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1.
J Biomol Tech ; 30(3): 36-44, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31452645

RESUMO

Shared scientific resources, also known as core facilities, support a significant portion of the research conducted at biomolecular research institutions. The Association of Biomolecular Resource Facilities (ABRF) established the Committee on Core Rigor and Reproducibility (CCoRRe) to further its mission of integrating advanced technologies, education, and communication in the operations of shared scientific resources in support of reproducible research. In order to first assess the needs of the scientific shared resource community, the CCoRRe solicited feedback from ABRF members via a survey. The purpose of the survey was to gain information on how U.S. National Institutes of Health (NIH) initiatives on advancing scientific rigor and reproducibility influenced current services and new technology development. In addition, the survey aimed to identify the challenges and opportunities related to implementation of new reporting requirements and to identify new practices and resources needed to ensure rigorous research. The results revealed a surprising unfamiliarity with the NIH guidelines. Many of the perceived challenges to the effective implementation of best practices (i.e., those designed to ensure rigor and reproducibility) were similarly noted as a challenge to effective provision of support services in a core setting. Further, most cores routinely use best practices and offer services that support rigor and reproducibility. These services include access to well-maintained instrumentation and training on experimental design and data analysis as well as data management. Feedback from this survey will enable the ABRF to build better educational resources and share critical best-practice guidelines. These resources will become important tools to the core community and the researchers they serve to impact rigor and transparency across the range of science and technology.

2.
Cell Metab ; 2018 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-30449685

RESUMO

Identification of cell-surface markers specific to human pancreatic ß cells would allow in vivo analysis and imaging. Here we introduce a biomarker, ectonucleoside triphosphate diphosphohydrolase-3 (NTPDase3), that is expressed on the cell surface of essentially all adult human ß cells, including those from individuals with type 1 or type 2 diabetes. NTPDase3 is expressed dynamically during postnatal human pancreas development, appearing first in acinar cells at birth, but several months later its expression declines in acinar cells while concurrently emerging in islet ß cells. Given its specificity and membrane localization, we utilized an NTPDase3 antibody for purification of live human ß cells as confirmed by transcriptional profiling, and, in addition, for in vivo imaging of transplanted human ß cells. Thus, NTPDase3 is a cell-surface biomarker of adult human ß cells, and the antibody directed to this protein should be a useful new reagent for ß cell sorting, in vivo imaging, and targeting.

3.
J Clin Invest ; 127(12): 4462-4476, 2017 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-29130932

RESUMO

p120-Catenin (p120) functions as a tumor suppressor in intestinal cancer, but the mechanism is unclear. Here, using conditional p120 knockout in Apc-sensitized mouse models of intestinal cancer, we have identified p120 as an "obligatory" haploinsufficient tumor suppressor. Whereas monoallelic loss of p120 was associated with a significant increase in tumor multiplicity, loss of both alleles was never observed in tumors from these mice. Moreover, forced ablation of the second allele did not further enhance tumorigenesis, but instead induced synthetic lethality in combination with Apc loss of heterozygosity. In tumor-derived organoid cultures, elimination of both p120 alleles resulted in caspase-3-dependent apoptosis that was blocked by inhibition of Rho kinase (ROCK). With ROCK inhibition, however, p120-ablated organoids exhibited a branching phenotype and a substantial increase in cell proliferation. Access to data from Sleeping Beauty mutagenesis screens afforded an opportunity to directly assess the tumorigenic impact of p120 haploinsufficiency relative to other candidate drivers. Remarkably, p120 ranked third among the 919 drivers identified. Cofactors α-catenin and epithelial cadherin (E-cadherin) were also among the highest scoring candidates, indicating a mechanism at the level of the intact complex that may play an important role at very early stages of of intestinal tumorigenesis while simultaneously restricting outright loss via synthetic lethality.

