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1.
Protein Sci ; 26(11): 2280-2290, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28857320

RESUMO

The expression of eukaryotic genes is precisely controlled by interactions between general transcriptional factors and promoter-specific transcriptional activators. The fourth element of TATA-box binding protein-associated factor (TAF4), an essential subunit of the general transcription factor TFIID, serves as a coactivator for various promoter-specific transcriptional regulators. Interactions between TAF4 and site-specific transcriptional activators, such as Sp1, are important for regulating the expression levels of genes of interest. However, only limited information is available on the molecular mechanisms underlying the interactions between these transcriptional regulatory proteins. We herein analyzed the interaction between the transcriptional factors Sp1 and TAF4 using high-resolution solution nuclear magnetic resonance spectroscopy. We found that four glutamine-rich (Q-rich) regions in TAF4 were largely disordered under nearly physiological conditions. Among them, the first Q-rich region in TAF4 was essential for the interaction with another Q-rich region in the Sp1 molecule, most of which was largely disordered. The residues responsible for this interaction were specific and highly localized in a defined region within a range of 20-30 residues. Nevertheless, a detailed analysis of 13 C-chemical shift values suggested that no significant conformational change occurred upon binding. These results indicate a prominent and exceptional binding mode for intrinsically disordered proteins other than the well-accepted concept of "coupled folding and binding."


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Fator de Transcrição Sp1/química , Fatores Associados à Proteína de Ligação a TATA/química , Fator de Transcrição TFIID/química , Motivos de Aminoácidos , Sítios de Ligação , Isótopos de Carbono , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular/métodos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Fator de Transcrição Sp1/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/metabolismo , Transcrição Gênica
2.
Protein Sci ; 25(11): 2006-2017, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27515574

RESUMO

The expression of eukaryotic genes is precisely controlled by specific interactions between general transcription initiation factors and gene-specific transcriptional activators. The general transcription factor TFIID, which plays an essential role in mediating transcriptional activation, is a multisubunit complex comprising the TATA box-binding protein (TBP) and multiple TBP-associated factors (TAFs). On the other hand, biochemical and genetic approaches have shown that the promoter-specific transcriptional activator Sp1 has the ability to interact with one of the components of TFIID, the TBP-associated factor TAF4. We herein report the structural details of the glutamine-rich domains (Q-domains) of Sp1 and TAF4 using circular dichroism (CD) and heteronuclear magnetic resonance (NMR) spectroscopy. We found that the two Q-domains of Sp1 and four Q-domains of TAF4 were disordered under physiological conditions. We also quantitatively analyzed the interaction between the Q-domains of Sp1 and TAF4 by NMR and surface plasmon resonance, and detected a weak but specific association between them. Nevertheless, a detailed analysis of CD spectra suggested that any significant conformational change did not occur concomitantly with this association, at least at the level of the overall secondary structure. These results may represent a prominent and exceptional binding mode for the IDPs, which are not categorized in a well-accepted concept of "coupled folding and binding."


Assuntos
Fator de Transcrição Sp1/química , Fatores Associados à Proteína de Ligação a TATA/química , Fator de Transcrição TFIID/química , Dicroísmo Circular , Humanos , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Estrutura Secundária de Proteína , Fator de Transcrição Sp1/metabolismo , Ressonância de Plasmônio de Superfície , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/metabolismo
3.
J Pharmacol Sci ; 130(1): 33-7, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26809377

RESUMO

We investigated the involvement of intracellular cAMP in endothelial cell injury induced by epirubicin. Epirubicin-induced decrease in cell viability and increase in caspase-3/7 activity were reversed by a cAMP analog dibutyryl cAMP (DBcAMP) or an activator of adenylate cyclase forskolin concomitant with a phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Moreover, epirubicin-induced elevation of lipid peroxide levels was attenuated by DBcAMP. Interestingly, the exposure of epirubicin decreased intracellular cAMP levels before the onset of epirubicin-induced production of lipid peroxidation. These results suggest that intracellular cAMP plays an important role in epirubicin-induced endothelial cell injury.


