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1.
Oncogene ; 35(20): 2574-83, 2016 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-26364611

RESUMO

RNA helicase DDX3 has oncogenic activity in breast and lung cancers and is required for translation of complex mRNA transcripts, including those encoding key cell-cycle regulatory proteins. We sought to determine the expression and function of DDX3 in sarcoma cells, and to investigate the antitumor activity of a novel small molecule DDX3 inhibitor, RK-33. Utilizing various sarcoma cell lines, xenografts and human tissue microarrays, we measured DDX3 expression at the mRNA and protein levels, and evaluated cytotoxicity of RK-33 in sarcoma cell lines. To study the role of DDX3 in Ewing sarcoma, we generated stable DDX3-knockdown Ewing sarcoma cell lines using DDX3-specific small hairpin RNA (shRNA), and assessed oncogenic activity. DDX3-knockdown and RK-33-treated Ewing sarcoma cells were compared with wild-type cells using an isobaric mass-tag quantitative proteomics approach to identify target proteins impacted by DDX3 inhibition. Overall, we found high expression of DDX3 in numerous human sarcoma subtypes compared with non-malignant mesenchymal cells, and knockdown of DDX3 by RNA interference inhibited oncogenic activity in Ewing sarcoma cells. Treatment with RK-33 was preferentially cytotoxic to sarcoma cells, including chemotherapy-resistant Ewing sarcoma stem cells, while sparing non-malignant cells. Sensitivity to RK-33 correlated with DDX3 protein expression. Growth of human Ewing sarcoma xenografts expressing high DDX3 was inhibited by RK-33 treatment in mice, without overt toxicity. DDX3 inhibition altered the Ewing sarcoma cellular proteome, especially proteins involved in DNA replication, mRNA translation and proteasome function. These data support further investigation of the role of DDX3 in sarcomas, advancement of RK-33 to Ewing sarcoma clinical trials and development of RNA helicase inhibition as a novel anti-neoplastic strategy.


Assuntos
RNA Helicases DEAD-box/metabolismo , Terapia de Alvo Molecular , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/enzimologia , Animais , Apoptose/efeitos dos fármacos , Azepinas/farmacologia , Linhagem Celular Tumoral , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Imidazóis/farmacologia , Camundongos , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Br J Cancer ; 110(1): 71-82, 2014 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-24322890

RESUMO

BACKGROUND: Heat shock protein 90 (HSP90) has a key role in the maintenance of the cellular proteostasis. However, HSP90 is also involved in stabilisation of oncogenic client proteins and facilitates oncogene addiction and cancer cell survival. The development of HSP90 inhibitors for cancer treatment is an area of growing interest as such agents can affect multiple pathways that are linked to all hallmarks of cancer. This study aimed to test the hypothesis that targeting cysteine residues of HSP90 will lead to degradation of client proteins and inhibition of cancer cell proliferation. METHODS: Combining chemical synthesis, biological evaluation, and structure-activity relationship analysis, we identified a new class of HSP90 inhibitors. Click chemistry and protease-mass spectrometry established the sites of modification of the chaperone. RESULTS: The mildly electrophilic sulphoxythiocarbamate alkyne (STCA) selectively targets cysteine residues of HSP90, forming stable thiocarbamate adducts. Without interfering with the ATP-binding ability of the chaperone, STCA destabilises the client proteins RAF1, HER2, CDK1, CHK1, and mutant p53, and decreases proliferation of breast cancer cells. Addition of a phenyl or a tert-butyl group in tandem with the benzyl substituent at nitrogen increased the potency. A new compound, S-4, was identified as the most robust HSP90 inhibitor within a series of 19 derivatives. CONCLUSION: By virtue of their cysteine reactivity, sulphoxythiocarbamates target HSP90, causing destabilisation of its client oncoproteins and inhibiting cell proliferation.


