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1.
Proc Natl Acad Sci U S A ; 109(5): 1685-90, 2012 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-22307631

RESUMO

The p53 transcription factor modulates gene expression programs that induce cell cycle arrest, senescence, or apoptosis, thereby preventing tumorigenesis. However, the mechanisms by which these fates are selected are unclear. Our objective is to understand p53 target gene selection and, thus, enable its optimal manipulation for cancer therapy. We have generated targeted transgenic reporter mice in which EGFP expression is driven by p53 transcriptional activity at a response element from either the p21 or Puma promoter, which induces cell cycle arrest/senescence and apoptosis, respectively. We demonstrate that we could monitor p53 activity in vitro and in vivo and detect variations in p53 activity depending on the response element, tissue type, and stimulus, thereby validating our reporter system and illustrating its utility for preclinical drug studies. Our results also show that the sequence of the p53 response element itself is sufficient to strongly influence p53 target gene selection. Finally, we use our reporter system to provide evidence for p53 transcriptional activity during early embryogenesis, showing that p53 is active as early as embryonic day 3.5 and that p53 activity becomes restricted to embryonic tissue by embryonic day 6.5. The data from this study demonstrate that these reporter mice could serve as powerful tools to answer questions related to basic biology of the p53 pathway, as well as cancer therapy and drug discovery.


Assuntos
Genes Reporter , Genes p53 , Regiões Promotoras Genéticas , Animais , Western Blotting , Desenvolvimento Embrionário , Citometria de Fluxo , Genes erbB-1 , Camundongos , Camundongos Transgênicos
2.
J Pathol ; 223(2): 116-26, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21125670

RESUMO

Mutations in the TP53 (p53) gene are present in a large fraction of human tumours, which frequently express mutant p53 proteins at high but heterogeneous levels. The clinical significance of this protein accumulation remains clouded. Mouse models bearing knock-in mutations of p53 have established that the mutant p53 proteins can drive tumour formation, invasion and metastasis through dominant negative inhibition of wild-type p53 as well as through gain of function or 'neomorphic' activities that can inhibit or activate the function of other proteins. These models have also shown that mutation alone does not confer stability, so the variable staining of mutant proteins seen in human cancers reflects tumour-specific activation of p53-stabilizing pathways. Blocking the accumulation and activity of mutant p53 proteins may thus provide novel cancer therapeutic and diagnostic targets, but their induction by chemotherapy may paradoxically limit the effectiveness of these treatments.


Assuntos
Genes p53/genética , Mutação , Neoplasias/genética , Animais , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Neoplasias/metabolismo , Proteína Supressora de Tumor p53/metabolismo
3.
BMC Biochem ; 9: 4, 2008 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-18234089

RESUMO

BACKGROUND: The delivery of ubiquitinated proteins to the proteasome for degradation is a key step in the regulation of the ubiquitin-proteasome pathway, yet the mechanisms underlying this step are not understood in detail. The Rad23 family of proteins is known to bind ubiquitinated proteins through its two ubiquitin-associated (UBA) domains, and may participate in the delivery of ubiquitinated proteins to the proteasome through docking via the Rad23 ubiquitin-like (UBL) domain. RESULTS: In this study, we investigate how the interaction between the UBL and UBA domains may modulate ubiquitin recognition and the delivery of ubiquitinated proteins to the proteasome by autoinhibition. We have explored a competitive binding model using specific mutations in the UBL domain. Disrupting the intramolecular UBL-UBA domain interactions in HHR23A indeed potentiates ubiquitin-binding. Additionally, the analogous surface on the Rad23 UBL domain overlaps with that required for interaction with both proteasomes and the ubiquitin ligase Ufd2. We have found that mutation of residues on this surface affects the ability of Rad23 to deliver ubiquitinated proteins to the proteasome. CONCLUSION: We conclude that the competition of ubiquitin-proteasome pathway components for surfaces on Rad23 is important for the role of the Rad23 family proteins in proteasomal targeting.


