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1.
Methods Enzymol ; 526: 231-50, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23791104

RESUMO

Disulfide bond formation in the endoplasmic reticulum (ER) requires the sequential transfer of electrons from thiol residues to protein disulfide isomerase and ER oxidase 1, with the final reduction of molecular oxygen to form hydrogen peroxide. Conditions that perturb correct protein folding lead to accumulation of misfolded proteins in the ER lumen, which induce ER stress and oxidative stress. Oxidative damage of cellular macromolecules is a common marker of aging and various pathological conditions including diabetes, cancer, and neurodegenerative disease. As accumulating evidence suggests a tight connection between the ER stress and oxidative stress, analysis of appropriate markers becomes particularly important. Here, we describe methods to analyze markers of oxidative damage associated with ER stress.


Assuntos
Retículo Endoplasmático/metabolismo , Estresse Oxidativo , Resposta a Proteínas não Dobradas , Animais , Bioquímica/métodos , Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Peroxidação de Lipídeos , Mitocôndrias/metabolismo , Carbonilação Proteica
2.
J Biol Chem ; 287(43): 35825-37, 2012 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-22942278

RESUMO

Polyamines are small organic polycations that are absolutely required for cell growth and proliferation; yet the basis for this requirement is mostly unknown. Here, we combined a genome-wide expression profiling with biochemical analysis to reveal the molecular basis for inhibited proliferation of polyamine-depleted cells. Transcriptional responses accompanying growth arrest establishment in polyamine-depleted cells or growth resumption following polyamine replenishment were monitored and compared. Changes in the expression of genes related to various fundamental cellular processes were established. Analysis of mirror-symmetric expression patterns around the G(1)-arrest point identified a set of genes representing a stress-response signature. Indeed, complementary biochemical analysis demonstrated activation of the PKR-like endoplasmic reticulum kinase arm of the unfolded protein response and of the stress-induced p38 MAPK. These changes were accompanied by induction of key growth-inhibitory factors such as p21 and Gadd45a and reduced expression of various cyclins, most profoundly cyclin D1, setting the basis for the halted proliferation. However, although the induced stress response could arrest growth, polyamine depletion also inhibited proliferation of PKR-like endoplasmic reticulum kinase and p38α-deficient cells and of cells harboring a nonphosphorylatable mutant eIF2α (S51A), suggesting that additional yet unidentified mechanisms might inhibit proliferation of polyamine-depleted cells. Despite lengthy persistence of the stress and activation of apoptotic signaling, polyamine-depleted cells remained viable, apparently due to induced expression of protective genes and development of autophagy.


Assuntos
Poliaminas Biogênicas/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Regulação da Expressão Gênica/fisiologia , Estresse Fisiológico/fisiologia , Transcrição Gênica/fisiologia , Animais , Apoptose/fisiologia , Autofagia/fisiologia , Perfilação da Expressão Gênica , Camundongos , Células NIH 3T3 , Transdução de Sinais/fisiologia , Resposta a Proteínas não Dobradas/fisiologia
3.
J Biol Chem ; 285(17): 12474-81, 2010 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-20181941

RESUMO

Polyamines are essential cell constituents whose depletion results in growth cessation. Here we have investigated potential mechanisms of action of polyamines in supporting mammalian cell proliferation. We demonstrate that polyamines regulate translation both at the initiation and at the elongation steps. L-alpha-difluoromethylornithine treatment resulting in polyamine depletion reduces protein synthesis via inhibition of translation initiation. N1-guanyl-diaminoheptane (GC7), a spermidine analogue that inhibits eukaryotic initiation factor 5A (eIF5A) hypusination, also caused inhibition of translation initiation. In contrast, depletion of eIF5A by short hairpin RNA inhibits translation elongation as was recently demonstrated in yeast and Drosophila. These results suggest that in addition to competing with spermidine in the hypusination reaction, GC7 also competes with spermidine at yet undefined sites required for translation initiation. Finally, we show that either polyamine depletion or GC7 treatment induced eIF2alpha phosphorylation and reduced phosphorylation of 4E-BP, thus setting the molecular basis for the observed inhibition of translation initiation.


