Explore the vast range of titles available.

The tumor suppressor p53 is mutated in approximately half of all human cancers. p53 can induce apoptosis through mitochondrial membrane permeabilization by interacting with and antagonizing the anti-apoptotic proteins BCL-xL and BCL-2. However, the mechanisms by which p53 induces mitochondrial apoptosis remain elusive. Here, we report a 2.5 Å crystal structure of human p53/BCL-xL complex. In this structure, two p53 molecules interact as a homodimer, and bind one BCL-xL molecule to form a ternary complex with a 2:1 stoichiometry. Mutations at the p53 dimer interface or p53/BCL-xL interface disrupt p53/BCL-xL interaction and p53-mediated apoptosis. Overall, our current findings of the bona fide structure of p53/BCL-xL complex reveal the molecular basis of the interaction between p53 and BCL-xL, and provide insight into p53-mediated mitochondrial apoptosis. The structure of human tumor suppressor p53 in complex with the antiapoptotic protein BCL-xL reveals the basis of the p53–BCL-xL interaction and provides insight into the mechanisms of p53-mediated mitochondrial apoptosis.

In response to genotoxic stress, the tumor suppressor p53 acts as a transcription factor by regulating the expression of genes critical for cancer prevention. Mutations in the gene encoding p53 are associated with cancer development. PRIMA-1 and eprenetapopt (APR-246/PRIMA-1 MET ) are small molecules that are converted into the biologically active compound, methylene quinuclidinone (MQ), shown to reactivate mutant p53 by binding covalently to cysteine residues. Here, we investigate the structural basis of mutant p53 reactivation by MQ based on a series of high-resolution crystal structures of cancer-related and wild-type p53 core domains bound to MQ in their free state and in complexes with their DNA response elements. Our data demonstrate that MQ binds to several cysteine residues located at the surface of the core domain. The structures reveal a large diversity in MQ interaction modes that stabilize p53 and its complexes with DNA, leading to a common global effect that is pertinent to the restoration of non-functional p53 proteins. The tumor suppressor p53 is mutated in more than half of human cancers and the compound methylene quinuclidinone (MQ) was shown to reactivate p53 mutants by binding covalently to cysteine residues. Here, the authors present crystal structures of wild-type and cancer related p53 mutant core domains bound to MQ alone and in complex with their DNA response elements and observe that MQ is bound to several cysteine residues located at the surface of the core domain.

Genetically engineered bacterial protein toxins are attractive systems for delivery of exogenous proteins into the cytosol of mammalian cells. The binary C2 toxin from C. botulinum has emerged as powerful delivery vehicle, which rests on its binding/translocation component C2IIa and the genetically modified adaptor domain C2IN that act in concert to trigger cell uptake. The p53 tumor suppressor protein has a crucial function in suppressing carcinogenesis and is frequently inactivated by diverse mechanisms in human tumor cells. Therefore, we constructed a C2IN-p53 fusion protein, which is internalized into cancer cells by C2IIa. To this end, the C2IN-p53 fusion construct was overexpressed in E. coli with good solubility, purified by heparin affinity chromatography and protein identity was confirmed by immunoblotting. We demonstrated that the fusion protein is capable of binding to the p53 consensus-DNA with high affinity in a p53-specific manner in vitro. Next, the internalization of C2IN-p53 was monitored in HeLa cells by cell fractionation and immunoblot analysis, which revealed a C2IIa-mediated translocation of the fusion protein into the cytosol. The uptake was also shown in A549 and Saos-2 cells with similar efficiency. These findings were further corroborated by confocal immunofluorescence analyses of C2IN-p53/C2IIa-treated HeLa and A549 cells, displaying predominantly cytoplasmic localization of the fusion construct.

An amperometric magnetoimmunosensor for the determination of human p53 protein is described in this work using a sandwich configuration involving the covalent immobilization of a specific capture antibody onto activated carboxylic-modified magnetic beads (HOOC-MBs) and incubation of the modified MBs with a mixture of the target protein and horseradish peroxidase-labeled antibody (HRP-anti-p53). The resulting modified MBs are captured by a magnet placed under the surface of a disposable carbon screen-printed electrode (SPCE) and the amperometric responses are measured at −0.20 V (vs. an Ag pseudo-reference electrode), upon addition of hydroquinone (HQ) as a redox mediator and H2O2 as the enzyme substrate. The magnetoimmunosensing platform was successfully applied for the detection of p53 protein in different cell lysates without any matrix effect after a simple sample dilution. The results correlated accurately with those provided by a commercial ELISA kit, thus confirming the immunosensor as an attractive alternative for rapid and simple determination of this protein using portable and affordable instrumentation.

