SummaryWe have carried out a cell-based screen aimed at discovering small molecules that activate p53 and have the potential to decrease tumor growth. Here, we describe one of our hit compounds, tenovin-1, along with a more water-soluble analog, tenovin-6. Via a yeast genetic screen, biochemical assays, and target validation studies in mammalian cells, we show that tenovins act through inhibition of the protein-deacetylating activities of SirT1 and SirT2, two important members of the sirtuin family. Tenovins are active on mammalian cells at one-digit micromolar concentrations and decrease tumor growth in vivo as single agents. This underscores the utility of these compounds as biological tools for the study of sirtuin function as well as their potential therapeutic interest.
The herpesvirus entry mediator C (HveC), previously known as poliovirus receptor-related protein 1 (PRR1), and the herpesvirus Ig-like receptor (HIgR) are the bona fide receptors employed by herpes simplex virus-1 and -2 (HSV-1 and -2) for entry into the human cell lines most frequently used in HSV studies. They share an identical ectodomain made of one V and two C2 domains and differ in transmembrane and cytoplasmic regions. Expression of their mRNA in the human nervous system suggests possible usage of these receptors in humans in the path of neuron infection by HSV. Glycoprotein D (gD) is the virion component that mediates HSV-1 entry into cells by interaction with cellular receptors. We report on the identification of the V domain of HIgR͞PRR1 as a major functional region in HSV-1 entry by several approaches. First, the epitope recognized by mAb R1.302 to HIgR͞PRR1, capable of inhibiting infection, was mapped to the V domain. Second, a soluble form of HIgR͞ PRR1 consisting of the single V domain competed with cell-bound full-length receptor and blocked virion infectivity. Third, the V domain was sufficient to mediate HSV entry, as an engineered form of PRR1 in which the two C2 domains were deleted and the V domain was retained and fused to its transmembrane and cytoplasmic regions was still able to confer susceptibility, although at reduced efficiency relative to full-length receptor. Consistently, transfer of the V domain of HIgR͞PRR1 to a functionally inactive structural homologue generated a chimeric receptor with virus-entry activity. Finally, the single V domain was sufficient for in vitro physical interaction with gD. The in vitro binding was specific as it was competed both by antibodies to the receptor and by a mAb to gD with potent neutralizing activity for HSV-1 infectivity.
We have previously reported that the human p53 gene encodes at least nine different p53 isoforms, including D133p53a, which can modulate p53 transcriptional activity and apoptosis. In this study, we aimed to investigate the regulation of D133p53a isoform expression and its physiological role in modulating cell cycle arrest and apoptosis. We report here that in response to a low dose of doxorubicin (which induces cell cycle arrest without promoting apoptosis), p53 directly transactivates the human p53 internal promoter, inducing D133p53a protein expression. The induced D133p53a then inhibits p53-dependent apoptosis and G1 arrest without inhibiting p53-dependent G2 arrest. Therefore, endogenous D133p53a does not exclusively function in a dominant-negative manner toward p53, but differentially regulates cell cycle arrest and apoptosis.
a b s t r a c t p53 gene expresses several protein isoforms modulating p53-mediated responses through regulation of gene expression. Here, we identify a novel p53 isoform, D160p53, lacking the first 159 residues. By knockdown experiments and site-directed mutagenesis, we show that D160p53 is encoded by D133p53 transcript using ATG160 as translational initiation site. This hypothesis is supported by endogenous expression of D160p53 in U2OS, T47D and K562 cells, the latter ones carrying a premature stop codon that impairs p53 and D133p53 protein expression but not the one of D160p53. Overall, these results show that the D133p53 transcript generates two different p53 isoforms, D133p53 and D160p53.
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