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1.
EMBO J ; 35(18): 1963-78, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27354364

RESUMO

Pre-B-cell leukemia homeobox (PBX) transcription factors are known to regulate organogenesis, but their molecular targets and function in midbrain dopaminergic neurons (mDAn) as well as their role in neurodegenerative diseases are unknown. Here, we show that PBX1 controls a novel transcriptional network required for mDAn specification and survival, which is sufficient to generate mDAn from human stem cells. Mechanistically, PBX1 plays a dual role in transcription by directly repressing or activating genes, such as Onecut2 to inhibit lateral fates during embryogenesis, Pitx3 to promote mDAn development, and Nfe2l1 to protect from oxidative stress. Notably, PBX1 and NFE2L1 levels are severely reduced in dopaminergic neurons of the substantia nigra of Parkinson's disease (PD) patients and decreased NFE2L1 levels increases damage by oxidative stress in human midbrain cells. Thus, our results reveal novel roles for PBX1 and its transcriptional network in mDAn development and PD, opening the door for new therapeutic interventions.


Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Neurônios Dopaminérgicos/fisiologia , Redes Reguladoras de Genes , Doença de Parkinson/patologia , Proteínas Proto-Oncogênicas/metabolismo , Substância Negra/patologia , Humanos , Fator de Transcrição 1 de Leucemia de Células Pré-B
2.
Biochem J ; 476(8): 1285-1302, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-30944155

RESUMO

αδ-Bungarotoxins, a novel group of long-chain α-neurotoxins, manifest different affinity to two agonist/competitive antagonist binding sites of muscle-type nicotinic acetylcholine receptors (nAChRs), being more active at the interface of α-δ subunits. Three isoforms (αδ-BgTx-1-3) were identified in Malayan Krait (Bungarus candidus) from Thailand by genomic DNA analysis; two of them (αδ-BgTx-1 and 2) were isolated from its venom. The toxins comprise 73 amino acid residues and 5 disulfide bridges, being homologous to α-bungarotoxin (α-BgTx), a classical blocker of muscle-type and neuronal α7, α8, and α9α10 nAChRs. The toxicity of αδ-BgTx-1 (LD50 = 0.17-0.28 µg/g mouse, i.p. injection) is essentially as high as that of α-BgTx. In the chick biventer cervicis nerve-muscle preparation, αδ-BgTx-1 completely abolished acetylcholine response, but in contrast with the block by α-BgTx, acetylcholine response was fully reversible by washing. αδ-BgTxs, similar to α-BgTx, bind with high affinity to α7 and muscle-type nAChRs. However, the major difference of αδ-BgTxs from α-BgTx and other naturally occurring α-neurotoxins is that αδ-BgTxs discriminate the two binding sites in the Torpedo californica and mouse muscle nAChRs showing up to two orders of magnitude higher affinity for the α-δ site as compared with α-ε or α-γ binding site interfaces. Molecular modeling and analysis of the literature provided possible explanations for these differences in binding mode; one of the probable reasons being the lower content of positively charged residues in αδ-BgTxs. Thus, αδ-BgTxs are new tools for studies on nAChRs.


Assuntos
Bungarotoxinas/química , Bungarus , Proteínas de Peixes/química , Proteínas Musculares/química , Receptores Nicotínicos/química , Animais , Sítios de Ligação , Bungarotoxinas/metabolismo , Feminino , Proteínas de Peixes/metabolismo , Masculino , Camundongos , Proteínas Musculares/metabolismo , Receptores Nicotínicos/metabolismo , Torpedo
3.
J Biol Chem ; 291(35): 18410-8, 2016 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-27365393