4.
Cold Spring Harb Protoc ; 2017(11): pdb.prot093831, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-29093202

RESUMO

For research groups needing to isotype a significant number of antibodies on a fairly regular basis, the protocol outlined here provides a cost-effective option. In this simple sandwich ELISA, the monoclonal antibody is first captured with antibodies that discriminate between heavy-chain isotypes and then is detected with antibodies specific for each light chain. This combination ensures that free light chain secreted by hybridomas does not yield a false-positive result, because in a true positive both the capture and detecting antibodies bind the monoclonal antibody.


Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/classificação , Ensaio de Imunoadsorção Enzimática/métodos , Isotipos de Imunoglobulinas/análise , Animais , Roedores
5.
Cold Spring Harb Protoc ; 2017(11): pdb.top093823, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-29093209

RESUMO

Perhaps because they are such commonly used tools, many researchers view antibodies one-dimensionally: Antibody Y binds antigen X. Although few techniques require a comprehensive understanding of any particular antibody's characteristics, well-executed experiments do require a basic appreciation of what is known and, equally as important, what is not known about the antibody being used. Ignorance of the relevant antibody characteristics critical for a particular assay can easily lead to loss of precious resources (time, money, and limiting amounts of sample) and, in worst-case scenarios, erroneous conclusions. Here, we describe various antibody characteristics to provide a more well-rounded perspective of these critical reagents. With this information, it will be easier to make informed decisions on how best to choose and use the available antibodies, as well as knowing when it is essential and how to determine a particular as yet-undefined characteristic.


Assuntos
Anticorpos/imunologia , Anticorpos/metabolismo , Antígenos/metabolismo , Epitopos/metabolismo , Imunoensaio/métodos
6.
CBE Life Sci Educ ; 16(3)2017.
Artigo em Inglês | MEDLINE | ID: mdl-28798213

RESUMO

Many biomedical PhD trainees lack exposure to business principles, which limits their competitiveness and effectiveness in academic and industry careers. To fill this training gap, we developed Business and Management Principles for Scientists, a semester-long program that combined didactic exposure to business fundamentals with practical team-based projects aimed at solving real business problems encountered by institutional shared--resource core facilities. The program also included a retreat featuring presentations by and networking with local life science entrepreneurs and final team presentations to expert judges. Quantitative and qualitative metrics were used to evaluate the program's impact on trainees. A pretest-posttest approach was used to assess trainees' baseline knowledge and mastery of module concepts, and each individual's pretest and posttest responses were compared. The mean score improved by more than 17 percentage points. Trainees also took an online survey to provide feedback about the module. Nearly all participants agreed or strongly agreed that the module was a valuable use of their time and will help guide their career decisions and that project work helped drive home module concepts. More than 75% of trainees reported discussing the module with their research advisors, and all of these participants reported supportive or neutral responses. Collectively, the trainee feedback about the module, improvement in test scores, and trainee perception of advisor support suggest that this short module is an effective method of providing scientists with efficient and meaningful exposure to business concepts.


Assuntos
Pesquisa Biomédica , Pesquisadores/educação , Desenvolvimento de Pessoal/métodos , Educação de Pós-Graduação , Humanos
7.
J Virol ; 91(4)2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-27928010

