Purified P53 Protein Research Articles

Recovery of recombinant zebrafish p53 protein from inclusion bodies and its binding activity to p53 mRNA in vitro

p53 protein is an important regulation factor that can bind to p53 mRNA to regulate its translation in human and murine. To determine if a similar interaction exists in zebrafish and if the interaction affects zebrafish development, we cloned and expressed p53 protein from zebrafish in Escherichia coli. Soluble p53 protein with high purity was successfully obtained using the optimized renaturation approach. Results of a UV-crosslinking experiment and immunoprecipitation:RT-PCR analysis confirmed that the purified p53 protein could bind specifically to its cognate mRNA. Our results suggest that selecting a suitable buffer is important for renaturing p53 protein from inclusion bodies. We also demonstrated a specific interaction between p53 and it own RNA in zebrafish. Measurement of the binding activity may be a useful approach for identifying the activity of recombinant p53 protein in vitro.

Regulation of Plasminogen Activator Inhibitor-1 Expression by Tumor Suppressor Protein p53

H1299 lung carcinoma cells lacking p53 (p53-/-) express minimal amounts of plasminogen activator inhibitor-1 (PAI-1) protein as well as mRNA. p53(-/-) cells express highly unstable PAI-1 mRNA. Transfection of p53 in p53(-/-) cells enhanced PAI-1 expression and stabilized PAI-1 mRNA. On the contrary, inhibition of p53 expression by RNA silencing in non-malignant human lung epithelial (Beas2B) cells decreased basal as well as urokinase-type plasminogen activator-induced PAI-1 expression because of accelerated degradation of PAI-1 mRNA. Purified p53 protein specifically binds to the PAI-1 mRNA 3'-un-translated region (UTR), and endogenous PAI-1 mRNA forms an immune complex with p53. Treatment of purified p53 protein with anti-p53 antibody abolished p53 binding to the 3'-UTR of PAI-1 mRNA. The p53 binding region maps to a 70-nucleotide PAI-1 mRNA 3'-UTR sequence, and insertion of the p53-binding sequence into beta-globin mRNA destabilized the chimeric transcript. Deletion experiments indicate that the carboxyl-terminal region (amino acid residues 296-393) of p53 protein interacts with PAI-1 mRNA. These observations demonstrate a novel role for p53 as an mRNA-binding protein that regulates increased PAI-1 expression and stabilization of PAI-1 mRNA in human lung epithelial and carcinoma cells.

Regulation of Urokinase Receptor Expression by p53: Novel Role in Stabilization of uPAR mRNA

We found that p53-deficient (p53(-/-)) lung carcinoma (H1299) cells express robust levels of cell surface uPAR and uPAR mRNA. Expression of p53 protein in p53(-/-) cells suppressed basal and urokinase (uPA)-induced cell surface uPAR protein and increased uPAR mRNA degradation. Inhibition of p53 by RNA silencing in Beas2B human airway epithelial cells conversely increased basal as well as uPA-mediated uPAR expression and stabilized uPAR mRNA. Purified p53 protein specifically binds to the uPAR mRNA 3' untranslated region (3'UTR), and endogenous uPAR mRNA associates with p53. The p53 binding region involves a 37-nucleotide uPAR 3'UTR sequence, and insertion of the p53 binding sequence into beta-globin mRNA destabilized beta-globin mRNA. Inhibition of p53 expression in these cells reverses decay of chimeric beta-globin-uPAR mRNA. These observations demonstrate a novel regulatory role for p53 as a uPAR mRNA binding protein that down-regulates uPAR expression, destabilizes uPAR mRNA, and thereby contributes to the viability of human airway epithelial or lung carcinoma cells.

Bcl2's flexible loop domain regulates p53 binding and survival.

p53 not only functions as a transcription factor but also has a direct, apoptogenic role at the mitochondria. We have discovered that DNA damage-induced p53-Bcl2 binding is associated with decreased Bcl2-Bax interaction and increased apoptotic cell death in a mechanism regulated by Bcl2's flexible loop regulatory domain (FLD), since purified p53 protein can disrupt the Bcl2/Bax complex by directly binding to a negative regulatory region of the FLD (amino acids [aa] 32 to 68). Deletion of the negative regulatory region (Delta32-68) abolishes Bcl2-p53 binding and enhances Bcl2's antiapoptotic function. Conversely, removal of a positive regulatory region (aa 69 to 87) of the FLD, which contains the Bcl2 phosphorylation site(s) T69, S70, and S87, enhances Bcl2-p53 binding and significantly abrogates Bcl2's survival activity. The phospho-mimetic T69E/S70E/S87E (EEE) but not the nonphosphorylatable T69A/S70A/S87A (AAA) Bcl2 mutant displays a reduced capacity to bind p53 and potently inhibits p53-induced cytochrome c release from isolated mitochondria. Furthermore, the FLD-only aa32-87 and aa32-68 peptides but not the aa69-87 peptide can directly bind p53 in vitro. p53-induced cytochrome c release occurs through a mechanism involving Bax's integral insertion into the outer mitochondrial membrane. Either DNA damage to cells or expression of p53 selectively targeted to the mitochondria results in Bcl2-p53 binding followed by exposure of Bcl2's BH3 domain in association with inactivation of Bcl2's antiapoptotic function, indicating a conformational change in Bcl2 can occur upon direct ligation of p53. Thus, Bcl2's FLD contains both positive and negative regulatory regions which functionally regulate Bcl2's antiapoptotic activity by affecting Bax or p53 binding.