Assuntos
Ligação Competitiva , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Ligação Competitiva/genética , Humanos , Modelos Biológicos , Mutação , Poliubiquitina/metabolismo , Estrutura Terciária de Proteína/genética , Ubiquitina/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/genética
4.
Biochim Biophys Acta ; 1695(1-3): 73-87, 2004 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-15571810

RESUMO

Many biological processes rely on targeted protein degradation, the dysregulation of which contributes to the pathogenesis of various diseases. Ubiquitin plays a well-established role in this process, in which the covalent attachment of polyubiquitin chains to protein substrates culminates in their degradation via the proteasome. The three-dimensional structural topology of ubiquitin is highly conserved as a domain found in a variety of proteins of diverse biological function. Some of these so-called "ubiquitin family proteins" have recently been shown to bind components of the 26S proteasome via their ubiquitin-like domains, thus implicating proteasome activity in pathways other than protein degradation. In this chapter, we provide a structural perspective of how the ubiquitin family of proteins interacts with the proteasome.


Assuntos
Complexo de Endopeptidases do Proteassoma/química , Ubiquitina/química , Sequência de Aminoácidos , Animais , Sequência Conservada , Cristalografia por Raios X , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Complexo de Endopeptidases do Proteassoma/fisiologia , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteínas/metabolismo , Eletricidade Estática , Ubiquitina/fisiologia
5.
Oncotarget ; 6(20): 17968-80, 2015 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-26255629

RESUMO

The tumour suppressor p53 is regulated primarily at the protein level. In normal tissues its levels are maintained at a very low level by the action of specific E3 ligases and the ubiquitin proteosome pathway. The mutant p53 protein contributes to transformation, metastasis and drug resistance. High levels of mutant p53 can be found in tumours and the accumulation of mutant p53 has previously been reported in pathologically normal cells in human skin. We show for the first time that similarly elevated levels of mutant p53 can be detected in apparently normal cells in a mutant p53 knock-in mouse model. In fact, in the small intestine, mutant p53 spontaneously accumulates in a manner dependent on gene dosage and cell type. Mutant p53 protein is regulated similarly to wild type p53, which can accumulate rapidly after induction by ionising radiation or Mdm2 inhibitors, however, the clearance of mutant p53 protein is much slower than wild type p53. The accumulation of the protein in the murine small intestine is limited to the cycling, crypt base columnar cells and proliferative zone and is lost as the cells differentiate and exit the cell cycle. Loss of Mdm2 results in even higher levels of p53 expression but p53 is still restricted to proliferating cells in the small intestine. Therefore, the small intestine of these p53 mutant mice is an experimental system in which we can dissect the molecular pathways leading to p53 accumulation, which has important implications for cancer prevention and therapy.


Assuntos
Ciclo Celular , Proliferação de Células , Intestino Delgado/metabolismo , Mutação , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Fatores Etários , Animais , Diferenciação Celular , Dano ao DNA , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Genótipo , Intestino Delgado/diagnóstico por imagem , Intestino Delgado/efeitos dos fármacos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Radiografia , Fatores de Tempo , Técnicas de Cultura de Tecidos
6.
J Clin Invest ; 124(7): 3263-73, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24911145

RESUMO

The number of newly formed neurons declines rapidly during aging, and this decrease in neurogenesis is associated with decreased function of neural stem/progenitor cells (NPCs). Here, we determined that a WIP1-dependent pathway regulates NPC differentiation and contributes to the age-associated decline of neurogenesis. Specifically, we found that WIP1 is expressed in NPCs of the mouse subventricular zone (SVZ) and aged animals with genetically enhanced WIP1 expression exhibited higher NPC numbers and neuronal differentiation compared with aged WT animals. Additionally, augmenting WIP1 expression in aged animals markedly improved neuron formation and rescued a functional defect in fine odor discrimination in aged mice. We identified the WNT signaling pathway inhibitor DKK3 as a key downstream target of WIP1 and found that expression of DKK3 in the SVZ is restricted to NPCs. Using murine reporter strains, we determined that DKK3 inhibits neuroblast formation by suppressing WNT signaling and Dkk3 deletion or pharmacological activation of the WNT pathway improved neuron formation and olfactory function in aged mice. We propose that WIP1 controls DKK3-dependent inhibition of neuronal differentiation during aging and suggest that regulating WIP1 levels could prevent certain aspects of functional decline of the aging brain.


