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1.
J Clin Endocrinol Metab ; 105(3)2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31652310

RESUMO

CONTEXT: Obesity-related insulin resistance (OIR) is one of the main contributors to type 2 diabetes and other metabolic diseases. Protein kinases are implicated in insulin signaling and glucose metabolism. Molecular mechanisms underlying OIR involving global kinase activities remain incompletely understood. OBJECTIVE: To investigate abnormal kinase activity associated with OIR in human skeletal muscle. DESIGN: Utilization of stable isotopic labeling-based quantitative proteomics combined with affinity-based active enzyme probes to profile in vivo kinase activity in skeletal muscle from lean control (Lean) and OIR participants. PARTICIPANTS: A total of 16 nondiabetic adults, 8 Lean and 8 with OIR, underwent hyperinsulinemic-euglycemic clamp with muscle biopsy. RESULTS: We identified the first active kinome, comprising 54 active protein kinases, in human skeletal muscle. The activities of 23 kinases were different in OIR muscle compared with Lean muscle (11 hyper- and 12 hypo-active), while their protein abundance was the same between the 2 groups. The activities of multiple kinases involved in adenosine monophosphate-activated protein kinase (AMPK) and p38 signaling were lower in OIR compared with Lean. On the contrary, multiple kinases in the c-Jun N-terminal kinase (JNK) signaling pathway exhibited higher activity in OIR vs Lean. The kinase-substrate-prediction based on experimental data further confirmed a potential downregulation of insulin signaling (eg, inhibited phosphorylation of insulin receptor substrate-1 and AKT1/2). CONCLUSIONS: These findings provide a global view of the kinome activity in OIR and Lean muscle, pinpoint novel specific impairment in kinase activities in signaling pathways important for skeletal muscle insulin resistance, and may provide potential drug targets (ie, abnormal kinase activities) to prevent and/or reverse skeletal muscle insulin resistance in humans.

2.
World J Stem Cells ; 11(8): 535-547, 2019 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-31523372

RESUMO

Human hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) promise a valuable source of cells with human genetic background, physiologically relevant liver functions, and unlimited supply. With over 10 years' efforts in this field, great achievements have been made. HLCs have been successfully derived and applied in disease modeling, toxicity testing and drug discovery. Large cohorts of induced pluripotent stem cells-derived HLCs have been recently applied in studying population genetics and functional outputs of common genetic variants in vitro. This has offered a new paradigm for genome-wide association studies and possibly in vitro pharmacogenomics in the nearly future. However, HLCs have not yet been successfully applied in bioartificial liver devices and have only displayed limited success in cell transplantation. HLCs still have an immature hepatocyte phenotype and exist as a population with great heterogeneity, and HLCs derived from different hPSC lines display variable differentiation efficiency. Therefore, continuous improvement to the quality of HLCs, deeper investigation of relevant biological processes, and proper adaptation of recent advances in cell culture platforms, genome editing technology, and bioengineering systems are required before HLCs can fulfill the needs in basic and translational research. In this review, we summarize the discoveries, achievements, and challenges in the derivation and applications of HLCs.

3.
Cell Rep ; 26(4): 884-892.e4, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30673611

RESUMO

DNA variants in the SLC16A11 coding region were identified to be strongly associated with type 2 diabetes (T2DM) in a Mexican population. Previous studies suggested that these variants disrupt SLC16A11 function and therefore proposed to revive SLC16A11 levels or activity to achieve therapeutic benefit. However, with knockout mouse models, here we show that Slc16a11 depletion has no significant metabolic defects. Further studies demonstrate that reconstitution of the mutant, but not the wild-type Slc16a11, in the liver of knockout mice causes more triglyceride accumulation and induction of insulin resistance via upregulation of lipin 1, suggesting gaining of aberrant functions of the mutant protein that affects lipid metabolism. Our findings offer a different explanation to the function of these diabetic variants, challenging the concept of enhancing SLC16A11 function to treat T2DM. The contradictory results by our and previous studies suggest that how the SLC16A11 locus contributes to human metabolism warrants further investigation.

