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1.
J Biol Chem ; : 107561, 2024 Jul 11.
Artículo en Inglés | MEDLINE | ID: mdl-39002674

RESUMEN

Protein phosphatase 1D (PPM1D, Wip1) is induced by the tumor suppressor p53 during DNA damage response signaling and acts as an oncoprotein in several human cancers. Although PPM1D is a potential therapeutic target, insights into its atomic structure were challenging due to flexible regions unique to this family member. Here we report the first crystal structure of the PPM1D catalytic domain to 1.8 Å resolution. The structure reveals the active site with two Mg2+ ions bound, similar to other structures. The flap subdomain and B-loop, which are crucial for substrate recognition and catalysis, were also resolved, with the flap forming two short helices and three short ß-strands that are followed by an irregular loop. Unexpectedly, a nitrogen-oxygen-sulfur bridge was identified in the catalytic domain. Molecular dynamics simulations and kinetic studies provided further mechanistic insights into the regulation of PPM1D catalytic activity. In particular, the kinetic experiments demonstrated a magnesium concentration-dependent lag in PPM1D attaining steady-state velocity, a feature of hysteretic enzymes that show slow transitions compared with catalytic turnover. All combined, these results advance the understanding of PPM1D function and will support the development of PPM1D-targeted therapeutics.

2.
Nucleic Acids Res ; 49(2): 713-725, 2021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33406227

RESUMEN

We report a series of synthetic, nucleic acid mimics with highly customizable thermodynamic binding to DNA. Incorporation of helix-promoting cyclopentanes into peptide nucleic acids (PNAs) increases the melting temperatures (Tm) of PNA+DNA duplexes by approximately +5°C per cyclopentane. Sequential addition of cyclopentanes allows the Tm of PNA + DNA duplexes to be systematically fine-tuned from +5 to +50°C compared with the unmodified PNA. Containing only nine nucleobases and an equal number of cyclopentanes, cpPNA-9 binds to complementary DNA with a Tm around 90°C. Additional experiments reveal that the cpPNA-9 sequence specifically binds to DNA duplexes containing its complementary sequence and functions as a PCR clamp. An X-ray crystal structure of the cpPNA-9-DNA duplex revealed that cyclopentanes likely induce a right-handed helix in the PNA with conformations that promote DNA binding.


Asunto(s)
Ciclopentanos/química , ADN/metabolismo , Conformación de Ácido Nucleico , Ácidos Nucleicos de Péptidos/química , Calorimetría , Dicroismo Circular , Cristalografía por Rayos X , Ciclopentanos/metabolismo , Modelos Moleculares , Desnaturalización de Ácido Nucleico , Ácidos Nucleicos de Péptidos/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrofotometría Ultravioleta , Termodinámica , Temperatura de Transición
3.
J Biol Chem ; 294(46): 17654-17668, 2019 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-31481464

RESUMEN

WT P53-Induced Phosphatase 1 (WIP1) is a member of the magnesium-dependent serine/threonine protein phosphatase (PPM) family and is induced by P53 in response to DNA damage. In several human cancers, the WIP1 protein is overexpressed, which is generally associated with a worse prognosis. Although WIP1 is an attractive therapeutic target, no potent, selective, and bioactive small-molecule modulator with favorable pharmacokinetics has been reported. Phosphatase enzymes are among the most challenging targets for small molecules because of the difficulty of achieving both modulator selectivity and bioavailability. Another major obstacle has been the availability of robust and physiologically relevant phosphatase assays that are suitable for high-throughput screening. Here, we describe orthogonal biochemical WIP1 activity assays that utilize phosphopeptides from native WIP1 substrates. We optimized an MS assay to quantify the enzymatically dephosphorylated peptide reaction product in a 384-well format. Additionally, a red-shifted fluorescence assay was optimized in a 1,536-well format to enable real-time WIP1 activity measurements through the detection of the orthogonal reaction product, Pi We validated these two optimized assays by quantitative high-throughput screening against the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection and used secondary assays to confirm and evaluate inhibitors identified in the primary screen. Five inhibitors were further tested with an orthogonal WIP1 activity assay and surface plasmon resonance binding studies. Our results validate the application of miniaturized physiologically relevant and orthogonal WIP1 activity assays to discover small-molecule modulators from high-throughput screens.