Assuntos
Carbamatos/farmacologia , Cisteína/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Proteínas de Choque Térmico HSP72/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Humanos , Células MCF-7 , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Terapia de Alvo Molecular , Compostos de Sulfidrila/farmacologia , Sulfóxidos/farmacologia , Regulação para Cima/efeitos dos fármacos
3.
Placenta ; 33(5): 424-32, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22385826

RESUMO

Characterization of normal changes in the serum proteome during pregnancy may enhance understanding of maternal physiology and lead to the development of new gestational biomarkers. In 23 Nepalese pregnant women who delivered at term, two-dimensional difference in-gel electrophoresis (DIGE) was used to assess changes in relative protein abundance between paired serum samples collected in the first and third trimesters. One-hundred and forty-five of over 700 protein spots in DIGE gels (pI 4.2-6.8) exhibited nominally significant (p < 0.05) differences in abundance across trimesters. Additional filtering using a Bonferroni correction reduced the number of significant (p < 0.00019) spots to 61. Mass spectrometric analysis detected 38 proteins associated with gestational age, cytoskeletal remodeling, blood pressure regulation, lipid and nutrient transport, and inflammation. One new protein, pregnancy-specific ß-glycoprotein 4 was detected. A follow-up isotope tagging for relative and absolute quantitation (iTRAQ) experiment of six mothers from the DIGE study revealed 111 proteins, of which 11 exhibited significant (p < 0.05) differences between trimesters. Four of these proteins: gelsolin, complement C1r subcomponent, α-1-acid glycoprotein, and α-1B-glycoprotein also changed in the DIGE analysis. Although not previously associated with normal pregnancy, gelsolin decreased in abundance by the third trimester (p < 0.01) in DIGE, iTRAQ and Western analyses. Changes in abundance of proteins in serum that are associated with syncytiotrophoblasts (gelsolin, pregnancy-specific ß-1 glycoprotein 1 and ß-2-glycoprotein I) probably reflect dynamics of a placental proteome shed into maternal circulation during pregnancy. Measurement of changes in the maternal serum proteome, when linked with birth outcomes, may yield biomarkers for tracking reproductive health in resource poor settings in future studies.


Assuntos
Primeiro Trimestre da Gravidez/sangue , Terceiro Trimestre da Gravidez/sangue , Proteoma , Western Blotting , Cromatografia Líquida , Feminino , Humanos , Desnutrição , Espectrometria de Massas , Nepal , Gravidez , População Rural , Eletroforese em Gel Diferencial Bidimensional
4.
Physiol Genomics ; 43(11): 674-84, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21427361

RESUMO

To identify additional potential functions for the multi-PDZ domain containing protein Na+/H+ exchanger regulatory factor 2 (NHERF2), which is present in the apical domain of intestinal epithelial cells, proteomic studies of mouse jejunal villus epithelial cell brush border membrane vesicles compared wild-type to homozygous NHERF2 knockout FVB mice by a two-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS)-iTRAQ approach. Jejunal architecture appeared normal in NHERF2 null in terms of villus length and crypt depth, Paneth cell number, and microvillus structure by electron microscopy. There was also no change in proliferative activity based on BrdU labeling. Four brush border membrane vesicles (BBMV) preparations from wild-type mouse jejunum were compared with four preparations from NHERF2 knockout mice. LC-MS/MS identified 450 proteins in both matched wild-type and NHERF2 null BBMV; 13 proteins were changed in two or more separate BBMV preparations (9 increased and 4 decreased in NHERF2 null mice), while an additional 92 proteins were changed in a single BBMV preparation (68 increased and 24 decreased in NHERF2 null mice). These proteins were categorized as 1) transport proteins (one increased and two decreased in NHERF2 null); 2) signaling molecules (2 increased in NHERF2 null); 3) cytoskeleton/junctional proteins (4 upregulated and 1 downregulated in NHERF2 null); and 4) metabolic proteins/intrinsic BB proteins) (2 upregulated and 1 downregulated in NHERF2 null). Immunoblotting of BBMV was used to validate or extend the findings, demonstrating increase in BBMV of NHERF2 null of MCT1, coronin 3, and ezrin. The proteome of the NHERF2 null mouse small intestinal BB demonstrates up- and downregulation of multiple transport proteins, signaling molecules, cytoskeletal proteins, tight junctional and adherens junction proteins, and proteins involved in metabolism, suggesting involvement of NHERF2 in multiple apical regulatory processes and interactions with luminal contents.


