a tumor suppressor and suggest a possible role for the relative levels of IRF3 and its dominant negative mutant in tumorigenesis.
