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1.
Oncotarget ; 9(15): 12079-12100, 2018 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-29552294

RESUMO

Thyroid ultrasound and ultrasound-guided fine-needle aspiration (USG/FNA) biopsy are currently used for diagnosing papillary thyroid carcinoma (PTC), but their detection limit could be improved by combining other biomarkers. To discover novel PTC biomarkers, we herein applied a GeLC-MS/MS strategy to analyze the proteome profiles of serum-abundant-protein-depleted FNA cystic fluid from benign and PTC patients, as well as two PTC cell line secretomes. From them, we identified 346, 488, and 2105 proteins, respectively. Comparative analysis revealed that 191 proteins were detected in the PTC but not the benign cystic fluid samples, and thus may represent potential PTC biomarkers. Among these proteins, 101 were detected in the PTC cell line secretomes, and seven of them (NPC2, CTSC, AGRN, GPNMB, DPP4, ERAP2, and SH3BGRL3) were reported in public PTC transcriptome datasets as having 4681 elevated mRNA expression in PTC. Immunoblot analysis confirmed the elevated expression levels of five proteins (NPC2, CTSC, GPNMB, DPP4, and ERAP2) in PTC versus benign cystic fluids. Immunohistochemical studies from near 100 pairs of PTC tissue and their adjacent non-tumor counterparts further showed that AGRN (n = 98), CTSC (n = 99), ERAP2 (n = 98) and GPNMB (n = 100) were significantly (p < 0.05) overexpressed in PTC and higher expression levels of AGRN and CTSC were also significantly associated with metastasis and poor prognosis of PTC patients. Collectively, our results indicate that an integrated analysis of FNA cystic fluid proteome, cancer cell secretome and tissue transcriptome datasets represents a useful strategy for efficiently discovering novel PTC biomarker candidates.

2.
Cell Death Dis ; 9(4): 425, 2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29556045

RESUMO

The overexpression of stomatin-like protein-2 (SLP-2) is commonly observed in non-small cell lung cancer (NSCLC) cells. In the present study, we transfected a number of NSCLC cells with an SLP-2 shRNA-expressing vector (AdSLP2i) and examined its possible effects on cell growth and apoptosis. We found that suppression of SLP-2 expression inhibited cell growth, and that the apoptosis induced by SLP-2 suppression was correlated with decreased survivin protein expression. Moreover, the reduced survivin expression was found to be associated with reduced ß-catenin nuclear localization and appeared not to be modulated through the AKT signaling pathway. By using immunoprecipitation and proteomics to analyze protein-protein interactions in A549 cells with SLP-2 overexpression, we found that annexin A2 interacted with SLP-2 and ß-catenin directly. Our data further suggested that the knockdown of SLP-2 gene affected the SLP-2/Annexin A2/ß-catenin cascade formation, reduced the translocation of cytoplasmic ß-catenin into nucleus, and downregulated downstream target genes. The results presented in this study, together with our previous findings, suggest that SLP-2 promotes NSCLC cell proliferation by enhancing survivin expression mediated via ß-catenin pathway.


Assuntos
Proteínas Sanguíneas/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais , Survivina/metabolismo , Apoptose , Proteínas Sanguíneas/antagonistas & inibidores , Proteínas Sanguíneas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Peptídeos/análise , Regiões Promotoras Genéticas , Ligação Proteica , Mapas de Interação de Proteínas , Proteômica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Survivina/genética , beta Catenina/metabolismo
3.
J Proteomics ; 188: 139-151, 2018 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-29524648

