Over-expression of the human MDM2 p53 binding domain by fusion to a p53 transactivation peptide

MDM2 binds to the tumor suppressor protein p53 and regulates the level of p53 in cells. Although it is possible to prepare a small amount of the region of MDM2 that binds to p53, the expression level of this fragment of MDM2 is relatively low, limiting the studies involving this protein. Here, we describe a construct for the optimized bacterial expression and purification of the MDM2 p53 binding domain. We found that the expression level of the soluble MDM2 p53 binding domain in bacteria was increased dramatically by fusing it to its interaction partner, the p53 transactivation peptide. Attachment of the p53 transactivation peptide (residues 17-29) to the N-terminus of MDM2 resulted in a more than 200-fold increase of soluble protein expression of the p53 binding domain in bacteria. To obtain the final MDM2 p53 binding domain (residues 5-109) we inserted a tobacco etch virus protease recognition site between the P53 peptide and the MDM2 p53 binding domain. To weaken the protein/peptide interaction and facilitate the separation of the protein from the complex, we introduced a point mutation of one of the key interaction residues (F19A or W23A) in the p53 peptide. The advantages of our new construct are high yield and easy purification of the MDM2 protein.     

Structural changes in the C-terminus of Ca2+-bound rat S100B (beta beta) upon binding to a peptide derived from the C-terminal regulatory domain of p53.

S100B(beta beta) is a dimeric Ca2+-binding protein that interacts with p53, inhibits its phosphorylation by protein kinase C (PKC) and promotes disassembly of the p53 tetramer. Likewise, a 22 residue peptide derived from the C-terminal regulatory domain of p53 has been shown to interact with S100B(beta beta) in a Ca2+-dependent manner and inhibits its phosphorylation by PKC. Hence, structural studies of Ca2+-loaded S100B(beta beta) bound to the p53 peptide were initiated to characterize this interaction. Analysis of nuclear Overhauser effect (NOE) correlations, amide proton exchange rates, 3J(NH-H alpha) coupling constants, and chemical shift index data show that, like apo- and Ca2+-bound S100B(beta beta), S100B remains a dimer in the p53 peptide complex, and each subunit has four helices (helix 1, Glu2-Arg20; helix 2, Lys29-Asn38; helix 3, Gln50-Asp61; helix 4, Phe70-Phe87), four loops (loop 1, Glu21-His25; loop 2, Glu39-Glu49; loop 3, Glu62-Gly66; loop 4, Phe88-Glu91), and two beta-strands (beta-strand 1, Lys26-Lys28; beta-strand 2, Glu67-Asp69), which forms a short antiparallel beta-sheet. However, in the presence of the p53 peptide helix 4 is longer by five residues than in apo- or Ca2+-bound S100B(beta beta). Furthermore, the amide proton exchange rates in helix 3 (K55, V56, E58, T59, L60, D61) are significantly slower than those of Ca2+-bound S100B(beta beta). Together, these observations plus intermolecular NOE correlations between the p53 peptide and S100B(beta beta) support the notion that the p53 peptide binds in a region of S100B(beta beta), which includes residues in helix 2, helix 3, loop 2, and the C-terminal loop, and that binding of the p53 peptide interacts with and induces the extension of helix 4.     

The effect of mutations on peptide models of the DNA binding helix of p53: Evidence for a correlation between structure and tumorigenesis

The tumor suppresser protein p53 has been called the “guardian of the genome.” DNA damage induces p53 to either halt the cell cycle, allowing for repair, or initiate apoptosis. P53 is mutated in over 50% of human tumors and it has been proposed that many tumorigenic mutations are deleterious to p53 because they induce local unfolding. To explore this hypothesis, peptide models have been developed to study tumorigenic mutations in the H2 helix of the p53 core domain. This helix is rich with charged residues and is a key component of the DNA binding region. A 16‐residue peptide corresponding to the H2 wild‐type sequence extended with an Ala‐rich C‐terminus was synthesized and studied by ¹H‐nmr (500 MHz) and CD. The nmr studies demonstrate that this peptide adopts helical structure in solution. Six additional peptides corresponding to subtle tumorigenic mutations were synthesized and CD was used to assess the relative stability of these “mutant analogues.” All six mutations studied are destabilizing relative to the wild type, with ΔΔG values in the range of 0.26 to 1.35 kcal mol⁻¹. Surprisingly, substitution of Asp 281 with Ala resulted in a peptide with the greatest destabilization even though Ala possesses the largest helix propensity of the common 20 amino acids. Because this helix appears to be stabilized mainly by local electrostatics, we conclude that its structure is susceptible to even the most conservative mutations. These results provide support for the hypothesis that tumorigenic mutations induce local unfolding of p53. © 1999 John Wiley & Sons, Inc. Biopoly 49: 215–224, 1999     