Assuntos
Jejuno/metabolismo , Fosfoproteínas/genética , Proteoma/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Animais , Caderinas/metabolismo , Proliferação de Células , Cromatografia Líquida , Citoesqueleto/metabolismo , Regulação para Baixo , Imunofluorescência , Masculino , Camundongos , Camundongos Knockout , Microvilosidades/genética , Microvilosidades/metabolismo , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , beta Catenina/metabolismo
5.
Physiol Genomics ; 42A(3): 200-10, 2010 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-20736413

RESUMO

Na/H exchanger regulatory factor 1 (NHERF1) is a scaffold protein made up of two PDZ domains and an ERM binding domain. It is in the brush border of multiple epithelial cells where it modulates 1) Na absorption by regulating NHE3 complexes and cytoskeletal association, 2) Cl secretion through trafficking of CFTR, and 3) Na-coupled phosphate absorption through membrane retention of NaPi2a. To further understand the role of NHERF1 in regulation of small intestinal Na absorptive cell function, with emphasis on apical membrane transport regulation, quantitative proteomic analysis was performed on brush border membrane vesicles (BBMV) prepared from wild-type (WT) and homozygous NHERF1 knockout mouse jejunal villus Na absorptive cells. Jejunal architecture appeared normal in NHERF1 null; however, there was increased proliferative activity, as indicated by increased crypt BrdU staining. LC-MS/MS analysis using iTRAQ to compare WT and NHERF1 null BBMV identified 463 proteins present in both WT and NHERF1 null BBMV of simultaneously prepared and studied samples. Seventeen proteins had an altered amount of expression between WT and NHERF1 null in two or more separate preparations, and 149 total proteins were altered in at least one BBMV preparation. The classes of the majority of proteins altered included transport proteins, signaling and trafficking proteins, and proteins involved in proliferation and cell division. Affected proteins also included tight junction and adherens junction proteins, cytoskeletal proteins, as well as metabolic and BB digestive enzymes. Changes in abundance of several proteins were confirmed by immunoblotting [increased CEACAM1, decreased ezrin (p-ezrin), NHERF3, PLCß3, E-cadherin, p120, ß-catenin]. The changes in the jejunal BBMV proteome of NHERF1 null mice are consistent with a more complex role of NHERF1 than just forming signaling complexes and anchoring proteins to the apical membrane and include at least alterations in proteins involved in transport, signaling, and proliferation.


Assuntos
Jejuno/metabolismo , Fosfoproteínas/genética , Proteoma/análise , Trocadores de Sódio-Hidrogênio/genética , Vesículas Transportadoras/metabolismo , Animais , Caderinas/análise , Cromatografia por Troca Iônica , Feminino , Immunoblotting , Imuno-Histoquímica , Jejuno/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Microvilosidades/metabolismo , Microvilosidades/ultraestrutura , Fosfoproteínas/metabolismo , Proteômica/métodos , Trocadores de Sódio-Hidrogênio/metabolismo , Espectrometria de Massas em Tandem , beta Catenina/análise
6.
J Physiol ; 588(Pt 17): 3217-29, 2010 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-20603331

RESUMO

The postsynaptic muscle-specific kinase (MuSK) coordinates formation of the neuromuscular junction (NMJ) during embryonic development. Here we have studied the effects of MuSK autoantibodies upon the NMJ in adult mice. Daily injections of IgG from four MuSK autoantibody-positive myasthenia gravis patients (MuSK IgG; 45 mg day(1)i.p. for 14 days) caused reductions in postsynaptic ACh receptor (AChR) packing as assessed by fluorescence resonance energy transfer (FRET). IgG from the patients with the highest titres of MuSK autoantibodies caused large (51-73%) reductions in postsynaptic MuSK staining (cf. control mice; P < 0.01) and muscle weakness. Among mice injected for 14 days with control and MuSK patient IgGs, the residual level of MuSK correlated with the degree of impairment of postsynaptic AChR packing. However, the loss of postsynaptic MuSK preceded this impairment of postsynaptic AChR. When added to cultured C2 muscle cells the MuSK autoantibodies caused tyrosine phosphorylation of MuSK and the AChR beta-subunit, and internalization of MuSK from the plasma membrane. The results suggest a pathogenic mechanism in which MuSK autoantibodies rapidly deplete MuSK from the postsynaptic membrane leading to progressive dispersal of postsynaptic AChRs. Moreover, maintenance of postsynaptic AChR packing at the adult NMJ would appear to depend upon physical engagement of MuSK with the AChR scaffold, notwithstanding activation of the MuSK-rapsyn system of AChR clustering.