Assuntos
Proliferação de Células , Elongação Traducional da Cadeia Peptídica/fisiologia , Iniciação Traducional da Cadeia Peptídica/fisiologia , Espermidina/metabolismo , Animais , Drosophila/metabolismo , Eflornitina/farmacologia , Inibidores Enzimáticos/farmacologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Guanina/análogos & derivados , Guanina/farmacologia , Humanos , Camundongos , Células NIH 3T3 , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Espermidina/antagonistas & inibidores , Leveduras/metabolismo , Fator de Iniciação de Tradução Eucariótico 5A
4.
J Biol Chem ; 283(8): 4528-34, 2008 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-18089576

RESUMO

Mammalian antizyme (mAz) is a central element of a feedback circuit regulating cellular polyamines by accelerating ornithine decarboxylase (ODC) degradation and inhibiting polyamine uptake. Although yeast antizyme (yAz) stimulates the degradation of yeast ODC (yODC), we show here that it has only a minor effect on polyamine uptake by yeast cells. A segment of yODC that parallels the Az binding segment of mammalian ODC (mODC) is required for its binding to yAz. Although demonstrating minimal homology to mAz, our results suggest that yAz stimulates yODC degradation via a similar mechanism of action. We demonstrate that interaction with yAz provokes degradation of yODC by yeast but not by mammalian proteasomes. This differential recognition may serve as a tool for investigating proteasome functions.


Assuntos
Ornitina Descarboxilase/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Linhagem Celular , Humanos , Ornitina Descarboxilase/genética , Inibidores da Ornitina Descarboxilase , Poliaminas/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Proteínas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Especificidade da Espécie
5.
Glycobiology ; 18(1): 28-41, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18006589

RESUMO

NKp30 is a natural cytotoxicity receptor expressed by human NK cells and involved in NK lytic activity. We previously published that membranal heparan sulfate serves as a coligand for human NKp30. In the present study, we complement our results by showing direct binding of recombinant NKp30 to immobilized heparin. The heparan sulfate epitope(s) on target tumor cells and the heparin epitope(s) recognized by NKp30 share similar characteristics. Warren and colleagues (Warren HS, Jones AL, Freeman C, Bettadapura J, Parish CR. 2005. Evidence that the cellular ligand for the human NK cell activation receptor NKp30 is not a heparan sulfate glycosaminoglycan. J Immunol. 175:207-212) published that NKp30 does not bind to membranal heparan sulfate on target cells and that heparan sulfate is not involved in NKp30-mediated lysis. In the current study, we examine the binding of six different recombinant NKp30s to membranal heparan sulfate and conclude that NKp30 does interact with membranal heparan sulfate. Yet, two of the six recombinant NKp30s, including the commercially available recombinant NKp30 (employed by Warren et al.) did not show heparan sulfate-dependent binding. We demonstrate that this is due to an altered glycosylation of these two recombinant NKp30s. Upon removal of its N-linked glycans, heparan sulfate-dependent binding to tumor cells and direct binding to heparin were restored. Overall, our results emphasize the importance of proper glycosylation for analysis of NKp30 binding to its ligand and that membranal heparan sulfate could serve as a coligand for NKp30. At the cellular level, soluble heparan sulfate enhanced the secretion of IFNgamma by NK-92 natural killer cells activated with anti-NKp30 monoclonal antibody. We discuss the involvement of heparan sulfate binding to NKp30 in NKp30-mediated activation of NK cells.


Assuntos
Heparitina Sulfato/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Sítios de Ligação , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Glicosilação , Células HeLa , Humanos , Receptor 3 Desencadeador da Citotoxicidade Natural , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Polissacarídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
6.
Biochemistry ; 46(25): 7426-36, 2007 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-17536787

RESUMO

NKp44 is a natural cytotoxicity receptor expressed by human NK cells upon activation. In this study, we demonstrate that cell surface heparan sulfate proteoglycans (HSPGs), expressed by target cells, are involved in the recognition of tumor cells by NKp44. NKp44 showed heparan sulfate-dependent binding to tumor cells; this binding was partially blocked with an antibody to heparan sulfate. In addition, direct binding of NKp44 to heparin was observed, and soluble heparin/heparan sulfate enhanced the secretion of IFNgamma by NK92 cells activated with anti-NKp44 monoclonal antibody. Basic amino acids, predicted to constitute the putative heparin/heparan sulfate binding site of NKp44, were mutated. Tumor cell recognition of the mutated NKp44 proteins was significantly reduced and correlated with their lower recognition of heparin. We previously reported that NKp44 recognizes the hemagglutinin of influenza virus (IV). Nevertheless, the ability of the mutated NKp44 proteins to bind viral hemagglutinin expressed by IV-infected cells was not affected. Thus, we suggest that heparan sulfate epitope(s) are ligands/co-ligands of NKp44 and are involved in its tumor recognition ability.