Assuntos
Envelhecimento/metabolismo , Envelhecimento/patologia , Neurogênese/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Via de Sinalização Wnt , Proteínas Adaptadoras de Transdução de Sinal , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/patologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Diferenciação Celular , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Neurogênese/genética , Percepção Olfatória/fisiologia , Fosfoproteínas Fosfatases/genética , Proteína Fosfatase 2C , Proteína Supressora de Tumor p53/metabolismo
7.
ACS Chem Biol ; 8(3): 506-12, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23214419

RESUMO

By using a phage display derived peptide as an initial template, compounds have been developed that are highly specific against Mdm2/Mdm4. These compounds exhibit greater potency in p53 activation and protein-protein interaction assays than a compound derived from the p53 wild-type sequence. Unlike Nutlin, a small molecule inhibitor of Mdm2/Mdm4, the phage derived compounds can arrest cells resistant to p53 induced apoptosis over a wide concentration range without cellular toxicity, suggesting they are highly suitable for cyclotherapy.


Assuntos
Peptídeos/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Proteínas de Ciclo Celular , Humanos , Modelos Moleculares , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Peptídeos/química , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/química
8.
Transcription ; 3(5): 240-4, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22885979

RESUMO

Understanding how the tumor suppressor p53 induces cell cycle arrest or apoptosis is critical for developing chemotherapeutic strategies. We have generated targeted transgenic reporter mice with which we can study p53 activity at specific promoters, and propose a model in which p53 protein conformation is key to target gene selection.


Assuntos
Proteína Supressora de Tumor p53/genética , Animais , Pontos de Checagem do Ciclo Celular , Morte Celular , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Transgênicos , Conformação Proteica , Elementos de Resposta , Proteína Supressora de Tumor p53/química
9.
Cell Cycle ; 10(7): 1100-8, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21422812

RESUMO

Embryonic stem (ES) cells are invaluable for their therapeutic potential as well as for the study of early development. Their clinical use demands an understanding of ES cell differentiation, particularly with respect to cell proliferation and the maintenance of genomic integrity, processes for which the transcription factor p53 is essential. However, although the function of p53 as a tumor suppressor has been extensively studied, its role in ES cell biology has not been clearly elucidated. To study p53 activity and regulation in differentiating ES cells, we used knock-in constructs to create a novel reporter system that provides a direct readout of p53 transcriptional activity. We thereby determine that the p53 pathway is active in ES cells, but that p53 activity and the p53-dependent stress response decrease upon differentiation. Although p53 protein levels and activity are usually primarily controlled by the ubiquitin ligase MDM2, we identify the MDM2 homolog MDM4 as the key modulator of p53 activity in differentiating ES cells. Our results provide a better understanding of ES cell regulation and could help to optimize ES cell differentiation protocols for their use in regenerative medicine.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/citologia , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Western Blotting , Primers do DNA/genética , Células-Tronco Embrionárias/metabolismo , Técnicas de Introdução de Genes , Genes Reporter/genética , Proteínas de Fluorescência Verde , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
10.
Cancer Cell ; 18(3): 201-2, 2010 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-20832747
11.
Biochemistry ; 41(6): 1767-77, 2002 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-11827521