Molecular dynamics of the full-length p53 monomer

The p53 protein is frequently mutated in a very large proportion of human tumors, where it seems to acquire gain-of-function activity that facilitates tumor onset and progression. A possible mechanism is the ability of mutant p53 proteins to physically interact with other proteins, including members of the same family, namely p63 and p73, inactivating their function. Assuming that this interaction might occurs at the level of the monomer, to investigate the molecular basis for this interaction, here, we sample the structural flexibility of the wild-type p53 monomeric protein. The results show a strong stability up to 850 ns in the DNA binding domain, with major flexibility in the N-terminal transactivations domains (TAD1 and TAD2) as well as in the C-terminal region (tetramerization domain). Several stable hydrogen bonds have been detected between N-terminal or C-terminal and DNA binding domain, and also between N-terminal and C-terminal. Essential dynamics analysis highlights strongly correlated movements involving TAD1 and the proline-rich region in the N-terminal domain, the tetramerization region in the C-terminal domain; Lys120 in the DNA binding region. The herein presented model is a starting point for further investigation of the whole protein tetramer as well as of its mutants.     

Effect of Alanine Replacement of L17 and F19 on the Aggregation and Neurotoxicity of Arctic-Type Aβ40

Alzheimer’s disease is the most common form of neurodegenerative disease. Beta-amyloid peptides (Aβ) are responsible for neuronal death both in vitro and in vivo. Previously, L17 and F19 residues were identified as playing key roles in the stabilization of the Aβ₄₀ conformation and in the reduction of its neurotoxicity. In this study, the effects of L17A/F19A mutations on the neurotoxicity of Aβ genetic mutant Arctic-type Aβ₄₀(E22G) were tested. The results showed that compared to Aβ₄₀(E22G), Aβ₄₀(L17A/F19A/E22G) reduced the rate of conformation conversion, aggregation, and cytotoxicity, suggesting that L17 and F19 are critical residues responsible for conformational changes which may trigger the neurotoxic cascade of Aβ. Aβ₄₀(L17A/F19A/E22G) also had decreased damage due to reactive oxygen species. The results are consistent with the discordant helix hypothesis, and confirm that residues 17–25 are in the discordant helix region. Compared to Aβ₄₀(L17A/F19A), reduction in aggregation of Aβ₄₀(L17A/F19A/E22G) was less significantly decreased. This observation provides an explanation based on the discordant helix hypothesis that the mutation of E22 to G22 of Aβ₄₀(E22G) alters the propensity of the discordant helix. Arctic-type Aβ₄₀(E22G) aggregates more severely than wild-type Aβ₄₀, with a consequential increase in toxicity.     

Skp2B attenuates p53 function by inhibiting prohibitin

The F‐box protein Skp2 and its isoform Skp2B are both overexpressed in breast cancers. Skp2 alters the activity of p53 by inhibiting its interaction with p300 and by promoting p300 degradation. Here, we report that Skp2B also attenuates the activity of p53; however, this effect is independent of p300, suggesting that another mechanism might be involved. Prohibitin, a protein reported to activate p53, was isolated in a two‐hybrid screen with the carboxy‐terminal domain unique to Skp2B. We observed that prohibitin is a new substrate of Skp2B and that the degradation of prohibitin is responsible for the attenuated activity of p53 in cells overexpressing Skp2B. Furthermore, we show that the activity of p53 is reduced in the mammary glands of Skp2B transgenic mice. This study indicates that both Skp2 and Skp2B attenuate p53 activity through different pathways, suggesting that amplification of the Skp2 locus represents a powerful mechanism to attenuate p53 function in cancer.     

The N-terminal domain of p53 is natively unfolded

p53 is one of the key molecules regulating cell proliferation, apoptosis and tumor suppression by integrating a wide variety of signals. The structural basis for this function is still poorly understood. p53 appears to exercise its function as a modular protein in which different functions are associated with distinct domains. Presumably, p53 contains both folded and partially structured parts. Here, we have investigated the structure of the isolated N-terminal part of p53 (amino acid residues 1-93) using biophysical techniques. We demonstrate that this domain is devoid of tertiary structure and largely missing secondary structure elements. It exhibits a large hydrodynamic radius, typical for unfolded proteins. These findings suggest strongly that the entire N-terminal part of p53 is natively unfolded under physiological conditions. Furthermore, the binding affinity to its functional antagonist Mdm2 was investigated. A comparison of the binding of human Mdm2 to the N-terminal part of p53 and full-length p53 suggests that unfolded and folded parts of p53 function synergistically.     