RESUMO

Reovirus attachment protein σ1 engages glycan receptors and junctional adhesion molecule-A (JAM-A) and is thought to undergo a conformational change during the proteolytic disassembly of virions to infectious subvirion particles (ISVPs) that accompanies cell entry. The σ1 protein is also the primary target of neutralizing antibodies. Here, we present a structural and functional characterization of two neutralizing antibodies that target σ1 of serotype 1 (T1) and serotype 3 (T3) reoviruses. The crystal structures revealed that each antibody engages its cognate σ1 protein within the head domain via epitopes distinct from the JAM-A-binding site. Surface plasmon resonance and cell-binding assays indicated that both antibodies likely interfere with JAM-A engagement by steric hindrance. To define the interplay between the carbohydrate receptor and antibody binding, we conducted hemagglutination inhibition assays using virions and ISVPs. The glycan-binding site of T1 σ1 is located in the head domain and is partly occluded by the bound Fab in the crystal structure. The T1-specific antibody inhibited hemagglutination by virions and ISVPs, probably via direct interference with glycan engagement. In contrast to T1 σ1, the carbohydrate-binding site of T3 σ1 is located in the tail domain, distal to the antibody epitope. The T3-specific antibody inhibited hemagglutination by T3 virions but not ISVPs, indicating that the antibody- and glycan-binding sites in σ1 are in closer spatial proximity on virions than on ISVPs. Our results provide direct evidence for a structural rearrangement of σ1 during virion-to-ISVP conversion and contribute new information about the mechanisms of antibody-mediated neutralization of reovirus. IMPORTANCE: Virus attachment proteins mediate binding to host cell receptors, serve critical functions in cell and tissue tropism, and are often targeted by the neutralizing antibody response. The structural investigation of antibody-antigen complexes can provide valuable information for understanding the molecular basis of virus neutralization. Studies with enveloped viruses, such as HIV and influenza virus, have helped to define sites of vulnerability and guide vaccination strategies. By comparison, less is known about antibody binding to nonenveloped viruses. Here, we structurally investigated two neutralizing antibodies that bind the attachment protein σ1 of reovirus. Furthermore, we characterized the neutralization efficiency, the binding affinity for σ1, and the effect of the antibodies on reovirus receptor engagement. Our analysis defines reovirus interactions with two neutralizing antibodies, allows us to propose a mechanism by which they block virus infection, and provides evidence for a conformational change in the σ1 protein during viral cell entry.


Assuntos
Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Reoviridae , Proteínas Virais/química , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Anticorpos Antivirais/farmacologia , Sítios de Ligação , Células CHO , Linhagem Celular , Cricetulus , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Testes de Neutralização , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Reoviridae/efeitos dos fármacos , Reoviridae/fisiologia , Relação Estrutura-Atividade , Proteínas Virais/imunologia , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos
8.
Med Sci Educ ; 27(4): 759-765, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29326856

RESUMO

As future physicians, nearly all medical students will be required to provide face-to-face feedback. Moreover, receiving high quality feedback from multiple perspectives is particularly valuable during the pre-clerkship training period. To address these needs, we developed a straightforward, easy to implement exercise that affords students the opportunity to practice giving and receiving feedback with peers. We describe how this exercise has been tailored to fit within the case-based learning small groups of our first-year curriculum and how to enhance the activity by weaving the basic principles of quality feedback into preparation sessions. This exercise has been valued greatly by students.

9.
Clin Transl Med ; 4(1): 63, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26154059

RESUMO

BACKGROUND: Activation of coagulation by expression of tissue factor (TF) in the airspace is a hallmark of acute lung injury (ALI) but the timing of TF activation in relationship to increases in lung permeability and inflammation are unknown. METHODS: To test the hypothesis that TF is upregulated early in the course of acute bleomycin lung injury and precedes increased permeability and inflammation we studied the early course of bleomycin-induced ALI in mice. Mice were treated with 0.04U intratracheal bleomycin or vehicle control and bronchoalveolar lavage (BAL) and lung tissue were collected daily for 7 days. Whole lung TF mRNA was determined by QT-PCR. TF protein was assessed by ELISA and immunostaining. BAL procoagulant activity was measured by BAL clot time and thrombin-antithrombin complexes. Inflammation was assessed by BAL cell count, differentials and CXCL1/KC concentration. Lung permeability was assessed by BAL protein and lung wet to dry weight ratio. RESULTS: Expression of CXCL1 occurred by day 1. BAL protein and lung wet-to-dry weight ratio increased significantly by day 3. TF mRNA and BAL procoagulant activity peaked on day 4 while whole lung TF protein peaked on day 6. Changes in permeability and procoagulant activity preceded inflammatory cell influx which was maximal at day 6 while whole lung TF protein peaked along with inflammation. CONCLUSION: These data demonstrate that cytokine upregulation is the earliest response to bleomycin administration, followed by increased lung permeability, upregulation of TF, and recruitment of inflammatory cells.