RESUMO

Hypoxia is associated with poor prognosis in most solid tumors due to its multiple effects on therapy resistance, angiogenesis, apoptotic resistance, and tumor invasion/metastasis. Here we used a comprehensive omics profiling to investigate hypoxia-regulated gene expression in HCT116 colon cancer cells. Quantitative analyses of proteome and secretome were performed in HCT116 cells cultured under hypoxic or normoxic conditions. A total of 5700 proteins were quantified in proteome analysis and 722 proteins were quantified in secretome analysis. Both datasets were combined with the transcriptome and translatome datasets for further analysis. Verification of candidate proteins/genes in this integrated omics analysis was performed using immunoblotting and quantitative real-time RT-PCR analyses. We also performed polysome profiling to assess changes in translational efficiency of hypoxia-induced genes. Notably, several genes were differently regulated at the transcriptional and translational levels in HCT116 cells during hypoxia. Bioinformatics analysis suggested that hypoxia regulates translation of genes involved in extracellular matrix organization, extracellular exosomes, and protein processing in endoplasmic reticulum. Aberrations in these metabolic pathways appear to be correlated with an increased risk of tumor invasion/metastasis. BIOLOGICAL SIGNIFICANCE: This study integrates transcriptome/translatome and proteome/secretome to analyze gene expression changes in human colon cancer cells under hypoxic conditions. Candidate proteins/genes in this integrated omics analysis were further validated by immunoblotting, quantitative real-time RT-PCR, and polysome profiling. The datasets would be useful to uncover the molecular mechanisms of hypoxia-induced gene regulation in colorectal cancer.


Assuntos
Neoplasias do Colo/patologia , Regulação Neoplásica da Expressão Gênica , Hipóxia/genética , Proteoma/análise , Neoplasias do Colo/química , Neoplasias do Colo/genética , Perfilação da Expressão Gênica , Células HCT116 , Humanos , Proteínas de Neoplasias/análise , Biossíntese de Proteínas
4.
Expert Rev Proteomics ; 14(9): 737-756, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28695748

RESUMO

INTRODUCTION: Cancer represents one of the major causes of human deaths. Identification of proteins as biomarkers for early detection of cancer and therapeutic targets for cancer treatment are important issues in precision medicine. Secretome of cancer cells represents the collection of proteins secreted or shed from cancer cells. Proteomic profiling of the cancer cell secretome has been proven to be a convenient and efficient way to discover cancer biomarker and/or therapeutic targets. Areas covered: There have been numerous reviews describing the history and application of secretome analysis in cancer biomarker/therapeutic target research. The present review focuses on the technological advancement for profiling low-molecular-mass proteins in secretome, the latest information regarding the new candidate biomarkers and molecular mechanisms discovered on the basis of cancer cell secretome analysis, as well as the previously discovered candidate biomarkers that enter into clinical trials. Expert commentary: Current technologies for protein sample preparation/separation and MS-based protein identification have allowed in-depth analysis of cancer cell secretome. Future efforts should focus on the comprehensiveness of cancer cell secretome, meta-analysis of different secretome datasets and integrated analysis via combining other omics datasets, as well as the incorporation of MS-based biomarker verification pipeline into both preclinical studies and clinical trials.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias/genética , Proteoma/genética , Proteômica , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/patologia
5.
Nature ; 547(7664): 458-462, 2017 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-28723894

RESUMO

The radiation-induced bystander effect (RIBE) refers to a unique process in which factors released by irradiated cells or tissues exert effects on other parts of the animal not exposed to radiation, causing genomic instability, stress responses and altered apoptosis or cell proliferation. Although RIBEs have important implications for radioprotection, radiation safety and radiotherapy, the molecular identities of RIBE factors and their mechanisms of action remain poorly understood. Here we use Caenorhabditis elegans as a model in which to study RIBEs, and identify the cysteine protease CPR-4, a homologue of human cathepsin B, as the first RIBE factor in nematodes, to our knowledge. CPR-4 is secreted from animals irradiated with ultraviolet or ionizing gamma rays, and is the major factor in the conditioned medium that leads to the inhibition of cell death and increased embryonic lethality in unirradiated animals. Moreover, CPR-4 causes these effects and stress responses at unexposed sites distal to the irradiated tissue. The activity of CPR-4 is regulated by the p53 homologue CEP-1 in response to radiation, and CPR-4 seems to exert RIBEs by acting through the insulin-like growth factor receptor DAF-2. Our study provides crucial insights into RIBEs, and will facilitate the identification of additional RIBE factors and their mechanisms of action.


