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1.
Int J Mol Sci ; 23(10)2022 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-35628290

RESUMO

Inflammatory responses by the innate and adaptive immune systems protect against infections and are essential to health and survival. Many diseases including atherosclerosis, osteoarthritis, rheumatoid arthritis, psoriasis, and obesity involve persistent chronic inflammation. Currently available anti-inflammatory agents, including non-steroidal anti-inflammatory drugs, steroids, and biologics, are often unsafe for chronic use due to adverse effects. The development of effective non-toxic anti-inflammatory agents for chronic use remains an important research arena. We previously reported that oral administration of Oxy210, a semi-synthetic oxysterol, ameliorates non-alcoholic steatohepatitis (NASH) induced by a high-fat diet in APOE*3-Leiden.CETP humanized mouse model of NASH and inhibits expression of hepatic and circulating levels of inflammatory cytokines. Here, we show that Oxy210 also inhibits diet-induced white adipose tissue inflammation in APOE*3-Leiden.CETP mice, evidenced by the inhibition of adipose tissue expression of IL-6, MCP-1, and CD68 macrophage marker. Oxy210 and related analogs exhibit anti-inflammatory effects in macrophages treated with lipopolysaccharide in vitro, mediated through inhibition of toll-like receptor 4 (TLR4), TLR2, and AP-1 signaling, independent of cyclooxygenase enzymes or steroid receptors. The anti-inflammatory effects of Oxy210 are correlated with the inhibition of macrophage polarization. We propose that Oxy210 and its structural analogs may be attractive candidates for future therapeutic development for targeting inflammatory diseases.


Assuntos
Hepatopatia Gordurosa não Alcoólica , Oxisteróis , Animais , Apolipoproteínas E/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Camundongos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/metabolismo , Oxisteróis/metabolismo , Oxisteróis/farmacologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo
2.
J Biol Chem ; 295(52): 18485-18493, 2020 12 25.
Artigo em Inglês | MEDLINE | ID: mdl-33097595

RESUMO

Timely repair of DNA double-strand breaks (DSBs) is essential to maintaining genomic integrity and preventing illnesses induced by genetic abnormalities. We previously demonstrated that the E3 ubiquitin ligase SMURF2 plays a critical tumor suppressing role via its interaction with RNF20 (ring finger protein 20) in shaping chromatin landscape and preserving genomic stability. However, the mechanism that mobilizes SMURF2 in response to DNA damage remains unclear. Using biochemical approaches and MS analysis, we show that upon the onset of the DNA-damage response, SMURF2 becomes phosphorylated at Ser384 by ataxia telangiectasia mutated (ATM) serine/threonine kinase, and this phosphorylation is required for its interaction with RNF20. We demonstrate that a SMURF2 mutant with an S384A substitution has reduced capacity to ubiquitinate RNF20 while promoting Smad3 ubiquitination unabatedly. More importantly, mouse embryonic fibroblasts expressing the SMURF2 S384A mutant show a weakened ability to sustain the DSB response compared with those expressing WT SMURF2 following etoposide treatment. These data indicate that SMURF2-mediated RNF20 ubiquitination and degradation controlled by ataxia telangiectasia mutated-induced phosphorylation at Ser384 constitutes a negative feedback loop that regulates DSB repair.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Cromatina/metabolismo , Dano ao DNA , Reparo do DNA , Retroalimentação Fisiológica , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Cromatina/genética , Instabilidade Genômica , Humanos , Camundongos , Fosforilação , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
3.
PLoS Biol ; 16(12): e3000091, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30566427