RESUMO

Leukotriene C4 synthase (LTC4S) catalyzes the formation of the proinflammatory lipid mediator leukotriene C4 (LTC4). LTC4 is the parent molecule of the cysteinyl leukotrienes, which are recognized for their pathogenic role in asthma and allergic diseases. Cellular LTC4S activity is suppressed by PKC-mediated phosphorylation, and recently a downstream p70S6k was shown to play an important role in this process. Here, we identified Ser(36) as the major p70S6k phosphorylation site, along with a low frequency site at Thr(40), using an in vitro phosphorylation assay combined with mass spectrometry. The functional consequences of p70S6k phosphorylation were tested with the phosphomimetic mutant S36E, which displayed only about 20% (20 µmol/min/mg) of the activity of WT enzyme (95 µmol/min/mg), whereas the enzyme activity of T40E was not significantly affected. The enzyme activity of S36E increased linearly with increasing LTA4 concentrations during the steady-state kinetics analysis, indicating poor lipid substrate binding. The Ser(36) is located in a loop region close to the entrance of the proposed substrate binding pocket. Comparative molecular dynamics indicated that Ser(36) upon phosphorylation will pull the first luminal loop of LTC4S toward the neighboring subunit of the functional homotrimer, thereby forming hydrogen bonds with Arg(104) in the adjacent subunit. Because Arg(104) is a key catalytic residue responsible for stabilization of the glutathione thiolate anion, this phosphorylation-induced interaction leads to a reduction of the catalytic activity. In addition, the positional shift of the loop and its interaction with the neighboring subunit affect active site access. Thus, our mutational and kinetic data, together with molecular simulations, suggest that phosphorylation of Ser(36) inhibits the catalytic function of LTC4S by interference with the catalytic machinery.


Assuntos
Glutationa Transferase/química , Substituição de Aminoácidos , Animais , Sítios de Ligação , Catálise , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Leucotrieno A4/biossíntese , Leucotrieno A4/química , Leucotrieno A4/genética , Camundongos , Mutação de Sentido Incorreto , Fosforilação , Estrutura Secundária de Proteína , Proteínas Quinases S6 Ribossômicas 70-kDa/química , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina/química , Serina/genética , Serina/metabolismo
4.
Proc Natl Acad Sci U S A ; 109(7): 2325-9, 2012 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-22308375

RESUMO

BRICHOS domains are encoded in > 30 human genes, which are associated with cancer, neurodegeneration, and interstitial lung disease (ILD). The BRICHOS domain from lung surfactant protein C proprotein (proSP-C) is required for membrane insertion of SP-C and has anti-amyloid activity in vitro. Here, we report the 2.1 Å crystal structure of the human proSP-C BRICHOS domain, which, together with molecular dynamics simulations and hydrogen-deuterium exchange mass spectrometry, reveals how BRICHOS domains may mediate chaperone activity. Observation of amyloid deposits composed of mature SP-C in lung tissue samples from ILD patients with mutations in the BRICHOS domain or in its peptide-binding linker region supports the in vivo relevance of the proposed mechanism. The results indicate that ILD mutations interfering with proSP-C BRICHOS activity cause amyloid disease secondary to intramolecular chaperone malfunction.


Assuntos
Amiloide/antagonistas & inibidores , Pulmão/metabolismo , Chaperonas Moleculares/metabolismo , Proteína C Associada a Surfactante Pulmonar/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Chaperonas Moleculares/química , Dados de Sequência Molecular , Conformação Proteica , Proteína C Associada a Surfactante Pulmonar/química
5.
Biochem Biophys Res Commun ; 446(2): 519-22, 2014 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-24613831

RESUMO

An 84-residue bactericidal peptide, PSK, was purified from a Chrysomya megacephala fly larvae preparation. Its amino acid sequence is similar to that of a previously reported larval peptide of the Drosophila genus (SK84) noticed for its anticancer and antimicrobial properties. The PSK sequence is also homologous to mitochondrial ATPase inhibitors from insects to humans (35-65% sequence identity), indicating an intracellular protein target and possible mechanism for PSK. It contains a cluster of six glycine residues, and has several two- and three-residue repeats. It is active against both Gram-positive and Gram-negative bacteria via a mechanism apparently involving cell membrane disintegration and inhibition of ATP hydrolysis. In addition, PSK induces an inward cationic current in pancreatic ß cells. Together, the findings identify a bioactive peptide of the ATPase inhibitor family with specific effects on both prokaryotic and mammalian cells.


