The degradation of p53 and its major E3 ligase Mdm2 is differentially dependent on the proteasomal ubiquitin receptor S5a.

p53 and its major E3 ligase Mdm2 are both ubiquitinated and targeted to the proteasome for degradation. Despite the importance of this in regulating the p53 pathway, little is known about the mechanisms of proteasomal recognition of ubiquitinated p53 and Mdm2. In this study, we show that knockdown of the proteasomal ubiquitin receptor S5a/PSMD4/Rpn10 inhibits p53 protein degradation and results in the accumulation of ubiquitinated p53. Overexpression of a dominant-negative deletion of S5a lacking its ubiquitin-interacting motifs (UIM)s, but which can be incorporated into the proteasome, also causes the stabilization of p53. Furthermore, small-interferring RNA (siRNA) rescue experiments confirm that the UIMs of S5a are required for the maintenance of low p53 levels. These observations indicate that S5a participates in the recognition of ubiquitinated p53 by the proteasome. In contrast, targeting S5a has no effect on the rate of degradation of Mdm2, indicating that proteasomal recognition of Mdm2 can be mediated by an S5a-independent pathway. S5a knockdown results in an increase in the transcriptional activity of p53. The selective stabilization of p53 and not Mdm2 provides a mechanism for p53 activation. Depletion of S5a causes a p53-dependent decrease in cell proliferation, demonstrating that p53 can have a dominant role in the response to targeting S5a. This study provides evidence for alternative pathways of proteasomal recognition of p53 and Mdm2. Differences in recognition by the proteasome could provide a means to modulate the relative stability of p53 and Mdm2 in response to cellular signals. In addition, they could be exploited for p53-activating therapies. This work shows that the degradation of proteins by the proteasome can be selectively dependent on S5a in human cells, and that this selectivity can extend to an E3 ubiquitin ligase and its substrate

CP-31398, a putative p53-stabilizing molecule tested in mammalian cells and in yeast for its effects on p53 transcriptional activity

BACKGROUND: CP-31398 is a small molecule that has been reported to stabilize the DNA-binding core domain of the human tumor suppressor protein p53 in vitro. The compound was also reported to function as a potential anti-cancer drug by rescuing the DNA-binding activity and, consequently, the transcription activation function of mutant p53 protein in mammalian tissue culture cells and in mice. RESULTS: We performed a series of gene expression experiments to test the activity of CP-31398 in yeast and in human cell cultures. With these cell-based assays, we were unable to detect any specific stimulation of mutant p53 activity by this compound. Concentrations of CP-31398 that were reported to be active in the published work were highly toxic to the human H1299 lung carcinoma and Saos-2 cell lines in our experiments. CONCLUSION: In our experiments, the small molecule CP-31398 was unable to reactivate mutant p53 protein. The results of our in vivo experiments are in agreement with the recently published biochemical analysis of CP-31398 showing that this molecule does not bind p53 as previously claimed, but intercalates into DNA

Silencing of PTK7 in Colon Cancer Cells: Caspase-10-Dependent Apoptosis via Mitochondrial Pathway

Protein tyrosine kinase-7 (PTK7) is a catalytically inactive receptor tyrosine kinase (RTK). PTK7 is upregulated in many common human cancers, including colon cancer, lung cancer, gastric cancer and acute myeloid leukemia. The reason for this up-regulation is not yet known. To explore the functional role of PTK7, the expression of PTK7 in HCT 116 cells was examined using small interference (siRNA)-mediated gene silencing. Following transfection, the siRNA successfully suppressed PTK7 mRNA and protein expression. Knocking down of PTK7 in HCT 116 cells inhibited cell proliferation compared to control groups and induced apoptosis. Furthermore, this apoptosis was characterized by decreased mitochondrial membrane potential and activation of caspase-9 and -10. Addition of a caspase-10 inhibitor totally blocked this apoptosis, suggesting that caspase-10 may play a critical role in PTK7-knockdown-induced apoptosis, downstream of mitochondria. These observations may indicate a role for PTK7 in cell proliferation and cell apoptosis and may provide a potential therapeutic pathway for the treatment of a variety of cancers

Role of the p53/p21 system in the response of human colon carcinoma cells to Doxorubicin