10.
Am J Respir Cell Mol Biol ; 53(5): 719-27, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25884207

RESUMO

Tissue factor (TF) initiates the extrinsic coagulation cascade in response to tissue injury, leading to local fibrin deposition. Low levels of TF in mice are associated with increased severity of acute lung injury (ALI) after intratracheal LPS administration. However, the cellular sources of the TF required for protection from LPS-induced ALI remain unknown. In the current study, transgenic mice with cell-specific deletions of TF in the lung epithelium or myeloid cells were treated with intratracheal LPS to determine the cellular sources of TF important in direct ALI. Cell-specific deletion of TF in the lung epithelium reduced total lung TF expression to 39% of wild-type (WT) levels at baseline and to 29% of WT levels after intratracheal LPS. In contrast, there was no reduction of TF with myeloid cell TF deletion. Mice lacking myeloid cell TF did not differ from WT mice in coagulation, inflammation, permeability, or hemorrhage. However, mice lacking lung epithelial TF had increased tissue injury, impaired activation of coagulation in the airspace, disrupted alveolar permeability, and increased alveolar hemorrhage after intratracheal LPS. Deletion of epithelial TF did not affect alveolar permeability in an indirect model of ALI caused by systemic LPS infusion. These studies demonstrate that the lung epithelium is the primary source of TF in the lung, contributing 60-70% of total lung TF, and that lung epithelial, but not myeloid, TF may be protective in direct ALI.


Assuntos
Lesão Pulmonar Aguda/genética , Coagulação Sanguínea/genética , Permeabilidade Capilar/genética , Hemorragia/genética , Síndrome do Desconforto Respiratório do Adulto/genética , Tromboplastina/genética , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Animais , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Expressão Gênica , Hemorragia/induzido quimicamente , Hemorragia/metabolismo , Hemorragia/patologia , Lipopolissacarídeos , Camundongos , Camundongos Knockout , Células Mieloides/metabolismo , Células Mieloides/patologia , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologia , Síndrome do Desconforto Respiratório do Adulto/induzido quimicamente , Síndrome do Desconforto Respiratório do Adulto/metabolismo , Síndrome do Desconforto Respiratório do Adulto/patologia , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Tromboplastina/deficiência
11.
PLoS One ; 7(12): e51205, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23251453

RESUMO

Myeloid translocation genes (MTGs) are transcriptional corepressors originally identified in acute myelogenous leukemia that have recently been linked to epithelial malignancy with non-synonymous mutations identified in both MTG8 and MTG16 in colon, breast, and lung carcinoma in addition to functioning as negative regulators of WNT and Notch signaling. A yeast two-hybrid approach was used to discover novel MTG binding partners. This screen identified the Zinc fingers, C2H2 and BTB domain containing (ZBTB) family members ZBTB4 and ZBTB38 as MTG16 interacting proteins. ZBTB4 is downregulated in breast cancer and modulates p53 responses. Because ZBTB33 (Kaiso), like MTG16, modulates Wnt signaling at the level of TCF4, and its deletion suppresses intestinal tumorigenesis in the Apc(Min) mouse, we determined that Kaiso also interacted with MTG16 to modulate transcription. The zinc finger domains of Kaiso as well as ZBTB4 and ZBTB38 bound MTG16 and the association with Kaiso was confirmed using co-immunoprecipitation. MTG family members were required to efficiently repress both a heterologous reporter construct containing Kaiso binding sites (4×KBS) and the known Kaiso target, Matrix metalloproteinase-7 (MMP-7/Matrilysin). Moreover, chromatin immunoprecipitation studies placed MTG16 in a complex occupying the Kaiso binding site on the MMP-7 promoter. The presence of MTG16 in this complex, and its contributions to transcriptional repression both required Kaiso binding to its binding site on DNA, establishing MTG16-Kaiso binding as functionally relevant in Kaiso-dependent transcriptional repression. Examination of a large multi-stage CRC expression array dataset revealed patterns of Kaiso, MTG16, and MMP-7 expression supporting the hypothesis that loss of either Kaiso or MTG16 can de-regulate a target promoter such as that of MMP-7. These findings provide new insights into the mechanisms of transcriptional control by ZBTB family members and broaden the scope of co-repressor functions for the MTG family, suggesting coordinate regulation of transcription by Kaiso/MTG complexes in cancer.


