RESUMEN
Candidalysin, a cytolytic peptide toxin secreted by the human fungal pathogen Candida albicans, is critical for fungal pathogenesis. Yet, its intracellular targets have not been extensively mapped. Here, we performed a high-throughput enhanced yeast two-hybrid (HT-eY2H) screen to map the interactome of all eight Ece1 peptides with their direct human protein targets and identified a list of potential interacting proteins, some of which were shared between the peptides. CCNH, a regulatory subunit of the CDK-activating kinase (CAK) complex involved in DNA damage repair, was identified as one of the host targets of candidalysin. Mechanistic studies revealed that candidalysin triggers a significantly increased double-strand DNA breaks (DSBs), as evidenced by the formation of γ-H2AX foci and colocalization of CCNH and γ-H2AX. Importantly, candidalysin binds directly to CCNH to activate CAK to inhibit DNA damage repair pathway. Loss of CCNH alleviates DSBs formation under candidalysin treatment. Depletion of candidalysin-encoding gene fails to induce DSBs and stimulates CCNH upregulation in a murine model of oropharyngeal candidiasis. Collectively, our study reveals that a secreted fungal toxin acts to hijack the canonical DNA damage repair pathway by targeting CCNH and to promote fungal infection.
Asunto(s)
Candida albicans , Proteínas Fúngicas , Humanos , Ratones , Animales , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Candida albicans/metabolismo , Péptidos/metabolismoRESUMEN
Cadherins play a crucial role in epithelial morphogenesis and mediate intercellular adhesion. These receptors bind catenins and are involved in signal transduction pathways that regulate cell growth and apoptosis, and are frequently down-regulated in invasive and metastatic carcinomas. In order to assess the role of E-cadherin in cell adhesion and growth, we transfected MCF-7 cells, a human breast cancer cell line, with a dominant-negative construct of E-cadherin (H-2kd-E-cad). The dominant-negative form of E-cadherin disrupted cell-cell adhesion of monolayer cells and induced an epithelial-to-fibroblastic conversion without any significant change in integrin profiles. Whereas control cells rapidly formed multicellular aggregates that tightly compacted into spheroids, dominant-negative transfected cells failed to compact and remained as loosely-associated cells. The transfectants exhibited down-regulation and redistribution of endogenous E-cadherin as well as increased levels of alpha- and beta-catenin. Importantly, the H-2kd-E-cad-transfected cells, when grown as multicellular aggregates, showed an increase in cell proliferation rate, compared to control cells. Overall, these observations suggest that in breast carcinoma, disruption of E-cadherin and catenin function modulates both cell-cell adhesion and permits escape from cell-cell contact-involved inhibition of cell growth.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cadherinas/fisiología , Adhesión Celular/fisiología , Inhibición de Contacto/fisiología , División Celular , Movimiento Celular , Proteínas del Citoesqueleto/metabolismo , Regulación hacia Abajo , Femenino , Genes Dominantes , Antígenos H-2/genética , Antígenos H-2/metabolismo , Humanos , Mutación , Proteínas Recombinantes de Fusión/metabolismo , Transactivadores/metabolismo , Transfección , Células Tumorales Cultivadas , Regulación hacia Arriba , alfa Catenina , beta CateninaRESUMEN
Postnatal constriction of the full-term ductus arteriosus produces hypoxia of the muscle media. This is associated with anatomic remodeling (including smooth muscle death) that prevents subsequent reopening. We used late-gestation fetal and neonatal lambs to determine which factors are responsible for the postnatal hypoxia. Hypoxia [measured by 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide technique] and cell death (measured by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique) were observed in regions of the constricted ductus wall within 4 h after delivery. Although there was a decrease in ductus luminal flow during the first 6 h after delivery (measured by Doppler transducer), the amount of oxygen delivered to the ductus lumen (3070 +/- 1880 micromol O2 x min(-1) x g(-1)) far exceeded the amount of oxygen consumed by the constricted ductus (0.052 +/- 0.021 micromol O2 x min(-1) x g(-1), measured in vitro). Postnatal constriction increased the effective oxygen diffusion distance across the ductus wall to >3x the limit that can be tolerated for normal tissue homeostasis. This was owing to both an increase in the thickness of the ductus (fetus, 1.12 +/- 0.20 mm; newborn, 1.60 +/- 0.17 mm; p < 0.01) and a marked reduction in vasa vasorum flow (fetus, 0.99 +/- 0.44 mL x min(-1) x g(-1); newborn, 0.21 +/- 0.08 mL x min(-1) x g(-1); p < 0.01). These findings suggest that hypoxic cell death in the full-term ductus is caused primarily by changes in vasa vasorum flow and muscle media thickness and can occur before luminal flow has been eliminated. We speculate that in contrast with the full-term ductus, the preterm ductus is much less likely to develop the degree of hypoxia needed for vessel remodeling inasmuch as it only is capable of increasing its oxygen diffusion distance to 1.3x the maximally tolerated limit.