Assuntos
Apoptose/efeitos dos fármacos , AMP Cíclico/fisiologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Epirubicina/efeitos adversos , Epirubicina/antagonistas & inibidores , 1-Metil-3-Isobutilxantina/farmacologia , Combinação Albuterol e Ipratrópio , Animais , Bucladesina/farmacologia , Caspase 3/metabolismo , Caspase 7/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Peróxidos Lipídicos/metabolismo , Suínos
4.
Biol Pharm Bull ; 38(9): 1410-4, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26328498

RESUMO

The anti-tumor effects of selective serotonin reuptake inhibitors (SSRIs) and serotonin and norepinephrine reuptake inhibitors (SNRIs) on several types of cancer cells have been reported. However, comparison of the anti-tumor effects of these drugs on human hepatocellular carcinoma (HepG2) cells has not been studied. We compared the anti-tumor effects of four SSRIs and two SNRIs on HepG2 cells. SSRIs and duloxetine dose-dependently decreased cell viability. Milnacipran had no effect on cell viability. The half-maximal inhibitory concentration was lower in the order of: sertraline, paroxetine, duloxetine, fluvoxamine, escitalopram, and milnacipran. Exposure to sertraline (2 µM) significantly increased caspase-3/7 activity. These results suggest that, of the agents tested here, sertraline had the highest sensitivity to HepG2 cells, and activation of the caspase pathway is involved in the anti-tumor effects of sertraline in HepG2 cells.


Assuntos
Antineoplásicos/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Inibidores da Recaptação de Serotonina e Norepinefrina/farmacologia , Carcinoma Hepatocelular , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citalopram/farmacologia , Ciclopropanos/farmacologia , Cloridrato de Duloxetina/farmacologia , Fluvoxamina/farmacologia , Células Hep G2 , Humanos , Neoplasias Hepáticas , Milnaciprano , Paroxetina/farmacologia , Sertralina/farmacologia
5.
Biosens Bioelectron ; 39(1): 334-7, 2013 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-22902533

RESUMO

In this communication, we describe a novel and facile method for the immobilization of NAD(+)/NADH on an electrode surface using a hydrophobic ionic liquid, 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ([C4mim][Tf(2)N]). By taking advantage of the insolubility of NAD(+)/NADH in hydrophobic ionic liquids, it is expected that NAD(+)/NADH can be retained on the electrode's surface. Alcohol dehydrogenase (ADH) and NAD(+)/NADH were immobilized with a gelatin hydrogel on an electrode that was modified with an electropolymerized ruthenium complex containing 5-amino-1,10-phenanthroline (pAPRu) as a mediator for NADH oxidation. The (ADH, NAD(+))/pAPRu-immobilized electrode exhibited the electrocatalytic oxidation of ethanol in [C4mim][Tf(2)N]. The obtained catalytic current in [C4mim][Tf(2)N] was comparable to that in buffer solution containing NAD(+). It was confirmed by UV-vis spectroscopy that NAD(+) did not dissolve in the [C4mim][Tf(2)N] and was retained on the electrode's surface. Furthermore, we succeeded in constructing an ethanol/O(2) biofuel cell comprised of an (ADH, NAD(+))/pAPRu anode and a bilirubin oxidase cathode using [C4mim][Tf(2)N] as an electrolyte.


Assuntos
Fontes de Energia Bioelétrica , Imidazóis/química , Imidas/química , Líquidos Iônicos/química , NAD/química , Álcool Desidrogenase/química , Álcool Desidrogenase/metabolismo , Técnicas Biossensoriais , Catálise , Eletrodos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Etanol/metabolismo , Gelatina/química , NAD/metabolismo , Fenantrolinas/química , Saccharomyces cerevisiae/enzimologia
6.
Protein Sci ; 21(10): 1481-8, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22855260

RESUMO

The promoter-specific transcription factor Sp1 is expressed ubiquitously, and plays a primary role in the regulation of the expression of many genes. Domains A and B located in the N-terminal half of the protein are characterized by glutamine-rich (Q-rich) sequences. These Q-rich domains have been shown to be involved in the interaction between Sp1 and different classes of nuclear proteins, such as TATA-binding protein associated factors. Furthermore, the self-association of Sp1 via Q-rich domains is also important for the regulation of transcriptional activity. It has been considered that an Sp1 molecule bound to a "distal" GC-box synergistically interacts with another Sp1 molecule at a "proximal" binding site. Although the formation of multimers via Q-rich domains seems functionally important for Sp1, little is known about the structural and physicochemical nature of the interaction between Q-rich domains. We analyzed the structural details of isolated glutamine-rich B (QB) domains of Sp1 by circular dichroism (CD), analytical ultracentrifugation, and heteronuclear magnetic resonance spectroscopy (NMR). We found the isolated QB domains to be disordered under all conditions examined. Nevertheless, a detailed analysis of NMR spectra clearly indicated interaction between the domains. In particular, the C-terminal half was responsible for the self-association. Furthermore, analytical ultracentrifugation demonstrated weak but significant interaction between isolated QB domains. The self-association between QB domains would be responsible, at least in part, for the formation of multimers by full-length Sp1 molecules that has been proposed to occur during transcriptional activation.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Fator de Transcrição Sp1/química , Fator de Transcrição Sp1/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Glutamina/química , Humanos , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Ultracentrifugação
7.
Biochem Biophys Res Commun ; 403(2): 161-6, 2010 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-20946882

