RESUMEN
ß-barrel pore-forming toxins perforate cell membranes by forming oligomeric ß-barrel pores. The most crucial step is the membrane-insertion of the pore-forming motifs that create the transmembrane ß-barrel scaffold. Molecular mechanism that regulates structural reorganization of these pore-forming motifs during ß-barrel pore-formation still remains elusive. Using Vibrio cholerae cytolysin as an archetypical example of the ß-barrel pore-forming toxin, we show that a key tyrosine residue (Y321) in the hinge region of the pore-forming motif plays crucial role in this process. Mutation of Y321 abrogates oligomerization of the membrane-bound toxin protomers, and blocks subsequent steps of pore-formation. Our study suggests that the presence of Y321 in the hinge region of the pore-forming motif is crucial for the toxin molecule to sense membrane-binding, and to trigger essential structural rearrangements required for the subsequent oligomerization and pore-formation process. Such a regulatory mechanism of pore-formation by V. cholerae cytolysin has not been documented earlier in the structurally related ß-barrel pore-forming toxins.
Asunto(s)
Secuencias de Aminoácidos , Perforina/química , Perforina/fisiología , Tirosina/química , Vibrio cholerae/química , Vibrio cholerae/fisiología , Proteínas Bacterianas/química , Proteínas Bacterianas/fisiología , Línea Celular , Membrana Celular/metabolismo , Células Cultivadas , Citotoxinas/química , Citotoxinas/fisiología , Humanos , Microscopía Electrónica de Transmisión , Simulación de Dinámica Molecular , Mutación , Perforina/ultraestructura , Conformación Proteica , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vibrio cholerae/ultraestructuraRESUMEN
Exercise and caloric restriction improve health, including reducing risk of cardiovascular disease, neurological disease, and cancer. However, molecular mechanisms underlying these protections are poorly understood, partly due to the cost and time investment of mammalian long-term diet and exercise intervention studies. We subjected Caenorhabditis elegans nematodes to a 6-day, twice daily swimming exercise regimen, during which time the animals also experienced brief, transient food deprivation. Accordingly, we included a non-exercise group with the same transient food deprivation, a non-exercise control with ad libitum access to food, and a group that exercised in food-containing medium. Following these regimens, we assessed mitochondrial health and sensitivity to mitochondrial toxicants. Exercise protected against age-related decline in mitochondrial morphology in body-wall muscle. Food deprivation increased organismal basal respiration; however, exercise was the sole intervention that increased spare respiratory capacity and proton leak. We observed increased lifespan in exercised animals compared to both control and transiently food-deprived nematodes. Finally, exercised animals (and to a lesser extent, transiently food-deprived animals) were markedly protected against lethality from acute exposures to the mitotoxicants rotenone and arsenic. Thus, swimming exercise and brief food deprivation provide effective intervention in C. elegans, protecting from age-associated mitochondrial decline and providing resistance to mitotoxicant exposures.
Asunto(s)
Privación de Alimentos/fisiología , Mitocondrias/fisiología , Condicionamiento Físico Animal/fisiología , Animales , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Restricción Calórica/métodos , Citotoxinas/fisiología , Mitocondrias/efectos de los fármacos , Natación/fisiologíaRESUMEN
The Gram-negative, intracellular bacterium Chlamydia trachomatis causes acute and chronic urogenital tract infection, potentially leading to infertility and ectopic pregnancy. The only partially characterized cytotoxin CT166 of serovar D exhibits a DXD motif, which is important for the enzymatic activity of many bacterial and mammalian type A glycosyltransferases, leading to the hypothesis that CT166 possess glycosyltransferase activity. CT166-expressing HeLa cells exhibit actin reorganization, including cell rounding, which has been attributed to the inhibition of the Rho-GTPases Rac/Cdc42. Exploiting the glycosylation-sensitive Ras(27H5) antibody, we here show that CT166 induces an epitope change in Ras, resulting in inhibited ERK and PI3K signaling and delayed cell cycle progression. Consistent with the hypothesis that these effects strictly depend on the DXD motif, CT166 with the mutated DXD motif causes neither Ras-ERK inhibition nor delayed cell cycle progression. In contrast, CT166 with the mutated DXD motif is still capable of inhibiting cell migration, suggesting that CT166 with the mutated DXD motif cannot be regarded as inactive in any case. Taken together, CT166 affects various fundamental cellular processes, strongly suggesting its importance for the intracellular survival of chlamydia.