Assuntos
Autoanticorpos/fisiologia , Regiões de Interação com a Matriz/fisiologia , Miastenia Gravis/metabolismo , Junção Neuromuscular/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/deficiência , Receptores Colinérgicos/metabolismo , Sinapses/enzimologia , Animais , Autoanticorpos/toxicidade , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Imunoglobulina G/fisiologia , Imunoglobulina G/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Miastenia Gravis/enzimologia , Miastenia Gravis/etiologia , Junção Neuromuscular/enzimologia , Junção Neuromuscular/genética , Receptores Proteína Tirosina Quinases/imunologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Colinérgicos/química , Receptores Colinérgicos/deficiência , Receptores Colinérgicos/imunologia , Sinapses/genética , Sinapses/metabolismo
7.
Science ; 298(5602): 2390-2, 2002 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-12493915

RESUMO

Acetyl-coenzyme A (CoA) synthetase (Acs) is an enzyme central to metabolism in prokaryotes and eukaryotes. Acs synthesizes acetyl CoA from acetate, adenosine triphosphate, and CoA through an acetyl-adenosine monophosphate (AMP) intermediate. Immunoblotting and mass spectrometry analysis showed that Salmonella enterica Acs enzyme activity is posttranslationally regulated by acetylation of lysine-609. Acetylation blocks synthesis of the adenylate intermediate but does not affect the thioester-forming activity of the enzyme. Activation of the acetylated enzyme requires the nicotinamide adenine dinucleotide-dependent protein deacetylase activity of the CobB Sir2 protein from S. enterica. We propose that acetylation modulates the activity of all the AMP-forming family of enzymes, including nonribosomal peptide synthetases, luciferase, and aryl- and acyl-CoA synthetases. These findings extend our knowledge of the roles of Sir2 proteins in gene silencing, chromosome stability, and cell aging and imply that lysine acetylation is a common regulatory mechanism in eukaryotes and prokaryotes.


Assuntos
Acetato-CoA Ligase/metabolismo , Proteínas de Bactérias/metabolismo , Lisina/metabolismo , Salmonella enterica/enzimologia , Sirtuínas/metabolismo , Acetato-CoA Ligase/química , Acetato-CoA Ligase/genética , Acetilação , Acil Coenzima A/metabolismo , Monofosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Coenzima A/metabolismo , Sequência Conservada , Ativação Enzimática , Regulação Bacteriana da Expressão Gênica , Immunoblotting , Espectrometria de Massas , NAD/metabolismo , Mapeamento de Peptídeos , Salmonella enterica/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
8.
J Neurochem ; 79(5): 1080-9, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11739622

RESUMO

Phosphorylation plays a key role in regulating growth cone migration and protein trafficking in nerve terminals. Here we show that nerve terminal proteins contain another abundant post-translational modification: beta-N-acetylglucosamine linked to hydroxyls of serines or threonines (O-GlcNAc(1)). O-GlcNAc modifications are essential for embryogenesis and mounting evidence suggests that O-GlcNAc is a regulatory modification that affects many phosphorylated proteins. We show that the activity and expression of O-GlcNAc transferase (OGT) and N-acetyl-beta-D-glucosaminidase (O-GlcNAcase), the two enzymes regulating O-GlcNAc modifications, are present in nerve terminal structures (synaptosomes) and are particularily abundant in the cytosol of synaptosomes. Numerous synaptosome proteins are highly modified with O-GlcNAc. Although most of these proteins are present in low abundance, we identified by proteomic analysis three neuron-specific O-GlcNAc modified proteins: collapsin response mediator protein-2 (CRMP-2), ubiquitin carboxyl hydrolase-L1 (UCH-L1) and beta-synuclein. CRMP-2, which is involved in growth cone collapse, is a major O-GlcNAc modified protein in synaptosomes. All three proteins are implicated in regulatory cascades that mediate intracellular signaling or neurodegenerative diseases. We propose that O-GlcNAc modifications in the nerve terminal help regulate the functions of these and other synaptosome proteins, and that O-GlcNAc may play a role in neurodegenerative disease.