RESUMO

Transcription factor Sp1 is localized in the nucleus and regulates the expression of many cellular genes, but the nuclear transport mechanism of Sp1 is not well understood. In this study, we revealed that GST-fused Sp1 protein bound to endogenous importin α in HeLa cells via the Sp1 zinc finger domains, which comprise the DNA binding domain of Sp1. It was found that the Sp1 zinc finger domains directly interacted with a wide range of importin α including the armadillo (arm) repeat domain and the C-terminal acidic domain. Furthermore, it turned out that all three zinc fingers of Sp1 are essential for binding to importin α. Taken together, these results suggest that the Sp1 zinc finger domains play an essential role as a NLS and Sp1 can be transported into the nucleus in an importin-dependent manner even though it possesses no classical NLSs.


Assuntos
Núcleo Celular/metabolismo , Sinais de Localização Nuclear/metabolismo , Fator de Transcrição Sp1/metabolismo , Dedos de Zinco , Transporte Ativo do Núcleo Celular , Células HeLa , Humanos , Sinais de Localização Nuclear/genética , Deleção de Sequência , Fator de Transcrição Sp1/genética , alfa Carioferinas/metabolismo
8.
Biochem Biophys Res Commun ; 380(1): 28-32, 2009 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-19138671

RESUMO

Transcription factor Sp1 is localized in the nucleus and regulates gene expression. Our previous study demonstrated that the carboxyl terminal region of Sp1 containing 3-zinc finger region as DNA binding domain can also serve as nuclear localization signal (NLS). However, the nuclear transport mechanism of Sp1 has not been well understood. In this study, we performed a gene expression study on mutant Sp1 genes causing a set of amino acid substitutions in zinc finger domains to elucidate nuclear import activity. Nuclear localization of the GFP-fused mutant Sp1 proteins bearing concomitant substitutions in the first and third zinc fingers was highly inhibited. These mutant Sp1 proteins had also lost the binding ability as to the GC box sequence. The results suggest that the overall tertiary structure formed by the three zinc fingers is essential for nuclear localization of Sp1 as well as dispersed basic amino acids within the zinc fingers region.


Assuntos
Núcleo Celular/metabolismo , Sinais de Localização Nuclear/fisiologia , Fator de Transcrição Sp1/metabolismo , Dedos de Zinco/fisiologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Aminoquinolinas/farmacologia , Células HeLa , Humanos , Sinais de Localização Nuclear/genética , Fator de Transcrição Sp1/genética , Compostos de Tosil/farmacologia , Zinco/metabolismo , Dedos de Zinco/genética
9.
J Med Invest ; 53(1-2): 103-12, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16538002

RESUMO

The human GLB1 gene encodes a lysosomal beta-galactosidase (beta-Gal) and an elastin-binding protein (EBP). Defect of the EBP as a chaperon for tropoelastin and a component of receptor complex among neuraminidase-1 (NEU1) and protective protein/cathepsin A (PPCA) is suggested responsible for impaired elastogenesis in autosomal recessive beta-Gal, PPCA and NEU1 deficiencies. The purpose of this study is to determine effects of GLB1, PPCA and NEU1 gene mutations on elastogenesis in skin fibroblasts. Elastic fiber formation and the EBP mRNA expression were examined by immunofluorescence with an anti-tropoelastin antibody and RT-PCR selective for EBP in skin fibroblasts with these lysosomal enzyme deficiencies. Apparently normal elastogenesis and EBP mRNA expression were observed for fibroblasts from Morquio B disease cases with the GLB1 gene alleles (W273L/W273L, W273L/R482H and W273L/W509C substitutions, respectively), a galactosialidosis case with the PPCA allele (IVS7+3A/IVS7+3A) and a sialidosis case with the NEU1 allele (V217M/G243R) as well as normal subject. In this study, the W273L substitution in the EBP could impossibly cause the proposed defect of elastogenesis, and the typical PPCA splicing mutation and the V217M/G243R substitutions in the NEU1 might hardly have effects on elastic fiber formation in the dermal fibroblasts.