Assuntos
Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Fatores de Transcrição/fisiologia , Transcrição Genética , Proteínas Supressoras de Tumor/metabolismo , Sítios de Ligação , Imunoprecipitação da Cromatina , Imunofluorescência , Técnicas de Silenciamento de Genes , Células HEK293 , Células HT29 , Humanos , Células K562 , Metaloproteinase 7 da Matriz/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fatores de Transcrição/genética
12.
Hybridoma (Larchmt) ; 31(4): 246-54, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22894777

RESUMO

The coiled-coil domain-containing delta-interacting protein A (DIPA) is a transcription factor implicated in developmental regulation. DIPA is the first protein discovered to selectively interact with the p120-catenin (p120) isoform 1, an alternatively spliced form of p120 expressed preferentially in mesenchymal cells. Although a small fraction of p120 can be observed in the nucleus under certain circumstances, the vast majority of it associates with classical cadherins at adherens junctions. We observed for the first time that a discrete fraction of DIPA exists at cell-cell junctions, in addition to its predominantly nuclear localization. Thus, the p120-DIPA interaction may regulate cell signaling and/or transcriptional events, as has been described previously for ß-catenin and the LEF/TCF transcription factor family. To facilitate further study of DIPA and to determine the physiological relevance of its interaction with p120, we have generated and characterized a panel of five DIPA-specific monoclonal antibodies (MAbs) that function in immunoblotting, immunoprecipitation, and immunofluorescence assays.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Anticorpos Monoclonais Murinos/química , Cateninas/metabolismo , Imunoglobulina G/química , Proteínas Repressoras/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais Murinos/biossíntese , Especificidade de Anticorpos , Western Blotting , Linhagem Celular , Cães , Técnica Indireta de Fluorescência para Anticorpo , Técnicas de Silenciamento de Genes , Humanos , Imunoglobulina G/biossíntese , Imunoprecipitação , Camundongos , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Interferência de RNA , Ratos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
13.
Development ; 139(10): 1754-64, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22461563

RESUMO

Although p120-catenin (p120) is crucial for E-cadherin function, ablation experiments in epithelial tissues from different organ systems reveal markedly different effects. Here, we examine for the first time the consequences of p120 knockout during mouse mammary gland development. An MMTV-Cre driver was used to target knockout to the epithelium at the onset of puberty. p120 ablation was detected in approximately one-quarter of the nascent epithelium at the forth week post-partum. However, p120 null cells were essentially nonadherent, excluded from the process of terminal end bud (TEB) morphogenesis and lost altogether by week six. This elimination process caused a delay in TEB outgrowth, after which the gland developed normally from cells that had retained p120. Mechanistic studies in vitro indicate that TEB dysfunction is likely to stem from striking E-cadherin loss, failure of cell-cell adhesion and near total exclusion from the collective migration process. Our findings reveal an essential role for p120 in mammary morphogenesis.