Assuntos
Efeito Espectador/efeitos da radiação , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/efeitos da radiação , Catepsina B/metabolismo , Animais , Caenorhabditis elegans/citologia , Proteínas de Caenorhabditis elegans/metabolismo , Cisteína Proteases/metabolismo , Receptor de Insulina/metabolismo , Raios Ultravioleta
6.
Sci Rep ; 6: 36214, 2016 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-27796369

RESUMO

The inflammasome adaptor protein, ASC, contributes to both innate immune responses and inflammatory diseases via self-oligomerization, which leads to the activation of the protease, caspase-1. Here, we report that the cytosolic tyrosine kinases, FAK and Pyk2, are differentially involved in NLRP3 and AIM2 inflammasome activation. The inhibition of FAK and Pyk2 with RNA interference or chemical inhibitors dramatically abolished ASC oligomerization, caspase-1 activation, and IL-1ß secretion in response to NLRP3 or AIM2 stimulation. Pyk2 is phosphorylated by the kinase Syk and relocalizes to the ASC specks upon NLRP3 inflammasome activation. Pyk2, but not FAK, could directly phosphorylate ASC at Tyr146, and only the phosphorylated ASC could participate in speck formation and trigger IL-1ß secretion. Moreover, the clinical-trial-tested Pyk2/FAK dual inhibitor PF-562271 reduced monosodium urate-mediated peritonitis, a disease model used for studying the consequences of NLRP3 activation. Our results suggest that although Pyk2 and FAK are involved in inflammasome activation, only Pyk2 directly phosphorylates ASC and brings ASC into an oligomerization-competent state by allowing Tyr146 phosphorylation to participate ASC speck formation and subsequent NLRP3 inflammation.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/imunologia , Quinase 2 de Adesão Focal/imunologia , Inflamassomos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Peritonite/imunologia , Animais , Proteínas Adaptadoras de Sinalização CARD/genética , Quinase 2 de Adesão Focal/genética , Inflamassomos/genética , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Peritonite/induzido quimicamente , Peritonite/genética , Peritonite/patologia , Fosforilação/genética , Fosforilação/imunologia , Multimerização Proteica/efeitos dos fármacos , Multimerização Proteica/genética , Multimerização Proteica/imunologia , Ácido Úrico/toxicidade
7.
Biochim Biophys Acta ; 1843(11): 2513-27, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25014165

RESUMO

The PAK2/ßPIX/GIT1 (p21-activated kinase 2/PAK-interacting exchange factor-ß/G protein-coupled receptor kinase-interactor 1) complex has been shown to distribute to both membrane ruffles and focal adhesions of cells, where it plays an important role in regulating focal adhesion turnover. However, the detailed mechanism underlying this regulation is largely unknown. We previously reported that MYO18Aα interacts via its carboxyl terminus with the PAK2/ßPIX/GIT1 complex through direct binding to ßPIX, and that knockdown of MYO18Aα in epithelial cells causes accumulation of the complex in focal adhesions and decreased cell migration ability (Hsu et al., 2010). The current study characterized the detailed MYO18Aα-ßPIX interaction mechanism and the biological significance of this interaction. We found that deletion of the carboxyl-terminal globular domain of MYO18Aα profoundly altered the cellular localization of ßPIX and inhibited cell migration. ßPIX interacts through its most carboxyl-terminus, PAWDETNL (639-646), with MYO18Aα and partially colocalized with MYO18Aα in membrane ruffles of cells, whereas ßPIX(1-638), a mutant with deletion of PAWDETNL, accumulated in focal adhesions. Both focal adhesion numbers and area in ßPIX(1-638)-expressing cells were greater than those in cells expressing wild-type ßPIX(FL). Further experiments using deletion mutants of MYO18A and ßPIX showed that disruption of MYO18A-ßPIX interaction not only impaired cell motility but also decreased Rac1 activity. Collectively, our data unravel the interaction regions between MYO18A and ßPIX and provide evidence for the critical role of this interaction in regulating cellular localization of ßPIX, Rac1 activity, and adhesion and migration in epithelial cells.