Asunto(s)
Chlamydia trachomatis/enzimología , Citotoxinas/fisiología , Glucosiltransferasas/fisiología , Secuencias de Aminoácidos , Ciclo Celular/genética , Movimiento Celular/genética , Proliferación Celular , Chlamydia trachomatis/crecimiento & desarrollo , Citotoxinas/genética , Glucosiltransferasas/genética , Células HeLa , Humanos , Sistema de Señalización de MAP Quinasas/genética , Mutación , Transfección , Proteínas ras/antagonistas & inhibidoresRESUMEN
Cholesterol-dependent cytolysins (CDCs) constitute a family of pore forming toxins secreted by Gram-positive bacteria. These toxins form transmembrane pores by inserting a large ß-barrel into cholesterol-containing membrane bilayers. Binding of water-soluble CDCs to the membrane triggers the formation of oligomers containing 35-50 monomers. The coordinated insertion of more than seventy ß-hairpins into the membrane requires multiple structural conformational changes. Perfringolysin O (PFO), secreted by Clostridium perfringens, has become the prototype for the CDCs. In this chapter, we will describe current knowledge on the mechanism of PFO cytolysis, with special focus on cholesterol recognition, oligomerization, and the conformational changes involved in pore formation.
Asunto(s)
Toxinas Bacterianas/química , Membrana Celular/química , Citotoxinas , Proteínas Hemolisinas/química , Proteínas Hemolisinas/fisiología , Secuencia de Aminoácidos , Animales , Colesterol/química , Colesterol/metabolismo , Citotoxinas/química , Citotoxinas/fisiología , Humanos , Datos de Secuencia Molecular , Mutación , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
The cholesterol-dependent cytolysins (CDCs) are a large family of pore-forming toxins that are produced by numerous Gram-positive bacterial pathogens. These toxins are released in the extracellular environment as water-soluble monomers or dimers that bind to cholesterol-rich membranes and assemble into large pore complexes. Depending upon their concentration, the nature of the host cell and membrane (cytoplasmic or intracellular) they target, the CDCs can elicit many different cellular responses. Among the CDCs, listeriolysin O (LLO), which is a major virulence factor of the facultative intracellular pathogen Listeria monocytogenes, is involved in several stages of the intracellular lifecycle of the bacterium and displays unique characteristics. It has long been known that following L. monocytogenes internalization into host cells, LLO disrupts the internalization vacuole, enabling the bacterium to replicate into the host cell cytosol. LLO is then used by cytosolic bacteria to spread from cell to cell, avoiding bacterial exposure to the extracellular environment. Although LLO is continuously produced during the intracellular lifecycle of L. monocytogenes, several processes limit its toxicity to ensure the survival of infected cells. It was previously thought that LLO activity was limited to mediating vacuolar escape during bacterial entry and cell to cell spreading. This concept has been challenged by compelling evidence suggesting that LLO secreted by extracellular L. monocytogenes perforates the host cell plasma membrane, triggering important host cell responses. This chapter provides an overview of the well-established intracellular activity of LLO and the multiple roles attributed to LLO secreted by extracellular L. monocytogenes.
Asunto(s)
Proteínas de Choque Térmico/fisiología , Proteínas Hemolisinas/fisiología , Listeria monocytogenes/patogenicidad , Animales , Toxinas Bacterianas/química , Colesterol/metabolismo , Citotoxinas/química , Citotoxinas/metabolismo , Citotoxinas/fisiología , Regulación Bacteriana de la Expresión Génica , Proteínas de Choque Térmico/química , Proteínas Hemolisinas/química , Humanos , Listeria monocytogenes/genética , Modelos Moleculares , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/fisiología , Factores de Virulencia/química , Factores de Virulencia/fisiologíaRESUMEN
The opportunistic fungus Candida albicans causes oral thrush and vaginal candidiasis, as well as candidaemia in immunocompromised patients including those undergoing cancer chemotherapy, organ transplant and those with AIDS. We previously found that the AMPs (antimicrobial peptides) LL37 and hBD-3 (human ß-defensin-3) inhibited C. albicans viability and its adhesion to plastic. For the present study, the mechanism by which LL37 and hBD-3 reduced C. albicans adhesion was investigated. After AMP treatment, C. albicans adhesion to plastic was reduced by up to ~60% and was dose-dependent. Our previous study indicated that LL37 might interact with the cell-wall ß-1,3-exoglucanase Xog1p, which is involved in cell-wall ß-glucan metabolism, and consequently the binding of LL37 or hBD-3 to Xog1p might cause the decrease in adhesion. For the present study, Xog1p(41-438)-6H, an N-terminally truncated, active, recombinant construct of Xog1p and Xog1p fragments were produced and used in pull-down assays and ELISA in vitro, which demonstrated that all constructs interacted with both AMPs. Enzymatic analyses showed that LL37 and hBD-3 enhanced the ß-1,3-exoglucanase activity of Xog1p(41-438)-6H approximately 2-fold. Therefore elevated Xog1p activity might compromise cell-wall integrity and decrease C. albicans adhesion. To test this hypothesis, C. albicans was treated with 1.3 µM Xog1p(41-438)-6H and C. albicans adhesion to plastic decreased 47.7%. Taken together, the evidence suggests that Xog1p is one of the LL37/hBD-3 targets, and elevated ß-1,3-exoglucanase activity reduces C. albicans adhesion to plastic.
