Cytoplasmic pro-apoptotic function of the tumor suppressor p73 is mediated through a modified mode of recognition of the anti-apoptotic regulator Bcl-X.

In response to genotoxic stress, the tumor suppressor protein p73 induces apoptosis and cell cycle arrest. Despite extensive studies on p73-mediated apoptosis, little is known about the cytoplasmic apoptotic function of p73. Here, using H1299 lung cancer cells and diverse biochemical approaches, including colony formation, DNA fragmentation, GST pulldown, and apoptosis assays along with NMR spectroscopy, we show that p73 induces transcription-independent apoptosis via its transactivation domain (TAD) through a mitochondrial pathway and that this apoptosis is mediated by the interaction between p73-TAD and the anti-apoptotic protein B-cell lymphoma-extra large (Bcl-X or BCL2L1).

Probing the Small-Molecule Inhibition of an Anticancer Therapeutic Protein-Protein Interaction Using a Solid-State Nanopore.

Nanopore sensing is an emerging technology for the single-molecule-based detection of various biomolecules. In this study, we probed the anticancer therapeutic p53 transactivation domain (p53TAD)/MDM2 interaction and its inhibition with a small-molecule MDM2 antagonist, Nutlin-3, using low-noise solid-state nanopores. Although the translocation of positively charged MDM2 through a nanopore was detected at the applied negative voltage, this MDM2 translocation was almost completely blocked upon formation of the MDM2/GST-p53TAD complex owing to charge conversion.

Structural convergence of unstructured p53 family transactivation domains in MDM2 recognition.

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.

Structure and apoptotic function of p73.

p73 is a structural and functional homologue of the p53 tumor suppressor protein. Like p53, p73 induces apoptosis and cell cycle arrest and transactivates p53-responsive genes, conferring its tumor suppressive activity. In addition, p73 has unique roles in neuronal development and differentiation.

A conserved mechanism for binding of p53 DNA-binding domain and anti-apoptotic Bcl-2 family proteins.

The molecular interaction between tumor suppressor p53 and the anti-apoptotic Bcl-2 family proteins plays an essential role in the transcription-independent apoptotic pathway of p53. In this study, we investigated the binding of p53 DNA-binding domain (p53DBD) with the anti-apoptotic Bcl-2 family proteins, Bcl-w, Mcl-1, and Bcl-2, using GST pull-down assay and NMR spectroscopy. The GST pull-down assays and NMR experiments demonstrated the direct binding of the p53DBD with Bcl-w, Mcl-1, and Bcl-2.

Structural basis for the conserved binding mechanism of MDM2-inhibiting peptides and anti-apoptotic Bcl-2 family proteins.

The interaction between tumor suppressor p53 and the anti-apoptotic Bcl-2 family proteins serves a critical role in the transcription-independent apoptosis mechanism of p53. Our previous studies showed that an MDM2-inhibiting motif (residues 15-29) in the p53 transactivation domain (p53TAD) mediates the interaction with anti-apoptotic Bcl-2 family proteins. In this study, we provided structural models of the complexes between the MDM2-inhibiting p53TAD peptide and Mcl-1, Bcl-w, and Kaposi sarcoma-associated herpes virus (KSHV) Bcl-2 using NMR chemical shift perturbation data.

Targeting of p53 peptide analogues to anti-apoptotic Bcl-2 family proteins as revealed by NMR spectroscopy.

Inhibition of the interaction between the p53 tumor suppressor and its negative regulator MDM2 is of great importance to cancer therapy. The anti-apoptotic Bcl-2 family proteins are also attractive anti-cancer molecular targets, as they are key regulators of apoptotic cell death. Previously, we reported the interactions between the p53 transactivation domain (p53TAD) and diverse members of the anti-apoptotic Bcl-2 family proteins.

Dual-site interactions of p53 protein transactivation domain with anti-apoptotic Bcl-2 family proteins reveal a highly convergent mechanism of divergent p53 pathways.

Molecular interactions between the tumor suppressor p53 and the anti-apoptotic Bcl-2 family proteins play an important role in the transcription-independent apoptosis of p53. The p53 transactivation domain (p53TAD) contains two conserved ΦXXΦΦ motifs (Φ indicates a bulky hydrophobic residue and X is any other residue) referred to as p53TAD1 (residues 15-29) and p53TAD2 (residues 39-57). We previously showed that p53TAD1 can act as a binding motif for anti-apoptotic Bcl-2 family proteins.

Interaction of a putative BH3 domain of clusterin with anti-apoptotic Bcl-2 family proteins as revealed by NMR spectroscopy.

Clusterin (CLU) is a multifunctional glycoprotein that is overexpressed in prostate and breast cancers. Although CLU is known to be involved in the regulation of apoptosis and cell survival, the precise molecular mechanism underlying the pro-apoptotic function of nuclear CLU (nCLU) remains unclear. In this study, we identified a conserved BH3 motif in C-terminal coiled coil (CC2) region of nCLU by sequence analysis and characterized the molecular interaction of the putative nCLU BH3 domain with anti-apoptotic Bcl-2 family proteins by nuclear magnetic resonance (NMR) spectroscopy.

Molecular mimicry-based repositioning of nutlin-3 to anti-apoptotic Bcl-2 family proteins.

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.