Assuntos
Antibacterianos/farmacologia , Fenômenos Fisiológicos Bacterianos/efeitos dos fármacos , Dípteros/metabolismo , Células Secretoras de Insulina/fisiologia , Ativação do Canal Iônico/fisiologia , Proteínas/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/química , Linhagem Celular , Humanos , Hidrólise , Células Secretoras de Insulina/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Larva/metabolismo , Dados de Sequência Molecular , Proteínas/química , Inibidores da Bomba de Prótons/química , Inibidores da Bomba de Prótons/farmacologia , Relação Estrutura-Atividade , Proteína Inibidora de ATPase
6.
Mol Cell Proteomics ; 10(9): M110.006510, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21610101

RESUMO

A membrane cell for hydrogen and deuterium exchange on-line with mass spectrometry has been developed to monitor protein-protein interactions and protein conformations. It consists of two channels separated by a semipermeable membrane, where one channel carries the protein sample and the other deuterium oxide. The membrane allows transfer of deuterium oxide into the sample flow. The labeling time is controlled via the flow rate in the sample channel. This cell was validated against three models commonly used in hydrogen-deuterium exchange mass spectrometry: monitoring of folded and unfolded states in a protein, mapping the protein secondary structure at the peptide level, and detection of protein and antibody interactions. The system avoids the conventionally used sample dilution and handling, allowing for potential automation.


Assuntos
Medição da Troca de Deutério/métodos , Deutério/metabolismo , Hidrogênio/metabolismo , Espectrometria de Massas/métodos , Peptídeos , Proteínas , Proteômica/métodos , Anticorpos/metabolismo , Automação Laboratorial , Óxido de Deutério/metabolismo , Cinética , Peptídeos/análise , Peptídeos/química , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Desdobramento de Proteína , Proteínas/análise , Proteínas/química
7.
J Biol Chem ; 285(10): 7246-53, 2010 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-20018874

RESUMO

Wnts are secreted, lipidated proteins that regulate multiple aspects of brain development, including dopaminergic neuron development. In this study, we perform the first purification and signaling analysis of Wnt2 and define the function of Wnt2 in ventral midbrain precursor cultures, as well as in Wnt2-null mice in vivo. We found that purified Wnt2 induces the phosphorylation of both Lrp5/6 and Dvl-2/3, and activates beta-catenin in SN4741 dopaminergic cells. Moreover, purified Wnt2 increases progenitor proliferation, and the number of dopaminergic neurons in ventral midbrain precursor cultures. In agreement with these findings, analysis of the ventral midbrain of developing Wnt2-null mice revealed a decrease in progenitor proliferation and neurogenesis that lead to a decrease in the number of postmitotic precursors and dopaminergic neurons. Collectively, our observations identify Wnt2 as a novel regulator of dopaminergic progenitors and dopaminergic neuron development.


Assuntos
Proliferação de Células , Mesencéfalo , Células-Tronco/fisiologia , Proteína Wnt2/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Dopamina/metabolismo , Feminino , Mesencéfalo/citologia , Mesencéfalo/embriologia , Camundongos , Camundongos Knockout , Neurogênese/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Gravidez , Processamento de Proteína Pós-Traducional , Células-Tronco/citologia , Proteína Wnt2/genética , Proteína Wnt2/isolamento & purificação , beta Catenina/metabolismo
8.
Biochem Biophys Res Commun ; 396(1): 125-30, 2010 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-20494124

RESUMO

Two large gene and protein superfamilies, SDR and MDR (short- and medium-chain dehydrogenases/reductases), were originally defined from analysis of alcohol and polyol dehydrogenases. The superfamilies contain minimally 82 and 25 genes, respectively, in humans, minimally 324 and 86 enzyme families when known lines in other organisms are also included, and over 47,000 and 15,000 variants in existing sequence data bank entries. SDR enzymes have one-domain subunits without metal and MDR two-domain subunits without or with zinc, and these three lines appear to have emerged in that order from the universal cellular ancestor. This is compatible with their molecular architectures, present multiplicity, and overall distribution in the kingdoms of life, with SDR also of viral occurrence. An MDR-zinc, when present, is often, but not always, catalytic. It appears also to have a structural role in inter-domain interactions, coenzyme binding and substrate pocket formation, as supported by domain variability ratios and ligand positions. Differences among structural and catalytic zinc ions may be relative and involve several states. Combined, the comparisons trace evolutionary properties of huge superfamilies, with partially redundant enzymes in cellular redox functions.