Assuntos
Catepsina A/deficiência , Elastina/biossíntese , Neuraminidase/deficiência , beta-Galactosidase/deficiência , Sequência de Bases , Catepsina A/genética , Células Cultivadas , Fibroblastos/metabolismo , Gangliosidoses/genética , Gangliosidoses/metabolismo , Humanos , Mucopolissacaridose IV/genética , Mucopolissacaridose IV/metabolismo , Mutação , Neuraminidase/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Pele/metabolismo , beta-Galactosidase/genética
10.
Glycobiology ; 16(4): 271-80, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16361247

RESUMO

Sialidosis and galactosialidosis are lysosomal storage diseases caused by the genetic defects of lysosomal sialidase (neuraminidase-1; NEU1) and lysosomal protective protein/cathepsin A (PPCA), respectively, associated with a NEU1 deficiency, excessive accumulation of sialylglycoconjugates, and development of progressive neurosomatic manifestations; in addition, the latter disorder is accompanied by simultaneous deficiencies of beta-galactosidase and cathepsin A. We demonstrated that a few soluble N-glycosylated proteins carrying sialyloligosaccharides sensitive to glycopeptidase F (GPF) can be specifically detected in cultured fibroblasts from sialidosis and galactosialidosis cases by blotting with a Maackia amurensis (MAM) lectin. We also examined the therapeutic effects of normal gene transfer and enzyme replacement by evaluating the decreases in sialylglycoconjugates accumulated in fibroblasts with these NEU1 deficiencies. The specific N-glycosylated proteins detected on MAM lectin blotting as well as the granular lysosomal fluorescence due to an avidin-FITC/biotinylated MAM lectin conjugate in sialidosis and galactosialidosis fibroblasts disappeared in parallel with the restoration of the intracellular NEU1 activity after transfection of the recombinant NEU1 fused to HA tag sequence and the wild-type PPCA cDNA as well as administration of the recombinant PPCA precursor protein. The detection method for the abnormal sialylglycoproteins in cultured cells involving MAM lectin was demonstrated to be useful not only for biochemical and diagnostic analyses of NEU1 deficiencies but also for therapeutic evaluation of these conditions.


Assuntos
Fibroblastos/metabolismo , Mucolipidoses/genética , Neuraminidase/genética , Modificação Traducional de Proteínas/genética , Sialoglicoproteínas/metabolismo , Catepsina A/genética , Catepsina A/metabolismo , Células Cultivadas , Estudos de Avaliação como Assunto , Fibroblastos/patologia , Terapia Genética , Humanos , Mucolipidoses/metabolismo , Mucolipidoses/patologia , Mucolipidoses/terapia , Neuraminidase/metabolismo , Oligossacarídeos/genética , Oligossacarídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sialoglicoproteínas/genética , Transfecção
11.
J Neurochem ; 92(6): 1497-507, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15748167

RESUMO

Sandhoff disease is a lysosomal storage disease caused by simultaneous deficiencies of beta-hexosaminidase A (HexA; alphabeta) and B (HexB; betabeta), due to a primary defect of the beta-subunit gene (HEXB) associated with excessive accumulation of GM2 ganglioside (GM2) and oligosaccharides with N-acetylhexosamine residues at their non-reducing termini, and with neurosomatic manifestations. To elucidate the neuroinflammatory mechanisms involved in its pathogenesis, we analyzed the expression of chemokines in Sandhoff disease model mice (SD mice) produced by disruption of the murine Hex beta-subunit gene allele (Hexb-/-). We demonstrated that chemokine macrophage inflammatory protein-1 alpha (MIP-1alpha) was induced in brain regions, including the cerebral cortex, brain stem and cerebellum, of SD mice from an early stage of the pathogenesis but not in other systemic organs. On the other hand, little changes in other chemokine mRNAs, including those of RANTES (regulated upon activation, normal T expressed and secreted), MCP-1 (monocyte chemotactic protein-1), SLC (secondary lymphoid-tissue chemokine), fractalkine and SDF-1 (stromal derived factor-1), were detected. Significant up-regulation of MIP-1alpha mRNA and protein in the above-mentioned brain regions was observed in parallel with the accumulation of natural substrates of HexA and HexB. Immunohistochemical analysis revealed that MIP-1alpha-immunoreactivity (IR) in the above-mentioned brain regions of SD mice was co-localized in Iba1-IR-positive microglial cells and partly in glial fibrillary acidic protein (GFAP)-IR-positive astrocytes, in which marked accumulation of N-acetylglucosaminyl (GlcNAc)-oligosaccharides was observed from the presymptomatic stage of the disease. In contrast, little MIP-1alpha-IR was observed in neurons in which GM2 accumulated predominantly. These results suggest that specific induction of MIP-1alpha might coincide with the accumulation of GlcNAc-oligosaccharides due to a HexB deficiency in resident microglia and astrocytes in the brains of SD mice causing their activation and acceleration of the progressive neurodegeneration in SD mice.