Assuntos
Cateninas/metabolismo , Glândulas Mamárias Animais/metabolismo , Morfogênese/fisiologia , Animais , Western Blotting , Cateninas/genética , Linhagem Celular , Células Cultivadas , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Humanos , Imuno-Histoquímica , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/embriologia , Camundongos , Morfogênese/genética , Cicatrização/genética , Cicatrização/fisiologia
14.
PLoS One ; 6(1): e16206, 2011 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-21283770

RESUMO

The difficulty of maintaining intact protein complexes while minimizing non-specific background remains a significant limitation in proteomic studies. Labile interactions, such as the interaction between p120-catenin and the E-cadherin complex, are particularly challenging. Using the cadherin complex as a model-system, we have developed a procedure for efficient recovery of otherwise labile protein-protein interactions. We have named the procedure "ReCLIP" (Reversible Cross-Link Immuno-Precipitation) to reflect the primary elements of the method. Using cell-permeable, thiol-cleavable crosslinkers, normally labile interactions (i.e. p120 and E-cadherin) are stabilized in situ prior to isolation. After immunoprecipitation, crosslinked binding partners are selectively released and all other components of the procedure (i.e. beads, antibody, and p120 itself) are discarded. The end result is extremely efficient recovery with exceptionally low background. ReCLIP therefore appears to provide an excellent alternative to currently available affinity-purification approaches, particularly for studies of labile complexes.


Assuntos
Complexos Multiproteicos/análise , Reagentes para Ligações Cruzadas , Imunoprecipitação , Métodos , Complexos Multiproteicos/isolamento & purificação , Estabilidade Proteica , Compostos de Sulfidrila
15.
PLoS One ; 5(12): e15747, 2010 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-21209830

RESUMO

BACKGROUND: p120-catenin (p120) is the prototypical member of a subclass of armadillo-related proteins that includes δ-catenin/NPRAP, ARVCF, p0071, and the more distantly related plakophilins 1-3. In vertebrates, p120 is essential in regulating surface expression and stability of all classical cadherins, and directly interacts with Kaiso, a BTB/ZF family transcription factor. METHODOLOGY/PRINCIPAL FINDINGS: To clarify functional relationships between these proteins and how they relate to the classical cadherins, we have examined the proteomes of 14 diverse vertebrate and metazoan species. The data reveal a single ancient δ-catenin-like p120 family member present in the earliest metazoans and conserved throughout metazoan evolution. This single p120 family protein is present in all protostomes, and in certain early-branching chordate lineages. Phylogenetic analyses suggest that gene duplication and functional diversification into "p120-like" and "δ-catenin-like" proteins occurred in the urochordate-vertebrate ancestor. Additional gene duplications during early vertebrate evolution gave rise to the seven vertebrate p120 family members. Kaiso family members (i.e., Kaiso, ZBTB38 and ZBTB4) are found only in vertebrates, their origin following that of the p120-like gene lineage and coinciding with the evolution of vertebrate-specific mechanisms of epigenetic gene regulation by CpG island methylation. CONCLUSIONS/SIGNIFICANCE: The p120 protein family evolved from a common δ-catenin-like ancestor present in all metazoans. Through several rounds of gene duplication and diversification, however, p120 evolved in vertebrates into an essential, ubiquitously expressed protein, whereas loss of the more selectively expressed δ-catenin, p0071 and ARVCF are tolerated in most species. Together with phylogenetic studies of the vertebrate cadherins, our data suggest that the p120-like and δ-catenin-like genes co-evolved separately with non-neural (E- and P-cadherin) and neural (N- and R-cadherin) cadherin lineages, respectively. The expansion of p120 relative to δ-catenin during vertebrate evolution may reflect the pivotal and largely disproportionate role of the non-neural cadherins with respect to evolution of the wide range of somatic morphology present in vertebrates today.