4.
J Proteome Res ; 17(9): 2963-2977, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30014700

RESUMO

Charcoal-stripped fetal bovine serum (CS-FBS) is commonly used to study androgen responsiveness and androgen metabolism in cultured prostate cancer (CaP) cells. Switching CaP cells from FBS to CS-FBS may reduce the activity of androgen receptor (AR), inhibit cell proliferation, or modulate intracellular androgen metabolism. The removal of proteins by charcoal stripping may cause changes in biological functions and has not yet been investigated. Here we profiled proteins in FBS and CS-FBS using an ion-current-based quantitative platform consisting of reproducible surfactant-aided precipitation/on-pellet digestion, long-column nanoliquid chromatography separation, and ion-current-based analysis. A total of 143 proteins were identified in FBS, among which 14 proteins including insulin-like growth factor 2 (IGF-2) and IGF binding protein (IGFBP)-2 and -6 were reduced in CS-FBS. IGF-1 receptor (IGF1R) and insulin receptor were sensitized to IGFs in CS-FBS. IGF-1 and IGF-2 stimulation fully compensated for the loss of AR activity to maintain cell growth in CS-FBS. Endogenous production of IGF and IGFBPs was verified in CaP cells and clinical CaP specimens. This study provided the most comprehensive protein profiles of FBS and CS-FBS and offered an opportunity to identify new protein regulators and signaling pathways that regulate AR activity, androgen metabolism, and proliferation of CaP cells.


Assuntos
Proteínas Sanguíneas/isolamento & purificação , Células Epiteliais/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , Proteômica/métodos , Testosterona/farmacologia , Adsorção , Animais , Proteínas Sanguíneas/química , Bovinos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Carvão Vegetal/química , Meios de Cultura/química , Meios de Cultura/farmacologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feto , Expressão Gênica , Humanos , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/isolamento & purificação , Proteína 6 de Ligação a Fator de Crescimento Semelhante à Insulina/isolamento & purificação , Fator de Crescimento Insulin-Like I/isolamento & purificação , Fator de Crescimento Insulin-Like I/farmacologia , Fator de Crescimento Insulin-Like II/isolamento & purificação , Fator de Crescimento Insulin-Like II/farmacologia , Masculino , Próstata/efeitos dos fármacos , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Receptor IGF Tipo 1/isolamento & purificação , Receptor de Insulina/isolamento & purificação , Receptores Androgênicos/biossíntese , Receptores Androgênicos/genética , Testosterona/isolamento & purificação
5.
Stem Cell Reports ; 11(1): 22-31, 2018 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-29861165

RESUMO

Hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) offer a promising cell resource for disease modeling and transplantation. However, differentiated HLCs exhibit an immature phenotype and comprise a heterogeneous population. Thus, a better understanding of HLC differentiation will improve the likelihood of future application. Here, by taking advantage of CRISPR-Cas9-based genome-wide screening technology and a high-throughput hPSC screening platform with a reporter readout, we identified several potential genetic regulators of HLC differentiation. By using a chemical screening approach within our platform, we also identified compounds that can further promote HLC differentiation and preserve the characteristics of in vitro cultured primary hepatocytes. Remarkably, both screenings identified histone deacetylase 3 (HDAC3) as a key regulator in hepatic differentiation. Mechanistically, HDAC3 formed a complex with liver transcriptional factors, e.g., HNF4, and co-regulated the transcriptional program during hepatic differentiation. This study highlights a broadly useful approach for studying and optimizing hPSC differentiation.


Assuntos
Diferenciação Celular , Hepatócitos/citologia , Hepatócitos/metabolismo , Histona Desacetilases/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Sistemas CRISPR-Cas , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Células Cultivadas , Citometria de Fluxo , Edição de Genes , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Genes Reporter , Genes abl , Fator 4 Nuclear de Hepatócito/metabolismo , Histona Desacetilases/genética , Humanos , Modelos Biológicos , Fenilenodiaminas/farmacologia
6.
J Proteome Res ; 17(3): 1300-1308, 2018 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-29369637

RESUMO

Failure to properly repair damaged due to myocardial infarction is a major cause of heart failure. In contrast with adult mammals, zebrafish hearts show remarkable regenerative capabilities after substantial damage. To characterize protein dynamics during heart regeneration, we employed an HPLC-ESI-MS/MS (mass spectrometry) approach. Myocardium tissues were taken from sham-operated fish and ventricle-resected sample at three different time points (2, 7, and 14 days); dynamics of protein expression were analyzed by an ion-current-based quantitative platform. More than 2000 protein groups were quantified in all 16 experiments. Two hundred and nine heart-regeneration-related protein groups were quantified and clustered into six time-course patterns. Functional analysis indicated that multiple molecular function and metabolic pathways were involved in heart regeneration. Interestingly, Ingenuity Pathway Analysis revealed that P53 signaling was inhibited during the heart regeneration, which was further verified by real-time quantitative polymerase chain reaction (Q-PCR). In summary, we applied systematic proteomics analysis on regenerating zebrafish heart, uncovered the dynamics of regenerative genes expression and regulatory pathways, and provided invaluable insight into design regenerative-based strategies in human hearts.