Assuntos
Encéfalo/metabolismo , Glicoconjugados/metabolismo , Proteínas Inflamatórias de Macrófagos/genética , Neuroglia/metabolismo , Doença de Sandhoff/metabolismo , beta-N-Acetil-Hexosaminidases/genética , Acetilglucosamina/metabolismo , Animais , Astrócitos/metabolismo , Encéfalo/fisiopatologia , Proteínas de Ligação ao Cálcio/metabolismo , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Gangliosídeo G(M2)/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteína Glial Fibrilar Ácida/metabolismo , Hexosaminidase A , Hexosaminidase B , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos , Microglia/metabolismo , Neuroglia/patologia , Subunidades Proteicas/genética , RNA Mensageiro/metabolismo , Doença de Sandhoff/genética , Doença de Sandhoff/patologia , Regulação para Cima/fisiologia , beta-N-Acetil-Hexosaminidases/deficiência
12.
J Antibiot (Tokyo) ; 57(5): 316-25, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15303492

RESUMO

The actions of peptidase inhibitors derived from Streptomycete on human cathepsin A (hCath A), yeast carboxypeptidase Y (CPY), and wheat carboxypeptidase II (CPW) were analyzed comparatively. Lactacystin and omuralide (clasto-lactacystin beta-lactone), well-known cytoplasmic proteasome inhibitors, both had a potent and non-competitive inhibitory effect on these homologous serine carboxypeptidases, although they inhibited CPW and hCath A more effectively than CPY in vitro. Ebelactone B exhibited a mixed non-competitive inhibitory effect and selectivity for CPY. Piperastatin A showed competitive inhibition of CPY and hCath A but had little effect on CPW. In contrast, chymostatin inhibited CPW efficiently, while it had less effect on hCath A and CPY. In cell culture system, lactacystin was the most potent as to inactivation of the intralysosomal recombinant hCath A activity expressed in a genetically engineered fibroblastic cell line with galactosialidosis (hCath A deficiency). These results suggest that the specific inhibitory effects of lactacystin and its derivatives on hCath A might be applicable to elucidate the pathophysiological roles in the human deficinecy.


Assuntos
Carboxipeptidases/antagonistas & inibidores , Saccharomyces cerevisiae/enzimologia , Inibidores de Serina Proteinase/farmacologia , Triticum/enzimologia , Catepsina A/antagonistas & inibidores , Catepsina A/metabolismo , Linhagem Celular , Cisteína Endopeptidases/metabolismo , DNA Complementar/biossíntese , DNA Complementar/genética , Relação Dose-Resposta a Droga , Fibroblastos/enzimologia , Humanos , Immunoblotting , Complexos Multienzimáticos/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteínas de Saccharomyces cerevisiae , Especificidade da Espécie , Streptomyces/química
13.
Neurochem Int ; 44(6): 447-57, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-14687610

RESUMO

Human neuroblastoma GOTO cell lines were established that stably express recombinant human lysosomal protective protein/cathepsin A (PPCA) cDNA by transfection. Intracellular cathepsin A (acid serine carboxypeptidase) activity increased four-fold compared with in those of the parent and mock-transfected cell lines. The immunoreactive 54 kDa precursor/zymogen and mature 32/20 kDa two-chain forms were produced in the cells. The amount of the latter form expressed in the GOTO cells was significantly larger than those in the PPCA-overexpressing CHO cell lines previously established. The intracellular proteins showed a typical lysosomal granular distribution and the glycosylated 54 kDa precursor was secreted into the culture medium without the addition of an alkalizing agent. The PPCA-overexpressing cell lines also retained the ability to differentiate bi-directionally as well as the parent cells; into neuronal cells on induction by dibutyryl cAMP in serum-free medium and into Schwannian cells on induction by bromodeoxyuridine. During the course of differentiation into neuronal and Schwannian cells, the intracellular cathepsin A activity further increased two and five times, respectively, which was associated with an increase in the expression of the 32/20 kDa two-chain form. The glycosylated precursor proteins were taken up via the mannose 6-phosphate receptors, and the cathepsin A, alpha-neuraminidase and beta-galactosidase (beta-Gal) activities deficient in the fibroblasts derived from a patient with PPCA deficiency (galactosialidosis) were restored. These results suggest that the bi-directional differentiation of GOTO cell lines stably expressing the recombinant human PPCA gene could be a model system for analyzing the functions of PPCA in peripheral neuronal cells and Schwannian cells as well as the recombinant PPCA could be a useful source for enzyme replacement therapy (ERT) for galactosialidosis patients.


