S100A6 Competes with the TAZ2 Domain of p300 for Binding to p53 and Attenuates p53 Acetylation

S100A6 is a calcium binding protein that, like some other members of the S100 protein family, is able to bind p53. This interaction may be physiologically relevant considering the numerous connotations of S100 proteins and of S100A6, in particular, with cancer and metastasis. In this work, we show that the interaction with S100A6 is limited to unmodified or phosphorylated p53 and is inhibited by p53 acetylation. Using in vitro acetylation assay, we show that the presence of S100A6 attenuates p53 acetylation by p300. Furthermore, using ELISA, we show that S100A6 and the TAZ2 domain of p300 bind p53 with similar affinities and that S100A6 effectively competes with TAZ2 for binding to p53. Our results add another element to the complicated scheme of p53 activation.     

Disruption of an intermonomer salt bridge in the p53 tetramerization domain results in an increased propensity to form amyloid fibrils

We describe in molecular detail how disruption of an intermonomer salt bridge (Arg337-Asp352) leads to partial destabilization of the p53 tetramerization domain and a dramatically increased propensity to form amyloid fibrils. At pH 4.0 and 37 degrees C, a p53 tetramerization domain mutant (p53tet-R337H), associated with adrenocortical carcinoma in children, readily formed amyloid fibrils, while the wild-type (p53tet-wt) did not. We characterized these proteins by equilibrium denaturation, 13C(alpha) secondary chemical shifts, (1H)-15N heteronuclear NOEs, and H/D exchange. Although p53tet-R337H was thermodynamically less stable, NMR data indicated that the two proteins had similar secondary structure and molecular dynamics. NMR derived pK(a) values indicated that at low pH the R337H mutation partially disrupted an intermonomer salt bridge. Backbone H/D exchange results showed that for at least a small population of p53tet-R337H molecules disruption of this salt bridge resulted in partial destabilization of the protein. It is proposed that this decrease in p53tet-R337H stability resulted in an increased propensity to form amyloid fibrils.     

Targeting of p53 and its homolog p73 by protoporphyrin IX

The p53 tumor suppressor is recognized as a promising target for anti-cancer therapies. We previously reported that protoporphyrin IX (PpIX) disrupts the p53/murine double minute 2 (MDM2) complex and leads to p53 accumulation and activation of apoptosis in HCT 116 cells. Here we show the direct binding of PpIX to the N-terminal domain of p53. Furthermore, we addressed the induction of apoptosis in HCT 116 p53-null cells by PpIX and revealed interactions between PpIX and p73. We propose that PpIX disrupts the p53/MDM2 or MDMX and p73/MDM2 complexes and thereby activates the p53- or p73-dependent cancer cell death.     

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.     

Interaction of the p53 DNA-binding domain with its n-terminal extension modulates the stability of the p53 tetramer

The tetrameric tumor suppressor p53 plays a pivotal role in the control of the cell cycle and provides a paradigm for an emerging class of oligomeric, multidomain proteins with structured and intrinsically disordered regions. Many of its biophysical and functional properties have been extrapolated from truncated variants, yet the exact structural and functional role of certain segments of the protein is unclear. We found from NMR and X-ray crystallography that the DNA-binding domain (DBD) of human p53, usually defined as residues 94-292, extends beyond these domain boundaries. Trp91, in the hinge region between the disordered proline-rich N-terminal domain and the DBD, folds back onto the latter and has a cation-π interaction with Arg174. These additional interactions increase the melting temperature of the DBD by up to 2 °C and inhibit aggregation of the p53 tetramer. They also modulate the dissociation of the p53 tetramer. The absence of the Trp91/Arg174 packing presumably allows nonnative DBD-DBD interactions that both nucleate aggregation and stabilize the interface. These data have important implications for studies of multidomain proteins in general, highlighting the fact that weak ordered-disordered domain interactions can modulate the properties of proteins of complex structure.     