Asunto(s)
Candida albicans/fisiología , Catelicidinas/fisiología , Proteínas Fúngicas/metabolismo , Glucano 1,3-beta-Glucosidasa/metabolismo , beta-Defensinas/fisiología , Péptidos Catiónicos Antimicrobianos , Candida albicans/genética , Candida albicans/crecimiento & desarrollo , Candida albicans/metabolismo , Catelicidinas/genética , Catelicidinas/metabolismo , Catelicidinas/farmacología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Pared Celular/efectos de los fármacos , Pared Celular/genética , Pared Celular/metabolismo , Citotoxinas/genética , Citotoxinas/metabolismo , Citotoxinas/farmacología , Citotoxinas/fisiología , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Evaluación Preclínica de Medicamentos , Proteínas Fúngicas/genética , Proteínas Fúngicas/farmacología , Proteínas Fúngicas/fisiología , Glucano 1,3-beta-Glucosidasa/genética , Glucano 1,3-beta-Glucosidasa/farmacología , Glucano 1,3-beta-Glucosidasa/fisiología , Humanos , Pruebas de Sensibilidad Microbiana , Organismos Modificados Genéticamente , Plásticos , Unión Proteica/genética , beta-Defensinas/genética , beta-Defensinas/metabolismo , beta-Defensinas/farmacologíaRESUMEN
BACKGROUND/AIMS: Impaired mitochondrial function has been described in Alzheimer's disease. We previously reported that, in neuronal cells, ß-amyloid 1-42 (Aß(1-42)) is targeted to mitochondria. We have also reported that, when incubated with isolated rat brain mitochondria, Aß(1-42) inhibits complex IV, uncouples the mitochondrial respiratory chain, and promotes opening of the membrane permeability transition pore. Here, we further analyzed the targeting and mitotoxicity of Aß(1-42). METHODS AND RESULTS: Immunoelectron microscopy revealed that the mitochondrial targeting of Aß(1-42) was concentration- and time-dependent. Incubation of human neuroblastoma cells with Aß(1-42) increased the release of adenylate kinase, a mitochondrial enzyme released after membrane permeability transition pore opening. However, it failed to trigger DNA fragmentation and apoptosis, suggesting that the ability of this peptide to uncouple the respiratory chain underlies its mitotoxicity and cytotoxicity. Aß(1-42) targeting to mitochondria was blocked by caprospinol, a steroid derivative shown to protect neuronal cells against Aß(1-42)-induced neurotoxicity. Further experiments revealed that the mitotoxic effect of Aß(1-42) is specific to its primary amino acid sequence and suggested that it may be also related to its tertiary structure. Importantly, the mitotoxic effect of Aß(1-42) was not restricted to brain cells, indicating that it is not cell- or tissue-specific. CONCLUSION: Taken together, these results suggest that extracellular Aß(1-42) targets neuronal mitochondria to exert its toxic effects.
Asunto(s)
Péptidos beta-Amiloides/envenenamiento , Citotoxinas/envenenamiento , Sistemas de Liberación de Medicamentos/métodos , Mitocondrias/patología , Neuronas/patología , Fragmentos de Péptidos/envenenamiento , Péptidos beta-Amiloides/administración & dosificación , Péptidos beta-Amiloides/fisiología , Línea Celular Tumoral , Citotoxinas/administración & dosificación , Citotoxinas/fisiología , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Espacio Extracelular/fisiología , Células HEK293 , Células Hep G2 , Humanos , Mitocondrias/efectos de los fármacos , Neuronas/efectos de los fármacos , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/fisiologíaRESUMEN
Melia azedarach (cinnamon) and Azadirachta indica (neem) have a variety of biologically active ingredients against virus, bacteria and protozoan parasites; however, little is known about their action on Toxoplasma gondii intracellular development. Toxoplasma gondii infects all eukaryotic cells, where it establishes and multiplies inside a modified vacuole called the parasitophorous vacuole until the cell ruptures, re-infecting other cells and establishing the infection. There are no efficient chemotherapies for the elimination of T. gondii, minimizing side effects. In this study, we performed in vitro assays with neem and cinnamon aqueous extracts against the intracellular development of T. gondii tachyzoites. After treatment with neem and cinnamon for 24 h, the percentage of infected cells and the number of intracellular parasites drastically decreased. This effect was concentration-dependent. During the incubation of the extracts, progressive morphological and ultrastructure alterations led to intense vesiculation and complete elimination of the parasite from the intracellular medium. However, during the treatment with extracts, no morphological effects were observed in the structure of the host cell. These results suggest that the aqueous extracts of neem and cinnamon were capable of interfering with and eliminating the intracellular development of Toxoplasma gondii.