Assuntos
Acil-CoA Desidrogenase/classificação , Butiril-CoA Desidrogenase/classificação , Evolução Molecular , Metaloproteínas/classificação , Zinco/metabolismo , Acil-CoA Desidrogenase/química , Acil-CoA Desidrogenase/genética , Butiril-CoA Desidrogenase/química , Butiril-CoA Desidrogenase/genética , Humanos , Metaloproteínas/química , Metaloproteínas/genética , Filogenia , Conformação Proteica
9.
Nat Struct Mol Biol ; 12(3): 238-44, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15711563

RESUMO

To determine the role of actin-ribonucleoprotein complexes in transcription, we set out to identify novel actin-binding proteins associated with RNA polymerase II (Pol II). Using affinity chromatography on fractionated HeLa cells, we found that hnRNP U binds actin through a short amino acid sequence in its C-terminal domain. Post-transcriptional gene silencing of hnRNP U and nuclear microinjections of a short peptide encompassing the hnRNP U actin-binding sequence inhibited BrUTP incorporation in vivo. In living cells, we found that both actin and hnRNP U are associated with the phosphorylated C-terminal domain of Pol II, and antibodies to actin and hnRNP U blocked Pol II-mediated transcription. Taken together, our results indicate that a general actin-based mechanism is implicated in the transcription of most Pol II genes. Actin in complex with hnRNP U may carry out its regulatory role during the initial phases of transcription activation.


Assuntos
Actinas/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/fisiologia , RNA Polimerase II/metabolismo , Transcrição Gênica/fisiologia , Actinas/metabolismo , Sequência de Aminoácidos , Inativação Gênica , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/metabolismo , Humanos , Dados de Sequência Molecular , Fosforilação , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética
10.
J Gen Virol ; 90(Pt 11): 2821-2828, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19605588

RESUMO

Prions are infectious agents resulting from the conversion of a normal cellular protein, PrP(C), to a misfolded species, PrP(Sc). Iatrogenic transmission of prions is known from surgical procedures involving stainless steel materials. Here, it was shown that stainless steel containing nickel and molybdenum binds PrP(Sc) more efficiently and transmits infection to cells in culture to a higher degree than if these elements are not present. Furthermore, both nickel and molybdenum alone adsorbed PrP(Sc), and nickel powder could be used to extract PrP(Sc) from dilute solutions, thus providing a simple approach to concentration of PrP(Sc). The fact that nickel and molybdenum in steel alloys increased the binding affinity, and bound infectivity, of PrP(Sc) is an important issue to consider in the manufacture of surgical instruments and abattoir tools.


Assuntos
Cirurgia Geral/instrumentação , Molibdênio/metabolismo , Níquel/metabolismo , Doenças Priônicas/transmissão , Príons/metabolismo , Aço Inoxidável , Adsorção , Animais , Humanos , Ligação Proteica
11.
Rapid Commun Mass Spectrom ; 23(22): 3591-8, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19844966

RESUMO

The C-terminal domain of lung surfactant protein C (CTC) precursor (proSP-C) is involved in folding of the transmembrane segment of proSP-C. CTC includes a Brichos domain with homologs in cancer- and dementia-associated proteins. Mutations in the Brichos domain cause misfolding of proSP-C and hence amyloid fibril formation in interstitial lung disease. Electrospray ionization mass spectrometry (ESI-MS) with collision-induced dissociation (CID) experiments was applied to study non-covalent interactions between human recombinant CTC or its Brichos domain, and SP-C analogs, homotripeptides and peptides designed to model amyloid fibril formation. The results show that the Brichos domain contains the peptide-binding function of CTC. In titration experiments, apparent dissociation constants (KD) were in the micromolar range where triple-valine showed the lowest KD and triple-tyrosine the highest. Non-hydrophobic peptides failed to form complexes with Brichos. CID revealed that complexes with aromatic peptide ligands are more stable in the gas phase than complexes with non-aromatic ligands. The Brichos domain was also shown to bind fibril-forming peptides containing aromatic/hydrophobic residues.