Assuntos
Acetilglucosamina/metabolismo , Citosol/metabolismo , Terminações Nervosas/metabolismo , Serina/análogos & derivados , Serina/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Química Encefálica , Metabolismo dos Carboidratos , Citosol/enzimologia , Galactosiltransferases/metabolismo , Glicosilação , Hidrólise , Técnicas In Vitro , Espectrometria de Massas , Dados de Sequência Molecular , Terminações Nervosas/enzimologia , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/química , Neurônios/metabolismo , Processamento de Proteína Pós-Traducional , Ratos , Ratos Sprague-Dawley , Espectrometria de Fluorescência , Frações Subcelulares/metabolismo , Sinaptossomos/metabolismo , Tripsina
9.
J Clin Invest ; 107(4): 495-504, 2001 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11181649

RESUMO

Isolated biotin-resistant 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine catabolism that appears to be the most frequent organic aciduria detected in tandem mass spectrometry-based neonatal screening programs. The phenotype is variable, ranging from neonatal onset with severe neurological involvement to asymptomatic adults. MCC is a heteromeric mitochondrial enzyme composed of biotin-containing alpha subunits and smaller beta subunits. Here, we report cloning of MCCA and MCCB cDNAs and the organization of their structural genes. We show that a series of 14 MCC-deficient probands defines two complementation groups, CG1 and 2, resulting from mutations in MCCB and MCCA, respectively. We identify five MCCA and nine MCCB mutant alleles and show that missense mutations in each result in loss of function.


Assuntos
Carbono-Carbono Ligases/deficiência , Carbono-Carbono Ligases/genética , Alelos , Sequência de Aminoácidos , DNA Complementar/análise , Genes , Teste de Complementação Genética , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Mutação
10.
Curr Protoc Protein Sci ; Chapter 12: Unit 12.8, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18429112

RESUMO

First, a protocol for increasing the stoichiometry of O-GlcNAc on proteins is given. This is followed by simple techniques for the detection/screening of O-GlcNAc-modified proteins either by immunoblotting or lectin affinity chromatography. Separate protocols verify that the glycan is O-linked GlcNAc. These methods are followed by protocols for more comprehensive analysis of O-GlcNAc modified proteins, including labeling of O-GlcNAc residues with [3H]Gal, and subsequent product analysis. The final two protocols assay for O-GlcNAc transferase and O-GlcNAcase activity, respectively.


Assuntos
Acetilglucosamina/química , Proteínas/análise , Proteínas/química , Cromatografia de Afinidade , Immunoblotting , Lectinas/química , Processamento de Proteína Pós-Traducional , Proteínas/isolamento & purificação
11.
J Mol Biol ; 304(4): 633-44, 2000 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-11099385

RESUMO

Concanavalin A (Con A) kills procyclic (insect) forms of Trypanosoma brucei by binding to N-glycans on EP-procyclin, a major surface glycosyl phosphatidylinositol (GPI)-anchored protein which is rich in Glu-Pro repeats. We have previously isolated and studied two procyclic mutants (ConA 1-1 and ConA 4-1) that are more resistant than wild-type (WT) to Con A killing. Although both mutants express the same altered oligosaccharides compared to WT cells, ConA 4-1 is considerably more resistant to lectin killing than is ConA 1-1. Thus, we looked for other alterations to account for the differences in sensitivity. Using mass spectrometry, together with chemical and enzymatic treatments, we found that both mutants express types of EP-procyclin that are either poorly expressed or not found at all in WT cells. ConA 1-1 expresses mainly EP1-3, a novel procyclin that contains 18 EP repeats, is partially N-glycosylated, and bears hybrid-type glycans. On the other hand, ConA 4-1 cells express almost exclusively EP2-3, a novel non-glycosylated procyclin isoform with 23 EP repeats and no site for glycosylation. The predominance of EP2-3 in ConA 4-1 cells explains their high resistance to ConA killing. Thus, switching the procyclin repertoire, a process that could be relevant to parasite development in the insect vector, modulates the sensitivity of trypanosomes to cytotoxic lectins.


Assuntos
Concanavalina A/metabolismo , Concanavalina A/toxicidade , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Mutação/genética , Trypanosoma brucei brucei/efeitos dos fármacos , Sequência de Aminoácidos , Amino Açúcares/metabolismo , Animais , Resistência a Medicamentos/genética , Evolução Molecular , Glicosilação/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Hidrólise , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/isolamento & purificação , Dados de Sequência Molecular , Peptídeos/análise , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Sequências Repetitivas de Aminoácidos , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crescimento & desenvolvimento
12.
Biochemistry ; 39(38): 11609-20, 2000 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-10995228