Melia azedarach (canela) e Azadirachta indica (nim) apresenta grande variedade de ingredientes biologicamente ativos contra vírus, bactérias e protozoários, mas nenhum efeito sobre o desenvolvimento intracelular do Toxoplasma gondii é conhecido. Toxoplasma gondii infecta todos os tipos de células Eucarióticas, onde se estabelece no meio intracelular em vacúolo modificado conhecido como vacúolo parasitóforo. Neste vacúolo ocorre a replicação levando a ruptura da célula hospedeira e reinfecção de novas células, perpetuando a infecção. A quimioterapia utilizada não é capaz de eliminar o parasita além de induzir fortes efeitos colaterais. Neste estudo, nós demonstramos o efeito in vitro de extratos aquosos da canela e nim sobre o desenvolvimento intracelular do taquizoíto do Toxoplasma gondii. Após tratamento de nim e canela por 24 h, a porcentagem de infecção e o número de taquizoítos intracelulares decaiu drasticamente. Este efeito foi concentração-dependente. Durante incubação dos extratos, uma progressiva desorganização morfológica e ultraestrutural levaram a formação de intensa vesiculação e completa destruição do parasita, que passou a uma estrutura amorfa, antes da completa eliminação do meio intracelular. No entanto durante o tratamento com os extratos, efeitos morfológicos não foram observados nas estruturas da célula hospedeira. Estes resultados sugerem que os extratos aquosos de nim e canela foram capazes de interferir e eliminar o desenvolvimento intracelular do Toxoplasma gondii.
Asunto(s)
Azadirachta/análisis , Espacio Intracelular/parasitología , Extractos Vegetales/química , Hojas de la Planta/parasitología , Técnicas In Vitro , Toxoplasma/parasitología , Azadirachta/parasitología , Cinnamomum zeylanicum/parasitología , Citotoxinas/fisiología , Citotoxinas/químicaRESUMEN
Cholesterol is a major constituent of the plasma membrane in eukaryotic cells. It regulates the physical state of the phospholipid bilayer and is crucially involved in the formation of membrane microdomains. Cholesterol also affects the activity of several membrane proteins, and is the precursor for steroid hormones and bile acids. Here, methods are described that are used to explore the binding and/or interaction of proteins to cholesterol. For this purpose, a variety of cholesterol probes bearing radio-, spin-, photoaffinity- or fluorescent labels are currently available. Examples of proven cholesterol binding molecules are polyene compounds, cholesterol-dependent cytolysins, enzymes accepting cholesterol as substrate, and proteins with cholesterol binding motifs. Main topics of this report are the localization of candidate membrane proteins in cholesterol-rich microdomains, the issue of specificity of cholesterol- protein interactions, and applications of the various cholesterol probes for these studies.
Asunto(s)
Membrana Celular/metabolismo , Colesterol/metabolismo , Microdominios de Membrana/fisiología , Proteínas de la Membrana/metabolismo , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , Marcadores de Afinidad , Animales , Proteínas Portadoras , Colesterol/análogos & derivados , Colesterol 24-Hidroxilasa , Ciclodextrinas/farmacología , Citotoxinas/fisiología , Filipina/farmacología , Humanos , Fluidez de la Membrana , Elastasa Pancreática , Marcadores de Spin , Esteroide Hidroxilasas/metabolismo , Esterol O-Aciltransferasa/metabolismo , Sulfotransferasas/metabolismoRESUMEN
Successful host defense against bacteria such as Staphylococcus aureus (SA) depends on a prompt response by circulating polymorphonuclear leukocytes (PMN). Stimulated PMN create in their phagosomes an environment inhospitable to most ingested bacteria. Granules that fuse with the phagosome deliver an array of catalytic and noncatalytic antimicrobial peptides, while activation of the NADPH oxidase at the phagosomal membrane generates reactive oxygen species within the phagosome, including hypochlorous acid (HOCl), formed by the oxidation of chloride by the granule protein myeloperoxidase in the presence of H(2)O(2). In this study, we used SA-expressing cytosolic GFP to provide a novel probe of the fate of SA in human PMN. PMN bleaching of GFP in SA required phagocytosis, active myeloperoxidase, H(2)O(2) from the NADPH oxidase, and chloride. Not all ingested SA were bleached, and the number of cocci within PMN-retaining fluorescent GFP closely correlated with the number of viable bacteria remaining intracellularly. The percent of intracellular fluorescent and viable SA increased at higher multiplicity of infection and when SA presented to PMN had been harvested from the stationary phase of growth. These studies demonstrate that the loss of GFP fluorescence in ingested SA provides a sensitive experimental probe for monitoring biochemical events within individual phagosomes and for identifying subpopulations of SA that resist intracellular PMN cytotoxicity. Defining the molecular basis of SA survival within PMN should provide important insights into bacterial and host properties that limit PMN antistaphylococcal action and thus contribute to the pathogenesis of staphylococcal infection.