Pharmacological activation of a novel p53-dependent S-phase checkpoint involving CHK-1

We have recently shown that induction of the p53 tumour suppressor protein by the small-molecule RITA (reactivation of p53 and induction of tumour cell apoptosis; 2,5-bis(5-hydroxymethyl-2-thienyl)furan) inhibits hypoxia-inducible factor-1α and vascular endothelial growth factor expression in vivo and induces p53-dependent tumour cell apoptosis in normoxia and hypoxia. Here, we demonstrate that RITA activates the canonical ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related DNA damage response pathway. Interestingly, phosphorylation of checkpoint kinase (CHK)-1 induced in response to RITA was influenced by p53 status. We found that induction of p53, phosphorylated CHK-1 and γH2AX proteins was significantly increased in S-phase. Furthermore, we found that RITA stalled replication fork elongation, prolonged S-phase progression and induced DNA damage in p53 positive cells. Although CHK-1 knockdown did not significantly affect p53-dependent DNA damage or apoptosis induced by RITA, it did block the ability for DNA integrity to be maintained during the immediate response to RITA. These data reveal the existence of a novel p53-dependent S-phase DNA maintenance checkpoint involving CHK-1.     

Posttranslational Modifications Affect the Interaction of S100 Proteins with Tumor Suppressor p53

Proteins of the S100 family bind to the intrinsically disordered transactivation domain (TAD; residues 1-57) and C-terminus (residues 293-393) of the tumor suppressor p53. Both regions provide sites that are subject to posttranslational modifications, such as phosphorylation and acetylation, that can alter the affinity for interacting proteins such as p300 and MDM2. Here, we found that S100A1, S100A2, S100A4, S100A6, and S100B bound to two subdomains of the TAD (TAD1 and TAD2). Both subdomains were mandatory for high-affinity binding to S100 proteins. Phosphorylation of Ser and Thr residues increased the affinity for the p53 TAD. Conversely, acetylation and phosphorylation of the C-terminus of p53 decreased the affinity for S100A2 and S100B. In contrast, we found that nitrosylation of S100B caused a minor increase in binding to the p53 C-terminus, whereas binding to the TAD remained unaffected. As activation of p53 is usually accompanied by phosphorylation and acetylation at several sites, our results suggest that a shift in binding from the C-terminus in favor of the N-terminus occurs upon the modification of p53. We propose that binding to the p53 TAD might be involved in the stimulation of p53 activity by S100 proteins.     

Definition of the p53 functional domains necessary for inducing apoptosis

The p53 protein contains several functional domains necessary for inducing cell cycle arrest and apoptosis. The C-terminal basic domain within residues 364-393 and the proline-rich domain within residues 64-91 are required for apoptotic activity. In addition, activation domain 2 within residues 43-63 is necessary for apoptotic activity when the N-terminal activation domain 1 within residues 1-42 is deleted (DeltaAD1) or mutated (AD1(-)). Here we have discovered that an activation domain 2 mutation at residues 53-54 (AD2(-)) abrogates the apoptotic activity but has no significant effect on cell cycle arrest. We have also found that p53-(DeltaAD2), which lacks activation domain 2, is inert in inducing apoptosis. p53-(AD2(-)DeltaBD), which is defective in activation domain 2 and lacks the C-terminal basic domain, p53-(DeltaAD2DeltaBD), which lacks both activation domain 2 and the C-terminal basic domain, and p53-(DeltaPRDDeltaBD), which lacks both the proline-rich domain and the C-terminal basic domain, are also inert in inducing apoptosis. All four mutants are still capable of inducing cell cycle arrest, albeit to a lesser extent than wild-type p53. Interestingly, we have found that deletion of the N-terminal activation domain 1 alleviates the requirement of the C-terminal basic domain for apoptotic activity. Thus, we have generated a small but potent p53-(DeltaAD1DeltaBD) molecule. Furthermore, we have determined that at least two of the three domains (activation domain 1, activation domain 2, and the proline-rich domain), are required for inducing cell cycle arrest. Taken together, our results suggest that activation domain 2 and the proline-rich domain form an activation domain for inducing pro-apoptotic genes or inhibiting anti-apoptotic genes. The C-terminal basic domain is required for maintaining this activation domain competent for transactivation or transrepression.     

p53 regulates the proliferation, differentiation and spontaneous transformation of mesenchymal stem cells

Mesenchymal stem cells (MSC) have been extensively studied and gained wide popularity due to their therapeutic potential. Spontaneous transformation of MSC, from both human and murine origin, has been reported in many studies. MSC transformation depends on the culture conditions, the origin of the cells and the time on culture; however, the precise biological characteristics involved in this process have not been fully defined yet. In this study, we investigated the role of p53 in the biology and transformation of murine bone marrow (BM)-derived MSC. We demonstrate that the MSC derived from p53KO mice showed an augmented proliferation rate, a shorter doubling time and also morphologic and phenotypic changes, as compared to MSC derived from wild-type animals. Furthermore, the MSC devoid of p53 had an increased number of cells able to generate colonies. In addition, not only proliferation but also MSC differentiation is controlled by p53 since its absence modifies the speed of the process. Moreover, genomic instability, changes in the expression of c-myc and anchorage independent growth were also observed in p53KO MSC. In addition, the absence of p53 implicates the spontaneous transformation of MSC in long-term cultures. Our results reveal that p53 plays a central role in the biology of MSC.     