Asunto(s)
Activadores de Enzimas/química , Fosfopéptidos/química , Proteína Fosfatasa 2C/química , Bibliotecas de Moléculas Pequeñas/química , Activadores de Enzimas/aislamiento & purificación , Activadores de Enzimas/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Proteína Fosfatasa 2C/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/aislamiento & purificación , Bibliotecas de Moléculas Pequeñas/farmacología , Especificidad por Sustrato , Proteína p53 Supresora de Tumor/química
4.
J Biol Chem ; 293(21): 7993-8008, 2018 05 25.
Artículo en Inglés | MEDLINE | ID: mdl-29602904

RESUMEN

Metal-dependent protein phosphatases (PPM) are evolutionarily unrelated to other serine/threonine protein phosphatases and are characterized by their requirement for supplementation with millimolar concentrations of Mg2+ or Mn2+ ions for activity in vitro The crystal structure of human PPM1A (also known as PP2Cα), the first PPM structure determined, displays two tightly bound Mn2+ ions in the active site and a small subdomain, termed the Flap, located adjacent to the active site. Some recent crystal structures of bacterial or plant PPM phosphatases have disclosed two tightly bound metal ions and an additional third metal ion in the active site. Here, the crystal structure of the catalytic domain of human PPM1A, PPM1Acat, complexed with a cyclic phosphopeptide, c(MpSIpYVA), a cyclized variant of the activation loop of p38 MAPK (a physiological substrate of PPM1A), revealed three metal ions in the active site. The PPM1Acat D146E-c(MpSIpYVA) complex confirmed the presence of the anticipated third metal ion in the active site of metazoan PPM phosphatases. Biophysical and computational methods suggested that complex formation results in a slightly more compact solution conformation through reduced conformational flexibility of the Flap subdomain. We also observed that the position of the substrate in the active site allows solvent access to the labile third metal-binding site. Enzyme kinetics of PPM1Acat toward a phosphopeptide substrate supported a random-order, bi-substrate mechanism, with substantial interaction between the bound substrate and the labile metal ion. This work illuminates the structural and thermodynamic basis of an innate mechanism regulating the activity of PPM phosphatases.


Asunto(s)
Metales/metabolismo , Fosfopéptidos/metabolismo , Proteína Fosfatasa 2C/química , Proteína Fosfatasa 2C/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Conformación Proteica , Proteína Fosfatasa 2C/genética , Homología de Secuencia , Especificidad por Sustrato
5.
Biochemistry ; 56(21): 2676-2689, 2017 05 30.
Artículo en Inglés | MEDLINE | ID: mdl-28481111

RESUMEN

PPM serine/threonine protein phosphatases function in signaling pathways and require millimolar concentrations of Mn2+ or Mg2+ ions for activity. Whereas the crystal structure of human PP2Cα displayed two tightly bound Mn2+ ions in the active site, recent investigations of PPM phosphatases have characterized the binding of a third, catalytically essential metal ion. The binding of the third Mg2+ to PP2Cα was reported to have millimolar affinity and to be entropically driven, suggesting it may be structurally and catalytically important. Here, we report the use of hydrogen/deuterium exchange-mass spectrometry and molecular dynamics to characterize conformational changes in PP2Cα between the active and inactive states. In the presence of millimolar concentrations of Mg2+, metal-coordinating residues in the PP2Cα active site are maintained in a more rigid state over the catalytically relevant time scale of 30-300 s. Submillimolar Mg2+ concentrations or introduction of the D146A mutation increased the conformational mobility in the Flap subdomain and in buttressing helices α1 and α2. Residues 192-200, located in the Flap subdomain, exhibited the greatest interplay between effects of Mg2+ concentration and the D146A mutation. Molecular dynamics simulations suggest that the presence of the third metal ion and the D146A mutation each produce distinct conformational realignments in the Flap subdomain. These observations suggest that the binding of Mg2+ to the D146/D239 binding site stabilizes the conformation of the active site and the Flap subdomain.