Assuntos
Cateninas/genética , Animais , Caderinas/metabolismo , Cateninas/metabolismo , Evolução Molecular , Humanos , Modelos Biológicos , Família Multigênica , Neurônios/metabolismo , Filogenia , Estrutura Terciária de Proteína , Proteoma , Proteômica/métodos , Especificidade da Espécie
16.
Cell ; 127(5): 1027-39, 2006 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-17129786

RESUMO

Integration of receptor tyrosine kinase, integrin, and cadherin activities is crucial for normal cell growth, motility, and adhesion. Here, we describe roles for p120-catenin (p120) and p190RhoGAP that coordinate crosstalk between these systems and regulate cadherin function. Surprisingly, PDGFR-induced actin remodeling in NIH3T3 cells is blocked in the absence of p120, and the cells are partially transformed via constitutive activation of Rho. We have traced the mechanism to unexpected codependent roles for p120 and p190RhoGAP in regulating Rac-dependent antagonism of Rho. Receptor-induced Rac activity causes translocation of p190RhoGAP to adherens junctions (AJs), where it couples to the cadherin complex via interaction with p120. AJ formation is dependent on this p120-p190RhoGAP interaction and fails altogether if either of these proteins are compromised. We propose that Rac activation links diverse signaling systems to AJ assembly by controlling transient p190RhoGAP interactions with p120 and localized inhibition of Rho.


Assuntos
Moléculas de Adesão Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fosfoproteínas/metabolismo , Proteínas Repressoras/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Actinas/metabolismo , Junções Aderentes/metabolismo , Animais , Cateninas , Adesão Celular , Moléculas de Adesão Celular/deficiência , Linhagem Celular Transformada , Proliferação de Células , Extensões da Superfície Celular/metabolismo , Meios de Cultura Livres de Soro , Fibroblastos/citologia , Fibronectinas/metabolismo , Integrinas/metabolismo , Camundongos , Modelos Biológicos , Células NIH 3T3 , Fosfoproteínas/deficiência , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Fibras de Estresse/metabolismo , Proteínas rac de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/antagonistas & inibidores
17.
Exp Cell Res ; 312(17): 3336-48, 2006 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-16935280

RESUMO

p120-catenin (p120) regulates cadherin turnover and is required for cadherin stability. Extensive and dynamic phosphorylation on tyrosine, serine and threonine residues in the N-terminal regulatory domain has been postulated to regulate p120 function, possibly through modulation of the efficiency of p120/cadherin interaction. Here we have utilized novel phospho-specific monoclonal antibodies to four major p120 serine and threonine phosphorylation sites to monitor individual phosphorylation events and their consequences. Surprisingly, membrane-localization and not cadherin interaction is the main determinant in p120 serine and threonine phosphorylation and dephosphorylation. Furthermore, the phospho-status of these four residues had no obvious effect on p120's role in cadherin complex stabilization or cell-cell adhesion. Interestingly, dephosphorylation was dramatically induced by PKC activation, but PKC-independent pathways were also evident. The data suggest that p120 dephosphorylation at these sites is modulated by multiple cell surface receptors primarily through PKC-dependent pathways, but these changes do not seem to reduce p120/cadherin affinity.


Assuntos
Caderinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Fosfoproteínas/metabolismo , Serina/metabolismo , Treonina/metabolismo , Substituição de Aminoácidos , Animais , Cateninas , Moléculas de Adesão Celular/análise , Moléculas de Adesão Celular/imunologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cães , Humanos , Ligantes , Mutagênese Sítio-Dirigida , Fosfoproteínas/análise , Fosfoproteínas/imunologia , Fosforilação , Ligação Proteica , Proteína Quinase C/metabolismo , RNA Interferente Pequeno , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais
18.
Eukaryot Cell ; 4(3): 577-87, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15755920