Why is F19Ap53 unable to bind MDM2? Simulations suggest crack propagation modulates binding

Why doesn’t the F19A mutant of p53 bind to MDM2? Binding thermodynamics have suggested that the loss of packing interactions upon mutating Phe into Ala sidechain results in destabilizing the binding free energy between p53 and MDM2. Does this mutation also modulate the initial recognition between p53 and MDM2? We look at atomistic computer simulations of the process of the initial encounter between wild type p53 peptide and its F19A mutant with the N-terminal domain of MDM2. These simulations show that binding is characterized by a complex multistep process. It starts with the capture of F19 of wild type p53 by certain residues in the MDM2 binding pocket. This initial step anchors the peptide onto the surface of MDM2, and with the consequent reduction in the search space of the peptide, the peptide docks into the partially occluded surface of MDM2. This is similar to a crack forming in an otherwise occluded hydrophobic cavity in MDM2, and the peptide, docked through F19, modulates the propagation of this crack, which subsequently results in the stepwise docking of the rest of the peptide through insertions of W23 and L26. The lack of the bulky sidechain of F in the F19A mutant results in the absence of the initial “grasp” complex, and hence the mutant peptide diffuses randomly on the surface of MDM2 without binding. This is the first such demonstration of the possibility that a “kinetic” effect may partly underlie the destabilized thermodynamics of binding of F19A and is a feature that appears to be conserved in evolution. The observations by Wallace et al. (Mol Cell 2006; 23:251–63) that despite the inability of F19A to bind at the N-terminal domain of MDM2, it gets ubiquitinated, can now be partly understood based on a mechanism whereby the occupation of the binding pocket by ligands/peptides induces, via crack propagation and the dynamics of gatekeeper Y100, the ubiquitination signal for interactions between the acidic domain of MDM2 and the DNA binding domain of p53.     

p53过表达抑制同源盒基因NKX3.1启动子活性

NKX3.1是前列腺特异表达的同源盒基因,在前列腺癌的发生发展中起重要作用,而在前列腺癌进展中常会发生p53的基因突变.为研究两者之间的关系,构建NKX-3.1启动子(1 040bp)-荧光素酶报告基因重组质粒（pGL3-1040）及其缺失突变体,瞬时转染前列腺癌细胞LNCaP.通过荧光素酶表达活性分析,检测p53过表达对NKX3.1启动子活性的影响.结果表明：p53在LNCaP细胞中过表达可明显抑制NKX3.1启动子活性;RT-PCR及Western印迹检测p53过表达对NKX3.1表达的影响.结果表明,p53过表达可以明显抑制同源盒基因NKX3.1的表达.通过TRANSFAC软件分析,在NKX3.1基因上游-526至-507区存在一个p53反应元件的5′核心序列.缺失pGL3-1040中的p53反应元件核心序列并不能消除p53对NKX3.1启动子的抑制作用,表明p53不是通过p53反应元件直接抑制NKX3.1启动子活性.进一步通过5′缺失突变分析,发现NKX3.1启动子-140～+8 bp区仍受p53负调控.此148 bp区域中含有一个Sp1和一个CREB元件,瞬时共转染Sp1表达载体或CREB表达载体的结果表明,p53并不是通过与Sp1或CREB相互作用对NKX3.1启动子发挥抑制作用的.上述结果表明,p53过表达可以抑制同源盒基因NKX3.1启动子活性,下调NKX3.1基因的转录,其调控机制有待进一步研究.     

Multiple domains of the mouse p19ARF tumor suppressor are involved in p53-independent apoptosis

The ARF (p19ARF for the mouse ARF consisting of 169 amino acids and p14ARF for the human ARF consisting of 132 amino acids) genes upregulate p53 activities to induce cell cycle arrest and sensitize cells to apoptosis by inhibiting Mdm2 activity. p53-independent apoptosis also is induced by ectopic expression of p19ARF. We constructed various deletion mutants of p19ARF with a cre/loxP-regulated adenoviral vector to determine the regions of p19ARF which are responsible for p53-independent apoptosis. Ectopic expression of the C-terminal region (named C40) of p19ARF whose primary sequence is unique to the rodent ARF induced prominent apoptosis in p53-deficient mouse embryo fibroblasts. Relatively low-grade but significant apoptosis also was induced in p53-deficient mouse embryo fibroblasts by ectopic expression of p19ARF1-129, a p19ARF deletion mutant deficient in the C40 region. In contrast, ectopic expression of the wild-type p14ARF did not induce significant apoptosis in human cells. Taken together, we concluded that p53-independent apoptosis was mediated through multiple regions of the mouse ARF including C40, and the ability of the ARF gene to mediate p53-independent apoptosis has been not well conserved during mammalian evolution.     