RESUMO

The 26S proteasome is essential for the proteolysis of proteins that have been covalently modified by the attachment of polyubiquitinated chains. Although the 20S core particle performs the degradation, the 19S regulatory cap complex is responsible for recognition of polyubiquitinated substrates. We have focused on how the S5a component of the 19S complex interacts with different ubiquitin-like (ubl) modules, to advance our understanding of how polyubiquitinated proteins are targeted to the proteasome. To achieve this, we have determined the solution structure of the ubl domain of hPLIC-2 and obtained a structural model of hHR23a by using NMR spectroscopy and homology modeling. We have also compared the S5a binding properties of ubiquitin, SUMO-1, and the ubl domains of hPLIC-2 and hHR23a and have identified the residues on their respective S5a contact surfaces. We provide evidence that the S5a-binding surface on the ubl domain of hPLIC-2 is required for its interaction with the proteasome. This study provides structural insights into protein recognition by the proteasome, and illustrates how the protein surface of a commonly utilized fold has highly evolved for various biological roles.


Assuntos
Proteínas de Transporte/química , Proteínas de Ciclo Celular , Cisteína Endopeptidases/química , Complexos Multienzimáticos/química , Ubiquitinas/química , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Proteínas Relacionadas à Autofagia , Sítios de Ligação , Proteínas de Transporte/metabolismo , Cisteína Endopeptidases/metabolismo , Humanos , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos/metabolismo , Complexo de Endopeptidases do Proteassoma , Dobramento de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas , Proteínas de Ligação a RNA , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína SUMO-1/química , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Ubiquitinas/genética , Ubiquitinas/metabolismo
12.
Biochemistry ; 42(46): 13529-35, 2003 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-14621999

RESUMO

Ubiquitin is a prominent regulatory protein in numerous biological processes, including targeted protein degradation, endocytic sorting, transcriptional control, intranuclear localization, and retroviral virion budding. Ubiquitin-associated (UBA) domains, ubiquitin interacting motifs (UIM), and coupling of ubiquitin conjugation to ER degradation (CUE) motifs have been identified as ubiquitin receptors. The DNA repair protein hHR23a has two UBA domains that can each bind ubiquitin in addition to an N-terminal UBL domain that binds S5a and S2, two components of the 26S proteasome. Here we reveal hHR23a recognizes ubiquitin through a predominately hydrophobic surface formed by residues within alpha1 and alpha3 of each of its UBA domains. These two UBA surfaces bind a region on ubiquitin that includes K48. These findings have implications for published studies revealing that hHR23a inhibits K48-linked polyubiquitin chain formation. In addition, by using (15)N NMR relaxation experiments, we find that binding ubiquitin requires a structural change in hHR23a. HHR23 proteins are hypothesized to link ubiquitin to S5a, and we provide direct evidence that hHR23 could form a ternary complex with ubiquitin and S5a.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lisina/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Eletricidade Estática , Ubiquitina/genética
13.
Proc Natl Acad Sci U S A ; 100(22): 12694-9, 2003 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-14557549

RESUMO

The Rad23 family of proteins, including the human homologs hHR23a and hHR23b, stimulates nucleotide excision repair and has been shown to provide a novel link between proteasome-mediated protein degradation and DNA repair. In this work, we illustrate how the proteasomal subunit S5a regulates hHR23a protein structure. By using NMR spectroscopy, we have elucidated the structure and dynamic properties of the 40-kDa hHR23a protein and show it to contain four structured domains connected by flexible linker regions. In addition, we reveal that these domains interact in an intramolecular fashion, and by using residual dipolar coupling data in combination with chemical shift perturbation analysis, we present the hHR23a structure. By itself, hHR23a adopts a closed conformation defined by the interaction of an N-terminal ubiquitin-like domain with two ubiquitin-associated domains. Interestingly, binding of the proteasomal subunit S5a disrupts the hHR23a interdomain interactions and thereby causes it to adopt an opened conformation.


Assuntos
Proteínas de Transporte/química , Cisteína Endopeptidases/química , Reparo do DNA , Proteínas de Ligação a DNA/química , Complexos Multienzimáticos/química , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/metabolismo , Cisteína Endopeptidases/metabolismo , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos/metabolismo , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas de Ligação a RNA
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