Asunto(s)
Medición de Intercambio de Deuterio , Proteína Fosfatasa 2C/química , Proteína Fosfatasa 2C/metabolismo , Sitios de Unión , Humanos , Espectrometría de Masas , Conformación Proteica
6.
Biochemistry ; 54(11): 2001-10, 2015 Mar 24.
Artículo en Inglés | MEDLINE | ID: mdl-25753752

RESUMEN

The p53 tumor suppressor is a critical mediator of the cellular response to stress. The N-terminal transactivation domain of p53 makes protein interactions that promote its function as a transcription factor. Among those cofactors is the histone acetyltransferase p300, which both stabilizes p53 and promotes local chromatin unwinding. Here, we report the nuclear magnetic resonance solution structure of the Taz2 domain of p300 bound to the second transactivation subdomain of p53. In the complex, p53 forms an α-helix between residues 47 and 55 that interacts with the α1-α2-α3 face of Taz2. Mutational analysis indicated several residues in both p53 and Taz2 that are critical for stabilizing the interaction. Finally, further characterization of the complex by isothermal titration calorimetry revealed that complex formation is pH-dependent and releases a bound chloride ion. This study highlights differences in the structures of complexes formed by the two transactivation subdomains of p53 that may be broadly observed and play critical roles in p53 transcriptional activity.


Asunto(s)
Proteína p300 Asociada a E1A/metabolismo , Histona Acetiltransferasas/metabolismo , Modelos Moleculares , Proteína p53 Supresora de Tumor/metabolismo , Sustitución de Aminoácidos , Rastreo Diferencial de Calorimetría , Proteína p300 Asociada a E1A/química , Proteína p300 Asociada a E1A/genética , Histona Acetiltransferasas/química , Histona Acetiltransferasas/genética , Humanos , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Cinética , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genética
7.
Proc Natl Acad Sci U S A ; 109(2): E68-75, 2012 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-22065775

RESUMEN

The inactivation of the p53 tumor suppressor pathway in many cancers often increases their resistance to anticancer therapy. Here we show that a previously proposed strategy directed to Wip1 inhibition could be ineffective in tumors lacking p53. On the contrary, Wip1 overexpression sensitized these tumors to chemotherapeutic agents. This effect was mediated through interaction between Wip1 and RUNX2 that resulted, in response to anticancer treatment, in RUNX2-dependent transcriptional induction of the proapoptotic Bax protein. The potentiating effects of Wip1 overexpression on chemotherapeutic agents were directed only to tumor cells lacking p53. The overexpression of Wip1 in normal tissues provided protection from cisplatin-induced apoptosis through decreased strength of upstream signaling to p53. Thus, Wip1 phosphatase promotes apoptosis in p53-negative tumors and protects normal tissues during treatment with anticancer agents.


Asunto(s)
Antineoplásicos/farmacología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Neoplasias/tratamiento farmacológico , Fosfoproteínas Fosfatasas/metabolismo , Proteína p53 Supresora de Tumor/deficiencia , Animales , Antineoplásicos/metabolismo , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Western Blotting , Línea Celular , Cartilla de ADN/genética , Sinergismo Farmacológico , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Inmunohistoquímica , Inmunoprecipitación , Ratones , Neoplasias/metabolismo , Plásmidos/genética , Proteína Fosfatasa 2C , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/metabolismo
8.
Biochemistry ; 52(34): 5830-43, 2013 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-23906386