Asunto(s)
Proteínas Fluorescentes Verdes/fisiología , Neutrófilos/efectos de los fármacos , Neutrófilos/microbiología , Fagocitosis/inmunología , Fagosomas/microbiología , Staphylococcus aureus/crecimiento & desarrollo , Cloruros/farmacología , Citotoxinas/fisiología , Proteínas Fluorescentes Verdes/antagonistas & inhibidores , Proteínas Fluorescentes Verdes/biosíntesis , Humanos , Peróxido de Hidrógeno/farmacología , Ácido Hipocloroso/farmacología , Neutrófilos/inmunología , Peroxidasa/deficiencia , Peroxidasa/genética , Peroxidasa/farmacología , Fagocitosis/efectos de los fármacos , Fagosomas/efectos de los fármacos , Fagosomas/inmunología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/inmunologíaRESUMEN
Dopamine-induced neuronal cytotoxicity has been proposed as a leading pathological mechanism underlying many neuronal degenerative disorders including Parkinson disease. Various hypotheses have been proposed including oxidative stress and dopamine (DA)-induced intracellular signal disorder via DA D1 and D2 receptors. The exact mechanism involved in this process is far from clear. In this study, employing a neuronal blastoma cell line, SH-SY5Y, we tried to elucidate the roles of these different suggested mechanisms in this pathological process. The results showed that DA induced cell toxicity in a dose- and time-dependent way. Selective D1 and D2 DA receptor antagonist could not block the cytotoxic effects, whereas reductive reagent ascorbic acid but not GSH could effectively rescue the cell death, suggesting that DA-induced cell toxicity was caused by an extracellular oxidative stress. This was further supported by the enhancing effects of DA transporter blocker, GBR, which could increase the cell death when pretreated. Finally, ascorbic acid could also protect SY5Y cells from DA-induced cellular apoptotic signal changes including PARP and P53. Our studies suggested that DA exerted its cytotoxic effects via an extracellular metabolism, whereas intracellular transportation could reduce its oxidative stress. Cytotoxicity effects induced by extracellular DA could be protected by reductive agents as ascorbic acid. These results help to broaden our understanding of the mechanisms of DA-induced cell death and may provide potentially therapeutical alternative for the neurodegenerative disorders.
Asunto(s)
Citotoxinas/fisiología , Dopamina/toxicidad , Espacio Extracelular/fisiología , Estrés Oxidativo/fisiología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Ácido Ascórbico/fisiología , Transporte Biológico Activo/efectos de los fármacos , Transporte Biológico Activo/fisiología , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Línea Celular Tumoral , Citotoxinas/antagonistas & inhibidores , Ácido Deshidroascórbico/metabolismo , Antagonistas de Dopamina/metabolismo , Relación Dosis-Respuesta a Droga , Espacio Extracelular/efectos de los fármacos , Glutatión/fisiología , Humanos , Neuroblastoma/metabolismo , Neuroblastoma/patología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factores de TiempoRESUMEN
Type I IFN (IFN-I) signaling is detrimental to cells and mice infected with Listeria monocytogenes. In this study, we investigate the impact of IFN-I on the activity of listeriolysin O (LLO), a pore-forming toxin and virulence protein released by L. monocytogenes. Treatment of macrophages with IFN-beta increased the ability of sublytic LLO concentrations to cause transient permeability of the plasma membrane. At higher LLO concentrations, IFN-beta enhanced the complete breakdown of membrane integrity and cell death. This activity of IFN-beta required Stat1. Perturbation of the plasma membrane by LLO resulted in activation of the p38MAPK pathway. IFN-beta pretreatment enhanced LLO-mediated signaling through this pathway, consistent with its ability to increase membrane damage. p38MAPK activation in response to LLO was independent of TLR4, a putative LLO receptor, and inhibition of p38MAPK neither enhanced nor prevented LLO-induced death. IFN-beta caused cells to express increased amounts of caspase 1 and to produce a detectable caspase 1 cleavage product after LLO treatment. Contrasting recent reports with another pore-forming toxin, this pathway did not aid cell survival as caspase 1-deficient cells were equally sensitive to lysis by LLO. Key lipogenesis enzymes were suppressed in IFN-beta-treated cells, which may exacerbate the membrane damage caused by LLO.
