Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 5 de 5
Filtrar
Mais filtros











Base de dados
Intervalo de ano de publicação
1.
J Clin Invest ; 121(10): 4180-91, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21926462

RESUMO

Mast cell degranulation is important in the pathogenesis of anaphylaxis and allergic disorders. Many animal venoms contain components that can induce mast cell degranulation, and this has been thought to contribute to the pathology and mortality caused by envenomation. However, we recently reported evidence that mast cells can enhance the resistance of mice to the venoms of certain snakes and that mouse mast cell-derived carboxypeptidase A3 (CPA3) can contribute to this effect. Here, we investigated whether mast cells can enhance resistance to the venom of the Gila monster, a toxic component of that venom (helodermin), and the structurally similar mammalian peptide, vasoactive intestinal polypeptide (VIP). Using 2 types of mast cell-deficient mice, as well as mice selectively lacking CPA3 activity or the chymase mouse mast cell protease-4 (MCPT4), we found that mast cells and MCPT4, which can degrade helodermin, can enhance host resistance to the toxicity of Gila monster venom. Mast cells and MCPT4 also can limit the toxicity associated with high concentrations of VIP and can reduce the morbidity and mortality induced by venoms from 2 species of scorpions. Our findings support the notion that mast cells can enhance innate defense by degradation of diverse animal toxins and that release of MCPT4, in addition to CPA3, can contribute to this mast cell function.


Assuntos
Carboxipeptidases A/fisiologia , Mastócitos/enzimologia , Venenos de Escorpião/toxicidade , Serina Endopeptidases/fisiologia , Peptídeo Intestinal Vasoativo/toxicidade , Peçonhas/toxicidade , Sequência de Aminoácidos , Animais , Carboxipeptidases A/deficiência , Carboxipeptidases A/genética , Peptídeos e Proteínas de Sinalização Intercelular , Lagartos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Dados de Sequência Molecular , Peptídeos/antagonistas & inibidores , Peptídeos/genética , Peptídeos/toxicidade , Venenos de Escorpião/antagonistas & inibidores , Serina Endopeptidases/deficiência , Serina Endopeptidases/genética , Peptídeo Intestinal Vasoativo/antagonistas & inibidores , Peptídeo Intestinal Vasoativo/genética
2.
Nat Chem Biol ; 5(7): 469-78, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19465933

RESUMO

MARTX toxins modulate the virulence of a number of Gram-negative Vibrio species. This family of toxins is defined by the presence of a cysteine protease domain (CPD), which proteolytically activates the Vibrio cholerae MARTX toxin. Although recent structural studies of the CPD have uncovered a new allosteric activation mechanism, the mechanism of CPD substrate recognition or toxin processing is unknown. Here we show that interdomain cleavage of MARTXVc enhances effector domain function. We also identify the first small-molecule inhibitors of this protease domain and present the 2.35-A structure of the CPD bound to one of these inhibitors. This structure, coupled with biochemical and mutational studies of the toxin, reveals the molecular basis of CPD substrate specificity and underscores the evolutionary relationship between the CPD and the clan CD caspase proteases. These studies are likely to prove valuable for devising new antitoxin strategies for a number of bacterial pathogens.


Assuntos
Toxina da Cólera/química , Toxina da Cólera/metabolismo , Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Vibrio cholerae/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Western Blotting , Domínio Catalítico , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Alinhamento de Sequência , Espectroscopia de Infravermelho com Transformada de Fourier , Especificidade por Substrato , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/enzimologia
3.
J Proteome Res ; 4(6): 2412-9, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16335995

RESUMO

A double-vented serial tetraphasic capillary column approach is applied to proteomic MuDPIT-type analysis using extended length capillary reverse-phase columns. The heart of the tetraphasic device consists of a triphasic MuDPIT trap located upstream of a venting tee. The trap is followed by a 60 cm high-resolution capillary column. A conventional high-flow HPLC is used to develop gradients at standard flow rates and pressures. The double-vented triphasic MuDPIT trapping device relieves the capillary separation column from the salt burden during the on-line cation-exchange portion of the analysis. Two configurations are presented, a double-vented continuous column model and a discontinuous model in which the triphasic MuDPIT trap is installed on a six-port valve; both configurations were tested with 60 and 10 cm capillary columns. All four systems were challenged with a trypsin digest of undepleted human serum, and a matrix of proteomic results for the different models and column lengths are compared.


Assuntos
Proteômica/instrumentação , Proteômica/métodos , Proteínas Sanguíneas/química , Cátions , Cromatografia , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Eletroforese Capilar , Humanos , Espectrometria de Massas , Nanotecnologia , Peptídeos/química , Proteoma , Fatores de Tempo , Tripsina/farmacologia
4.
J Biol Chem ; 278(12): 10189-94, 2003 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-12524439

RESUMO

Akt (also called protein kinase B) is one of the major downstream targets of the phosphatidylinositol 3-kinase pathway. This protein kinase has been implicated in insulin signaling, stimulation of cellular growth, and inhibition of apoptosis as well as transformation of cells. Although a number of cellular proteins have been identified as putative targets of the enzyme, additional substrates may play a role in the varied responses elicited by this enzyme. We have used a combination of 14-3-3 binding and recognition by an antibody to the phosphorylation consensus of the enzyme to identify and isolate one of the major substrates of Akt, which is also a 14-3-3 binding protein. This 40-kDa protein, designated PRAS40, is a proline-rich Akt substrate. Demonstration that it is a substrate of Akt was accomplished by showing that 1) PRAS40 was phosphorylated in vitro by purified Akt on the same site that was phosphorylated in insulin-treated cells; 2) activation of an inducible Akt was alone sufficient to stimulate the phosphorylation of PRAS40; and 3) cells lacking Akt1 and Akt2 exhibit a diminished ability to phosphorylate this protein. Thus, PRAS40 is a novel substrate of Akt, the phosphorylation of which leads to the binding of this protein to 14-3-3.


Assuntos
Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Proteínas 14-3-3 , Sequência de Aminoácidos , Animais , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Linhagem Celular , Insulina/farmacologia , Camundongos , Dados de Sequência Molecular , Peso Molecular , Fosforilação , Prolina , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/análise , Treonina/metabolismo
5.
Rapid Commun Mass Spectrom ; 16(21): 2067-72, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12391582

RESUMO

A 60 microm internal diameter (i.d.) stainless-steel needle was adapted to the orthogonal ESI probe ( microESI) of a commercial ion trap mass spectrometer, and used for capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS) protein identification experiments. The modification allows for the use of nitrogen sheath gas which helps in the nebulization at LC flow rates exceeding 500 nL/min and eliminates problems caused by liquid junctions commonly used to initiate nanospray ionization (NSI). A comparison is made between the performance of a 75 microm i.d. column with a 15 microm pulled glass tip using a liquid junction, and that of a 150 microm i.d. column using the new microESI device. The combination of the 150 microm i.d. column and microESI gave sensitivity close to that of NSI (250 attomoles horse heart myoglobin digest on column), and proved to be more robust than the standard pulled glass tips of similar i.d. No evidence of metal needle catalyzed oxidation of methionine was observed during analysis of the tetrapeptide MRFA under a range of test conditions. Phosphorylated peptides in a beta-casein tryptic digest were also successfully identified using the microESI interface with a steel needle. In addition it was found that a mild sanding of the metal needle tip improved spray performance.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Nanotecnologia/métodos , Proteômica , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Cavalos , Metionina/química , Mioglobina/análise , Oxirredução , Peptídeos/análise , Peptídeos/química , Fosforilação , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização por Electrospray/instrumentação
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA