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1.
PLoS One ; 17(8): e0269208, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35969522

RESUMO

The Ajuba LIM protein Jub mediates regulation of Hippo signaling by cytoskeletal tension through interaction with the kinase Warts and participates in feedback regulation of junctional tension through regulation of the cytohesin Steppke. To investigate how Jub interacts with and regulates its distinct partners, we investigated the ability of Jub proteins missing different combinations of its three LIM domains to rescue jub phenotypes and to interact with α-catenin, Warts and Steppke. Multiple regions of Jub contribute to its ability to bind α-catenin and to localize to adherens junctions in Drosophila wing imaginal discs. Co-immunoprecipitation experiments in cultured cells identified a specific requirement for LIM2 for binding to Warts. However, in vivo, both LIM1 and LIM2, but not LIM3, were required for regulation of wing growth, Yorkie activity, and Warts localization. Conversely, LIM2 and LIM3, but not LIM1, were required for regulation of cell shape and Steppke localization in vivo, and for maximal Steppke binding in co-immunoprecipitation experiments. These observations identify distinct functions for the different LIM domains of Jub.


Assuntos
Proteínas de Drosophila/fisiologia , Drosophila/metabolismo , Proteínas com Domínio LIM/fisiologia , Animais , Citoesqueleto/química , Citoesqueleto/fisiologia , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/análise , Proteínas de Drosophila/genética , Proteínas com Domínio LIM/análise , Proteínas com Domínio LIM/genética , Proteínas com Homeodomínio LIM/análise , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/fisiologia , Transdução de Sinais , Fatores de Transcrição/metabolismo , Asas de Animais/crescimento & desenvolvimento , alfa Catenina/metabolismo
2.
Pharmacol Res ; 151: 104546, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31740385

RESUMO

The LIM protein Ajuba contains an unstructured proline/glycine-rich preLIM region in the N terminus and conserved tandem LIM motifs in the C terminus. Additionally, Ajuba contains both nuclear export sequences (NES) and nuclear localization sequences (NLS), which enable Ajuba shuttle between the cytoplasm and the nucleus. Thus, Ajuba can act as a versatile scaffold participating in assembly of variety of protein complexes to execute multiple cellular functions including cell adhesion, motility, mitosis, survival, gene expression, microRNA processing and mechanical force sensing. Numerous studies have demonstrated that Ajuba plays important roles in oncogenesis and progression by regulating major signalling pathways such as Wnt, RAS/ERK, JAK/STAT and Hippo, and by acting as a co-regulator of key transcription factors such as Snail, Sp1 and nuclear hormone receptors. Clinically, Ajuba is highly expressed in various types of tumors and can be a marker for prognosis and diagnosis. In this review, we aim to summarize the up-to-date literatures on the signaling pathways mediated by Ajuba and its associated protein complexes in oncogenesis, and to discuss Ajuba as a potential target for new therapeutics to treat cancers.


Assuntos
Carcinogênese/metabolismo , Proteínas com Domínio LIM/metabolismo , Transdução de Sinais , Animais , Apoptose , Carcinogênese/patologia , Adesão Celular , Humanos , Proteínas com Domínio LIM/análise , Mitose , Neoplasias/metabolismo , Neoplasias/patologia , Mapas de Interação de Proteínas
3.
Diagn Pathol ; 14(1): 100, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31484540

RESUMO

BACKGROUND: To evaluate the clinical utility of LIM Domain Only 2 (LMO2) negative and CD38 positive in diagnosis of Burkitt lymphoma (BL). METHODS: LMO2 and CD38 expression determined by immunohistochemistry in 75 BL, 12 High-grade B-cell lymphoma, NOS (HGBL,NOS) and 3 Burkitt-like lymphomas with the 11q aberration. RESULTS: The sensitivity and specificity of LMO2 negative for detecting BL were 98.67 and 100%, respectively; those of CD38 positive were 98.67 and 66.67%, respectively. The sensitivity and specificity of a combination of both for detecting BL were 97.33 and 100%, respectively. In our study, the combined LMO2 negative and CD38 positive results had a higher area under the curve than either LMO2 negative or CD38 positive alone. CONCLUSIONS: A combination of LMO2 negative and CD38 positive is useful for the diagnosis of Burkitt lymphoma.


Assuntos
ADP-Ribosil Ciclase 1/análise , Proteínas Adaptadoras de Transdução de Sinal/análise , Biomarcadores Tumorais/análise , Linfoma de Burkitt/diagnóstico , Proteínas com Domínio LIM/análise , Glicoproteínas de Membrana/análise , Proteínas Proto-Oncogênicas/análise , ADP-Ribosil Ciclase 1/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Humanos , Imuno-Histoquímica , Proteínas com Domínio LIM/biossíntese , Masculino , Glicoproteínas de Membrana/biossíntese , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas/biossíntese , Sensibilidade e Especificidade , Adulto Jovem
4.
Anal Chem ; 90(8): 4935-4939, 2018 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-29565561

RESUMO

Protein phosphorylation is a ubiquitous and critical post-translational modification (PTM) involved in numerous cellular processes. Mass spectrometry (MS)-based proteomics has emerged as the preferred technology for protein identification, characterization, and quantification. Whereas ionization/detection efficiency of peptides in electrospray ionization (ESI)-MS are markedly influenced by the presence of phosphorylation, the physicochemical properties of intact proteins are assumed not to vary significantly due to the relatively smaller modification on large intact proteins. Thus, the ionization/detection efficiency of intact phosphoprotein is hypothesized not to alter appreciably for subsequent MS quantification. However, this hypothesis has never been rigorously tested. Herein, we systematically investigated the impact of phosphorylation on ESI-MS quantification of mono- and multiply phosphorylated proteins. We verified that a single phosphorylation did not appreciably affect the ESI-MS quantification of phosphoproteins as demonstrated in the enigma homolog isoform 2 (28 kDa) with monophosphorylation. Moreover, different ionization and desolvation parameters did not impact phosphoprotein quantification. In contrast to monophosphorylation, multiphosphorylation noticeably affected ESI-MS quantification of phosphoproteins likely due to differential ionization/detection efficiency between unphosphorylated and phosphorylated proteoforms as shown in the pentakis-phosphorylated ß-casein (24 kDa).


Assuntos
Fosfoproteínas/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Caseínas/análise , Caseínas/metabolismo , Cromatografia Líquida de Alta Pressão , Humanos , Proteínas com Domínio LIM/análise , Proteínas com Domínio LIM/metabolismo , Fosfopeptídeos/análise , Fosfoproteínas/metabolismo , Fosforilação , Proteômica
5.
Cell Mol Life Sci ; 75(11): 1959-1971, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29428964

RESUMO

Nuclear receptors are ligand-activated transcription factors that partake in several biological processes including development, reproduction and metabolism. Over the last decade, evidence has accumulated that group 2, 3 and 4 LIM domain proteins, primarily known for their roles in actin cytoskeleton organization, also partake in gene transcription regulation. They shuttle between the cytoplasm and the nucleus, amongst other as a consequence of triggering cells with ligands of nuclear receptors. LIM domain proteins act as important coregulators of nuclear receptor-mediated gene transcription, in which they can either function as coactivators or corepressors. In establishing interactions with nuclear receptors, the LIM domains are important, yet pleiotropy of LIM domain proteins and nuclear receptors frequently occurs. LIM domain protein-nuclear receptor complexes function in diverse physiological processes. Their association is, however, often linked to diseases including cancer.


Assuntos
Proteínas com Domínio LIM/metabolismo , Mapas de Interação de Proteínas , Receptores Citoplasmáticos e Nucleares/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Humanos , Proteínas com Domínio LIM/análise , Proteínas com Domínio LIM/classificação , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/análise , Receptores Citoplasmáticos e Nucleares/classificação , Ativação Transcricional
6.
Am J Surg Pathol ; 42(3): 342-350, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29309299

RESUMO

Myocyte enhancer binding factor 2B (MEF2B) is a transcriptional activator of the BCL6 proto-oncogene in normal germinal center (GC) B-cells. Limited data exists concerning its expression in B-cell lymphomas, and comparison with other GC-associated antigens is lacking. Its role in the differential diagnosis of B-cell lymphomas, particularly in the distinction of follicular lymphoma (FL) versus marginal zone lymphoma (MZL), remains to be determined. We evaluated MEF2B expression, in comparison with additional GC markers, LIM domain-only transcription factor 2 (LMO2), and human GC-associated lymphoma (HGAL), in a variety of B-cell lymphomas, with particular emphasis on their utility in differentiating FL from MZL. MEF2B was positive in all FL and Burkitt lymphomas, 8/9 mantle cell lymphomas, 2/24 splenic MZL, 1/10 chronic lymphocytic leukemia/small lymphocytic lymphomas, and 38/44 diffuse large B-cell lymphoma (DLBCL), but was negative in all extranodal MZL of mucosa-associated lymphoid tissue, nodal MZL, and B-lymphoblastic lymphomas. Focusing on low-grade FL versus MZL, MEF2B was 100% sensitive and 95% specific for FL, which was similar to BCL6, but superior to LMO2 (sensitivity 87%, specificity 86%) and HGAL (sensitivity 97%, specificity 86%). Importantly, MEF2B was positive in 4/4 FL with plasmacytoid differentiation, which were CD10, only weakly BCL6, and included 1 case that lacked both LMO2 and HGAL expression. MEF2B was positive in 22/25 (88%) GC-type DLBCL, but was also positive in 16/19 (61%) non-GC-type DLBCL. MEF2B shows superior sensitivity and specificity than LMO2 and HGAL in the differential diagnosis of FL versus MZL and is particularly useful in FL with plasmacytoid differentiation, which may have morphologic and immunophenotypic overlap with MZL. MEF2B, however, is not specific for GC-derived B-cell lymphomas as it is also apparently positive in most mantle cell lymphoma and many non-GC-type DLBCL.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/análise , Biomarcadores Tumorais/análise , Centro Germinativo/imunologia , Proteínas com Domínio LIM/análise , Linfoma de Células B/imunologia , Proteínas de Neoplasias/análise , Proteínas Proto-Oncogênicas/análise , Diferenciação Celular , Diagnóstico Diferencial , Centro Germinativo/patologia , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Linfoma de Células B/classificação , Linfoma de Células B/patologia , Linfoma de Zona Marginal Tipo Células B/imunologia , Linfoma de Zona Marginal Tipo Células B/patologia , Linfoma Folicular/imunologia , Linfoma Folicular/patologia , Fatores de Transcrição MEF2/análise , Proteínas dos Microfilamentos , Neprilisina/análise , Valor Preditivo dos Testes , Proto-Oncogene Mas , Análise Serial de Tecidos
7.
Med Oncol ; 33(1): 8, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26708131

RESUMO

Endometrial cancer is the sixth most common cancer in women. Galectin-3 (GAL-3) and CRIP-1 are multifunctional proteins which seem to be involved in many neoplasias. This study aims to point out correlations between clinicopathological findings and endometrial cancer patient survival to GAL-3 and CRIP-1 expression in order to enfold their diagnostic/prognostic potential. Tissues from 46 patients diagnosed with endometrial cancer were studied by immunohistochemistry, using monoclonal antibodies for GAL-3 and CRIP-1, and expression levels were correlated with clinicopathological findings and survival. Analysis was performed at single protein level or as co-expression. High expression of GAL-3 and CRIP-1 was independently associated with tumor depth and histological grade, respectively. Also, there was a significant correlation between high co-expression of the two proteins and the histological grade (aOR 2.66), the tumor depth (aOR 0.32) and the histological type (aOR 1.32), but not with the patients' age. Moreover, high expression of both proteins was observed in patients with shorter survival times. Interestingly, the co-expression of the two proteins exhibited some degree of monotony (Spearman's ρ = 0.768), indicating a common molecular pathway. This study provides evidence for a prognostic clinical potential of the combined study of GAL-3 and CRIP-1 in endometrial cancer. These factors are poorly studied in endometrium, and their role in the carcinogenetic process and on effective therapy awaits further elucidation.


Assuntos
Biomarcadores Tumorais/análise , Proteínas de Transporte/biossíntese , Neoplasias do Endométrio/patologia , Galectina 3/biossíntese , Proteínas com Domínio LIM/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas Sanguíneas , Proteínas de Transporte/análise , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/mortalidade , Feminino , Galectina 3/análise , Galectinas , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Proteínas com Domínio LIM/análise , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais
8.
J Hepatol ; 64(1): 110-7, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26334580

RESUMO

BACKGROUND & AIM: Hydrogen peroxide-inducible clone-5 (Hic-5), also named as transforming growth factor beta-1-induced transcript 1 protein (Tgfb1i1), was found to be induced by TGF-ß. Previous studies have shown that TGF-ß is a principal mediator of hepatic stellate cell (HSC) activation in liver fibrosis. However, this process remains elusive. In this study, we aimed to define the role of Hic-5 in HSC activation and liver fibrosis. METHODS: We examined the expression levels of Hic-5 during HSCs activation and in fibrotic liver tissues by quantitative real-time reverse transcriptase polymerase chain reaction, Western blot and immunohistochemistry. Hic-5 knockout (KO) and wild-type (WT) mice were subjected to bile duct ligation (BDL) or carbon tetrachloride (CCl4) injection to induce liver fibrosis. RESULTS: Hic-5 expression was strongly upregulated in activated HSCs of the human fibrotic liver tissue and BDL or CCl4-induced mouse liver fibrosis. Hic-5 deficiency significantly attenuated mouse liver fibrosis and HSC activation. Furthermore, Hic-5 knockdown by siRNA in vivo repressed CCl4-induced liver fibrosis in mice. Mechanistically, the absence of Hic-5 significantly inhibited the TGF-ß/Smad2 signaling pathway, proved by increasing Smad7 expression, resulting in reduced collagen production and α-smooth muscle actin expression in the activated HSCs. CONCLUSION: Hic-5 deficiency attenuates the activation of HSCs and liver fibrosis though reducing the TGF-ß/Smad2 signaling by upregulation of Smad7. Thus, Hic-5 can be regarded as a potential therapeutic target for liver fibrosis.


Assuntos
Proteínas do Citoesqueleto/deficiência , Proteínas de Ligação a DNA/deficiência , Células Estreladas do Fígado/fisiologia , Proteínas com Domínio LIM/deficiência , Cirrose Hepática/etiologia , Proteína Smad7/fisiologia , Actinas/análise , Animais , Tetracloreto de Carbono , Células Cultivadas , Proteínas do Citoesqueleto/análise , Proteínas de Ligação a DNA/análise , Humanos , Proteínas com Domínio LIM/análise , Camundongos , Camundongos Endogâmicos C57BL , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Regulação para Cima
9.
Electrophoresis ; 36(24): 3097-100, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26383991

RESUMO

Myofibrillar myopathies (MFMs) are a group of inherited or sporadic neuromuscular disorders morphologically characterized by foci of myofibril dissolution, disintegration of the Z-disk, and insoluble protein aggregates within the muscle fibers. The diagnosis is based on muscle biopsy. Light and electron microscopy has a central role in the diagnostic work up, and immunohistochemistry shows abnormal deposition of several proteins including αB-crystallin, desmin, and myotilin. In contrast, immunoblotting does not have any diagnostic value because it does not highlight differences in the amount of involved proteins. We investigated the pattern and level expression of desmin, αB-crystallin, myotilin, and ZASP (Z-band alternatively spliced PDZ motif-containing protein) in muscle of seven patients with MFMs by immunoblotting after SDS-PAGE and 2D-PAGE using two different solubilizing solutions, one radioimmunoprecipitation assay (RIPA) buffer, and the other urea-containing buffer. Our data demonstrated that urea-containing buffer improves the solubilization and recovery of desmin, αB-crystallin, myotilin, and ZASP as compared with RIPA buffer and that the total content of these proteins is increased in muscles of patients. The present results provide evidence that immunoblotting is an additional tool for confirming diagnosis of MFMs.


Assuntos
Biomarcadores/análise , Biomarcadores/química , Immunoblotting/métodos , Miopatias Congênitas Estruturais/diagnóstico , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/química , Conectina/análise , Conectina/química , Cristalinas/análise , Cristalinas/química , Eletroforese em Gel Bidimensional , Humanos , Proteínas com Domínio LIM/análise , Proteínas com Domínio LIM/química , Proteínas dos Microfilamentos
10.
PLoS One ; 8(9): e73332, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24039914

RESUMO

BACKGROUND: Recent studies have highlighted the heterogeneity of gliomas and demonstrated that molecular and genetic analysis could help in their classification and in the design of treatment protocols. In a previous study we have identified a 4-gene signature highly correlated with survival of glioma patients. The aim of this study is to confirm and extend these findings by investigating the expression of these genes at the protein level and their association with outcome of patients with high grade gliomas. METHODOLOGY/PRINCIPAL FINDINGS: Immunohistochemical staining for EDN/RB, HJURP, p60/CAF-1 and PDLI4 was studied on archive materials from 96 patients (64 glioblastomas and 32 grade III gliomas). The levels of all four proteins differed significantly between grade III and grade IV tumours. The levels of the EDN/RB, HJURP and p60/CAF-1 proteins were strongly associated with overall survival (p<0.001, p<0.001 and p=0.002, respectively), whereas the one of PDLI4 was not (P=0.11). A risk criterion defined as high levels of at least two of the EDN/RB, HJURP and p60/CAF-1 proteins accurately predicted the prognosis of patients. Multivariate analysis confirmed that this criterion was an independent negative prognostic marker (hazard ratio = 2.225; 95% CI, 1.248 to 3.966, p=0.007). CONCLUSIONS: The expression of the EDN/RB, HJURP, p60/CAF-1 and PDLI4 proteins is disrupted in high grade gliomas and increases in the levels of these proteins are closely linked to tumour aggressiveness and poor outcome.


Assuntos
Neoplasias Encefálicas/diagnóstico , Fator 1 de Modelagem da Cromatina/análise , Proteínas de Ligação a DNA/análise , Glioblastoma/diagnóstico , Glioma/diagnóstico , Proteínas com Domínio LIM/análise , Receptores de Endotelina/análise , Biomarcadores Tumorais/análise , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/patologia , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Glioma/patologia , Humanos , Imuno-Histoquímica , Estadiamento de Neoplasias , Prognóstico , Receptor de Endotelina B
11.
Biomaterials ; 34(26): 6229-38, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23714244

RESUMO

Vascular endothelial growth factor (VEGF) gene therapy to promote therapeutic angiogenesis has been advanced as an alternative treatment for myocardial ischemia. The unregulated expression of VEGF and the use of viral vectors, however, have slowed the clinical development of angiogenic gene therapy. The development of clinically beneficial angiogenic gene therapy requires a disease-specific gene expression system and an efficient non-viral gene carrier. To address these requirements, we developed a new post-translationally regulated hypoxia-responsible VEGF plasmid, pß-SP-ODD-VEGF, and a dendrimer-type bio-reducible polymer, PAM-ABP. The efficacy of VEGF gene therapy with the PAM-ABP/pß-SP-ODD-VEGF was evaluated and compared to the RTP-VEGF plasmid, a previously constructed hypoxia-inducible plasmid, in an ischemia/reperfusion (I/R) rat model. Cine magnetic resonance imaging was used to analyze the ischemia/reperfusion rats treated with either the PAM-ABP/pß-SP-ODD-VEGF or the PAM-ABP/RTP-VEGF. The PAM-ABP/pß-SP-ODD-VEGF treatment more effectively protected cardiomyocytes against apoptosis, preserved left ventricular (LV) function, and prevented LV remodeling compared to the PAM-ABP/RTP-VEGF-treated rats. These results suggest that the pß-SP-ODD-VEGF with PAM-ABP may be efficacious in the treatment of acute ischemic heart disease.


Assuntos
Arginina/química , Dendrímeros/química , Isquemia Miocárdica/terapia , Plasmídeos/uso terapêutico , Poliaminas/química , Transfecção , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Apoptose , Linhagem Celular , Expressão Gênica , Terapia Genética , Proteínas com Domínio LIM/análise , Proteínas com Domínio LIM/genética , Masculino , Isquemia Miocárdica/genética , Isquemia Miocárdica/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Plasmídeos/administração & dosagem , Plasmídeos/genética , Sinais Direcionadores de Proteínas , Ratos , Ratos Sprague-Dawley , Fator A de Crescimento do Endotélio Vascular/análise
12.
Mol Cell Proteomics ; 12(1): 215-27, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23115302

RESUMO

Filaminopathy is a subtype of myofibrillar myopathy caused by mutations in FLNC, the gene encoding filamin C, and histologically characterized by pathologic accumulation of several proteins within skeletal muscle fibers. With the aim to get new insights in aggregate composition, we collected aggregates and control tissue from skeletal muscle biopsies of six myofibrillar myopathy patients harboring three different FLNC mutations by laser microdissection and analyzed the samples by a label-free mass spectrometry approach. A total of 390 proteins were identified, and 31 of those showed significantly higher spectral indices in aggregates compared with patient controls with a ratio >1.8. These proteins included filamin C, other known myofibrillar myopathy associated proteins, and a striking number of filamin C binding partners. Across the patients the patterns were extremely homogeneous. Xin actin-binding repeat containing protein 2, heat shock protein 27, nebulin-related-anchoring protein, and Rab35 could be verified as new filaminopathy biomarker candidates. In addition, further experiments identified heat shock protein 27 and Xin actin-binding repeat containing protein 2 as novel filamin C interaction partners and we could show that Xin actin-binding repeat containing protein 2 and the known interaction partner Xin actin-binding repeat containing protein 1 simultaneously associate with filamin C. Ten proteins showed significant lower spectral indices in aggregate samples compared with patient controls (ratio <0.56) including M-band proteins myomesin-1 and myomesin-2. Proteomic findings were consistent with previous and novel immunolocalization data. Our findings suggest that aggregates in filaminopathy have a largely organized structure of proteins also interacting under physiological conditions. Different filamin C mutations seem to lead to almost identical aggregate compositions. The finding that filamin C was detected as highly abundant protein in aggregates in filaminopathy indicates that our proteomic approach may be suitable to identify new candidate genes among the many MFM patients with so far unknown mutation.


Assuntos
Proteínas Contráteis/genética , Proteínas dos Microfilamentos/genética , Fibras Musculares Esqueléticas/metabolismo , Distrofias Musculares/metabolismo , Proteoma/análise , Adulto , Biomarcadores Tumorais/análise , Proteínas de Ligação a DNA/análise , Feminino , Filaminas , Proteínas de Choque Térmico HSP27/análise , Proteínas de Choque Térmico , Humanos , Proteínas com Domínio LIM/análise , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Chaperonas Moleculares , Proteínas Musculares/análise , Músculo Esquelético/metabolismo , Distrofias Musculares/etiologia , Distrofias Musculares/genética , Mutação , Proteínas Nucleares/análise , Proteômica , Proteínas rab de Ligação ao GTP/análise
13.
J Proteome Res ; 11(4): 2114-26, 2012 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-22250753

RESUMO

Diagnosis of acute coronary syndromes is based on protein biomarkers, such as the cardiac troponins (cTnI/cTnT) and creatine kinase (CK-MB) that are released into the circulation. Biomarker discovery is focused on identifying very low abundance tissue-derived analytes from within albumin-rich plasma, in which the wide dynamic range of the native protein complement hinders classical proteomic investigations. We employed an ex vivo rabbit model of myocardial ischemia/reperfusion (I/R) injury using Langendorff buffer perfusion. Nonrecirculating perfusate was collected over a temporal profile of 60 min reperfusion following brief, reversible ischemia (15 min; 15I/60R) for comparison with irreversible I/R (60I/60R). Perfusate proteins were separated using two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry (MS), revealing 26 tissue-specific proteins released during reperfusion post-15I. Proteins released during irreversible I/R (60I/60R) were profiled using gel-based (2-DE and one-dimensional gel electrophoresis coupled to liquid chromatography and tandem mass spectrometry; geLC-MS) and gel-free (LC-MS/MS) methods. A total of 192 tissue-specific proteins were identified during reperfusion post-60I. Identified proteins included those previously associated with I/R (myoglobin, CK-MB, cTnI, and cTnT), in addition to examples currently under investigation in large cohort studies (heart-type fatty acid binding protein; FABPH). The postischemic release profile of a novel cardiac-specific protein, cysteine and glycine-rich protein 3 (Csrp3; cardiac LIM domain protein) was validated by Western blot analysis. We also identified Csrp3 in serum from 6 of 8 patients postreperfusion following acute myocardial infarction. These studies indicate that animal modeling of biomarker release using ex vivo buffer perfused tissue to limit the presence of obfuscating plasma proteins may identify candidates for further study in humans.


Assuntos
Traumatismo por Reperfusão Miocárdica/metabolismo , Proteoma/análise , Proteômica/métodos , Sequência de Aminoácidos , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Western Blotting , Cromatografia Líquida , Modelos Animais de Doenças , Eletroforese em Gel Bidimensional , Humanos , Proteínas com Domínio LIM/análise , Proteínas com Domínio LIM/sangue , Proteínas com Domínio LIM/metabolismo , Masculino , Dados de Sequência Molecular , Proteínas Musculares/análise , Proteínas Musculares/sangue , Proteínas Musculares/metabolismo , Necrose/metabolismo , Proteoma/metabolismo , Coelhos , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem , Função Ventricular Esquerda
14.
Cancer Biomark ; 12(1): 1-9, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23321464

RESUMO

The pathogenesis of esophageal squamous cell carcinoma (ESCC) involves both genetic and environmental factors. Previously, we have carried out gene and protein expression profiling of ESCC using DNA microarrays and mass spectrometry-based quantitative proteomics, respectively. These studies resulted in identification of several potential biomarkers of ESCC, some with known reports of differential expression in the scientific literature and others that were novel observations from our studies. We report systematic validation of selected markers from our studies on a larger cohort of cancer tissue sections by immunohistochemical labeling of tissue microarrays. We have validated expression of insulin-like growth factor-binding protein 7 (IGFBP7), stanniocalcin 2 (STC2), inhibin beta A (INHBA) and four and a half LIM domains 1 (FHL1). Immunohistochemical labeling with anti-stanniocalcin 2 antibody demonstrated its overexpression in 132/140 (94%) cases, IGFBP7 showed overexpression in 127/140 (91%) cases and overexpression of INHBA was observed in 62/105 (59%) of ESCC cases. In contrast, FHL1 expression was observed only in 12/143 (8%) of ESCC cases suggesting its possible involvement in tumor suppression. These data suggest that IGFBP7, INHBA, STC2 and FHL1 might play an important role in ESCC tumorigenesis, which can be explored in future studies. Overall, our findings open up new avenues for development of novel therapeutics and/or diagnostic approaches in ESCC.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/metabolismo , Glicoproteínas/metabolismo , Subunidades beta de Inibinas/metabolismo , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas Musculares/metabolismo , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/análise , Glicoproteínas/genética , Humanos , Subunidades beta de Inibinas/análise , Subunidades beta de Inibinas/genética , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/análise , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Peptídeos e Proteínas de Sinalização Intercelular/análise , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/análise , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/análise , Proteínas com Domínio LIM/genética , Proteínas Musculares/análise , Proteínas Musculares/genética , Reprodutibilidade dos Testes , Análise Serial de Tecidos
15.
Int J Cancer ; 130(11): 2549-56, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-21702045

RESUMO

Four and a half Lin-11, Isl-1, Mac-3 (LIM) protein 1 (FHL1) has been linked to carcinogenesis. However, the role of FHL1 in lung cancer remains unclear and the detailed mechanism underlying its tumor suppressive role is poorly understood. The purpose of this study was to examine FHL1 expression in lung cancer patients and to investigate how it was associated with lung cancer cell growth. Immunoblotting and immunohistochemistry showed that FHL1 protein was downregulated in over 90% of 80 lung cancer patients. FHL1 expression was strongly correlated with tumor histological types (p < 10(-4) ) and the differentiation of the tumor (p = 0.002). FHL1 inhibited anchorage-dependent and -independent growth of human lung cancer cell lines. The inhibitory effects of FHL1 on lung cancer cell growth were associated with both the G1 and the G2/M cell cycle arrest concomitant with a marked inhibition of cyclin A, cyclin B1 and cyclin D as well as the induction of the cyclin dependent kinase inhibitors p21 (WAF1/CIP1) and p27 (Kip1). Direct intratumoral injection of an adenovirus expressing FHL1 dramatically suppressed the growth of A549 lung cancer cells in nude mice. Our data suggest that reduced expression of FHL1 may play an important role in the development and progression of lung cancer and that FHL1 may be a useful target for lung cancer gene therapy.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas com Domínio LIM/fisiologia , Neoplasias Pulmonares/prevenção & controle , Proteínas Musculares/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Adulto , Idoso , Animais , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/análise , Proteínas com Domínio LIM/análise , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Proteínas Musculares/análise
16.
Mol Cell Biol ; 32(1): 173-85, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22037766

RESUMO

ß-Catenin-independent Wnt signaling pathways have been implicated in the regulation of planar cell polarity (PCP) and convergent extension (CE) cell movements. Prickle, one of the core proteins of these pathways, is known to asymmetrically localize proximally at the adherens junction of Drosophila melanogaster wing cells and to locally accumulate within plasma membrane subdomains in cells undergoing CE movements during vertebrate development. Using mass spectrometry, we have identified the Ste20 kinase Mink1 as a Prickle-associated protein and found that they genetically interact during the establishment of PCP in the Drosophila eye and CE in Xenopus laevis embryos. We show that Mink1 phosphorylates Prickle on a conserved threonine residue and regulates its Rab5-dependent endosomal trafficking, a process required for the localized plasma membrane accumulation and function of Prickle. Mink1 also was found to be important for the clustering of Vangl within plasma membrane puncta. Our results provide a link between Mink and the Vangl-Prickle complex and highlight the importance of Prickle phosphorylation and endosomal trafficking for its function during Wnt-PCP signaling.


Assuntos
Proteínas com Domínio LIM/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endossomos/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/análise , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/análise , Proteínas de Membrana/análise , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Fosforilação , Proteínas Serina-Treonina Quinases/análise , Transporte Proteico , Proteínas Supressoras de Tumor/análise , Xenopus/embriologia , Xenopus/metabolismo , Proteínas de Xenopus/metabolismo
17.
Cardiovasc Res ; 92(1): 132-40, 2011 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-21636540

RESUMO

AIMS: Previous studies on the role of stem cell factor (SCF) in endothelial progenitor cell (EPC)-mediated neovascularization have focused on the EPC mobilization and homing process. However, the direct effects of SCF on neovascularization activity of EPCs have not been characterized. We sought to determine whether SCF regulates the neovascularization ability of EPCs by comparing its roles in mature endothelial cells. METHODS AND RESULTS: In vitro and in vivo assays revealed that SCF substantially increased the neovascularization activity of human EPCs through the c-Kit receptor. Notably, the SCF-induced increase in neovascularization activity was substantially greater in EPCs than that in human umbilical vein endothelial cells (HUVECs). SCF-induced phosphorylation of c-Kit and downstream signalling molecules was consistently found to be more potent and longer-lasting in EPCs than in HUVECs. This high responsiveness of EPCs to SCF was explained by the finding that the cell-surface expression of c-Kit is far higher in EPCs than in HUVECs. A c-Kit promoter assay revealed that the increased expression of c-Kit in EPCs could be attributed to the greater expression of stem cell leukaemia, LIM-only 2, and GATA-binding protein 2. CONCLUSION: In addition to its documented role in the mobilization and recruitment of EPCs, our findings show that SCF directly enhances the neovascularization activity of EPCs. Furthermore, the present study provides further evidence that EPCs exhibit differentially greater responsiveness to hypoxia-inducible cytokines, including SCF, than mature endothelial cells, suggesting that EPCs in ischaemic tissues function differently from mature endothelial cells, although they exhibit very similar phenotypes.


Assuntos
Células Endoteliais/citologia , Neovascularização Fisiológica , Fator de Células-Tronco/fisiologia , Células-Tronco/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/análise , Fatores de Transcrição Hélice-Alça-Hélice Básicos/análise , Fator de Transcrição GATA2 , Humanos , Proteínas com Domínio LIM/análise , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas c-kit/análise , Proteínas Proto-Oncogênicas c-kit/fisiologia , Transdução de Sinais , Proteína 1 de Leucemia Linfocítica Aguda de Células T
18.
Cell Cycle ; 10(11): 1726-30, 2011 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-21519191

RESUMO

The protection of chromosome ends requires the inhibition of DNA damage responses at telomeres. This inhibition is exerted in great part by the shelterin complex, known to prevent inappropriate ATM and ATR activation. The molecular mechanisms by which shelterin protects telomeres are incompletely understood. Recently, we have implicated for the first time a class of molecules, LIM domain proteins, in telomere protection. This protection occurred through interaction with shelterin, possibly through POT1, and required the pair of LIM proteins TRIP6 and LPP, themselves part of the Zyxin family. The domain similarity between TRIP6, LPP and Zyxin led us to ask whether the latter also interacted with telomeres. Here, we show that there is specificity in the association of LIM proteins with telomeres: Zyxin, despite a high degree of similarity with TRIP6 and LPP, was not detected at telomeres, nor found in a complex with shelterin. TRIP6 and LPP, however, were detected by immunofluorescence at a small subset of telomeres, perhaps those that are critically short. We speculate that specific LIM proteins are part of complex events occurring in the context of the telomere dysfunction response, and possibly at play during the induction of senescence.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas do Citoesqueleto/análise , Proteínas com Domínio LIM/análise , Telômero/química , Fatores de Transcrição/análise , Zixina/análise , ATPases Associadas a Diversas Atividades Celulares , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Dano ao DNA , Humanos , Proteínas com Domínio LIM/metabolismo , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Complexo Shelterina , Telômero/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Fatores de Transcrição/metabolismo , Zixina/metabolismo
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