Mutations in p97/VCP induce unfolding activity

A comparison of the protein sequences of various two-domain AAA+ ATPases revealed a striking difference in the residues lining the central pore of the D1 domain. The protein unfoldases of the bacterial Clp family and the archaeal VAT protein have at least one aromatic residue in the central D1 pore. In contrast, none of the members of the eukaryotic p97/VCP protein family has an aromatic residue in the D1 pore. The protein unfolding activity of VAT and other AAA+ ATPases is critically dependent on the presence of aromatic residues in this central pore. Unfoldase activity has not been demonstrated for the p97/VCP family in vitro. Thus, we exchanged the two aliphatic residues leucine and alanine of the D1 pore for aromatic tyrosine residues in full length p97 and in p97DeltaN, a truncated form of p97 lacking the N domain. We found that the mutant p97DeltaN variants with a single tyrosine or with two tyrosine residues in the central pore of D1 unfold the Clp family and VAT model substrate YFP-ssrA, whereas full length p97 with aromatic pore residues and wild-type p97 or p97DeltaN do not. Thus, p97 can exert unfoldase activity in vitro, provided that a single tyrosine residue is introduced into the D1 pore and that the N domain is deleted.     

Diphenyleneiodonium induces ROS-independent p53 expression and apoptosis in human RPE cells

The diphenyleneiodonium (DPI) is widely used as an inhibitor of flavoenzymes, particularly NADPH oxidase. In this study, we investigated the effect of DPI on the apoptosis of human RPE cells. DPI treatment in ARPE-19 cells evoked a dose- and time-dependent growth inhibition, and also induced DNA fragmentation and protein content of the proapoptotic factor Bax. In addition, DPI significantly induced the expression and phosphorylation of p53, which induces proapoptotic genes in response to DNA damage or irreparable cell cycle arrest. ROS have been implicated as a key factor in the activation of p53 by many chemotherapeutic drugs. Recent data on the regulation of intracellular ROS by DPI are controversial. Therefore, we analyzed whether DPI could contribute to the generation of intracellular ROS. Although there was increase in ROS level from cells treated for 24h with DPI, it was not detectable at early time points, required to induce p53 expression. And DPI-induced p53 expression was not affected by the ROS scavenger NAC. We conclude that DPI induces the expression of p53 by ROS-independent mechanism in ARPE-19 cells, and renders cells sensitive to drug-induced apoptosis by induction of p53 expression.     

The p53-inducible E3 ubiquitin ligase p53RFP induces p53-dependent apoptosis

Recently, it has been shown that really interesting new gene (RING)-in between ring finger (IBR)-RING domain-containing proteins, such as Parkin and Parc, are E3 ubiquitin ligases and are involved in regulation of apoptosis. In this report, we show that p53-inducible RING-finger protein (p53RFP), a p53-inducible E3 ubiquitin ligase, induces p53-dependent but caspase-independent apoptosis. p53RFP contains an N-terminal RING-IBR-RING domain and an uncharacterized, evolutionally highly conserved C-terminal domain. p53RFP interacts with E2 ubiquitin-conjugating enzymes UbcH7 and UbcH8 but not with UbcH5, and this interaction is mediated through the RING-IBR-RING domain of p53RFP. Interestingly, the conserved C-terminal domain of p53RFP is required and sufficient for p53RFP-mediated apoptosis, suggesting p53RFP-mediated apoptosis does not require its E3 ubiquitin ligase activity. Together with a recent report showing that p53RFP is involved in ubiquitination and degradation of p21, a p53 downstream protein promoting growth arrest and antagonizing apoptosis, our findings suggest that p53RFP is involved in switching a cell from p53-mediated growth arrest to apoptosis.