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is characterized by abnormal accumulation of triglycerides (TG) in the liver and other metabolic syndrome symptoms, but its molecular genetic causes are not completely understood. Here, we show that mice deficient for ubiquitin ligase (E3) Smad ubiquitin regulatory factor 1 (Smurf1) spontaneously develop hepatic steatosis as they age and exhibit the exacerbated phenotype under a high-fat diet (HFD). Our data indicate that loss of Smurf1 up-regulates the expression of peroxisome proliferator-activated receptor γ (PPARγ) and its target genes involved in lipid synthesis and fatty acid uptake. We further show that PPARγ is a direct substrate of Smurf1-mediated non-proteolytic lysine 63 (K63)-linked ubiquitin modification that suppresses its transcriptional activity, and treatment of Smurf1-deficient mice with a PPARγ antagonist, GW9662, completely reversed the lipid accumulation in the liver. Finally, we demonstrate an inverse correlation of low SMURF1 expression to high body mass index (BMI) values in human patients, thus revealing a new role of SMURF1 in NAFLD pathogenesis.


Assuntos
Fígado Gorduroso/prevenção & controle , PPAR gama/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Animais , Linhagem Celular , Dieta Hiperlipídica , Ácidos Graxos/metabolismo , Fígado Gorduroso/metabolismo , Fígado Gorduroso/fisiopatologia , Humanos , Fígado/metabolismo , Fígado/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/metabolismo , Triglicerídeos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
4.
J Biol Chem ; 292(10): 4302-4312, 2017 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-28154170

RESUMO

Transforming growth factor-ß (TGF-ß) signals through both SMAD and non-SMAD pathways to elicit a wide array of biological effects. Existing data have shown the association and coordination between STATs and SMADs in mediating TGF-ß functions in hepatic cells, but it is not clear how STATs are activated under these circumstances. Here, we report that JAK1 is a constitutive TGFßRI binding protein and is absolutely required for phosphorylation of STATs in a SMAD-independent manner within minutes of TGF-ß stimulation. Following the activation of SMADs, TGF-ß also induces a second phase of STAT phosphorylation that requires SMADs, de novo protein synthesis, and contribution from JAK1. Our global gene expression profiling indicates that the non-SMAD JAK1/STAT pathway is essential for the expression of a subset of TGF-ß target genes in hepatic stellate cells, and the cooperation between the JAK1-STAT3 and SMAD pathways is critical to the roles of TGF-ß in liver fibrosis.


Assuntos
Embrião de Mamíferos/patologia , Células Estreladas do Fígado/patologia , Janus Quinase 1/metabolismo , Cirrose Hepática/patologia , Fator de Transcrição STAT3/metabolismo , Proteínas Smad/fisiologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Células Cultivadas , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Janus Quinase 1/genética , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Camundongos , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Fator de Transcrição STAT3/genética , Transdução de Sinais/efeitos dos fármacos
5.
EMBO J ; 30(23): 4777-89, 2011 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-22045334

RESUMO

TGF-ß signalling is regulated by post-translational modifications of Smad proteins to translate quantitative difference in ligand concentration into proportional transcriptional output. Previous studies in cell culture systems suggested that Smad ubiquitination regulatory factors (Smurfs) act in this regulation by targeting Smads for proteasomal degradation, but whether this mechanism operates under physiological conditions is not clear. Here, we generated mice harbouring a target-disrupted Smurf2 allele. Using primary mouse embryonic fibroblasts and dermal fibroblasts, we show that TGF-ß-mediated, Smad-dependent transcriptional responses are elevated in the absence of Smurf2. Instead of promoting poly-ubiquitination and degradation, we show that Smurf2 actually induces multiple mono-ubiquitination of Smad3 in vivo. Phosphorylation of T179, immediately upstream of the Smad3 PY motif, enhances Smurf2 and Smad3 interaction and Smad3 ubiquitination. We have mapped Smurf2-induced Smad3 ubiquitination sites to lysine residues at the MH2 domain, and demonstrate that Smad3 ubiquitination inhibits the formation of Smad3 complexes. Thus, our data support a model in which Smurf2 negatively regulates TGF-ß signalling by attenuating the activity of Smad3 rather than promoting its degradation.