Bcl2 Flexible Loop Domain Positively and Negatively Regulates Survival Function through Direct Interaction with p53.

p53 not only functions as a transcription factor but also has a direct extranuclear apoptogenic role at the mitochondria. We have discovered that DNA damage-induced p53/Bcl2 binding is associated with decreased Bcl2/Bax interaction and increased apoptotic cell death in a mechanism regulated by Bcl2's flexible loop regulatory domain (FLD) in IL3-dependent myeloid NSF/N1.H7 cells. Purified p53 protein can disrupt the Bcl2/Bax complex by directly binding to a negative regulatory region of the FLD (aa32–68). Deletion of the negative regulatory region (aa32–68) abolishes Bcl2/ p53 binding and enhances its antiapoptotic function. Removal of aa69–87 of the FLD, which contains Bcl2's phosphorylation site(s) (T69, S70 and S87), enhances Bcl2/p53 binding and abrogates Bcl2's potent survival activity, indicating this is a positive regulatory region. Phosphorylation of Bcl2 in the FLD inhibits p53 binding since the phosphomimetic T69E/S70E/S87E (EEE) but not the nonphosphorylatable T69A/S70A/S87A (AAA) Bcl2 mutant displays a reduced capacity to bind p53 and more potently inhibits p53-induced cytochrome c release. A full-length Bcl2 loop-only protein (aa32–87) can directly bind p53 to impede Bcl2/p53 binding in vitro. Either DNA damage or expression of p53 targeted to mitochondria results in Bcl2/p53 binding followed by exposure of Bcl2's BH3 domain and inactivation of Bcl2's antiapoptotic function. Therefore, Bcl2's FLD contains both a positive and negative regulatory regions which functionally regulate Bcl2's antiapoptotic activity via differential binding of Bax and p53, respectively.

Rational Design of p53, an Intrinsically Unstructured Protein, for the Fabrication of Novel Molecular Sensors

The dominant paradigm of protein engineering is structure-based site-directed mutagenesis. This rational approach is generally more effective for the engineering of local properties, such as substrate specificity, than global ones such as allostery. Previous workers have modified normally unregulated reporter enzymes, including beta-galactosidase, alkaline phosphatase, and beta-lactamase, so that the engineered versions are activated (up to 4-fold) by monoclonal antibodies. A reporter that could easily be "reprogrammed" for the facile detection of novel effectors (binding or modifying activities) would be useful in high throughput screens for directed evolution or drug discovery. Here we describe a straightforward and general solution to this potentially difficult design problem. The transcription factor p53 is normally regulated by a variety of post-translational modifications. The insertion of peptides into intrinsically unstructured domains of p53 generated variants that were activated up to 100-fold by novel effectors (proteases or antibodies). An engineered p53 was incorporated into an existing high throughput screen for the detection of human immunodeficiency virus protease, an arbitrarily chosen novel effector. These results suggest that the molecular recognition properties of intrinsically unstructured proteins are relatively easy to engineer and that the absence of crystal structures should not deter the rational engineering of this class of proteins.