RESUMO

Estrogen receptor beta, a homologue to estrogen receptor alpha, is a new member of the steroid hormone receptor family. Recently, we documented that estrogen receptor alpha, like other transcription factors, is modified by O-linked N-acetylglucosamine (O-GlcNAc), a ubiquitous transitory posttranslational modification on nuclear and cytoplasmic proteins. Here, we report that estrogen receptor beta is alternatively modified by either O-GlcNAc or O-phosphate. Lectin chromatography of in vitro translated protein first suggested that murine estrogen receptor beta (mER-beta) is O-GlcNAcylated. Structural characterization of the carbohydrate moieties on mER-beta, overexpressed in insect Sf9 cells, confirmed the presence of O-GlcNAc. mER-beta, overexpressed in mammalian cells, is also O-GlcNAcylated. The major site of O-GlcNAc on mER-beta from Sf9 cells is Ser(16) near the N-terminus. Concomitant analyses also documented the O-phosphorylation of mER-beta at Ser(16). MALDI-TOF mass spectrometry showed alternative occupancy of this locus by these two abundant and dynamic posttranslational modifications. The localization of a major O-GlcNAc/O-phosphate site in proximity of the transactivation domain and as part of a PEST region (target sequences for rapid protein degradation) on mER-beta suggests that these modifications may play a role in regulating estrogen receptor beta transactivation and turnover.


Assuntos
Receptores de Estrogênio/química , Receptores de Estrogênio/metabolismo , Acetilglucosamina/isolamento & purificação , Acetilglucosamina/metabolismo , Acilação , Sequência de Aminoácidos , Animais , Células COS , Configuração de Carboidratos , Receptor beta de Estrogênio , Glicosilação , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação , Estrutura Terciária de Proteína/genética , Receptores de Estrogênio/biossíntese , Receptores de Estrogênio/genética , Serina/genética , Serina/metabolismo , Spodoptera/genética , Ativação Transcricional
13.
J Biol Chem ; 274(42): 29763-71, 1999 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-10514452

RESUMO

The surface of the insect stages of the protozoan parasite Trypanosoma brucei is covered by abundant glycosyl phosphatidylinositol (GPI)-anchored glycoproteins known as procyclins. One type of procyclin, the EP isoform, is predicted to have 22-30 Glu-Pro (EP) repeats in its C-terminal domain and is encoded by multiple genes. Because of the similarity of the EP isoform sequences and the heterogeneity of their GPI anchors, it has been impossible to separate and characterize these polypeptides by standard protein fractionation techniques. To facilitate their structural and functional characterization, we used a combination of matrix-assisted laser desorption ionization and electrospray mass spectrometry to analyze the entire procyclin repertoire expressed on the trypanosome cell. This analysis, which required removal of the GPI anchors by aqueous hydrofluoric acid treatment and cleavage at aspartate-proline bonds by mild acid hydrolysis, provided precise information about the glycosylation state and the number of Glu-Pro repeats in these proteins. Using this methodology we detected in a T. brucei clone the glycosylated products of the EP3 gene and two different products of the EP1 gene (EP1-1 and EP1-2). Furthermore, only low amounts of the nonglycosylated products of the GPEET and EP2 genes were detected. Because all procyclin genes are transcribed polycistronically, the latter finding indicates that the expression of the GPEET and EP2 genes is post-transcriptionaly regulated. This is the first time that the whole procyclin repertoire from procyclic trypanosomes has been characterized at the protein level.


Assuntos
Glicina/análise , Glicoproteínas de Membrana/metabolismo , Peptídeos/metabolismo , Prolina/análise , Proteínas de Protozoários , Trypanosoma brucei brucei/metabolismo , Sequência de Aminoácidos , Animais , Ácido Aspártico/metabolismo , Glicina/metabolismo , Glicosilação , Hidrólise , Dados de Sequência Molecular , Peptídeos/química , Fosforilação , Prolina/metabolismo , Conformação Proteica , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
14.
J Neurochem ; 73(1): 418-28, 1999 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10386995

RESUMO

Synapsin I is concentrated in nerve terminals, where it appears to anchor synaptic vesicles to the cytoskeleton and thereby ensures a steady supply of fusion-competent synaptic vesicles. Although phosphorylation-dependent binding of synapsin I to cytoskeletal elements and synaptic vesicles is well characterized, little is known about synapsin I's O-linked N-acetylglucosamine (O-GlcNAc) modifications. Here, we identified seven in vivo O-GlcNAcylation sites on synapsin I by analysis of HPLC-purified digests of rat brain synapsin I. The seven O-GlcNAcylation sites (Ser55, Thr56, Thr87, Ser516, Thr524, Thr562, and Ser576) in synapsin I are clustered around its five phosphorylation sites in domains B and D. The proximity of phosphorylation sites to O-GlcNAcylation sites in the regulatory domains of synapsin I suggests that O-GlcNAcylation may modulate phosphorylation and indirectly affect synapsin I interactions. With use of synthetic peptides, however, the presence of an O-GlcNAc at sites Thr562 and Ser576 resulted in only a 66% increase in the Km of calcium/calmodulin-dependent protein kinase II phosphorylation of site Ser566 with no effect on its Vmax. We conclude that O-GlcNAcylation likely plays a more direct role in synapsin I interactions than simply modulating the protein's phosphorylation.