Asunto(s)
Permeabilidad de la Membrana Celular/inmunología , Proteínas de Choque Térmico/fisiología , Proteínas Hemolisinas/fisiología , Interferón beta/fisiología , Macrófagos/citología , Macrófagos/inmunología , Regulación hacia Arriba/inmunología , Adyuvantes Inmunológicos/fisiología , Adyuvantes Inmunológicos/toxicidad , Animales , Toxinas Bacterianas/toxicidad , Muerte Celular/inmunología , Células Cultivadas , Citotoxinas/fisiología , Proteínas de Choque Térmico/toxicidad , Proteínas Hemolisinas/toxicidad , Interferón beta/toxicidad , Células L , Listeria monocytogenes/crecimiento & desarrollo , Listeria monocytogenes/inmunología , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/inmunologíaRESUMEN
The tumor suppressor p14ARF is widely deregulated in many types of cancers and is believed to function as a failsafe mechanism, inhibiting proliferation and inducing apoptosis as cellular response to a high oncogene load. We have found that a 22-amino-acid-long peptide derived from the N-terminal part of p14ARF, denoted ARF(1-22), which has previously been shown to mimic the function of p14ARF, has cell-penetrating properties. This peptide is internalized to the same extent as the cell-penetrating peptide (CPP) TP10 and dose-dependently decreases proliferation in MCF-7 and MDA MB 231 cells. Uptake of the ARF(1-22) peptide is associated with low membrane disturbance, measured by deoxyglucose and lactate dehydrogenase (LDH) leakage, as compared to its scrambled peptide. Also, flow cytometric analysis of annexin V/propidium iodide (PI) binding and Hoechst staining of nuclei suggest that ARF(1-22) induces apoptosis, whereas scrambled or inverted peptide sequences have no effect. The ARF(1-22) peptide mainly translocates cells through endocytosis, and is found intact inside cells for at least 3 hours. To our knowledge, this is the first time a CPP having pro-apoptopic activity has been designed from a protein.
Asunto(s)
Permeabilidad de la Membrana Celular , Citotoxinas/síntesis química , Citotoxinas/fisiología , Péptidos/síntesis química , Péptidos/fisiología , Proteína p14ARF Supresora de Tumor/química , Secuencia de Aminoácidos , Apoptosis/genética , Línea Celular Tumoral , Permeabilidad de la Membrana Celular/genética , Proliferación Celular , Citotoxinas/genética , Células HeLa , Humanos , Imitación Molecular/genética , Datos de Secuencia Molecular , Ácidos Nucleicos de Péptidos/síntesis química , Ácidos Nucleicos de Péptidos/genética , Ácidos Nucleicos de Péptidos/fisiología , Péptidos/genética , Proteína p14ARF Supresora de Tumor/genética , Proteína p14ARF Supresora de Tumor/metabolismoRESUMEN
AIMS: To understand the mechanism(s) of alcoholic pancreatitis and role of fatty acid ethyl esters (FAEEs, non-oxidative metabolites of ethanol) in ethanol-induced pancreatic injury. METHODS: A time- and concentration-dependent synthesis of FAEEs and the cytotoxicity of ethanol and its predominant fatty acid esters were studied in rat pancreatic tumour (AR42J) cells in cultures. Role of FAEEs in ethanol-induced cytotoxicity was investigated by measuring the synthesis of FAEEs, injury markers and apoptosis in cells incubated simultaneously with ethanol and FAEE synthase inhibitor, 3-benzyl-6-chloro-2-pyrone. The cells were pre-incubated with caspase-3 inhibitor (N-acetyl-DEVD-CHO) to measure the effect of caspase-3 inhibition on ethanol-induced apoptosis. RESULTS: The levels of FAEEs synthesized in cell cultures incubated with 800 mg% ethanol for 6 h were approximately 10-fold higher (60 nmol/25 x 10(6) cells) than those in cells incubated with 100 mg% ethanol (5.4 nmol/25 x 10(6) cells). Ethanol exposure resulted in a concentration-dependent apoptosis (10, 12 and 13% at 200, 400 and 800 mg% ethanol, respectively, vs 5% in controls). A similar concentration-dependent apoptosis was also found in the cells incubated with ethyl oleate (one of the predominant FAEEs reported in alcoholic patients). Inhibition of FAEE synthesis and resultant apoptosis was found in the cells incubated simultaneously with pancreatic FAEE synthase inhibitor and ethanol. Ethanol-induced apoptosis was significantly inhibited in cells pre-incubated with caspase-3 inhibitor. CONCLUSIONS: These results support our hypothesis that ethanol-induced cytotoxicity in AR42J cells is mediated by the non-oxidative metabolite(s) of ethanol, and caspase-3 mediated apoptosis could be one of the mechanisms involved in ethanol-induced pancreatic injury.