Function of Polo-like Kinase 3 in NF-κB-mediated Proapoptotic Response

RelA, the p65 subunit of NF-kappaB transcription factors, plays a key role in regulation of antiapoptotic and proapoptotic responses. However, the downstream target genes regulated by RelA-NF-kappaB in the initiation of proapoptotic signaling were not identified. We previously showed that RelA-NF-kappaB functioned as a proapoptotic factor by activating the p53-signaling pathway in response to doxycycline-induced superoxide. In the present study, we demonstrate that the ability of doxycycline/superoxide to induce expression of polo-like kinase 3 (Plk3) depends on NF-kappaB activity. We identified a kappaB binding site in the promoter of Plk3, and this kappaB site is directly involved in its induction by the RelA-NF-kappaB complex. Plk3 formed a complex with p53 and was involved in the phosphorylation of p53 on Ser-20 in response to superoxide. Inhibition of Plk3 expression by Plk3 small interfering RNA suppressed the doxycycline/superoxide-mediated apoptosis. Overexpression of wild-type Plk3 in HCT116 p53+/+ cells induced rapid apoptosis, whereas overexpression of wild-type Plk3 in HCT116 p53-/- cells and the kinase-defective mutant Plk3(K91R) in p53+/+ cells induced delayed onset of apoptosis. Furthermore, mutagenesis of Plk3 showed that the N-terminal domain (amino acids 1-26) is essential for the induction of delay onset of apoptosis. These data show that Plk3 is a RelA-NF-kappaB-regulated gene that induces apoptosis in both p53-dependent and -independent signaling pathways, suggesting a possible mechanism for RelA-NF-kappaB-regulated proapoptotic responses.

Human Monocyte-Derived Dendritic Cells Pulsed with Wild-Type p53 Protein Efficiently Induce CTLs against p53 Overexpressing Human Cancer Cells

Dendritic cells are the most potent antigen-presenting cells for initiating cellular immune responses. Dendritic cells are attractive immunoregulatory cells for cancer immunotherapy, and their efficacy has been investigated in clinical trials. The tumor suppressor gene p53 is pivotal in the regulation of apoptosis, and p53-based immunization is an attractive approach to cancer immunotherapy because of the accumulation of p53 protein in malignant but not in normal cells. It has been shown that dendritic cells transduced with an adenoviral wild-type p53 (wt-p53) construct mediate the antitumor immune responses against p53-overexpressing tumor cells. We examined whether monocyte-derived human dendritic cells pulsed with the purified full-length wt-p53 protein were also capable of inducing the specific antitumor responses against p53-overexpressing tumors in vitro. Immature dendritic cells generated in the presence of interleukin-4 and granulocyte/macrophage colony-stimulating factor from monocytes of HLA-A2- or HLA-A24-positive healthy individuals were pulsed with the purified p53 protein. Uptake of p53 protein by human dendritic cells was assessed by Western blotting and immunohistochemical staining using anti-p53 antibody. Induction of p53-specific CTL response was also evaluated by the cytotoxic assay against p53-overexpressing human tumor cells. Both Western blot and immunohistochemical analysis showed the accumulation of p53 protein in human immature dendritic cells. T cells obtained from HLA-A2- or HLA-A24-positive healthy donors were stimulated twice with p53 protein-pulsed dendritic cells and then applied to the cytotoxicity assay against p53-overexpressing target cells. The CTL activity was specific for p53-overexpressing tumor cells and MHC class I restricted. Moreover, the CTL activity generated by p53 protein-pulsed dendritic cells was nearly identical with that induced by adenoviral wt-p53-transduced dendritic cells. Our results indicate that monocyte-derived human dendritic cells pulsed with the wt-p53 protein could induce the specific antitumor effect against p53-overexpressing tumors and that this in vitro model offers a new and more simple approach to the development of p53-based immunotherapy.

P53 Regulates the Expression of the Tumor Suppressor Gene Maspin

Maspin has been shown to inhibit tumor cell invasion and metastasis in breast tumor cells. Maspin expression was detected in normal breast and prostate epithelial cells, whereas tumor cells exhibited reduced or no expression. However, the regulatory mechanism of maspin expression remains unknown. We report here a rapid and robust induction of maspin expression in prostate cancer cells (LNCaP, DU145, and PC3) and breast tumor cells (MCF7) following wild type p53 expression from an adenovirus p53 expression vector (AdWTp53). p53 activates the maspin promoter by binding directly to the p53 consensus-binding site present in the maspin promoter. DNA-damaging agents and cytotoxic drugs induced endogenous maspin expression in cells containing the wild type p53. Maspin expression was refractory to the DNA-damaging agents in cells containing mutant p53. These results, combined with recent studies of the tumor metastasis suppressor gene KAI1 and plasminogen activator inhibitor 1 (PAI1), define a new category of molecular targets of p53 that have the potential to negatively regulate tumor invasion and/or metastasis.

Surface plasmon resonance measurements reveal stable complex formation between p53 and DNA polymerase alpha.