8.
BMC Biochem ; 14: 18, 2013 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-23870088

RESUMO

BACKGROUND: Lamins A and C, two major structural components of the nuclear lamina that determine nuclear shape and size, are phosphoproteins. Phosphorylation of lamin A/C is cell cycle-dependent and is involved in regulating the assembly-disassembly of lamin filaments during mitosis. We previously reported that P-STM, a phosphoepitope-specific antibody raised against the autophosphorylation site of p21-activated kinase 2, recognizes a number of phosphoproteins, including lamins A and C, in mitotic HeLa cells. RESULTS: Here, using recombinant proteins and synthetic phosphopeptides containing potential lamin A/C phosphorylation sites in conjunction with in vitro phosphorylation assays, we determined the lamin A/C phosphoepitope(s) recognized by P-STM. We found that phosphorylation of Thr-19 is required for generating the P-STM phosphoepitope in lamin A/C and showed that it could be created in vitro by p34cdc2/cyclin B kinase (CDK1)-catalyzed phosphorylation of lamin A/C immunoprecipitated from unsynchronized HeLa S3 cells. To further explore changes in lamin A/C phosphorylation in living cells, we precisely quantified the phosphorylation levels of Thr-19 and other sites in lamin A/C isolated from HeLa S3 cells at interphase and mitosis using the SILAC method and liquid chromatography-tandem mass spectrometry. The results showed that the levels of phosphorylated Thr-19, Ser-22 and Ser-392 in both lamins A and C, and Ser-636 in lamin A only, increased -2- to 6-fold in mitotic HeLa S3 cells. CONCLUSIONS: Collectively, our results demonstrate that P-STM is a useful tool for detecting Thr-19-phosphorylated lamin A/C in cells and reveal quantitative changes in the phosphorylation status of major lamin A/C phosphorylation sites during mitosis.


Assuntos
Anticorpos/imunologia , Lamina Tipo A/metabolismo , Fosfopeptídeos/imunologia , Sequência de Aminoácidos , Proteína Quinase CDC2/metabolismo , Isótopos de Carbono/química , Cromatografia Líquida de Alta Pressão , Células HeLa , Humanos , Imunoprecipitação , Marcação por Isótopo , Lamina Tipo A/química , Mitose , Dados de Sequência Molecular , Fosfopeptídeos/análise , Fosfopeptídeos/isolamento & purificação , Fosforilação , Espectrometria de Massas em Tandem
9.
J Sep Sci ; 36(9-10): 1582-9, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23494885

RESUMO

This study reported a pH-mediated stacking CE coupled with ESI MS/MS method to determine the phosphorylation sites of three synthetic phosphopeptides containing structural isomers. These phosphopeptides mimic the phosphopeptides (amino acid residues 12-25) derived from the trypsin-digested products of human lamin A/C protein. The LODs were determined to be 118, 132 and 1240 fmol for SGAQASS(19)TpPL(22)SPTR, SGAQASS(19)TPL(22)SpPTR, and SGAQASS(19)TpPL(22)SpPTR, respectively. The established method was employed to analyze the phosphorylation sites of the trypsin-digested products of glutathione S-transferase-lamin A/C (1-57) fusion protein that had been phosphorylated in vitro by cyclin-dependent kinase 1. The results indicated that this method is feasible to specifically determine the phosphorylation site from phosphopeptide isomers in the trypsin-digested products of a kinase-catalyzed phosphoprotein, which should benefit the investigation of protein kinase-mediated cellular signal transduction.


Assuntos
Eletroforese Capilar/métodos , Glutationa Transferase/química , Fosfopeptídeos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteína Quinase CDC2/metabolismo , Eletroforese Capilar/instrumentação , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Isomerismo , Laminas/química , Laminas/genética , Laminas/metabolismo , Dados de Sequência Molecular , Fosfopeptídeos/genética , Fosfopeptídeos/isolamento & purificação , Fosfopeptídeos/metabolismo , Fosforilação , Espectrometria de Massas em Tandem/métodos
10.
J Mol Med (Berl) ; 90(2): 187-200, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21997591

RESUMO

Colorectal cancer (CRC) is one of the most common cancers worldwide. More than half of all CRC patients will develop metastases, which represents the major cause of death for CRC patients. CRC metastases confined in other organs are potentially resectable, and patients who receive curative resections appear to have better outcomes. Thus, the early detection of metastasis in CRC patients could improve their survival rate after curative surgery. Here, we report the use of Cy-dye labeling combined with multi-dimensional fractionation and mass spectrometry as a proteomics-based approach for identifying CRC metastasis-associated biomarker(s) in plasma samples collected from three CRC patients upon diagnosis of their primary and metastatic tumors. Among the eight identified proteins, we used Western blot analysis and an in-house-developed ELISA to validate the increased plasma levels of one, secretory (plasma) gelsolin, in >80% of CRC patients with distal metastases in a larger sample cohort (32 patients). We also found a significant increase of secretory gelsolin in plasma samples of stage IV versus stages I-III CRC patients before treatment. Furthermore, immunohistochemistry showed that secretory gelsolin was highly overexpressed in CRC tissue specimens compared to adjacent normal tissues, and a cell model study showed that secretory gelsolin may help regulate CRC cell migration.