Asunto(s)
Citotoxinas/toxicidad , Etanol/toxicidad , Ácidos Grasos/toxicidad , Páncreas Exocrino/efectos de los fármacos , Páncreas Exocrino/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Línea Celular Tumoral , Citotoxinas/biosíntesis , Citotoxinas/fisiología , Relación Dosis-Respuesta a Droga , Ésteres , Ácidos Grasos/biosíntesis , Ácidos Grasos/fisiología , Humanos , Páncreas Exocrino/citología , RatasRESUMEN
The data of pathogenicity factors of opportunistic enterobacteria, including Klebsiella, Enterobacter, Citrobacter, Proteus, Providencia and Hafnia species are submitted. The genetic control and a role of pathogenicity factors of opportunistic enterobacteria in development of diarrhea syndrome are presented. Data about adhesins, hemolysins, cytotoxic necrotizing factors and bacterial modulins are described. The characteristic of cytotonic and cytotoxic enterotoxins, including LT, ST, Shiga-like and cytolethal toxins, and mechanisms of diarrheagenic action are analysed. The role of bacterial lipopolysaccharide (endotoxin) and induction of locally synthesized proinflammatory cytokins in pathogenisis of diarrhea are discussed.
Asunto(s)
Diarrea/microbiología , Infecciones por Enterobacteriaceae/microbiología , Enterobacteriaceae/patogenicidad , Factores de Virulencia/fisiología , Adhesinas Bacterianas/genética , Proteínas Bacterianas/fisiología , Citotoxinas/fisiología , Endotoxinas/fisiología , Enterobacteriaceae/metabolismo , Enterotoxinas/fisiología , Proteínas Hemolisinas/fisiología , HumanosRESUMEN
How the HIV1 Vpr protein initiates the host cell response leading to cell cycle arrest in G(2) has remained unknown. Here, we show that recruitment of DCAF1/VprBP by Vpr is essential for its cytostatic activity, which can be abolished either by single mutations of Vpr that impair DCAF1 binding, or by siRNA-mediated silencing of DCAF1. Furthermore, DCAF1 bridges Vpr to DDB1, a core subunit of Cul4 ubiquitin ligases. Altogether these results point to a mechanism where Vpr triggers G(2) arrest by hijacking the Cul4/DDB1(DCAF1) ubiquitin ligase. We further show that, Vpx, a non-cytostatic Vpr-related protein acquired by HIV2 and SIV, also binds DCAF1 through a conserved motif. Thus, Vpr from HIV1 and Vpx from SIV recruit DCAF1 with different physiological outcomes for the host cell. This in turn suggests that both proteins have evolved to preserve interaction with the same Cul4 ubiquitin ligase while diverging in the recognition of host substrates targeted for proteasomal degradation.
Asunto(s)
Ciclo Celular/fisiología , Proteínas Cullin/metabolismo , Proteínas de Unión al ADN/metabolismo , Productos del Gen vpr/fisiología , VIH-1/fisiología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Proteínas Cullin/fisiología , Citotoxinas/fisiología , Citotoxinas/toxicidad , Proteínas de Unión al ADN/fisiología , Productos del Gen vpr/toxicidad , Células HeLa , Humanos , Datos de Secuencia Molecular , Transporte de Proteínas/fisiología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
A number of bacterial pathogens utilize the type III secretion pathway to deliver effector proteins directly into the host cell cytoplasm. Certain strains of Pseudomonas aeruginosa associated with acute infections express a potent cytotoxin, exoenzyme U (ExoU), that is delivered via the type III secretion pathway directly into contacting host cells. Once inside the mammalian cell, ExoU rapidly lyses the intoxicated cells via its phospholipase A(2) (PLA(2)) activity. A high-throughput cell-based assay was developed to screen libraries of compounds for those capable of protecting cells against the cytotoxic effects of ExoU. A number of compounds were identified in this screen, including one group that blocks the intracellular activity of ExoU. In addition, these compounds specifically inhibited the PLA(2) activity of ExoU in vitro, whereas eukaryotic secreted PLA(2) and cytosolic PLA(2) were not inhibited. This novel inhibitor of ExoU-specific PLA(2) activity, named pseudolipasin A, may provide a new lead for virulence factor-based therapeutic design.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Citotoxinas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Fluorenos/farmacología , Fosfolipasas A/antagonistas & inhibidores , Pseudomonas aeruginosa/enzimología , Animales , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Células CHO , Cricetinae , Cricetulus , Citotoxinas/biosíntesis , Citotoxinas/genética , Citotoxinas/fisiología , Fluorenos/química , Fosfolipasas A2 , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiología , Factores de Virulencia/antagonistas & inhibidores , Factores de Virulencia/biosíntesis , Factores de Virulencia/genética , Factores de Virulencia/fisiologíaRESUMEN