Assuntos
Proteínas de Peixes/genética , Traumatismos Cardíacos/genética , Ventrículos do Coração/metabolismo , Miocárdio/metabolismo , Proteômica/métodos , Regeneração/genética , Animais , Cromatografia Líquida de Alta Pressão , Proteínas de Peixes/metabolismo , Ontologia Genética , Traumatismos Cardíacos/metabolismo , Traumatismos Cardíacos/reabilitação , Ventrículos do Coração/lesões , Redes e Vias Metabólicas/genética , Anotação de Sequência Molecular , Proteômica/instrumentação , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas por Ionização por Electrospray , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Peixe-Zebra
7.
Anal Chem ; 90(4): 2434-2439, 2018 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-29376338

RESUMO

Membrane proteins may act as transporters, receptors, enzymes, and adhesion-anchors, accounting for nearly 70% of pharmaceutical drug targets. Difficulties in efficient enrichment, extraction, and solubilization still exist because of their relatively low abundance and poor solubility. A simplified membrane protein extraction approach with advantages of user-friendly sample processing procedures, good repeatability and significant effectiveness was developed in the current research for enhancing enrichment and identification of membrane proteins. This approach combining centrifugation and detergent along with LC-MS/MS successfully identified higher proportion of membrane proteins, integral proteins and transmembrane proteins in membrane fraction (76.6%, 48.1%, and 40.6%) than in total cell lysate (41.6%, 16.4%, and 13.5%), respectively. Moreover, our method tended to capture membrane proteins with high degree of hydrophobicity and number of transmembrane domains as 486 out of 2106 (23.0%) had GRAVY > 0 in membrane fraction, 488 out of 2106 (23.1%) had TMs ≥ 2. It also provided for improved identification of membrane proteins as more than 60.6% of the commonly identified membrane proteins in two cell samples were better identified in membrane fraction with higher sequence coverage. Data are available via ProteomeXchange with identifier PXD008456.

8.
Obesity (Silver Spring) ; 24(7): 1506-14, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27345962

RESUMO

OBJECTIVE: To provide a more global view of adipocyte changes in human insulin resistance by proteomics analyses. METHODS: Baseline biopsies of abdominal subcutaneous adipose tissue were obtained from 23 subjects without diabetes. Euglycemic clamps were used to divide subjects into an insulin-resistant group (IR, N = 10) and an insulin-sensitive (IS, N = 13) group, which were of similar age and gender but unequal adiposity (greater in IR). Proteins of isolated adipocytes were quantified by mass spectrometry using normalized spectral abundance factors. RESULTS: Of 1,245 proteins assigned, 30 were detected in at least 12 of the 23 subjects that differed significantly in abundance ≥1.5-fold between IR and IS. IR displayed a pattern of increased cytoskeletal proteins and decreased mitochondrial proteins and FABP4 and FABP5. In subgroup analyses of adiposity-matched subjects, several of these changes were less pronounced in IR, but the abundance of proteins related to lipid metabolism and the unfolded/misfolded protein response were significantly and unfavorably altered. CONCLUSIONS: These results confirm lower abundance of mitochondrial proteins and suggest increased cytoskeletal proteins and decreased FABP4 and FABP5 in subcutaneous adipocytes of typical IR individuals. Changes in proteins related to lipid metabolism and the unfolded/misfolded protein may discriminate IR and IS individuals of equal adiposity.