Hsp70 family member, mot-2/mthsp70/GRP75, binds to the cytoplasmic sequestration domain of the p53 protein

Hsp70 family member mot-2/mthsp70/GRP75/PBP74 was shown to bind to the tumor suppressor protein p53. In this study, by in vivo coimmunoprecipitation of mot-2 with p53 and its deletion mutants, the mot-2 binding site of p53 was mapped to its C-terminal amino acid residues 312-352, a region of p53 that includes its cytoplasmic sequestration domain. These data demonstrate that cytoplasmic sequestration and inactivation of p53 by mot-2 occurs by its binding to the cytoplasmic sequestration domain. Therefore, perturbation of mot-p53 interactions can be employed to abrogate cytoplasmic retention of wild-type p53 in tumors.     

Down-regulation of Tripartite-motif containing 22 expression in breast cancer is associated with a lack of p53-mediated induction

Tripartite-motif containing 22 (TRIM22) is a direct p53 target gene and inhibits the clonogenic growth of leukemic cells. Its expression in Wilms tumors is negatively associated with disease relapse. This study addresses if TRIM22 expression is de-regulated in breast carcinoma. Western blotting analysis of a panel of 10 breast cancer cell lines and 3 non-malignant mammary epithelial cell lines with a well-characterized TRIM22 monoclonal antibody showed that TRIM22 protein is greatly under-expressed in breast cancer cells as compared to non-malignant cell lines. Similarly, TRIM22 protein is significantly down-regulated in breast tumors as compared to matched normal breast tissues. Study of cell lines with methylation inhibitor and bisulfite sequencing indicates that TRIM22 promoter hypermethylation may not be the cause for TRIM22 under-expression in breast cancer. Instead, we found that TRIM22 protein level correlates strongly (R = 0.79) with p53 protein level in normal breast tissue, but this correlation is markedly impaired (R = 0.48) in breast cancer tissue, suggesting that there is some defects in p53 regulation of TRIM22 gene in breast cancer. This notion is supported by cell line studies, which showed that TRIM22 was no longer inducible by p53-activating genotoxic drugs in breast cancer cell lines and in a p53 null cell line H1299 transfected with wild type p53. In conclusion, this study shows that TRIM22 is greatly under-expressed in breast cancer. p53 dysfunction may be one of the mechanisms for TRIM22 down-regulation.     

Structural elements of human parathyroid hormone and their possible relation to biological activities.

Human parathyroid hormone (hPTH) and several deletion analogues were examined for the presence of secondary structure using circular dichroism spectroscopy. The spectra of hPTH and the deletion analogues 8-84, 34-53, 53-84, 1-34, 13-34, 1-19, and 20-34, in neutral, aqueous buffer, gave no evidence for extensive secondary structure. An alpha-helical-like spectral contribution was found to arise from a region within peptide 13-34. This spectral contribution was speculated to arise from partial stability of a helix consisting of residues 17-29. Molecular dynamics simulations of peptide 1-34 suggested that this peptide tends to fold with a bend defined by residues 10-14, with the amino-terminal and carboxyl-terminal residues tending to be in more extended forms and the other residues in helical-like conformations. The addition of trifluoroethanol promoted the formation of alpha-helix, mainly in the 1-34 region. The putative helix comprised of residues 17-29 was stabilized by the addition of 10-20% TFE, while a second putative helix proximal to the amino terminus, and comprised of residues 3-11, was stabilized by slightly higher concentrations of TFE. An amphiphilic sequence was identified within the 20-34 fragment. The development of alpha-helix on binding this fragment, and other analogues containing this sequence, to palmitoyloleoylphosphatidylserine vesicles provided experimental evidence for the potential role of this amphiphilic sequence in binding to membranes or to a membrane receptor. The relationships between these alpha-helical regions in 1-34, either potentiated by trifluoroethanol or lipid vesicles, are discussed in terms of different receptor-binding regions within hPTH.