Assuntos
Transdução de Sinais/fisiologia , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases , Ubiquitinação/fisiologia , Animais , Western Blotting , Fibroblastos/metabolismo , Camundongos , Modelos Biológicos , Ligação Proteica , Transcrição Gênica/fisiologia , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
6.
J Biol Chem ; 288(30): 21784-92, 2013 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-23760265

RESUMO

Smad ubiquitin regulatory factors (Smurfs) are HECT-domain ubiquitin E3 ligases that regulate diverse cellular processes, including normal and tumor cell migration. However, the underlying mechanism of the Smurfs' role in cell migration is not fully understood. Here we show that Smurf1 induces ubiquitination of tumor necrosis factor receptor-associated factor 4 (TRAF4) at K190. Using the K190R mutant of TRAF4, we demonstrate that Smurf1-induced ubiquitination is required for proper localization of TRAF4 to tight junctions in confluent epithelial cells. We further show that TRAF4 is essential for the migration of both normal mammary epithelial and breast cancer cells. The ability of TRAF4 to promote cell migration is also dependent on Smurf1-mediated ubiquitination, which is associated with Rac1 activation by TRAF4. These results reveal a new regulatory circuit for cell migration, consisting of Smurf1-mediated ubiquitination of TRAF4 and Rac1 activation.


Assuntos
Movimento Celular , Células Epiteliais/metabolismo , Fator 4 Associado a Receptor de TNF/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular , Células Epiteliais/citologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Lisina/genética , Lisina/metabolismo , Células MCF-7 , Glândulas Mamárias Humanas/citologia , Camundongos , Microscopia Confocal , Mutação , Interferência de RNA , Fator 4 Associado a Receptor de TNF/genética , Junções Íntimas/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Proteínas rac1 de Ligação ao GTP/metabolismo
7.
Cell Biosci ; 12(1): 119, 2022 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-35908024

RESUMO

BACKGROUND: Developmental signaling pathways such as those of Hedgehog (HH) and WNT play critical roles in cancer stem cell self-renewal, migration, and differentiation. They are often constitutively activated in many human malignancies, including non-small cell lung cancer (NSCLC). Previously, we reported that two oxysterol derivatives, Oxy186 and Oxy210, are potent inhibitors of HH/GLI signaling and NSCLC cancer cell growth. In addition, we also showed that Oxy210 is a potent inhibitor of TGF-ß/SMAD signaling. In this follow-up study, we further explore the mechanism of action by which these oxysterols control NSCLC cell proliferation and tumor growth. RESULTS: Using a GLI-responsive luciferase reporter assay, we show here that HH ligand could not mount a signaling response in the NSCLC cell line A549, even though Oxy186 and Oxy210 still inhibited non-canonical GLI activity and suppressed the proliferation of A549 cells. Further, we uncover an unexpected activity of these two oxysterols in inhibiting the WNT/ß-catenin signaling at the level of LRP5/6 membrane receptors. We also show that in a subcutaneous xenograft tumor model generated from A549 cells, Oxy186, but not Oxy210, exhibits strong inhibition of tumor growth. Subsequent RNA-seq analysis of the xenograft tumor tissue reveal that the WNT/ß-catenin pathway is the target of Oxy186 in vivo. CONCLUSION: The oxysterols Oxy186 and Oxy210 both possess inhibitory activity towards WNT/ß-catenin signaling, and Oxy186 is also a potent inhibitor of NSCLC tumor growth.