Assuntos
Adipócitos/química , Resistência à Insulina , Proteômica , Gordura Subcutânea Abdominal/citologia , Adiposidade , Adulto , Proteínas do Citoesqueleto/análise , Proteínas de Ligação a Ácido Graxo/análise , Feminino , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Mitocondriais/análise
9.
Mol Cell Endocrinol ; 424: 1-11, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26780722

RESUMO

Protein phosphatase 2A (PP2A) is one of the major serine/threonine phosphatases. We hypothesize that PP2A regulates signaling cascades in pancreatic ß-cells in the context of glucose-stimulated insulin secretion (GSIS). Using co-immunoprecipitation (co-IP) and tandem mass spectrometry, we globally identified the protein interaction partners of the PP2A catalytic subunit (PP2Ac) in insulin-secreting pancreatic ß-cells. Among the 514 identified PP2Ac interaction partners, 476 were novel. This represents the first global view of PP2Ac protein-protein interactions caused by hyperglycemic conditions. Additionally, numerous PP2Ac partners were found involved in a variety of signaling pathways in the ß-cell function, such as insulin secretion. Our data suggest that PP2A interacts with various signaling proteins necessary for physiological insulin secretion as well as signaling proteins known to regulate cell dysfunction and apoptosis in the pancreatic ß-cells.


Assuntos
Redes Reguladoras de Genes , Células Secretoras de Insulina/fisiologia , Proteína Fosfatase 2/metabolismo , Proteômica/métodos , Células Cultivadas , Glucose/farmacologia , Humanos , Mapeamento de Interação de Proteínas/métodos , Espectrometria de Massas em Tandem
10.
PLoS One ; 10(10): e0140255, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26465754

RESUMO

Insulin resistance and Type 2 diabetes are marked by an aberrant response in the insulin signaling network. The phosphoinositide-dependent serine/threonine kinase, Akt2, plays a key role in insulin signaling and glucose uptake, most notably within skeletal muscle. Protein-protein interaction regulates the functional consequence of Akt2 and in turn, Akt2's role in glucose uptake. However, only few insulin-responsive Akt2 interaction partners have been identified in skeletal muscle cells. In the present work, rat L6 myoblasts, a widely used insulin sensitive skeletal muscle cell line, were used to examine endogenous, insulin-stimulated Akt2 protein interaction partners. Akt2 co-immunoprecipitation was coupled with 1D-SDS-PAGE and fractions were analyzed by HPLC-ESI-MS/MS to reveal Akt2 protein-protein interactions. The pull-down assay displayed specificity for the Akt2 isoform; Akt1 and Akt3 unique peptides were not detected. A total of 49 were detected with a significantly increased (47) or decreased (2) association with Akt2 following insulin administration (n = 4; p<0.05). Multiple pathways were identified for the novel Akt2 interaction partners, such as the EIF2 and ubiquitination pathways. These data suggest that multiple new endogenous proteins may associate with Akt2 under basal as well as insulin-stimulated conditions, providing further insight into the insulin signaling network. Data are available via ProteomeXchange with identifier PXD002557.


Assuntos
Proteínas de Transporte/metabolismo , Mioblastos/metabolismo , Mapeamento de Interação de Proteínas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Células Cultivadas , Ligação Proteica , Ratos , Transdução de Sinais , Quinases Associadas a rho/metabolismo
11.
J Proteome Res ; 14(11): 4662-73, 2015 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-26390080

RESUMO

The two key steps for analyzing proteomic data generated by high-resolution MS are database searching and postprocessing. While the two steps are interrelated, studies on their combinatory effects and the optimization of these procedures have not been adequately conducted. Here, we investigated the performance of three popular search engines (SEQUEST, Mascot, and MS Amanda) in conjunction with five filtering approaches, including respective score-based filtering, a group-based approach, local false discovery rate (LFDR), PeptideProphet, and Percolator. A total of eight data sets from various proteomes (e.g., E. coli, yeast, and human) produced by various instruments with high-accuracy survey scan (MS1) and high- or low-accuracy fragment ion scan (MS2) (LTQ-Orbitrap, Orbitrap-Velos, Orbitrap-Elite, Q-Exactive, Orbitrap-Fusion, and Q-TOF) were analyzed. It was found combinations involving Percolator achieved markedly more peptide and protein identifications at the same FDR level than the other 12 combinations for all data sets. Among these, combinations of SEQUEST-Percolator and MS Amanda-Percolator provided slightly better performances for data sets with low-accuracy MS2 (ion trap or IT) and high accuracy MS2 (Orbitrap or TOF), respectively, than did other methods. For approaches without Percolator, SEQUEST-group performs the best for data sets with MS2 produced by collision-induced dissociation (CID) and IT analysis; Mascot-LFDR gives more identifications for data sets generated by higher-energy collisional dissociation (HCD) and analyzed in Orbitrap (HCD-OT) and in Orbitrap Fusion (HCD-IT); MS Amanda-Group excels for the Q-TOF data set and the Orbitrap Velos HCD-OT data set. Therefore, if Percolator was not used, a specific combination should be applied for each type of data set. Moreover, a higher percentage of multiple-peptide proteins and lower variation of protein spectral counts were observed when analyzing technical replicates using Percolator-associated combinations; therefore, Percolator enhanced the reliability for both identification and quantification. The analyses were performed using the specific programs embedded in Proteome Discoverer, Scaffold, and an in-house algorithm (BuildSummary). These results provide valuable guidelines for the optimal interpretation of proteomic results and the development of fit-for-purpose protocols under different situations.


