The p53, p63, and p73 proteins belong to the p53 family of transcription factors, which play key roles in tumor suppression. Although the transactivation domains (TADs) of the p53 family are intrinsically disordered, these domains are commonly involved in the regulatory interactions with mouse double minute 2 (MDM2). In this study, we determined the solution structure of the p73TAD peptide in complex with MDM2 using NMR spectroscopy and biophysically characterized the interactions between the p53 family TAD peptides and MDM2. In combination with mutagenesis data, the complex structures revealed remarkably close mimicry of the MDM2 recognition mechanism among the p53 family TADs. Upon binding with MDM2, the intrinsically disordered p73TAD and p63TAD peptides adopt an amphipathic a-helical conformation, which is similar to the conformation of p53TAD, although the a-helical content induced by MDM2 binding varies. With isothermal titration calorimetry (ITC) and circular dichroism (CD) data, our biophysical characterization showed that p73TAD resembles p53TAD more closely than p63TAD in terms of helical stability, MDM2 binding affinity, and phosphorylation effects on MDM2 binding. Therefore, our structural information may be useful in establishing alternative anticancer strategies that exploit the activation of the p73 pathway against human tumors bearing p53 mutations.

The identification of off-target binding of drugs is a key to repositioning drugs to new therapeutic categories. Here we show the universal interactions of the p53 transactivation domain (p53TAD) with various anti-apoptotic Bcl-2 family proteins via a mouse double minute 2 (MDM2) binding motif, which play an important role in transcription-independent apoptotic pathways of p53. Interestingly, our structural studies reveal that the anti-apoptotic Bcl-2 family proteins and MDM2 share a similar mode of interaction with the p53TAD. On the basis of this close molecular mimicry, our NMR results demonstrate that the potent MDM2 antagonists Nutlin-3 and PMI bind to the anti-apoptotic Bcl-2 family proteins in a manner analogous to that with the p53TAD.

Human papillomaviruses (HPVs) have been recognized as the primary cause of cervical cancer. HPV 16 E7 binds to tumor suppressor retinoblastoma protein, and interferes with its function, causing release of the transcription factor E2F, which influences expression of cell cycle-related genes. This study was performed to identify the genes and proteins modulated by the HPV E7 oncogene. An HPV-negative cervical cancer cell line (C33A) was prepared to establish a stable cell line expressing E7. In order to analyze the target molecules modulated by E7 expression, we used two approaches: matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and DNA microarrays. Forty-seven spots were identified in C33A/E7 by two-dimensional electrophoresis and MALDI/TOF MS. Protein disulfide isomerase A3, integrase interactor 1 protein, growth inhibitory protein, glutathione S-transferase P, and vav proto-oncogene were down-regulated, whereas heat shock 60 kDa protein 1, Ku70 binding protein, alpha enolase, 26S proteasome subunit were up-regulated. A genomic approach using a microarray kit showed that IL-12R beta 1, cytochrome c, and tumor necrosis factor receptor II were induced by the E7 oncogene. These results suggest that E7 can evade immune surveillance by suppressing or inducing these cell signaling factors, cell cycle regulators, and chaperones.