Assuntos
Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Receptores Imunológicos/química , Receptores Imunológicos/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Sítios de Ligação , Células CHO , Carcinoma Ductal/patologia , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Epitopos , Células HeLa , Heparitina Sulfato/metabolismo , Humanos , Imunoglobulinas/genética , Interferon gama/metabolismo , Ligantes , Masculino , Melanoma/patologia , Dados de Sequência Molecular , Mutação , Receptor 2 Desencadeador da Citotoxicidade Natural , Neoplasias Pancreáticas/patologia , Neoplasias da Próstata/patologia , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Imunológicos/genética , Proteínas Recombinantes de Fusão/metabolismo
7.
Nat Immunol ; 7(5): 517-23, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16565719

RESUMO

The elimination of viruses and tumors by natural killer cells is mediated by specific natural killer cell receptors. To study the in vivo function of a principal activating natural killer cell receptor, NCR1 (NKp46 in humans), we replaced the gene encoding this receptor (Ncr1) with a green fluorescent protein reporter cassette. There was enhanced spread of certain tumors in 129/Sv but not C57BL/6 Ncr1(gfp/gfp) mice, and influenza virus infection was lethal in both 129/Sv and C57BL/6 Ncr1(gfp/gfp) mice. We noted accumulation of natural killer cells at the site of influenza infection by tracking the green fluorescent protein. Our results demonstrate a critical function for Ncr1 in the in vivo eradication of influenza virus.


Assuntos
Vírus da Influenza A/imunologia , Células Matadoras Naturais/imunologia , Infecções por Orthomyxoviridae/imunologia , Receptores Imunológicos/genética , Animais , Linhagem Celular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imunidade Inata/imunologia , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Knockout , Receptor 1 Desencadeador da Citotoxicidade Natural , Receptores Imunológicos/deficiência , Receptores Imunológicos/metabolismo , Especificidade da Espécie , Taxa de Sobrevida
8.
Biochemistry ; 44(44): 14477-85, 2005 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-16262248

RESUMO

NKp46 is a member of a group of receptors collectively termed natural cytotoxicity receptors (NCRs) that are expressed by natural killer (NK) cells. NCRs are capable of mediating direct killing of tumor and virus-infected cells by NK cells. We have recently shown that NKp46 recognizes the heparan sulfate moieties of membranal heparan sulfate proteoglycans (HSPGs), thus enabling lysis of tumor cells by NK cells. In the current study, we further examined the residues in NKp46 that may be involved in heparan sulfate binding on tumor cells. On the basis of both the electrostatic potential map and comparison to the heparin binding site on human fibronectin, we predicted a continuous region containing the basic amino acids K133, R136, H139, R142, and K146 to be involved in NKp46 binding to heparan sulfate. Mutating these amino acids on NKp46D2 to noncharged amino acids retained its virus binding capacity but reduced its binding to tumor cells with a 10-100 fold lower K(D) when tested for direct binding to heparin. The minimal length of the heparin/heparan sulfate epitope recognized by NKp46 was eight saccharides as predicted from the structure and proven by testing heparin oligomers. Testing selectively monodesulfated heparin oligomers emphasized the specific contributions of O-sulfation, N-sulfation, and N-acetylation to epitope recognition by NKp46. The characterization of heparan sulfate binding region in NKp46 offers further insight into the identity of the ligands for NKp46 and the interaction of NK and cancers.


Assuntos
Anticoagulantes/metabolismo , Heparitina Sulfato/metabolismo , Glicoproteínas de Membrana/química , Receptores Imunológicos/química , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Heparina , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Receptor 1 Desencadeador da Citotoxicidade Natural , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Alinhamento de Sequência
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