8.
Nat Commun ; 13(1): 6069, 2022 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-36241625

RESUMO

Interleukin-9 (IL-9)-producing CD4+ T helper cells (Th9) have been implicated in allergy/asthma and anti-tumor immunity, yet molecular insights on their differentiation from activated T cells, driven by IL-4 and transforming growth factor-beta (TGF-ß), is still lacking. Here we show opposing functions of two transcription factors, D-binding protein (DBP) and E2F8, in controlling Th9 differentiation. Specifically, TGF-ß and IL-4 signaling induces phosphorylation of the serine 213 site in the linker region of the Smad3 (pSmad3L-Ser213) via phosphorylated p38, which is necessary and sufficient for Il9 gene transcription. We identify DBP and E2F8 as an activator and repressor, respectively, for Il9 transcription by pSmad3L-Ser213. Notably, Th9 cells with siRNA-mediated knockdown for Dbp or E2f8 promote and suppress tumor growth, respectively, in mouse tumor models. Importantly, DBP and E2F8 also exhibit opposing functions in regulating human TH9 differentiation in vitro. Thus, our data uncover a molecular mechanism of Smad3 linker region-mediated, opposing functions of DBP and E2F8 in Th9 differentiation.


Assuntos
Interleucina-4 , Interleucina-9 , Animais , Humanos , Camundongos , Diferenciação Celular/genética , Interleucina-4/metabolismo , Proteínas Repressoras/genética , RNA Interferente Pequeno/metabolismo , Serina/metabolismo , Linfócitos T Auxiliares-Indutores , Fator de Crescimento Transformador beta/metabolismo , Fatores de Crescimento Transformadores/metabolismo
9.
Cells ; 8(10)2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31652618

RESUMO

Non-Small Cell Lung Cancer (NSCLC) is a common malignancy and leading cause of death by cancer. Metastasis and drug resistance are serious clinical problems encountered in NSCLC therapy. Aberrant activation of the Transforming Growth Factor beta (TGFß) and Hedgehog (Hh) signal transduction cascades often associate with poor prognosis and aggressive disease progression in NSCLC, as these signals can drive cell proliferation, angiogenesis, metastasis, immune evasion and emergence of drug resistance. Therefore, simultaneous inhibition of TGFß and Hh signaling, by a single agent, or in combination with other drugs, could yield therapeutic benefits in NSCLC and other cancers. In the current study, we report on the biological and pharmacological evaluation of Oxy210, an oxysterol-based dual inhibitor of TGFß and Hh signaling. In NSCLC cells, Oxy210 inhibits proliferation, epithelial-mesenchymal transition (EMT) and invasive activity. Combining Oxy210 with Carboplatin (CP) increases the anti-proliferative response to CP and inhibits TGFß-induced resistance to CP in A549 NSCLC cells. In addition, Oxy210 displays encouraging drug-like properties, including chemical scalability, metabolic stability and oral bioavailability in mice. Unlike other known inhibitors, Oxy210 antagonizes TGFß and Hh signaling independently of TGFß receptor kinase inhibition and downstream of Smoothened, respectively.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteínas Hedgehog/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Oxisteróis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Células A549 , Animais , Carboplatina/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Células NIH 3T3
10.
Int J Biol Sci ; 14(5): 542-548, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29805305

RESUMO

Smad ubiquitin regulatory factor 2 (Smurf2) is a HECT domain-containing E3 ubiquitin ligase. Together with its closely related homolog Smurf1, Smurf2 was initially recognized as a negative regulator of transforming growth factor-ß (TGF-ß) and bone morphogenetic protein (BMP) signaling, but subsequent studies have expanded its function to regulate many different signaling pathways and play important roles in genomic stability, cell polarity, tissue homeostasis and carcinogenesis. Mice with conventional knockout of Smurf1 or Smurf2 alleles are viable, but conventional Smurf1 and Smurf2 double knockout mice were early embryonic lethal. In order to study the physiological function of Smurfs during late stage of embryonic development or in adult animals, we generated Smurf2flox/flox mice carrying a targeted mutation for conditional Smurf2 gene inactivation. We demonstrated that Cre-mediated recombination using Alb-Cre, a Cre line expressed in hepatocyte, results in specific deletion of the gene in liver tissue. We also showed that Cre-mediated recombination in mouse embryonic fibroblasts (MEFs) with Smurf2flox/flox genotype resulted in generation of Smurf2 knockout MEFs, and Smurf2 deficiency affects multiple signaling pathways. Therefore, this animal model will be useful to study the distinct roles of Smurf2 in different tissues at different ages.