RESUMEN

The PPM phosphatases require millimolar concentrations of Mg²âº or Mn²âº to activate phosphatase activity in vitro. The human phosphatases PP2Cα (PPM1A) and Wip1 (PPM1D) differ in their physiological function, substrate specificity, and apparent metal affinity. A crystallographic structure of PP2Cα shows only two metal ions in the active site. However, recent structural studies of several bacterial PP2C phosphatases have indicated three metal ions in the active site. Two residues that coordinate the third metal ion are highly conserved, suggesting that human PP2C phosphatases may also bind a third ion. Here, isothermal titration calorimetry analysis of Mg²âº binding to PP2Cα distinguished binding of two ions to high affinity sites from the binding of a third ion with a millimolar affinity, similar to the apparent metal affinity required for catalytic activity. Mutational analysis indicated that Asp239 and either Asp146 or Asp243 was required for low-affinity binding of Mg²âº, but that both Asp146 and Asp239 were required for catalysis. Phosphatase activity assays in the presence of MgCl2, MnCl2, or mixtures of the two, demonstrate high phosphatase activity toward a phosphopeptide substrate when Mg²âº was bound to the low-affinity site, whether Mg²âº or Mn²âº ions were bound to the high affinity sites. Mutation of the corresponding putative third metal ion-coordinating residues of Wip1 affected catalytic activity similarly both in vitro and in human cells. These results suggest that phosphatase activity toward phosphopeptide substrates by PP2Cα and Wip1 requires the binding of a Mg²âº ion to the low-affinity site.


Asunto(s)
Dominio Catalítico , Magnesio/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Alanina/química , Secuencia de Aminoácidos , Ácido Aspártico/química , Catálisis , Células HEK293 , Humanos , Manganeso/metabolismo , Fosfopéptidos/metabolismo , Fosfoproteínas Fosfatasas/genética , Proteína Fosfatasa 2C , Alineación de Secuencia
9.
JACS Au ; 3(7): 1952-1964, 2023 Jul 24.
Artículo en Inglés | MEDLINE | ID: mdl-37502163

RESUMEN

Selective incorporation of conformational constraints into thyclotides can be used to modulate their binding to complementary oligonucleotides, increase polarity, and optimize uptake into HCT116 cells without assistance from moieties known to promote cell uptake. The X-ray structure and biophysical studies of a thyclotide-DNA duplex reveal that incorporation of tetrahydrofurans into an aegPNA backbone promotes a helical conformation that enhances binding to complementary DNA and RNA. Selective incorporation of tetrahydrofurans into the aegPNA backbone allows polarity to be increased incrementally so that uptake into HCT116 cells can be optimized. The enhanced binding, polarity, and cellular uptake properties of thyclotides were used to demonstrate effective inhibition of microRNA-21 in HCT116 cells.

10.
Carcinogenesis ; 33(8): 1441-9, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22505655

RESUMEN

The p53 tumor suppressor is a critical component of the cellular response to stress. As it can inhibit cell growth, p53 is mutated or functionally inactivated in most tumors. A multitude of protein-protein interactions with transcriptional cofactors are central to p53-dependent responses. In its activated state, p53 is extensively modified in both the N- and C-terminal regions of the protein. These modifications, especially phosphorylation of serine and threonine residues in the N-terminal transactivation domain, affect p53 stability and activity by modulating the affinity of protein-protein interactions. Here, we review recent findings from in vitro and in vivo studies on the role of p53 N-terminal phosphorylation. These modifications can either positively or negatively affect p53 and add a second layer of complex regulation to the divergent interactions of the p53 transactivation domain.