Asunto(s)
Conducto Arterial/anatomía & histología , Corazón/anatomía & histología , Hipoxia/fisiopatología , Flujo Sanguíneo Regional , Vasa Vasorum/fisiología , Animales , Animales Recién Nacidos , Anticuerpos Monoclonales , Muerte Celular , Hipoxia de la Célula , Conducto Arterial/fisiología , Etanidazol/análogos & derivados , Etanidazol/metabolismo , Edad Gestacional , Corazón/crecimiento & desarrollo , Humanos , Hidrocarburos Fluorados/metabolismo , Etiquetado Corte-Fin in Situ , Indicadores y Reactivos/metabolismo , Modelos Biológicos , Oxígeno/metabolismo , OvinosRESUMEN
Indomethacin produces constriction and hypoxia of the fetal ductus arteriosus. This is associated with death of smooth muscle cells in the ductus wall and an increased incidence of patent ductus arteriosus in the newborn period. We used fetal sheep to determine which factors are responsible for indomethacin-induced hypoxic cell death. Cell death in the ductus wall is directly related to the degree of indomethacin-induced ductus constriction and is present at both moderate and marked degrees of constriction. Both moderate and marked degrees of ductus constriction reduce vasa vasorum flow to the ductus (moderate = 69 +/- 25%; marked = 30 +/- 16% of preinfusion values) and increase the thickness of the ductus wall. In contrast, ductus luminal blood flow is not affected by moderate degrees of constriction and is reduced only after marked constriction. Although indomethacin increases ductus tone, it has no effect on ductus oxygen consumption. These findings suggest that the hypoxic cell death that occurs during the early stages of indomethacin-induced constriction is primarily due to changes in vasa vasorum blood flow and muscle media thickness.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Conducto Arterial/fisiología , Indometacina/farmacología , Oxígeno/metabolismo , Efectos Tardíos de la Exposición Prenatal , Animales , Muerte Celular/efectos de los fármacos , Conducto Arterioso Permeable/fisiopatología , Femenino , Etiquetado Corte-Fin in Situ , Microesferas , Consumo de Oxígeno/efectos de los fármacos , Embarazo , Ovinos , Tocólisis , Vasa Vasorum/efectos de los fármacosRESUMEN
Anatomic remodeling and permanent closure of the newborn ductus arteriosus appears to require the development of intense hypoxia within the constricted vessel wall. Hypoxic ductus smooth muscle cells express vascular endothelial cell growth factor (VEGF). We studied premature baboons and sheep to determine the effects of VEGF inhibition (in baboons) and VEGF stimulation (in sheep) on ductus remodeling in vivo. For study of VEGF inhibition, 13 premature newborn baboons (68% gestation) were treated with inhibitors of both prostaglandin and nitric oxide production to constrict the ductus and induce ductus wall hypoxia. Six received a neutralizing monoclonal antibody against VEGF (A.4.6.1, mAbVEGF), while seven did not. Both groups developed the same degree of ductus constriction, tissue hypoxia, and VEGF expression. The mAbVEGF treatment produced a significant (P < 0.05) reduction in ductus vasa vasorum ingrowth and neointima formation (due to both a decrease in luminal endothelial cell proliferation and a decrease in smooth muscle cell migration into the neointima). For study of VEGF stimulation, nine sheep fetuses (70% gestation) had their ductus wall injected with either VEGF (n = 6) or vehicle (n = 4) in vivo. VEGF administration produced a significant (P < 0.05) increase in vasa vasorum ingrowth and neointima formation. We conclude that VEGF plays an important role in the formation of neointimal mounds and vasa vasorum ingrowth during permanent ductus closure.