BACKGROUND: Colon adenocarcinomas are refractory to a number of widely used anticancer agents. Multifactorial mechanisms have been implicated in this intrinsically resistant phenotype, including deregulation of cell death pathways. In this regard, the p53 protein has a well established role in the control of tumor cell response to DNA damaging agents; however, the relationship between p53-driven genes and drug sensitivity remains controversial. The present study investigates the role of the p53/p21 system in the response of human colon carcinoma cells to treatment with the cytotoxic agent doxorubicin (DOX) and the possibility to modify the therapeutic index of DOX by modulation of p53 and/or p21 protein levels. METHODS: The relationship between p53 and p21 protein levels and the cytotoxic effect of DOX was investigated, by MTT assay and western blot analysis, in HCT116 (p53-positive) and HT29 (p53-negative) colon cancer cells. We then assessed the effects of DOX in two isogenic cell lines derived from HCT116 by abrogating the expression and/or function of p53 and p21 (HCT116-E6 and HCT116 p21-/-, respectively). Finally, we evaluated the effect of pre-treatment with the piperidine nitroxide Tempol (TPL), an agent that was reported to induce p21 expression irrespective of p53 status, on the cytotoxicity of DOX in the four cell lines. Comparisons of IC50 values and apoptotic cell percentages were performed by ANOVA and Bonferroni's test for independent samples. C.I. calculations were performed by the combination Index method. RESULTS: Our results indicate that, in the colon carcinoma cell lines tested, sensitivity to DOX is associated with p21 upregulation upon drug exposure, and DOX cytotoxicity is potentiated by pre-treatment with TPL, but only in those cell lines in which p21 can be upregulated. CONCLUSIONS: p21 induction may significantly contribute to the response of colon adenocarcinomas cells to DOX treatment; and small molecules that can exploit p53-independent pathways for p21 induction, such as TPL, may find a place in chemotherapeutic protocols for the clinical management of colorectal cancer, where p53 function is often lost, due to genetic or epigenetic defects or to post-transcriptional inactivating mechanisms

Probing the Functional Impact of Sequence Variation on p53-DNA Interactions Using a Novel Microsphere Assay for Protein-DNA Binding with Human Cell Extracts

The p53 tumor suppressor regulates its target genes through sequence-specific binding to DNA response elements (REs). Although numerous p53 REs are established, the thousands more identified by bioinformatics are not easily subjected to comparative functional evaluation. To examine the relationship between RE sequence variation—including polymorphisms—and p53 binding, we have developed a multiplex format microsphere assay of protein-DNA binding (MAPD) for p53 in nuclear extracts. Using MAPD we measured sequence-specific p53 binding of doxorubicin-activated or transiently expressed p53 to REs from established p53 target genes and p53 consensus REs. To assess the sensitivity and scalability of the assay, we tested 16 variants of the p21 target sequence and a 62-multiplex set of single nucleotide (nt) variants of the p53 consensus sequence and found many changes in p53 binding that are not captured by current computational binding models. A group of eight single nucleotide polymorphisms (SNPs) was examined and binding profiles closely matched transactivation capability tested in luciferase constructs. The in vitro binding characteristics of p53 in nuclear extracts recapitulated the cellular in vivo transactivation capabilities for eight well-established human REs measured by luciferase assay. Using a set of 26 bona fide REs, we observed distinct binding patterns characteristic of transiently expressed wild type and mutant p53s. This microsphere assay system utilizes biologically meaningful cell extracts in a multiplexed, quantitative, in vitro format that provides a powerful experimental tool for elucidating the functional impact of sequence polymorphism and protein variation on protein/DNA binding in transcriptional networks

Active Zone Protein Bassoon Co-Localizes with Presynaptic Calcium Channel, Modifies Channel Function, and Recovers from Aging Related Loss by Exercise