Intracellular protein and proteomic studies using mass spectrometry, imaging microscopy, flow cytometry, or western blotting techniques require genetic manipulation, cell permeabilization, and/or cell lysis. We present a biophysical method that employs a nanoaspirator to ‘fish’ native cytoplasmic or nuclear proteins from single mammalian cells, without compromising cell viability, followed by ex cellulo quantitative detection. Our work paves the way for spatiotemporally-controlled, quantitative, live, single-cell proteomics.

p300 acetylates and activates the tumor suppressor p53 after DNA damage. Here, we show that MDM2, a negative-feedback regulator of p53, inhibited p300-mediated p53 acetylation by complexing with these two proteins. First, we purified a p300-MDM2-p53 protein complex from HeLa nuclear extracts, which was inactive in p53 acetylation, but active in histone acetylation. Also, wild-type, but not N-terminally deleted, MDM2 inhibited p53 acetylation by p300 in vitro and in vivo. This inhibition was specific for p53, because MDM2 did not affect acetylation of histones or the C terminus of p73 by p300. Consequently, wild-type, but not the mutant, MDM2 repressed the p300-stimulated sequence-specific DNA-binding and transcriptional activities of p53. These results demonstrate that an additional mechanism of p53 inactivation by MDM2 is to inhibit p53 acetylation by p300.

Little is known about factors that stimulate transcription of the p53 tumor suppressor gene. Here, we report that the human pituitary homeobox 1 (hPitx1) transcription factor increases the expression of p53 at the mRNA and protein levels in human mammary carcinoma (MCF-7) cells. Increased p53 mRNA expression was due to activation of the p53 promoter by hPitx1. hPitx1 bound directly to the p53 promoter and functionally utilized two hPitx1 consensus elements. The predominant consensus element utilized by hPitx1 to stimulate p53 transcription was located within the first exon of the p53 gene. A hPitx1 mutant (hPitx1-R141P) acting as a dominant inhibitor repressed p53 transcription. Forced expression of hPitx1 resulted in cell-cycle arrest and p53-dependent apoptosis in p53-replete MCF-7 cells. Furthermore, hPitx1 stimulated the transcription of p53 target genes involved in cell-cycle arrest and apoptosis (p21 and PTGF- β ), again in a p53-dependent manner. Depletion of endogenous hPitx1 by small interfering RNA (siRNA) in MCF-7 cells resulted in decreased basal expression of p53 and consequently of p21 and placental transforming growth factor β (PTGF- β ). Depletion of p53 by siRNA dramatically attenuated hPitx1-induced apoptosis in MCF-7 cells. Thus, p53 is a direct transcriptional target gene of hPitx1. This observation is concordant with the recent identification of hPitx1 as a tumor suppressor gene.

P53 is an attractive target in molecular cancer therapeutics because of its critical role in regulating cell cycle arrest and apoptosis. The limitations in the development of p53-based cancer therapeutic strategy include its inefficient transmission through cell membrane of tumor cells and low protein yields in the expression system. In the present study, p53 was fused with HIV TAT protein, which can cross cell membranes, and expressed by Pichia pastoris . Stable production of Tat-p53 was achieved. After being transduced with Tat-p53 protein, the growth of cancer cell line, HepG2, was inhibited by increased apoptosis in culture. This expression system could thus be utilized to produce human Tat-p53 fusion protein.

Progress toward understanding the biology of prostate cancer has been slow due to the few animal research models available to study the spectrum of this uniquely human disease. To develop an animal model for prostate cancer, several lines of transgenic mice were generated by using the prostate-specific rat probasin promoter to drive expression of the simian virus 40 large tumor antigen-coding region. Mice expressing high levels of the transgene display progressive forms of prostatic disease that histologically resemble human prostate cancer, ranging from mild intraepithelial hyperplasia to large multinodular malignant neoplasia. Prostate tumors have been detected specifically in the prostate as early as 10 weeks of age. Immunohistochemical analysis of tumor tissue has demonstrated that dorsolateral prostate-specific secretory proteins were confined to well-differentiated ductal epithelial cells adjacent to, or within, the poorly differentiated tumor mass. Prostate tumors in the mice also display elevated levels of nuclear p53 and a decreased heterogeneous pattern of androgen-receptor expression, as observed in advanced human prostate cancer. The establishment of breeding lines of transgenic mice that reproducibly develop prostate cancer provides an animal model system to study the molecular basis of transformation of normal prostatic cells and the factors influencing the progression to metastatic prostate cancer.