Assuntos
Algoritmos , Peptídeos/análise , Proteoma/análise , Proteômica/métodos , Ferramenta de Busca/métodos , Software , Linhagem Celular Tumoral , Bases de Dados de Proteínas , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteoma/genética , Proteoma/metabolismo , Proteômica/instrumentação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas em Tandem
12.
J Proteomics ; 109: 63-75, 2014 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-24972320

RESUMO

UNLABELLED: Serine/threonine protein phosphatase 1 regulatory subunit 12A (PPP1R12A) modulates the activity and specificity of the catalytic subunit of protein phosphatase 1, regulating various cellular processes via dephosphorylation. Nonetheless, little is known about phosphorylation events controlled by PPP1R12A in skeletal muscle insulin signaling. Here, we used quantitative phosphoproteomics to generate a global picture of phosphorylation events regulated by PPP1R12A in a L6 skeletal muscle cell line, which were engineered for inducible PPP1R12A knockdown. Phosphoproteomics revealed 3876 phosphorylation sites (620 were novel) in these cells. Furthermore, PPP1R12A knockdown resulted in increased overall phosphorylation in L6 cells at the basal condition, and changed phosphorylation levels for 698 sites (assigned to 295 phosphoproteins) at the basal and/or insulin-stimulated conditions. Pathway analysis on the 295 phosphoproteins revealed multiple significantly enriched pathways related to insulin signaling, such as mTOR signaling and RhoA signaling. Moreover, phosphorylation levels for numerous regulatory sites in these pathways were significantly changed due to PPP1R12A knockdown. These results indicate that PPP1R12A indeed plays a role in skeletal muscle insulin signaling, providing novel insights into the biology of insulin action. This new information may facilitate the design of experiments to better understand mechanisms underlying skeletal muscle insulin resistance and type 2 diabetes. BIOLOGICAL SIGNIFICANCE: These results identify a large number of potential new substrates of serine/threonine protein phosphatase 1 and suggest that serine/threonine protein phosphatase 1 regulatory subunit 12A indeed plays a regulatory role in multiple pathways related to insulin action, providing novel insights into the biology of skeletal muscle insulin signaling. This information may facilitate the design of experiments to better understand the molecular mechanism responsible for skeletal muscle insulin resistance and associated diseases, such as type 2 diabetes and cardiovascular diseases.


Assuntos
Insulina/metabolismo , Proteínas Musculares/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Proteína Fosfatase 1/metabolismo , Proteômica , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Resistência à Insulina , Fosforilação/fisiologia , Ratos , Serina-Treonina Quinases TOR/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
13.
Diabetes ; 63(6): 1933-47, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24584551

RESUMO

Insulin receptor substrate 1 (IRS1) is a key mediator of insulin signal transduction. Perturbations involving IRS1 complexes may lead to the development of insulin resistance and type 2 diabetes (T2D). Surprisingly little is known about the proteins that interact with IRS1 in humans under health and disease conditions. We used a proteomic approach to assess IRS1 interaction partners in skeletal muscle from lean healthy control subjects (LCs), obese insulin-resistant nondiabetic control subjects (OCs), and participants with T2D before and after insulin infusion. We identified 113 novel endogenous IRS1 interaction partners, which represents the largest IRS1 interactome in humans and provides new targets for studies of IRS1 complexes in various diseases. Furthermore, we generated the first global picture of IRS1 interaction partners in LCs, and how they differ in OCs and T2D patients. Interestingly, dozens of proteins in OCs and/or T2D patients exhibited increased associations with IRS1 compared with LCs under the basal and/or insulin-stimulated conditions, revealing multiple new dysfunctional IRS1 pathways in OCs and T2D patients. This novel abnormality, increased interaction of multiple proteins with IRS1 in obesity and T2D in humans, provides new insights into the molecular mechanism of insulin resistance and identifies new targets for T2D drug development.