Assuntos
Biomarcadores Tumorais/sangue , Neoplasias Colorretais/sangue , Gelsolina/sangue , Regulação Neoplásica da Expressão Gênica , Idoso , Sequência de Aminoácidos , Linhagem Celular Tumoral , Movimento Celular , Neoplasias Colorretais/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Metástase Neoplásica , Estadiamento de Neoplasias , Proteômica/métodos
11.
Proc Natl Acad Sci U S A ; 107(42): 18022-7, 2010 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-20921392

RESUMO

Translational repression mediated by RNA-binding proteins or micro RNAs has emerged as a major regulatory mechanism for fine-tuning important biological processes. In Caenorhabditis elegans, translational repression of the key sex-determination gene tra-2 (tra, transformer) is controlled by a 28-nucleotide repeat element, the TRA-2/GLI element (TGE), located in its 3' untranslated region (UTR). Mutations that disrupt TGE or the germline-specific TGE-binding factor GLD-1 increase TRA-2 protein expression and inhibit sperm production in hermaphrodites. Here we report the characterization of the sup-26 gene, which regulates sex determination in the soma and encodes an RNA recognition motif (RRM)-containing protein. We show that SUP-26 regulates the level of the TRA-2 protein through TGE in vivo and binds directly to TGE in vitro through its RRM domain. Interestingly, SUP-26 associates with poly(A)-binding protein 1 (PAB-1) in vivo and may repress tra-2 expression by inhibiting the translation-stimulating activity of PAB-1. Taken together, our results provide further insight into how mRNA-binding factors repress translation and modulate sexual development in different tissues of C. elegans.


Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Biossíntese de Proteínas , Proteínas de Ligação a RNA/fisiologia , Processos de Determinação Sexual , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Clonagem Molecular , Dados de Sequência Molecular , Mutação , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética
12.
J Cell Biochem ; 110(3): 660-70, 2010 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-20512926

RESUMO

Increased macrophage vulnerability is associated with progression of systemic lupus erythematosus. Our previous studies have shown that cystamine, an inhibitor of transglutaminase 2 (TG2), alleviated the apoptosis of hepatocyte and brain cell in lupus-prone mice NZB/W-F1. In present study, we further investigated the effects of cystamine on apoptosis-prone macrophages (APMs) in the lupus mice. Using two-dimensional gel electrophoresis (2-DE) analysis, we found that cystamine induced a differential protein expression pattern of APM as comparing to the PBS control. The protein spots presenting differential level between cystamine and PBS treatment were then identified by peptide-mass fingerprinting (PMF). After bioinformatic analysis, these identified proteins were found involved in mitochondrial apoptotic pathway, oxidative stress, and mitogen-activated protein (MAP) kinase-mediated pathway. Further investigation revealed that cystamine significantly decreased the levels of apoptotic Bax and Apaf-1 and the activity of caspase-3, and increased the levels of anti-apoptotic Bcl-2 in APM. We also found that these apoptotic mediators were up-regulated in a correlation with the progression of lupus severity in NZB/W-F1, which were little affected in BALB/c mice. We also found that the reduced serum glutathione was restored by cystamine in NZB/W-F1. Interestingly, the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in APM and the phagocytic ability was diminished in presence of cystamine. In conclusion, our findings indicate that cystamine significantly inhibited mitochondrial pathway, induced antioxidant proteins, and diminished phosphorylation of extracellular ERK1/2, which may alleviate the apoptosis and the phagocytic ability of APM.


Assuntos
Apoptose/efeitos dos fármacos , Cistamina/farmacologia , Inibidores Enzimáticos/farmacologia , Lúpus Eritematoso Sistêmico/metabolismo , Macrófagos/efeitos dos fármacos , Animais , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Feminino , Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica , Immunoblotting , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NZB , Mapeamento de Peptídeos , Proteômica/métodos
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