Assuntos
Catepsina A/metabolismo , Diferenciação Celular , Neuroblastoma/metabolismo , Neurônios/citologia , Células de Schwann/citologia , Transformação Celular Neoplásica , Eletroforese em Gel de Poliacrilamida , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Lisossomos/enzimologia , Neuroblastoma/patologia , Proteínas Recombinantes/metabolismo
14.
Bioorg Med Chem Lett ; 12(16): 2069-72, 2002 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-12127506

RESUMO

Synthetic intermediates of alkaloid halichlorine with the azaspiro core structure have been found to induce apoptosis of cultured human cells including an acute monocytic leukemia cell line (THP-1) at micromolar concentrations. The novel biological activity of the intermediates was suggested to depend on the skeletal structure and silyloxymethyl functionality on the five-membered ring.


Assuntos
Alcaloides/síntese química , Alcaloides/farmacologia , Apoptose/efeitos dos fármacos , Compostos de Espiro/síntese química , Compostos de Espiro/farmacologia , Alcaloides/química , Animais , Caspase 3 , Caspase 7 , Caspases/metabolismo , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Humanos , Estrutura Molecular , Compostos de Espiro/química , Fatores de Tempo , Células Tumorais Cultivadas
15.
J Am Chem Soc ; 124(23): 6526-7, 2002 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-12047160

RESUMO

In this communication, a novel strategy for the design of a zinc finger peptide on the basis of alpha-helix substitution has been demonstrated. Sp1HM is a helix-substituted mutant for the wild-type Sp1(zf123) and its alpha-helix of each finger is replaced by that of fingers 4-6 of CF2-II. The circular dichroism spectrum of Sp1HM suggests that Sp1HM has an ordered secondary structure similar to that of Sp1(zf123). From the analyses of the DNA binding affinity and specificity by gel mobility shift assay, it is clearly indicated that Sp1HM specifically binds to the AT-rich sequence (5'-GTA TAT ATA-3') with 3.2 nM dissociation constants. Moreover, the zinc finger peptides for the sequence alternating between the AT- and GC-rich subsites can also be created by the alpha-helix substitution. This strategy is evidently effective and is also more convenient than the phage display method. Consequently, our design method is widely applicable to creating zinc finger peptides with novel binding specificities.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Drosophila , Fator de Transcrição Sp1/química , Fatores de Transcrição/química , Dedos de Zinco , Adenina/metabolismo , Sequência de Aminoácidos , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Cinética , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Fator de Transcrição Sp1/metabolismo , Especificidade por Substrato , Timidina/metabolismo , Fatores de Transcrição/metabolismo
16.
Nucleic Acids Res Suppl ; (2): 67-8, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12903108

RESUMO

A novel strategy for the design of a zinc finger peptide on the basis of alpha-helix substitution has been demonstrated. Sp1HM is a helix-substituted mutant for the wild-type Sp1(zf123) and its alpha-helix of each finger is replaced by that of fingers 4-6 of CF2-II. The circular dichroism spectrum of Sp1HM suggests that Sp1HM has an ordered secondary structure similar to Sp1(zf123). From the analyses of the DNA binding affinity and specificity by gel mobility shift assay, it is clearly indicated that Sp1HM specifically binds to the AT-rich sequence (5'-GTA TAT ATA-3') with 3 nM dissociation constants. Moreover, the zinc finger peptides for the sequence alternating between the AT- and GC-rich subsites can also be created by the alpha-helix substitution. This strategy is evidently effective and is also more convenient than the phage display method. Consequently, our design method is widely applicable to creating zinc finger peptides with novel binding specificities.


Assuntos
Adenina/química , Timina/química , Dedos de Zinco , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos
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