Assuntos
Peptídeos/química , Proteína C Associada a Surfactante Pulmonar/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Cinética , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteína C Associada a Surfactante Pulmonar/genética , Proteína C Associada a Surfactante Pulmonar/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade da Espécie
12.
Biochem Biophys Res Commun ; 373(4): 482-7, 2008 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-18571501

RESUMO

The reversible thermal unfolding of the archaeal histone-like protein Ssh10b from the extremophile Sulfolobus shibatae was studied using differential scanning calorimetry and circular dichroism spectroscopy. Analytical ultracentrifugation and gel filtration showed that Ssh10b is a stable dimer in the pH range 2.5-7.0. Thermal denaturation data fit into a two-state unfolding model, suggesting that the Ssh10 dimer unfolds as a single cooperative unit with a maximal melting temperature of 99.9 degrees C and an enthalpy change of 134 kcal/mol at pH 7.0. The heat capacity change upon unfolding determined from linear fits of the temperature dependence of DeltaH(cal) is 2.55 kcal/(mol K). The low specific heat capacity change of 13 cal/(mol K residue) leads to a considerable flattening of the protein stability curve (DeltaG (T)) and results in a maximal DeltaG of only 9.5 kcal/mol at 320 K and a DeltaG of only 6.0 kcal/mol at the optimal growth temperature of Sulfolobus.


Assuntos
Proteínas Arqueais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Temperatura Alta , Proteínas de Ligação a RNA/metabolismo , Sulfolobus/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/química , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Proteínas de Ligação a DNA/química , Histonas/química , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Desnaturação Proteica , Dobramento de Proteína , Proteínas de Ligação a RNA/química , Termodinâmica
13.
FEBS J ; 274(3): 751-9, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17288555

RESUMO

A 37 residue peptide, aglycin, has been purified from porcine intestine. The sequence is identical to that of residues 27-63 of plant albumin 1 B precursor (PA1B, chain b) from pea seeds. Aglycin resists in vitro proteolysis by pepsin, trypsin and Glu-C protease, compatible with its intestinal occurrence and an exogenous origin from plant food. When subcutaneously injected into mice (at 10 microg.g(-1) body weight), aglycin has a hyperglycemic effect resulting in a doubling of the blood glucose level within 60 min. Using surface plasmon resonance biosensor technology, an aglycin binding protein with an apparent molecular mass of 34 kDa was detected in membrane protein extracts from porcine and mice pancreas. The polypeptide was purified by affinity chromatography and identified through peptide mass fingerprinting as the voltage-dependent anion-selective channel protein 1. The results indicate that aglycin has the potential to interfere with mammalian physiology.


Assuntos
Glicemia/análise , Peptídeos/química , Proteínas de Plantas/química , Animais , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Camundongos , Peso Molecular , Pâncreas/química , Pisum sativum/química , Mapeamento de Peptídeos , Peptídeos/metabolismo , Peptídeos/farmacologia , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacologia , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ressonância de Plasmônio de Superfície/métodos , Suínos , Canal de Ânion 1 Dependente de Voltagem/metabolismo
14.
Biochimie ; 89(8): 950-60, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17475390

RESUMO

Sphingolipids are degraded by sphingomyelinase and ceramidase in the gut to ceramide and sphingosine, which may inhibit cell proliferation and induce apoptosis, and thus have anti-tumour effects in the gut. Although previous rodent studies including experiments on knockout mice indicate a role of neutral ceramidase in ceramide digestion, the human enzyme has never been purified and characterized in its purified form. We here report the purification and characterization of neutral ceramidase from human ileostomy content, using octanoyl-[(14)C]sphingosine as substrate. After four chromatographic steps, a homogeneous protein band with 116kDa was obtained. MALDI mass spectrometry identified 16 peptide masses similar to human ceramidase previously cloned by El Bawab et al. [Molecular cloning and characterization of a human mitochondrial ceramidase, J. Biol. Chem. 275 (2000) 21508-21513] and Hwang et al. [Subcellular localization of human neutral ceramidase expressed in HEK293 cells, Biochem. Biophys. Res. Commun. 331 (2005) 37-42]. By RT-PCR and 5'-RACE methods, a predicted partial nucleotide sequence of neutral ceramidase was obtained from a human duodenum biopsy sample, which was homologous to that of known neutral/alkaline ceramidases. The enzyme has neutral pH optimum and catalyses both hydrolysis and formation of ceramide without distinct bile salt dependence. It is inhibited by Cu(2+) and Zn(2+) ions and by low concentrations of cholesterol. The enzyme is a glycoprotein but deglycosylation does not affect its activity. Our study indicates that neutral ceramidase is expressed in human intestine, released in the intestinal lumen and plays a major role in ceramide metabolism in the human gut.