Asunto(s)
Proteína p53 Supresora de Tumor/metabolismo , Animales , Humanos , Ratones , Modelos Moleculares , Fosforilación , Proteína p53 Supresora de Tumor/química
11.
Nat Cell Biol ; 7(2): 165-71, 2005 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-15619621

RESUMEN

The tumour suppressor p53 becomes activated in response to upstream stress signals, such as DNA damage, and causes cell-cycle arrest or apoptosis. Here we report a novel role for p53 in the differentiation of mouse embryonic stem cells (ESCs). p53 binds to the promoter of Nanog, a gene required for ESC self-renewal, and suppresses Nanog expression after DNA damage. The rapid down-regulation of Nanog mRNA during ESC differentiation correlates with the induction of p53 transcriptional activity and Ser 315 phosphorylation. The importance of Ser 315 phosphorylation was revealed by the finding that induction of p53 activity is impaired in p53(S315A) knock-in ESCs during differentiation, leading to inefficient suppression of Nanog expression. The decreased inhibition of Nanog expression in p53(S315A) ESCs during differentiation is due to an impaired recruitment of the co-repressor mSin3a to the Nanog promoter. These findings indicate an alternative mechanism for p53 to maintain genetic stability in ESCs, by inducing the differentiation of ESCs into other cell types that undergo efficient p53-dependent cell-cycle arrest and apoptosis.


Asunto(s)
Diferenciación Celular/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Células Madre/citología , Proteína p53 Supresora de Tumor/fisiología , Animales , Regulación hacia Abajo , Embrión de Mamíferos/citología , Regulación de la Expresión Génica , Ratones , Proteína Homeótica Nanog , Fosforilación , Regiones Promotoras Genéticas , Tretinoina
12.
Eur J Med Chem ; 243: 114763, 2022 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-36179402

RESUMEN

The wild-type p53 induced phosphatase 1 (Wip1), a member of the serine/threonine-specific PP2C family, is overexpressed in numerous human cancers. Wip1 dephosphorylates p53 as well as several kinases (such as p38 MAPK, ATM, Chk1, and Chk2) in the DNA damage response pathway that are responsible for maintaining genomic stability and preventing oncogenic transformation. As a result, Wip1 is an attractive target for synthetic inhibitors that could be further developed into therapeutics to treat some cancers. In this study, we report a series of alkyl-substituted N-methylaryl-N'-aryl-4-aminobenzamides and their inhibitory activity of the Wip1 phosphatase. A straightforward synthetic route was developed to synthesize the target compounds from commercially available starting materials. Three different portions (R1, R2, R3) of the core scaffold were extensively modified to examine structure-activity relationships. This study revealed interesting trends about a new molecular scaffold to inhibit Wip1.


Asunto(s)
Fosfoproteínas Fosfatasas , Proteína p53 Supresora de Tumor , Humanos , Proteína Fosfatasa 2C/genética , Proteína Fosfatasa 2C/metabolismo , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Serina-Treonina Quinasas , Daño del ADN , Fosforilación
13.
Front Oncol ; 12: 1094210, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36713582

RESUMEN

TP53 is the most commonly mutated gene in cancer, and gain-of-function mutations have wide-ranging effects. Efforts to reactivate wild-type p53 function and inhibit mutant functions have been complicated by the variety of TP53 mutations. Identified from a screen, the NSC59984 compound has been shown to restore activity to mutant p53 in colorectal cancer cells. Here, we investigated its effects on esophageal adenocarcinoma cells with specific p53 hot-spot mutations. NSC59984 treatment of cells reactivated p53 transcriptional regulation, inducing mitochondrial intrinsic apoptosis. Analysis of its effects on cellular metabolism demonstrated increased utilization of the pentose phosphate pathway and inhibition of glycolysis at the fructose-1,6-bisphosphate to fructose 6-phosphate junction. Furthermore, treatment of cells with NSC59984 increased reactive oxygen species production and decreased glutathione levels; these effects were enhanced by the addition of buthionine sulfoximine and inhibited by N-acetyl cysteine. We found that the effects of NSC59984 were substantially greater in cells harboring the p53 R248W mutation. Overall, these findings demonstrate p53-dependent effects of NSC59984 on cellular metabolism, with increased activity in cells harboring the p53 R248W mutation. This research highlights the importance of defining the mutational status of a particular cancer to create a patient-centric strategy for the treatment of p53-driven cancers.