Evidence is accumulating that a growing number of bacterial toxins act by modulating the eukaryotic cell cycle machinery. In this context, we provide evidence that a protein toxin named cytotoxic necrotizing factor 1 (CNF1) from uropathogenic Escherichia coli is able to block cell cycle G(2)/M transition in the uroepithelial cell line T24. CNF1 permanently activates the small GTP-binding proteins of the Rho family that, beside controlling the actin cytoskeleton organization, also play a pivotal role in a large number of other cellular processes, including cell cycle regulation. The results reported here show that CNF1 is able to induce the accumulation of cells in the G(2)/M phase by sequestering cyclin B1 in the cytoplasm and down-regulating its expression. The possible role played by the Rho GTPases in the toxin-induced cell cycle deregulation has been investigated and discussed. The activity of CNF1 on cell cycle progression can offer a novel view of E. coli pathogenicity.
Asunto(s)
División Celular/fisiología , Citotoxinas/fisiología , Proteínas de Escherichia coli/fisiología , Escherichia coli/fisiología , Fase G2/fisiología , Inhibidores de Crecimiento/fisiología , Vejiga Urinaria/citología , Vejiga Urinaria/microbiología , Toxinas Bacterianas , Línea Celular Tumoral , Ciclina B/metabolismo , Ciclina B1 , Escherichia coli/patogenicidad , Humanos , Urotelio/microbiología , Proteínas de Unión al GTP rho/fisiologíaRESUMEN
The Helicobacter pylori VacA toxin is an 88-kDa secreted protein that causes multiple alterations in mammalian cells and is considered an important virulence factor in the pathogenesis of peptic ulcer disease and gastric cancer. We have shown previously that a VacA mutant protein lacking amino acids 6 to 27 (Delta6-27p88 VacA) is able to inhibit many activities of wild-type VacA in a dominant-negative manner. Analysis of a panel of C-terminally truncated Delta6-27p88 VacA proteins indicated that a fragment containing amino acids 1 to 478 (Delta6-27p48) exhibited a dominant-negative phenotype similar to that of the full-length Delta6-27p88 VacA protein. In contrast, a shorter VacA fragment lacking amino acids 6 to 27 (Delta6-27p33) did not exhibit detectable inhibitory activity. The Delta6-27p48 protein physically interacted with wild-type p88 VacA, whereas the Delta6-27p33 protein did not. Mutational analysis indicated that amino acids 351 to 360 are required for VacA protein-protein interactions and for dominant-negative inhibitory activity. The C-terminal portion (p55 domain) of wild-type p88 VacA could complement either Delta6-27p33 or Delta(6-27/351-360)p48, reconstituting dominant-negative inhibitory activity. Collectively, our data provide strong evidence that the inhibitory properties of dominant-negative VacA mutant proteins are dependent on interactions between the mutant VacA proteins and wild-type VacA, and they allow mapping of a domain involved in the formation of oligomeric VacA complexes.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Citotoxinas/toxicidad , Helicobacter pylori/genética , Mutación , Mapeo de Interacción de Proteínas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidad , Citotoxinas/genética , Citotoxinas/fisiología , Células HeLa , Helicobacter pylori/metabolismo , Helicobacter pylori/patogenicidad , Humanos , Líquido Intracelular/microbiología , Peso Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/fisiología , Fragmentos de Péptidos/toxicidad , Fenotipo , Estructura Terciaria de Proteína/genética , Transporte de Proteínas/fisiología , Vacuolas/microbiología , Vacuolas/patologíaRESUMEN
A remarkable group of proteins challenge the notions that protein sequence determines a unique three-dimensional structure, and that membrane and soluble proteins are very distinct. The pore-forming toxins typically transform from soluble, monomeric proteins to oligomers that form transmembrane channels. Recent structural studies provide ideas about how these changes take place. The recently solved structures of the beta-pore-forming toxins LukS, epsilon-toxin and intermedilysin confirm that the pore-forming regions are initially folded up on the surfaces of the soluble precursors. To create the transmembrane pores, these regions must extend and refold into membrane-inserted beta-barrels.