Assuntos
Acetilglucosamina/análise , Sinapsinas/química , Sinapsinas/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Configuração de Carboidratos , Cromatografia Líquida de Alta Pressão , Glicosilação , Espectrometria de Massas , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Fosforilação , Ratos , Ratos Sprague-Dawley
15.
Biochemistry ; 37(47): 16828-38, 1998 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-9843453

RESUMO

Triosephosphate isomerase (TIM) catalyzes the reversible interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP), with Glu-165 removing the pro-R proton from C1 of DHAP and neutral His-95 polarizing the carbonyl group of the substrate. During the TIM reaction, approximately 2% of the pro-R tritium from C1 of DHAP is conserved and appears at C2 of GAP [Nickbarg, E. B., and Knowles, J. R. (1988) Biochemistry 27, 5939]. In the "classical" mechanism, 98% of the pro-R tritium exchanges with solvent from Glu-165 at the intermediate state and the remaining 2% is transferred by Glu-165 to C2 of the same substrate molecule. This intramolecular transfer of tritium is therefore predicted to be independent of DHAP concentration. On the basis of NMR detection of a strong hydrogen bond between Glu-165 and the 1-OH of an analogue of the enediol intermediate [Harris, T. K., Abeygunawardana, C., and Mildvan, A. S. (1997) Biochemistry 36, 14661], we have suggested a "criss-cross" mechanism for TIM in which Glu-165 transfers a proton from C1 of DHAP to O2 of the enediol, and subsequently from O1 of the enediol to C2 of the product GAP. Since the pro-R proton is transferred to O2 instead of C2 in the criss-cross mechanism, no intramolecular transfer of label from substrate to product would be expected to occur. However, intermolecular transfer of label could occur if the label exchanges from O2 into a group on the protein and is transferred to GAP in subsequent turnovers. The extent of intermolecular tritium transfer in the criss-cross mechanism would be predicted to be dependent on DHAP concentration. The extent of tritium transfer was studied as a function of initial DHAP concentration using DHAP highly tritiated at the pro-R position. At 50% conversion to GAP, triphasic tritium transfer behavior was found. For phase 1, between 0.03 and 0.3 mM DHAP, a constant extent of tritium transfer of 1.19 +/- 0.03% occurred. For phase 2, between 0.3 and 1.0 mM DHAP, the extent of transfer progressively increased as a function of DHAP concentration to 2.17 +/- 0.15%. For phase 3, between 1.0 and 7.0 mM DHAP, the extent of transfer slightly decreased to 1.68 +/- 0.17%. In a direct test for intermolecular isotope transfer, doubly labeled [1(R)-D, 13C3]DHAP and 13C-depleted [1(R)-H,12C3]DHAP were synthesized, mixed in equal amounts, and incubated at 1 mM total DHAP with TIM, GAP dehydrogenase, NAD+, and arsenate until 50% conversion to 3-phosphoglycerate occurred. Electrospray ionization mass spectral analysis of the stable 3-phosphoglycerate product detected an extent of 1.4 +/- 0.4% of intramolecular D transfer from [13C3]DHAP to the 13C3 product, but no intermolecular transfer (

Assuntos
Prótons , Triose-Fosfato Isomerase/química , Animais , Deutério/química , Deutério/metabolismo , Fosfato de Di-Hidroxiacetona/química , Fosfato de Di-Hidroxiacetona/metabolismo , Transporte de Elétrons , Gliceraldeído 3-Fosfato/química , Gliceraldeído 3-Fosfato/metabolismo , Músculo Esquelético/enzimologia , Coelhos , Especificidade por Substrato , Temperatura , Triose-Fosfato Isomerase/metabolismo , Trítio/química , Trítio/metabolismo
16.
J Biol Chem ; 271(46): 28741-4, 1996 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-8910513