RESUMO

Schizosaccharomyces pombe Dim1p is required for maintaining the steady-state level of the anaphase-promoting complex or cyclosome (APC/C) component Lid1p and thus for maintaining the steady-state level and activity of the APC/C. To gain further insight into Dim1p function, we have investigated the mechanism whereby Dim1p influences Lid1p levels. We show that S. pombe cells lacking Dim1p or Saccharomyces cerevisiae cells lacking its ortholog, Dib1p, are defective in generalized pre-mRNA splicing in vivo, a result consistent with the identification of Dim1p as a component of the purified yeast U4/U6.U5 tri-snRNP complex. Moreover, we find that Dim1p is part of a complex with the splicing factor Prp1p. However, although Dim1p is required for efficient splicing of lid1(+) pre-mRNA, circumventing the necessity for this particular function of Dim1p is insufficient for restoring normal Lid1p levels. Finally, we provide evidence that Dim1p also participates in the nuclear export of lid1(+) mRNA and that it is likely the combined loss of both of these two Dim1p functions which compromises Lid1p levels in the absence of proper Dim1p function. These data indicate that a mechanism acting at the level of mRNA impacts the functioning of the APC/C, a critical complex in controlling mitotic progression.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Processamento de RNA , RNA Mensageiro/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Subunidade Apc4 do Ciclossomo-Complexo Promotor de Anáfase , Proteínas de Ciclo Celular/genética , Humanos , Substâncias Macromoleculares , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Técnicas do Sistema de Duplo-Híbrido , Complexos Ubiquitina-Proteína Ligase/química
19.
Semin Cell Dev Biol ; 15(6): 657-63, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15561585

RESUMO

The strength of cadherin based cell-cell adhesion is modulated by signaling events that control the amount of cadherin present at the cell surface, and the clustering of cadherins into strong adhesive junctions. p120ctn has been indirectly implicated in clustering for some time, but it now appears that its main function is to regulate cadherin turnover. Forced p120 downregulation (e.g., by siRNA targeting) results in a striking dose-dependant loss of endogenous cadherins, indicating that p120 is essential for cadherin stability. These data challenge some important paradigms and suggest novel interpretations of existing data. For example, most of the effects of DN-cadherin expression can be accounted for by sequestration of p120. Thus, DN-cadherins phenocopy p120-downregulation, and a significant literature exists already that suggests consequences of p120-deficiency in disease and cancer. Moreover, p120 downregulation occurs frequently in essentially all of the major carcinoma types. Thus, it is possible that the classic observation of E-cadherin-deficiency in metastatic cancer may in some cases be due to p120 downregulation rather than better understood mechanisms acting at the level of E-cadherin transcription.


Assuntos
Caderinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular/fisiologia , Regulação Neoplásica da Expressão Gênica , Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiologia , Animais , Cateninas , Adesão Celular , Progressão da Doença , Regulação para Baixo , Genes Dominantes , Humanos , Modelos Biológicos , Metástase Neoplásica , Transdução de Sinais
20.
J Cell Biol ; 162(5): 851-62, 2003 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-12939254

RESUMO

Cytokinetic actin ring (CAR) formation in Schizosaccharomyces pombe requires two independent actin nucleation pathways, one dependent on the Arp2/3 complex and another involving the formin Cdc12p. Here we investigate the role of the S. pombe Cdc15 homology family protein, Cdc15p, in CAR assembly and find that it interacts with proteins from both of these nucleation pathways. Cdc15p binds directly to the Arp2/3 complex activator Myo1p, which likely explains why actin patches and the Arp2/3 complex fail to be medially recruited during mitosis in cdc15 mutants. Cdc15p also binds directly to Cdc12p. Cdc15p and Cdc12p not only display mutual dependence for CAR localization, but also exist together in a ring-nucleating structure before CAR formation. The disruption of these interactions in cdc15 null cells is likely to be the reason for their complete lack of CARs. We propose a model in which Cdc15p plays a critical role in recruiting and coordinating the pathways essential for the assembly of medially located F-actin filaments and construction of the CAR.


Assuntos
Actinas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiologia , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas Recombinantes de Fusão/metabolismo , Schizosaccharomyces/citologia , Proteínas de Schizosaccharomyces pombe/genética , Temperatura Ambiente , Técnicas do Sistema de Duplo-Híbrido
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