Assuntos
Amidoidrolases/química , Intestinos/enzimologia , Amidoidrolases/genética , Amidoidrolases/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Ceramidases , Ceramidas/metabolismo , Colesterol/metabolismo , Duodeno , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Dados de Sequência Molecular , Ceramidase Neutra , Células Tumorais Cultivadas
15.
Mol Biol Cell ; 13(12): 4195-205, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12475945

RESUMO

The biochemical properties and spatial localization of the protein alpha-dystrobrevin and other isoforms were investigated in cells of the human promyelocytic leukemia line HL-60 granulocytic differentiation as induced by retinoic acid (RA). Alpha-dystrobrevin was detected both in the cytosol and the nuclei of these cells, and a short isoform (gamma-dystrobrevin) was modified by tyrosine phosphorylation soon after the onset of the RA-triggered differentiation. Varying patterns of distribution of alpha-dystrobrevin and its isoforms could be discerned in HL-60 promyelocytes, RA-differentiated mature granulocytes, and human neutrophils. Moreover, the gamma-dystrobrevin isoform was found in association with actin and myosin light chain. The results provide new information about potential involvement of alpha-dystrobrevin and its splice isoforms in signal transduction in myeloid cells during induction of granulocytic differentiation and/or at the commitment stage of differentiation or phagocytic cells.


Assuntos
Proteínas do Citoesqueleto/química , Proteínas Associadas à Distrofina , Granulócitos/citologia , Proteínas de Membrana/química , Actinas/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Citosol/metabolismo , Eletroforese em Gel de Poliacrilamida , Granulócitos/metabolismo , Células HL-60 , Humanos , Immunoblotting , Proteínas de Membrana/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Cadeias Leves de Miosina/metabolismo , Neutrófilos/metabolismo , Fagocitose , Fosforilação , Testes de Precipitina , Isoformas de Proteínas , Transdução de Sinais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo , Tripsina/farmacologia , Tirosina/metabolismo
16.
Biochim Biophys Acta ; 1687(1-3): 94-102, 2005 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-15708357

RESUMO

Intestinal alkaline sphingomyelinase (alk-SMase) digests sphingomyelin and the process may influence colonic tumorigenesis and cholesterol absorption. We recently identified the gene of human alk-SMase and cloned the cDNA. Cross-species screening of homology in GenBank found a hypothetical rat protein, XP_221184, with 491 amino acid residues, which shares 73% identity with human alk-SMase. Based on the cDNA sequence of this protein, we cloned a cDNA from rat intestinal mucosa by RT-PCR. The cloned cDNA encodes a protein with 439 amino acid residues and higher (85%) identity with human alk-SMase. The cloned cDNA differed from the XP_221184 cDNA in splice sites linking exons 2 and 3, and exons 3 and 4, respectively. In the sequence of the cloned protein, the predicted activity motif, sphingomyelin binding sites, and potential glycosylation sites in human alk-SMase are all conserved. To confirm the cloned protein is the real form of alk-SMase, native alk-SMase was purified from rat intestine and subjected to proteolytic digestion followed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and electrospray ionization (ESI) tandem mass spectrometry. Seven tryptic peptides were found to match the cloned protein sequence. Transient expression of the cloned cDNA linked with a myc tag in COS-7 cells demonstrated high SMase activity, with an optimal pH at 9.0 and a specific dependence on taurocholate and taurochenodeoxycholate. The expressed protein reacted with both anti-myc and anti-human alk-SMase antibodies. Northern blotting of rat tissues revealed high levels of mRNA in jejunum but not in other tissues. In conclusion, we cloned rat alk-SMase cDNA from rat intestine, adjusted the putative rat alk-SMase protein in GenBank, and confirmed the specific expression of the gene in the small intestine.