Assuntos
Glicemia/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Resistência à Insulina/genética , Músculo Esquelético/metabolismo , Obesidade/metabolismo , Magreza/metabolismo , Índice de Massa Corporal , Diabetes Mellitus Tipo 2/genética , Feminino , Predisposição Genética para Doença , Variação Genética , Técnica Clamp de Glucose , Humanos , Insulina/administração & dosagem , Proteínas Substratos do Receptor de Insulina/genética , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Obesidade/genética , Regiões Promotoras Genéticas , RNA Mensageiro , Fatores de Risco , Transdução de Sinais , Magreza/genética
14.
Oncotarget ; 4(9): 1427-37, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23965803

RESUMO

Mineral dust-induced gene (mdig) had been linked to the development of human lung cancers associated with environmental exposure to mineral dust, tobacco smoke or other carcinogens. In the present studies, we demonstrated that the overexpression of mdig in A549 adenocarcinomic human alveolar type II epithelial cells decreases the heterochromatin conformation of the cells and de-represses the transcription of genes in the tandemly repeated DNA regions. Although mdig can only cause a marginal decrease of the total histone H3 lysine 9 trimethylation (H3K9me3), a significant reduction of H3K9me3 in the promoter region of H19, the paternally imprinted but maternally expressed gene transcribing a large intergenic non-coding RNA (lincRNA), was observed in the cells with mdig overexpression. Silencing mdig by either shRNA or siRNA not only increased the level of H3K9me3 in the promoter region of H19 but also attenuated the transcription of H19 long non-coding RNA. Demethylation assays using immunoprecipitated mdig and histone H3 peptide substrate suggested that mdig is able to remove the methyl groups from H3K9me3. Clinically, we found that higher levels of mdig and H19 expression correlate with poorer survival of the lung cancer patients. Taken together, our results imply that mdig is involved in the regulation of H3K9me3 to influence the heterochromatin structure of the genome and the expression of genes important for cell growth or transformation.


Assuntos
Heterocromatina/genética , Histonas/genética , Proteínas Nucleares/genética , RNA Longo não Codificante/genética , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma de Pulmão , Linhagem Celular Tumoral , Dioxigenases , Regulação para Baixo , Heterocromatina/metabolismo , Histona Desmetilases , Histonas/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Metilação , Proteínas Nucleares/metabolismo , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Análise de Sobrevida , Transcrição Genética , Transfecção
15.
Exp Hematol Oncol ; 2: 18, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23890079

RESUMO

Oxythiamine (OT), an analogue of anti-metabolite, can suppress the nonoxidative synthesis of ribose and induce cell apoptosis by causing a G1 phase arrest in vitro and in vivo. However, the molecular mechanism remains unclear yet. In the present study, a quantitative proteomic analysis using the modified SILAC method (mSILAC) was performed to determine the effect of metabolic inhibition on dynamic changes of protein expression in MIA PaCa-2 cancer cells treated with OT at various doses (0 µM, 5 µM, 50 µM and 500 µM) and time points (0 h, 12 h and 48 h). A total of 52 differential proteins in MIA PaCa-2 cells treated with OT were identified, including 14 phosphorylated proteins. Based on the dynamic expression pattern, these proteins were categorized in three clusters, straight down-regulation (cluster 1, 37% of total proteins), upright "V" shape expression pattern (cluster 2, 47.8% total), and downright "V" shape pattern (cluster 3, 15.2% total). Among them, Annexin A1 expression was significantly down-regulated by OT treatment in time-dependent manner, while no change of this protein was observed in OT dose-dependent fashion. Pathway analysis suggested that inhibition of transketolase resulted in changes of multiple cellular signaling pathways associated with cell apoptosis. The temporal expression patterns of proteins revealed that OT altered dynamics of protein expression in time-dependent fashion by suppressing phosphor kinase expression, resulting in cancer cell apoptosis. Results from this study suggest that interference of single metabolic enzyme activity altered multiple cellular signaling pathways.