The P/Q-type voltage-dependent calcium channels (VDCCs) are essential for synaptic transmission at adult mammalian neuromuscular junctions (NMJs); however, the subsynaptic location of VDCCs relative to active zones in rodent NMJs, and the functional modification of VDCCs by the interaction with active zone protein Bassoon remain unknown. Here, we show that P/Q-type VDCCs distribute in a punctate pattern within the NMJ presynaptic terminals and align in three dimensions with Bassoon. This distribution pattern of P/Q-type VDCCs and Bassoon in NMJs is consistent with our previous study demonstrating the binding of VDCCs and Bassoon. In addition, we now show that the interaction between P/Q-type VDCCs and Bassoon significantly suppressed the inactivation property of P/Q-type VDCCs, suggesting that the Ca2+ influx may be augmented by Bassoon for efficient synaptic transmission at NMJs. However, presynaptic Bassoon level was significantly attenuated in aged rat NMJs, which suggests an attenuation of VDCC function due to a lack of this interaction between VDCC and Bassoon. Importantly, the decreased Bassoon level in aged NMJs was ameliorated by isometric strength training of muscles for two months. The training increased Bassoon immunoreactivity in NMJs without affecting synapse size. These results demonstrated that the P/Q-type VDCCs preferentially accumulate at NMJ active zones and play essential role in synaptic transmission in conjunction with the active zone protein Bassoon. This molecular mechanism becomes impaired by aging, which suggests altered synaptic function in aged NMJs. However, Bassoon level in aged NMJs can be improved by muscle exercise

Repressive LTR Nucleosome Positioning by the BAF Complex Is Required for HIV Latency

The SWI/SNF BAF chromatin remodeling complex generates a repressive nucleosome structure at the HIV LTR conducive to establishment and maintenance of HIV latency, while PBAF augments HIV transcription

Synaptic E3 Ligase SCRAPPER in Contextual Fear Conditioning: Extensive Behavioral Phenotyping of Scrapper Heterozygote and Overexpressing Mutant Mice

SCRAPPER, an F-box protein coded by FBXL20, is a subunit of SCF type E3 ubiquitin ligase. SCRAPPER localizes synapses and directly binds to Rab3-interacting molecule 1 (RIM1), an essential factor for synaptic vesicle release, thus it regulates neural transmission via RIM1 degradation. A defect in SCRAPPER leads to neurotransmission abnormalities, which could subsequently result in neurodegenerative phenotypes. Because it is likely that the alteration of neural transmission in Scrapper mutant mice affect their systemic condition, we have analyzed the behavioral phenotypes of mice with decreased or increased the amount of SCRAPPER. We carried out a series of behavioral test batteries for Scrapper mutant mice. Scrapper transgenic mice overexpressing SCRAPPER in the hippocampus did not show any significant difference in every test argued in this manuscript by comparison with wild-type mice. On the other hand, heterozygotes of Scrapper knockout [SCR (+/−)] mice showed significant difference in the contextual but not cued fear conditioning test. In addition, SCR (+/−) mice altered in some tests reflecting anxiety, which implies the loss of functions of SCRAPPER in the hippocampus. The behavioral phenotypes of Scrapper mutant mice suggest that molecular degradation conferred by SCRAPPER play important roles in hippocampal-dependent fear memory formation

IL-10 transcription is negatively regulated by BAF180, a component of the SWI/SNF chromatin remodeling enzyme

<p>Abstract</p> <p>Background</p> <p>SWI/SNF chromatin remodeling enzymes play a critical role in the development of T helper lymphocytes, including Th2 cells, and directly program chromatin structure at Th2 cytokine genes. Different versions of SWI/SNF complexes, including BAF and PBAF, have been described based on unique subunit composition. However, the relative role of BAF and PBAF in Th cell function and cytokine expression has not been reported.</p> <p>Results</p> <p>Here we examine the role of the PBAF SWI/SNF complex in Th cell development and gene expression using mice deficient for a PBAF-specific component, BAF180. We find that T cell development in the thymus and lymphoid periphery is largely normal when the BAF180 gene is deleted late in thymic development. However, BAF180-deficient Th2 cells express high levels of the immunoregulatory cytokine IL-10. BAF180 binds directly to regulatory elements in the Il-10 locus but is replaced by BAF250 BAF complexes in the absence of BAF180, resulting in increased histone acetylation and CBP recruitment to the IL-10 locus.</p> <p>Conclusions</p> <p>These results demonstrate that BAF180 is a repressor of IL-10 transcription in Th2 cells and suggest that the differential recruitment of different SWI/SNF subtypes can have direct consequences on chromatin structure and gene transcription.</p