Surface plasmon resonance measurements were used for detecting and quantifying protein-protein interactions between the tumor suppressor protein p53, the SV40 large T antigen (T-ag), the cellular DNA polymerase alpha-primase complex (pol-prim), and the cellular single-strand DNA binding protein RPA. Highly purified p53 protein bound to immobilized T-ag with an apparent binding constant of 2 x 10(8) M(-1). Binding of p53 to RPA was in the same order of magnitude with a binding constant of 4 x 10(8) M(-1), when RPA was coupled to the sensor chip via its smallest subunit, and 1 x 10(8) M(-1), when RPA was coupled via its p70 subunit. Furthermore, p53 bound human DNA polymerase alpha-primase complex (pol-prim) with a K(A) value of 1 x 10(10) m(-1). Both the p68 subunit and the p180 subunit of pol-prim could interact with p53 displaying binding constants of 2 x 10(10) m1(-1) and 5 X 10(9) M(-1), respectively. Complex formation was also observed with a p180/p68 heterodimer, and again with a binding constant similar. Hence, there was no synergistic effect when p53 bound to higher order complexes of pol-prim. A truncated form of p53, consisting of amino acids 1-320, bound pol-prim by four orders of magnitude less efficiently. Therefore, an intact C-terminus of p53 seems to be important for efficient binding to pol-prim. It was also tried to measure complex formation between p53, pol-prim, and T-ag. However there was no evidence for the existence of a ternary complex consisting of T-ag, pol-prim, and p53.

Production of a polyclonal antibody raised against recombinant flounder p53 protein

The cDNA encoding the wild type p53 protein from flounder, Platichthys flesus, was expressed in Escherichia coli using the GST fusion protein system. Several milligrams of recombinant p53 protein were purified. This protein displayed an apparent molecular weight of 45 000 Da, a value which is very similar to Xenopus p53, but significantly greater than was expected based on the length of the open reading frame. Immunization of rabbits against purified p53 protein allowed the production of high titre polyclonal antiserum. This new polyclonal antibody recognized recombinant flounder p53 protein in Western blot. Cross reaction was also observed with recombinant Xenopus p53 protein but not with human p53 protein. Immunoblotting of the total protein extract from normal flounder ovaries did not reveal any p53 expression.

The expression of the KAI1 gene, a tumor metastasis suppressor, is directly activated by p53.

KAI1 is a tumor metastasis suppressor gene that is capable of inhibiting the metastatic process in animals. The expression of the KAI1 gene also is found to be down-regulated during the tumor progression of prostate, breast, lung, bladder, and pancreatic cancers in humans, and this down-regulation appears to be at or posttranscription level. We have found that the tumor suppressor gene p53 can directly activate the KAI1 gene by interacting with the 5' upstream region. The p53 responding region is located at approximately 860 bases upstream of the transcriptional initiation site, and it contains a typical tandem repeat of the p53 consensus-binding sequence. A gel-shift mobility analysis showed that this sequence indeed had the ability to bind to the purified p53 protein. Mutations of this sequence abolished the responsiveness to p53 and also the binding ability to the p53 protein. Furthermore, immunohistochemical analysis of 177 samples of human prostate tumors revealed that the expression of the KAI1 gene was correlated strongly to that of the p53 gene and that the loss of these two markers resulted in poor survivals of patients. Our data indicate a direct relationship between p53 and KAI1 genes and suggest that the loss of p53 function, which is commonly observed in many types of cancer, leads to the down-regulation of the KAI1 gene, which may result in the progression of metastasis.

P53 Protein Exhibits 3′-to-5′ Exonuclease Activity

Highly purified p53 protein from different sources was able to degrade DNA with a 3′-to-5′ polarity, yielding deoxynucleoside monophosphates as reaction products. This exonuclease activity was dependent on Mg 2+ and inhibited by addition of 5 mM nucleoside monophosphates. This exonuclease activity is intrinsic to the wild-type p53 protein: it copurified with p53 during p53 preparation; only purified wild-type p53, but not identically purified mutant p53 proteins displayed exonuclease activity; the exonuclease activity could be reconstituted from SDS gel-purified and urea-renatured p53 protein and mapped to the core domain of the p53 molecule; and finally, purified p53 protein could be UV cross-linked to GMP. A p53-intrinsic exonuclease activity should substantially extend our view on the role of p53 as a “guardian of the genome.”

A DNA binding domain is contained in the C-terminus ofwild typep53 protein

In the present study we evaluated the DNA binding activity of wild type and mutant p53 proteins that were isolated from bacterial expression vectors. A comparison of the binding activities of the various purified p53 proteins, assessed by their ability to bind DNA cellulose columns, indicated that wild type p53 has a higher affinity to DNA than have mutant p53 forms. Furthermore, only wild type p53 was able to bind genomic DNA upon electrophoretic protein blotting. As specific deletion of the C-terminal region of wild type p53 totally abolished binding to genomic DNA, it was concluded that the 47 C-terminal amino acids contain the DNA binding region. The fact that the N-terminus contains a transcription activation region whereas the C-terminus contains a DNA binding domain places p53 in the family of typical transcription factors. Our experiments show that the topographical positioning of these domains plays an important role in the activity of wild type p53.