16.
Elife ; 2: e00444, 2013 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-23580231

RESUMO

The secretory pathway of eukaryotic cells packages cargo proteins into COPII-coated vesicles for transport from the endoplasmic reticulum (ER) to the Golgi. We now report that complete genetic deficiency for the COPII component SEC24A is compatible with normal survival and development in the mouse, despite the fundamental role of SEC24 in COPII vesicle formation and cargo recruitment. However, these animals exhibit markedly reduced plasma cholesterol, with mutations in Apoe and Ldlr epistatic to Sec24a, suggesting a receptor-mediated lipoprotein clearance mechanism. Consistent with these data, hepatic LDLR levels are up-regulated in SEC24A-deficient cells as a consequence of specific dependence of PCSK9, a negative regulator of LDLR, on SEC24A for efficient exit from the ER. Our findings also identify partial overlap in cargo selectivity between SEC24A and SEC24B, suggesting a previously unappreciated heterogeneity in the recruitment of secretory proteins to the COPII vesicles that extends to soluble as well as trans-membrane cargoes. DOI:http://dx.doi.org/10.7554/eLife.00444.001.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/enzimologia , Colesterol/sangue , Dislipidemias/enzimologia , Retículo Endoplasmático/enzimologia , Fígado/enzimologia , Pró-Proteína Convertases/sangue , Serina Endopeptidases/sangue , Proteínas de Transporte Vesicular/deficiência , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Dislipidemias/sangue , Dislipidemias/genética , Retículo Endoplasmático/metabolismo , Epistasia Genética , Genótipo , Células HEK293 , Humanos , Fígado/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Fenótipo , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/metabolismo , Transporte Proteico , Receptores de LDL/genética , Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , Transfecção , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
17.
Insect Biochem Mol Biol ; 43(12): 1181-1188, 2013 12.
Artigo em Inglês | MEDLINE | ID: mdl-24446544

RESUMO

The silks of arthropods have an elementary role in the natural history of the organisms that spin them, yet they are coded by rapidly evolving genes leading some authors to speculate that silk proteins are non-homologous proteins co-opted multiple times independently for similar functions. However, some general structural patterns are emerging. In this work we identified three major silk gland proteins using a combined biochemical, proteomic, next-generation sequencing and bioinformatic approach. Biochemical characterization determined that they were phosphorylated with multiple isoforms and potentially differential phosphorylation. Structural characterization showed that their structure was more similar to silk proteins from distantly related aquatic Trichopteran species than more closely related terrestrial or aquatic Diptera. Overall, our approach is easily transferable to any non-model species and if used across a larger number of aquatic species, we will be able to better understand the processes involved in linking the secondary structure of silk proteins with their function between in an organisms and its habitat.


Assuntos
Proteômica , Seda/química , Animais , Fosforilação , Estrutura Secundária de Proteína , Seda/genética , Simuliidae/química , Simuliidae/genética
18.
J Proteome Res ; 11(7): 3548-60, 2012 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-22616666

RESUMO

Mechanism underlying smoke-induced loss of bone mass is unknown. In this study, we hypothesized that protein signals induced by smoking in bone marrow may be associated with the loss of bone mass. Using a proteomics approach, we identified 38 proteins differentially expressed in bone marrow cells from low-density lipoprotein receptor-related protein 5 (Lrp5) mice exposed to cigarette smoking. Smoking effects on protein expression in bone marrow among three genotypes (Lrp5(+/+), Lrp5(G171V), and Lrp5(-/-)) varied. On the basis of the ratio of protein expression induced by smoking versus nonsmoking, smoke induced protein expression significantly in wild-type mice compared to the other two genotypes (Lrp5(G171V) and Lrp5(-/-)). These proteins include inhibitors of ß-catenin and proteins associated with differentiation of osteoclasts. We observed that S100A8 and S100A9 were overexpressed in human smokers compared to nonsmokers, which confirmed the effect of smoking on the expression of two proteins in Lrp5 mice, suggesting the role of these proteins in bone remodeling. Smoke induced expression of S100A8 and S100A9 in a time-dependent fashion, which was opposite of the changes in the ratio of OPG/RANKL in bone marrow cells, suggesting that the high levels of S100A8 and S100A9 may be associated with smoke-induced bone loss by increasing bone resorption.