Assuntos
Bases de Dados de Ácidos Nucleicos , Mucosa Intestinal/enzimologia , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Clonagem Molecular , Feminino , Humanos , Dados de Sequência Molecular , Ratos , Ratos Sprague-Dawley , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Distribuição Tecidual
17.
Nucleic Acids Res ; 30(8): 1725-34, 2002 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-11937625

RESUMO

Pre-mRNP complexes were isolated from rat liver nuclei as 40S hnRNP particles, and actin-binding proteins were collected by DNase I affinity chromatography. The bound proteins were analyzed by 2D gel electrophoresis, and the following five hnRNP A/B-type proteins were identified by tandem mass spectrometry: DBP40/CBF-A (CArG binding factor A), a minor hnRNP A2 variant and three minor hnRNP A3 (mBx) variants. DBP40 was chosen for further analysis of the association of actin with the pre-mRNP complex. It was shown in vitro that purified actin binds to recombinant DBP40 suggesting that the interaction between actin and DBP40 is direct in the pre-mRNP particles. The association of actin with DBP40 was further explored in vivo. It was shown in a transfection study that DBP40 appears both in the nucleus and cytoplasm. Microinjection experiments revealed that DBP40 is exported from the nucleus to the cytoplasm. Finally, RNA-protein and protein-protein cross-linking experiments showed that DBP40 interacts with poly(A)(+) RNA as well as actin, both in the nucleus and cytoplasm. We propose that actin associated with DBP40, and perhaps with additional hnRNP A/B-type proteins, is transferred from nucleus to cytoplasm bound to mRNA.


Assuntos
Actinas/metabolismo , Núcleo Celular/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B , Ribonucleoproteínas/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Células COS , Eletroforese em Gel Bidimensional , Ribonucleoproteínas Nucleares Heterogêneas , Espectrometria de Massas , Microscopia de Fluorescência , Dados de Sequência Molecular , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ribonucleoproteínas/química
18.
J Biomol Tech ; 16(4): 392-7, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16522861

RESUMO

A large-surface biosensor technique using surface plasmon resonance (SPR) was tested for protein purification by recovery of a monoclonal antibody against human proinsulin C-peptide. Notably, both reversible attachment/desorption and actual purification of the antibody from a multi-component protein mixture was shown. For initial chip attachment of the peptide ligand, C-peptide was biotinylated and attached to neutravidin on plastic chips with a large gold surface (effective area 26 mm(2)). Antibody binding and desorption was monitored in real-time SPR, and for elution different conditions were employed. Five percent formic acid (in contact with the chip surface for 3 min) in a 60-mul segment between air bubbles was efficient for subsequent analysis. In this manner, protein amounts up to 35 pmoles were recovered in a single capture/elution cycle. Evaluation by SDS-PAGE showed essentially no carryover between fractions in this elution process, and also not with other proteins in the mixture after purification. Compared to existing commercial instruments, this technique gives higher recovery and makes it possible to monitor monitor protein binding/desorption. Recovery of affinity partners at the multi-pmole level is demonstrated for protein purification in SPR approaches.


Assuntos
Técnicas Biossensoriais , Biotecnologia/métodos , Proteínas/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Adsorção , Anticorpos Monoclonais/metabolismo , Avidina/farmacocinética , Biotinilação , Eletroforese em Gel de Poliacrilamida , Humanos , Ligantes , Espectrometria de Massas , Peso Molecular , Proinsulina/análise , Proinsulina/isolamento & purificação , Proinsulina/metabolismo , Ligação Proteica , Proteínas/análise , Proteínas/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/farmacologia
19.
Biochem J ; 380(Pt 2): 571-9, 2004 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-15025560