RESUMO

Tau is a family of phosphoproteins that are important in modulating microtubule stability in neurons. In Alzheimer's disease tau is abnormally hyperphosphorylated, no longer binds microtubules, and self-assembles to form paired helical filaments that likely contribute to neuron death. Here we demonstrate that normal bovine tau is multiply modified by Ser(Thr)-O-linked N-acetylglucosamine, a dynamic and abundant post-translational modification that is often reciprocal to Ser(Thr)-phosphorylation. O-GlcNAcylation of tau was demonstrated by blotting with succinylated wheat germ agglutinin and by probing with bovine milk beta(1,4)galactosyltransferase. Structural analyses confirm the linkage and the saccharide structure. Tau splicing variants are multiply O-GlcNAcylated at similar sites, with an average stoichiometry of greater than 4 mol of O-linked N-acetylglucosamine/mol of tau. However, the number of sites occupied appears to be greater than 12, suggesting substoichiometric occupancy at any given site. A similar relationship between average stoichiometry and site-occupancy has also been described for the phosphorylation of tau. Site-specific or stoichiometric changes in O-GlcNAcylation may not only modulate tau function but may also play a role in the formation of paired helical filaments.


Assuntos
Acetilglucosamina/química , Proteínas tau/química , Sequência de Aminoácidos , Animais , Química Encefálica , Bovinos , Galactosiltransferases/química , Glicosilação , Dados de Sequência Molecular , Fosforilação
18.
Plant Cell ; 7(9): 1459-71, 1995 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8589629

RESUMO

Only a few nuclear pore complex (NPC) proteins, mainly in vertebrates and yeast but none in plants, have been well characterized. As an initial step to identify plant NPC proteins, we examined whether NPC proteins from tobacco are modified by N-acetylglucosamine (GlcNAc). Using wheat germ agglutinin, a lectin that binds specifically to GlcNAc in plants, specific labeling was often found associated with or adjacent to NPCs. Nuclear proteins containing GlcNAc can be partially extracted by 0.5 M salt, as shown by a wheat germ agglutinin blot assay, and at least eight extracted proteins were modified by terminal GlcNAc, as determined by in vitro galactosyltransferase assays. Sugar analysis indicated that the plant glycans with terminal GlcNAc differ from the single O-linked GlcNAc of vertebrate NPC proteins in that they consist of oligosaccharides that are larger in size than five GlcNAc residues. Most of these appear to be bound to proteins via a hydroxyl group. This novel oligosaccharide modification may convey properties to the plant NPC that are different from those of vertebrate NPCs.


Assuntos
Acetilglucosamina/química , Membrana Nuclear/química , Proteínas Nucleares/química , Oligossacarídeos/química , Proteínas de Plantas/química , Células Cultivadas , Plantas Tóxicas , Nicotiana/química , Nicotiana/citologia
20.
J Neurosci ; 14(7): 4481-93, 1994 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-7517997

RESUMO

Carbohydrate recognition plays an important role in the development of normal projections of sensory afferent neurons in the leech CNS. Four different carbohydrate epitopes are expressed by sensory afferents on their 130 kDa surface proteins: all sensory afferents share a common carbohydrate epitope (CE0) that helps them to enter and project diffusely across the synaptic neuropil; a restricted expression of three other carbohydrate epitopes (CE1, CE2, and CE3) serves to distinguish three subsets of sensory afferents. We examined the subsets of sensory afferents defined by their subset carbohydrate epitopes in the leech lip, skin, gut, and CNS. We established that the CE1, CE2, and CE3 subset epitopes define disjoint subsets of neurons by double labeling sensory afferents with monoclonal antibodies for different pairs of subset epitopes. We found that CE2 and CE3 afferents populate the lip and skin, but not the gut, and that these two subsets of sensory afferents have convergent projection patterns in the CNS. We found that CE1 afferents populate the gut and skin, but not lips; furthermore, their CNS projections diverge from those of CE2 and CE3 afferents. Our data fit the hypothesis that these carbohydrate epitopes are related to sensory modality of afferent subsets.


Assuntos
Metabolismo dos Carboidratos , Carboidratos/imunologia , Epitopos , Neurônios Aferentes/metabolismo , Animais , Sistema Nervoso Central/citologia , Sanguessugas , Rede Nervosa/fisiologia , Pele/inervação , Transmissão Sináptica
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