14.
Structure ; 17(2): 202-10, 2009 Feb 13.
Artículo en Inglés | MEDLINE | ID: mdl-19217391

RESUMEN

Coactivators CREB-binding protein and p300 play important roles in mediating the transcriptional activity of p53. Until now, however, no detailed structural information has been available on how any of the domains of p300 interact with p53. Here, we report the NMR structure of the complex of the Taz2 (C/H3) domain of p300 and the N-terminal transactivation domain of p53. In the complex, p53 forms a short alpha helix and interacts with the Taz2 domain through an extended surface. Mutational analyses demonstrate the importance of hydrophobic residues for complex stabilization. Additionally, they suggest that the increased affinity of Taz2 for p53(1-39) phosphorylated at Thr(18) is due in part to electrostatic interactions of the phosphate with neighboring arginine residues in Taz2. Thermodynamic experiments revealed the importance of hydrophobic interactions in the complex of Taz2 with p53 phosphorylated at Ser(15) and Thr(18).


Asunto(s)
Dominios y Motivos de Interacción de Proteínas , Proteínas Quinasas/metabolismo , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Factores de Transcripción p300-CBP/química , Factores de Transcripción p300-CBP/metabolismo , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Fosforilación/fisiología , Unión Proteica , Mapeo de Interacción de Proteínas , Proteínas Quinasas/fisiología , Estructura Cuaternaria de Proteína , Serina/química , Serina/metabolismo , Termodinámica , Treonina/química , Treonina/metabolismo
15.
Nat Commun ; 12(1): 3622, 2021 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-34131120

RESUMEN

PPM1D/Wip1 is a negative regulator of the tumor suppressor p53 and is overexpressed in several human solid tumors. Recent reports associate gain-of-function mutations of PPM1D in immune cells with worse outcomes for several human cancers. Here we show that mice with genetic knockout of Ppm1d or with conditional knockout of Ppm1d in the hematopoietic system, in myeloid cells, or in neutrophils all display significantly reduced growth of syngeneic melanoma or lung carcinoma tumors. Ppm1d knockout neutrophils infiltrate tumors extensively. Chemical inhibition of Wip1 in human or mouse neutrophils increases anti-tumor phenotypes, p53-dependent expression of co-stimulatory ligands, and proliferation of co-cultured cytotoxic T cells. These results suggest that inhibition of Wip1 in neutrophils enhances immune anti-tumor responses.


Asunto(s)
Daño del ADN , Inmunidad , Neutrófilos/metabolismo , Proteína Fosfatasa 2C/genética , Proteína Fosfatasa 2C/metabolismo , Animales , Antineoplásicos , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Pulmón , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Linfocitos T , Microambiente Tumoral , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo
16.
Mol Cell Proteomics ; 7(4): 716-27, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18178582

RESUMEN

The tumor suppressor protein p53 is a sequence-specific transcription factor that has crucial roles in apoptosis, cell cycle arrest, cellular senescence, and DNA repair. Following exposure to a variety of stresses, p53 becomes post-translationally modified with concomitant increases in activity and stability. To better understand the role of acetylation of Lys-317 in mouse p53, the effect of ionizing radiation (IR) on the thymocytes of p53(K317R) knock-in mice was studied at the global level. Using cleavable ICAT quantitative mass spectrometry, the effect of IR on protein levels in either the wild type or p53(K317R) thymocytes was determined. We found 102 proteins to be significantly affected by IR in the wild type thymocytes, including several whose expression has been shown to be directly regulated by p53. When the effects of IR in the wild type and p53(K317R) samples were compared, 46 proteins were found to be differently affected (p < 0.05). The p53(K317R) mutation has widespread effects on specific protein levels following IR, including the levels of proteins involved in apoptosis, transcription, and translation. Pathway analysis of the differently regulated proteins suggests an increase in p53 activity in the p53(K317R) thymocytes as well as a decrease in tumor necrosis factor alpha signaling. These results suggest that acetylation of Lys-317 modulates the functions of p53 and influences the cross-talk between the DNA damage response and other signaling pathways.