Assuntos
Recombinação Genética , Ubiquitina-Proteína Ligases/genética , Alelos , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Feminino , Fibroblastos/metabolismo , Genótipo , Heterozigoto , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Genéticos , Transdução de Sinais , Distribuição Tecidual , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases/fisiologia
11.
Elife ; 32014 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-24925320

RESUMO

Cell surface reception of Sonic hedgehog (Shh) must ensure that the graded morphogenic signal is interpreted accordingly in neighboring cells to specify tissue patterns during development. Here, we report endocytic sorting signals for the receptor Patched1 (Ptch1), comprising two 'PPXY' motifs, that direct it to degradation in lysosomes. These signals are recognized by two HECT-domain ubiquitin E3 ligases, Smurf1 and Smurf2, which are induced by Shh and become enriched in Caveolin-1 lipid rafts in association with Ptch1. Smurf-mediated endocytic turnover of Ptch1 is essential for its clearance from the primary cilium and pathway activation. Removal of both Smurfs completely abolishes the ability of Shh to sustain the proliferation of postnatal granule cell precursors in the cerebellum. These findings reveal a novel step in the Shh pathway activation as part of the Ptch1 negative feedback loop that precisely controls the signaling output in response to Shh gradient signal.


Assuntos
Regulação da Expressão Gênica , Proteínas Hedgehog/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Células 3T3 , Alelos , Motivos de Aminoácidos , Animais , Encéfalo/metabolismo , Endocitose , Éxons , Fibroblastos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Microscopia de Fluorescência , Receptores Patched , Receptor Patched-1 , RNA Interferente Pequeno/metabolismo , Receptores de Superfície Celular/genética , Recombinação Genética , Ubiquitina-Proteína Ligases/genética
12.
Cell Biosci ; 1(1): 43, 2011 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-22204598

RESUMO

Transforming growth factor-ß (TGF-ß) signaling is tightly regulated at the level of post-translational modification to transmit quantitative difference in ligand concentration into proportional transcriptional output. Ubiquitination is one such modification with several E3 ubiquitin ligases implicated in TGF-ß signaling in marking crucial pathway components for proteasomal degradation. However, ubiquitination, particularly in the mono- or oligo-ubiquitin modifying form, is also known to regulate incorporation of substrate proteins into signaling complexes that involved in DNA repair, kinase activation, and endocytosis. This review focuses on recent advances in understanding the role of such non-degradative ubiquitination in TGF-ß signaling.

13.
Mol Cell Proteomics ; 6(11): 1952-67, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17693683

RESUMO

The complexity of canonical Wnt signaling comes not only from the numerous components but also from multiple post-translational modifications. Protein phosphorylation is one of the most common modifications that propagates signals from extracellular stimuli to downstream effectors. To investigate the global phosphorylation regulation and uncover novel phosphoproteins at the early stages of canonical Wnt signaling, HEK293 cells were metabolically labeled with two stable isotopic forms of lysine and were stimulated for 0, 1, or 30 min with purified Wnt3a. After phosphoprotein enrichment and LC-MS/MS analysis, 1057 proteins were identified in all three time points. In total 287 proteins showed a 1.5-fold or greater change in at least one time point. In addition to many known Wnt signaling transducers, other phosphoproteins were identified and quantitated, implicating their involvement in canonical Wnt signaling. k-Means clustering analysis showed dynamic patterns for the differential phosphoproteins. Profile pattern and interaction network analysis of the differential phosphoproteins implicated the possible roles for those unreported components in Wnt signaling. Moreover 100 unique phosphorylation sites were identified, and 54 of them were quantitated in the three time points. Site-specific phosphopeptide quantitation revealed that Ser-20 phosphorylation on RRM2 increased upon 30-min Wnt3a stimulation. Further studies with mutagenesis, the Wnt reporter gene assay, and RNA interference indicated that RRM2 functioned downstream of beta-catenin as an inhibitor of Wnt signaling and that Ser-20 phosphorylation of RRM2 counteracted its inhibition effect. Our systematic profiling of dynamic phosphorylation changes responding to Wnt3a stimulation not only presented a comprehensive phosphorylation network regulated by canonical Wnt signaling but also found novel molecules and phosphorylation involved in Wnt signaling.