Assuntos
Células da Medula Óssea/metabolismo , Reabsorção Óssea/etiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Fumaça/efeitos adversos , Tabaco/efeitos adversos , Animais , Remodelação Óssea , Reabsorção Óssea/metabolismo , Calgranulina A/genética , Calgranulina A/metabolismo , Calgranulina B/genética , Calgranulina B/metabolismo , Diferenciação Celular , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucócitos Mononucleares/metabolismo , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteoma/genética , Proteoma/metabolismo , Ligante RANK/genética , Ligante RANK/metabolismo , Radiografia , Fumar/efeitos adversos
19.
J Proteomics ; 75(11): 3342-50, 2012 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-22516431

RESUMO

Protein phosphatase 1 (PP1) is one of the major phosphatases responsible for protein dephosphorylation in eukaryotes. So far, only few specific phosphorylation sites of PP1 regulatory subunit 12A (PPP1R12A) have been shown to regulate the PP1 activity. The effect of insulin on PPP1R12A phosphorylation is largely unknown. Utilizing a mass spectrometry based phosphorylation identification and quantification approach, we identified 21 PPP1R12A phosphorylation sites (7 novel sites, including Ser20, Thr22, Thr453, Ser478, Thr671, Ser678, and Ser680) and quantified 16 of them under basal and insulin stimulated conditions in hamster ovary cells overexpressing the insulin receptor (CHO/IR), an insulin sensitive cell model. Insulin stimulated the phosphorylation of PPP1R12A significantly at Ser477, Ser478, Ser507, Ser668, and Ser695, while simultaneously suppressing the phosphorylation of PPP1R12A at Ser509 (more than 2-fold increase or decrease compared to basal). Our data demonstrate that PPP1R12A undergoes insulin stimulated/suppressed phosphorylation, suggesting that PPP1R12A phosphorylation may play a role in insulin signal transduction. The novel PPP1R12A phosphorylation sites as well as the new insulin-responsive phosphorylation sites of PPP1R12A in CHO/IR cells provide targets for investigation of the regulation of PPP1R12A and the PPP1R12A-PP1cδ complex in insulin action and other signaling pathways in other cell models, animal models, and humans.


Assuntos
Hipoglicemiantes/farmacologia , Insulina/farmacologia , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Receptor de Insulina/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Fosfatase de Miosina-de-Cadeia-Leve/genética , Fosforilação/efeitos dos fármacos , Receptor de Insulina/genética
20.
Pancreas ; 41(3): 397-408, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22158071

RESUMO

OBJECTIVES: Novel quantitative proteomic approaches were used to study the effects of inhibition of glycogen phosphorylase on proteome and signaling pathways in MIA PaCa-2 pancreatic cancer cells. METHODS: We performed quantitative proteomic analysis in MIA PaCa-2 cancer cells treated with a stratified dose of CP-320626 (5-chloro-1H-indole-2-carboxylic acid [1-(4-fuorobenzyl)-2-(4-hydroxypiperidin-1-yl)-2 oxoethyl] amide) (25, 50, and 100 µM). The effect of metabolic inhibition on cellular protein turnover dynamics was also studied using the modified SILAC (stable isotope labeling with amino acids in cell culture) method. RESULTS: A total of 22 protein spots and 4 phosphoprotein spots were quantitatively analyzed. We found that dynamic expression of total proteins and phosphoproteins was significantly changed in MIA PaCa-2 cells treated with an incremental dose of CP-320626. Functional analyses suggested that most of the proteins differentially expressed were in the pathways of mitogen-activated protein kinase/extracellular signal-regulated kinase and tumor necrosis factor α/nuclear factor κB. CONCLUSIONS: Signaling pathways and metabolic pathways share many common cofactors and substrates forming an extended metabolic network. The restriction of substrate through 1 pathway such as inhibition of glycogen phosphorylation induces pervasive metabolomic and proteomic changes manifested in protein synthesis, breakdown, and posttranslational modification of signaling molecules. Our results suggest that quantitative proteomic is an important approach to understand the interaction between metabolism and signaling pathways.


Assuntos
Amidas/farmacologia , Inibidores Enzimáticos/farmacologia , Glicogênio Fosforilase/antagonistas & inibidores , Glicogênio/metabolismo , Indóis/farmacologia , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/enzimologia , Transdução de Sinais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Eletroforese em Gel Bidimensional , Glicogênio Fosforilase/metabolismo , Humanos , Proteínas de Neoplasias/biossíntese , Neoplasias Pancreáticas/patologia , Mapeamento de Peptídeos , Fosforilação , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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