Asunto(s)
Lisina/metabolismo , Biosíntesis de Proteínas/efectos de la radiación , Proteómica , Linfocitos T/efectos de la radiación , Proteína p53 Supresora de Tumor/metabolismo , Acetilación , Sustitución de Aminoácidos/genética , Animales , Arginina/metabolismo , Espectrometría de Masas/métodos , Ratones , Ratones Mutantes , Mutación , Biosíntesis de Proteínas/genética , Proteínas/análisis , Proteínas/genética , Proteínas/metabolismo , Radiación Ionizante , Transducción de Señal/efectos de la radiación , Linfocitos T/química , Linfocitos T/metabolismo , Transcripción Genética/efectos de la radiación , Proteína p53 Supresora de Tumor/genética
17.
Nucleic Acids Res ; 36(22): 7168-80, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19015127

RESUMEN

PPM1D (Wip1), a type PP2C phosphatase, is expressed at low levels in most normal tissues but is overexpressed in several types of cancers. In cells containing wild-type p53, the levels of PPM1D mRNA and protein increase following exposure to genotoxic stress, but the mechanism of regulation by p53 was unknown. PPM1D also has been identified as a CREB-regulated gene due to the presence of a cyclic AMP response element (CRE) in the promoter. Transient transfection and chromatin immunoprecipitation experiments in HCT116 cells were used to characterize a conserved p53 response element located in the 5' untranslated region (UTR) of the PPM1D gene that is required for the p53-dependent induction of transcription from the human PPM1D promoter. CREB binding to the CRE contributes to the regulation of basal expression of PPM1D and directs transcription initiation at upstream sites. Following exposure to ultraviolet (UV) or ionizing radiation, the abundance of transcripts with short 5' UTRs increased in cells containing wild-type p53, indicating increased utilization of downstream transcription initiation sites. In cells containing wild-type p53, exposure to UV resulted in increased PPM1D protein levels even when PPM1D mRNA levels remained constant, indicating post-transcriptional regulation of PPM1D protein levels.


Asunto(s)
Daño del ADN , Fosfoproteínas Fosfatasas/genética , Elementos de Respuesta , Sitio de Iniciación de la Transcripción , Proteína p53 Supresora de Tumor/metabolismo , Regiones no Traducidas 5' , Animales , Secuencia de Bases , Línea Celular Tumoral , Cromatina/metabolismo , Secuencia Conservada , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Fosfoproteínas Fosfatasas/biosíntesis , Filogenia , Regiones Promotoras Genéticas , Proteína Fosfatasa 2C , ARN Mensajero/biosíntesis , Radiación Ionizante , Rayos Ultravioleta
18.
Biochemistry ; 48(6): 1244-55, 2009 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-19166313

RESUMEN

The tumor suppressor p53 functions as a transcriptional activator for many genes, including several key genes involved in cell cycle arrest and apoptosis. Following DNA damage-induced stress, p53 undergoes extensive posttranslational modification, resulting in increased stability and activity. Two critical cofactors for p53-mediated transactivation are the histone acetyltransferase paralogues CREB-binding protein (CBP) and p300. The N-terminal transactivation domain of p53 interacts with several domains of CBP/p300, including the Taz2 domain. Here, we report the effects of specific p53 phosphorylations on its interaction with the Taz2 domain of p300. Using a competitive fluorescence anisotropy assay, we determined that monophosphorylation of p53 at Ser(15) or Thr(18) increased the affinity of p53(1-39) for Taz2, and diphosphorylations at Ser(15) and Ser(37) or Thr(18) and Ser(20) further increased the affinity. In addition, we identified a second binding site for Taz2 within p53 residues 35-59. This second site bound Taz2 with a similar affinity as the first site, but the binding was unaffected by phosphorylation. Thus, p53 posttranslational modification modulates only one of the two binding sites for p300 Taz2. Further investigation of Taz2 binding to p53(1-39) or p53(35-59) by isothermal titration calorimetry indicated that upon complex formation, the change in heat capacity at constant pressure, DeltaC(p), was negative for both sites, suggesting the importance of hydrophobic interactions. However, the more negative value of DeltaC(p) for Taz2 binding to the first (-330 cal/(mol.K)) compared to the second site (-234 cal/(mol.K)) suggests that the importance of nonpolar and polar interactions differs between the two sites.