Assuntos
Fosfoproteínas/análise , Proteoma/análise , Ribonucleosídeo Difosfato Redutase/metabolismo , Proteínas Wnt/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Humanos , Marcação por Isótopo , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Fosforilação , Proteoma/efeitos dos fármacos , Proteoma/metabolismo , Ribonucleosídeo Difosfato Redutase/genética , Serina/química , Serina/metabolismo , Transdução de Sinais , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/farmacologia , Proteína Wnt3 , Proteína Wnt3A , beta Catenina/metabolismo
14.
Electrophoresis ; 26(23): 4540-62, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16315178

RESUMO

Mesangial cells (MC) play an important role in maintaining the structure and function of the glomerulus. The proliferation of MC is a prominent feature of many kinds of glomerular disease. The first reference 2-DE maps of rat mesangial cells (RMC), stained with silver staining or Pro-Q Diamond dye, have been established here to describe the proteome and phosphoproteome of RMC, respectively. A total of 157 selected protein spots, corresponding to 118 unique proteins, have been identified by MALDI-TOF-MS or LC-ESI-IT-MS/MS, in which 37 protein spots representing 28 unique proteins have also been stained with Pro-Q Diamond, indicating that they are in phosphorylated forms. All the identified proteins were bioinformatically annotated in detail according to their physiochemical characteristics, subcellular location, and function. Most of the separated or identified protein spots are distributed in the area of mass 10-70 kDa and pI 5.0-8.0. The identified proteins include mainly cytoplasmic and nuclear proteins and some mitochondrial, endoplasmic reticulum, and membrane proteins. These proteins are classified into different functional groups such as structure and mobility proteins (21.2%), metabolic enzymes (16.9%), protein folding and metabolism proteins (13.6%), signaling proteins (14.4%), heat-shock proteins (7.6%), and other functional proteins (12.7%). While structure and mobility proteins are mostly represented by protein spots with high abundance, signaling proteins are mostly represented by protein spots with relatively low abundance. Such a 2-DE database for RMC, especially with many signaling proteins and phosphoproteins characterized, will provide a valuable resource for comparative proteomics analysis of normal and pathologic conditions affecting MC function or pathologic progress.


Assuntos
Células Mesangiais/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Animais , Células Cultivadas , Biologia Computacional , Eletroforese em Gel Bidimensional , Masculino , Células Mesangiais/citologia , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
15.
Mol Cell Proteomics ; 4(7): 902-13, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15784933

RESUMO

We present the first proteomic analysis on the cellular response to severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection. The differential proteomes of Vero E6 cells with and without infection of the SARS-CoV were resolved and quantitated with two-dimensional differential gel electrophoresis followed by ESI-MS/MS identification. Moreover isotope-coded affinity tag technology coupled with two-dimensional LC-MS/MS were also applied to the differential proteins of infected cells. By combining these two complementary strategies, 355 unique proteins were identified and quantitated with 186 of them differentially expressed (at least 1.5-fold quantitative alteration) between infected and uninfected Vero E6 cells. The implication for cellular responses to virus infection was analyzed in depth according to the proteomic results. Thus, the present work provides large scale protein-related information to investigate the mechanism of SARS-CoV infection and pathogenesis.


Assuntos
Proteoma/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Eletroforese em Gel Bidimensional , Marcação por Isótopo , Dados de Sequência Molecular , Espectrometria de Massas por Ionização por Electrospray , Células Vero
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