RESUMO

We have previously shown that ScN2a cells (scrapie-infected neuroblastoma N2a cells) express 2-fold- and 4-fold-increased levels of IR (insulin receptor) and IGF-1R (insulin-like growth factor-1 receptor) respectively. In addition, the IR alpha- and beta-subunits are aberrantly processed, with apparent molecular masses of 128 and 85 kDa respectively, as compared with 136 and 95 kDa in uninfected N2a cells. Despite the 2-fold increase in IR protein, the number of (125)I-insulin-binding sites was slightly decreased in ScN2a cells [Ostlund, Lindegren, Pettersson and Bedecs (2001) Brain Res. 97, 161-170]. In order to determine the cellular localization of IR in ScN2a cells, surface biotinylation was performed, showing a correct IR trafficking and localization to the cell surface. The present study shows for the first time that neuroblastoma N2a cells express significant levels of IR-IGF-1R hybrid receptors, and in ScN2a cells the number of hybrid receptors was 2-fold higher than that found in N2a cells, potentially explaining the apparent loss of insulin-binding sites due to a lower affinity for insulin compared with the homotypic IR. Furthermore, the decreased molecular mass of IR subunits in ScN2a cells is not caused by altered phosphorylation or proteolytic processing, but rather by altered glycosylation. Enzymic deglycosylation of immunoprecipitated IR from N2a and ScN2a cells with endoglycosidase H, peptide N-glycosidase F and neuraminidase all resulted in subunits with increased electrophoretic mobility; however, the 8-10 kDa shift remained. Combined enzymic or chemical deglycosylation using anhydrous trifluoromethane sulphonic acid treatment ultimately showed that the IR alpha- and beta-subunits from ScN2a cells are aberrantly glycosylated. The increased formation of IR-IGF-1R hybrids in ScN2a cells may be part of a neuroprotective response to prion infection. The degree and functional significance of aberrantly glycosylated proteins in ScN2a cells remain to be determined.


Assuntos
Neuroblastoma/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Scrapie/metabolismo , Animais , Sítios de Ligação/genética , Linhagem Celular Tumoral , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Glicosilação , Insulina/metabolismo , Camundongos , Peso Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neuroblastoma/química , Neuroblastoma/patologia , Proteínas PrPSc/metabolismo , Proteínas PrPSc/patogenicidade , Ligação Proteica/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor IGF Tipo 1/química , Receptor IGF Tipo 1/genética , Receptor de Insulina/química , Receptor de Insulina/genética , Recombinação Genética/genética , Recombinação Genética/fisiologia , Scrapie/genética , Scrapie/patologia
20.
J Pharm Sci ; 91(10): 2116-21, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12226839

RESUMO

The alkaline degradation of the chemotherapeutic agent oxaliplatin has been studied using liquid chromatography. The oxalato ligand is lost in two consecutive steps. First, the oxalato ring is opened, forming an oxalato monodentate intermediate, as identified by electrospray ionization mass spectrometry. Subsequently, the oxalato ligand is lost and the dihydrated oxaliplatin complex is formed. The observed rate constants for the first step (k(1)) and the second step (k(2)) follow the equation k(1) or k(2) = k(0) + k(OH(-) )[OH(-)], where k(0) is the rate constant for the degradation catalyzed by water and k(OH(-) ) represents the second-order rate constant for the degradation catalyzed by the hydroxide ion. At 37 degrees C the rate constants for the first step are k(OH(-) ) = 5.5 x 10(-2) min(-1) M(-1) [95% confidence interval (CI), 2.7 x 10(-2) to 8.4 x 10(-2) min(-1) M(-1)] and k(0) = 4.3 x 10(-2) min(-1) (95% CI, 4.0 x 10(-2) to 4.7 x 10(-2) min(-1)). For the second step the rate constants are k(OH(-) ) = 1.1 x 10(-3) min(-1) M(-1) (95% CI, -1.1 x 10(-3) to 3.3 x 10(-3)) min(-1) M(-1) and k(0) = 7.5 x 10(-3) min(-1) (95% CI, 7.2 x 10(-3) to 7.8 x 10(-3) min(-1)). Thus, the ring-opening step is nearly six times faster than the step involving the loss of the oxalato ligand.


Assuntos
Antineoplásicos/química , Compostos Organoplatínicos/química , Compostos Organoplatínicos/síntese química , Compostos Organoplatínicos/farmacologia , Algoritmos , Cromatografia Líquida , Hidrólise , Indicadores e Reagentes , Cinética , Oxaliplatina , Platina/química , Hidróxido de Sódio/química , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria Ultravioleta
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