Asunto(s)
Proteína p300 Asociada a E1A/química , Proteína p300 Asociada a E1A/metabolismo , Activación Transcripcional/genética , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Calorimetría , Dicroismo Circular , Humanos , Cinética , Datos de Secuencia Molecular , Complejos Multiproteicos/metabolismo , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Termodinámica
19.
Mol Cell Biol ; 26(18): 6859-69, 2006 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16943427

RESUMEN

Posttranslational modifications of p53, including phosphorylation and acetylation, play important roles in regulating p53 stability and activity. Mouse p53 is acetylated at lysine 317 by PCAF and at multiple lysine residues at the extreme carboxyl terminus by CBP/p300 in response to genotoxic and some nongenotoxic stresses. To determine the physiological roles of p53 acetylation at lysine 317, we introduced a Lys317-to-Arg (K317R) missense mutation into the endogenous p53 gene of mice. p53 protein accumulates to normal levels in p53(K317R) mouse embryonic fibroblasts (MEFs) and thymocytes after DNA damage. While p53-dependent gene expression is largely normal in p53(K317R) MEFs after various types of DNA damage, increased p53-dependent apoptosis was observed in p53(K317R) thymocytes, epithelial cells from the small intestine, and cells from the retina after ionizing radiation (IR) as well as in E1A/Ras-expressing MEFs after doxorubicin treatment. Consistent with these findings, p53-dependent expression of several proapoptotic genes was significantly increased in p53(K317R) thymocytes after IR. These findings demonstrate that acetylation at lysine 317 negatively regulates p53 apoptotic activities after DNA damage.


Asunto(s)
Apoptosis , Daño del ADN , Lisina/metabolismo , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Acetilación , Proteínas E1A de Adenovirus/metabolismo , Animales , Células Cultivadas , Células Epiteliales/citología , Células Epiteliales/efectos de la radiación , Fibroblastos/citología , Fibroblastos/efectos de la radiación , Intestino Delgado/citología , Intestino Delgado/efectos de la radiación , Ratones , Ratones Mutantes , Análisis por Micromatrices , Proteínas Mutantes/metabolismo , Proteína Oncogénica p21(ras)/metabolismo , Radiación Ionizante , Retina/citología , Retina/efectos de la radiación , Termodinámica , Timo/citología , Timo/efectos de la radiación
20.
ChemMedChem ; 13(9): 894-901, 2018 05 08.
Artículo en Inglés | MEDLINE | ID: mdl-29476592

RESUMEN

The wild-type p53 induced phosphatase 1, Wip1 (PP2Cδ), is a protein phosphatase 2C (PP2C) family serine/threonine phosphatase that negatively regulates the function of the tumor suppressor p53 and several of its positive regulators such as ATM, Chk1, Chk2, Mdm2, and p38 MAPK. Wip1 dephosphorylates and inactivates its protein targets, which are critical for cellular stress responses. Additionally, Wip1 is frequently amplified and overexpressed in several human cancer types. Because of its negative role in regulating the function of tumor suppressor proteins, Wip1 has been identified as a potential therapeutic target in various types of cancers. Based on a recently reported Wip1 inhibitor (G-1), we performed an extensive structure-activity relationship (SAR) analysis. This led us to interesting findings in SAR trends and to the discovery of new chemical analogues with good specificity and bioavailability.


Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Proteína Fosfatasa 2C/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Humanos , Células MCF-7 , Proteína Fosfatasa 2C/metabolismo , Relación Estructura-Actividad , Células Tumorales Cultivadas
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