RESUMEN
The innate and adaptive immune responses involve the stimulation of nuclear factor κB (NF-κB) transcription factors through the Lys(63) (K(63))-linked ubiquitylation of specific components of NF-κB signaling pathways. We found that ubiquitylated components of the NF-κB pathway accumulated on the cytosolic leaflet of the endoplasmic reticulum (ER) membrane after the engagement of cell-surface, proinflammatory cytokine receptors or antigen receptors. Through mass spectrometric analysis, we found that the ER-anchored protein metadherin (MTDH) was a partner for these ubiquitylated activators of NF-κB and that it directly bound to K(63)-linked polyubiquitin chains. Knockdown of MTDH inhibited the accumulation of ubiquitylated NF-κB signaling components at the ER, reduced the extent of NF-κB activation, and decreased the amount of proinflammatory cytokines produced. Our observations highlight an unexpected facet of the ER as a key subcellular gateway for NF-κB activation.
Asunto(s)
Moléculas de Adhesión Celular/inmunología , Retículo Endoplásmico/inmunología , FN-kappa B/inmunología , Poliubiquitina/inmunología , Transducción de Señal/inmunología , Ubiquitinación/inmunología , Inmunidad Adaptativa/fisiología , Moléculas de Adhesión Celular/genética , Citocinas/genética , Citocinas/inmunología , Retículo Endoplásmico/genética , Células HEK293 , Células HeLa , Humanos , Inmunidad Innata/fisiología , Células Jurkat , Proteínas de la Membrana , FN-kappa B/genética , Poliubiquitina/genética , Proteínas de Unión al ARN , Transducción de Señal/genética , Ubiquitinación/genéticaRESUMEN
Glioblastoma constitutes the most aggressive and deadly of brain tumors. As yet, both conventional and molecular-based therapies have met with limited success in treatment of this cancer. Among other explanations, the heterogeneity of glioblastoma and the associated microenvironment contribute to its development, as well as resistance and recurrence in response to treatments. Increased vascularity suggests that tumor angiogenesis plays an important role in glioblastoma progression. However, the molecular crosstalk between endothelial and glioblastoma cells requires further investigation. To examine the effects of glioblastoma-derived signals on endothelial homeostasis, glioblastoma cell secretions were collected and used to treat brain endothelial cells. Here, we present evidence that the glioblastoma secretome provides pro-angiogenic signals sufficient to disrupt VE-cadherin-mediated cell-cell junctions and promote endothelial permeability in brain microvascular endothelial cells. An unbiased angiogenesis-specific antibody array screen identified the chemokine, interleukin-8, which was further demonstrated to function as a key factor involved in glioblastoma-induced permeability, mediated through its receptor CXCR2 on brain endothelia. This underappreciated interface between glioblastoma cells and associated endothelium may inspire the development of novel therapeutic strategies to induce tumor regression by preventing vascular permeability and inhibiting angiogenesis.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Permeabilidad Capilar , Células Endoteliales/metabolismo , Glioblastoma/metabolismo , Interleucina-8/metabolismo , Receptores de Interleucina-8B/metabolismo , Neoplasias Encefálicas/genética , Permeabilidad Capilar/efectos de los fármacos , Línea Celular Tumoral , Medios de Cultivo Condicionados/farmacología , Células Endoteliales/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Humanos , Interleucina-8/farmacología , Receptores de Interleucina-8B/genéticaRESUMEN
Toxoplasma gondii infection triggers host microtubule rearrangement and organelle recruitment around the parasite vacuole. Factors affecting initial stages of microtubule remodeling are unknown. To illuminate the mechanism, we tested the hypothesis that the parasite actively remodels host microtubules. Utilizing heat-killed parasites and time-lapse analysis, we determined microtubule rearrangement requires living parasites and is time dependent. We discovered a novel aster of microtubules (MTs) associates with the vacuole within 1h of infection. This aster lacks the concentrated foci of gamma (gamma)-tubulin normally associated with MT nucleation sites. Unexpectedly, vacuole enlargement does not correlate with an increase in MT staining around the vacuole. We conclude microtubule remodeling does not result from steric constraints. Using nocodazole washout studies, we demonstrate the vacuole nucleates host microtubule growth in-vivo via gamma-tubulin-associated sites. Moreover, superinfected host cells display multiple gamma-tubulin foci. Microtubule dynamics are critical for cell cycle control in uninfected cells. Using non-confluent monolayers, we show host cells commonly fail to finish cytokinesis resulting in larger, multinucleated cells. Our data suggest intimate interactions between T. gondii and host microtubules result in suppression of cell division and/or cause a mitotic defect, thus providing a larger space for parasite duplication.
Asunto(s)
Microtúbulos/metabolismo , Toxoplasma/fisiología , Animales , División Celular , Línea Celular , Chlorocebus aethiops , Ratones , Tubulina (Proteína)/metabolismo , Vacuolas/parasitologíaRESUMEN
Receptor for activated C kinase 1 (RACK1) has been implicated in multiple protein-protein interactions including functioning as a scaffolding protein for signaling molecules. We report the cloning and cellular localization of a RACK1 ortholog (TgRACK1) in the opportunistic pathogen Toxoplasma gondii. The full-length transcript possesses a predicted ORF of 966 bp and codes for a protein of approximately 35 kDa molecular weight. Molecular analysis of TgRACK1 reveals the presence of seven WD40 repeat motifs. TgRACK1 was tagged with a FLAG epitope and stably expressed in RH parasites. FLAG-TgRACK1 localizes to the parasite cytoplasm and nucleus. Immunoprecipitation (IP) of FLAG-TgRACK1 from highly purified extracellular parasites followed by immunoblot analysis reveals an interaction between TgbetaCOP and FLAG-TgRACK1. This is the first demonstration of an interaction between a betaCOP subunit and the RACK1 protein. This result is of interest given that a signaling event precedes protein secretion and parasite invasion.
Asunto(s)
Proteínas Protozoarias/metabolismo , Receptores de Superficie Celular/metabolismo , Toxoplasma/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Epítopos/genética , Epítopos/metabolismo , Microscopía Fluorescente , Datos de Secuencia Molecular , Peso Molecular , Oligopéptidos , Péptidos/genética , Péptidos/metabolismo , Proteínas Protozoarias/genética , Receptores de Cinasa C Activada , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Toxoplasma/genética , TransfecciónRESUMEN
Coatomer coated (COPI) vesicles play a pivotal role for multiple membrane trafficking steps throughout the eukaryotic cell. Our focus is on betaCOP, one of the most well known components of the COPI multi-protein complex. Amino acid differences in betaCOP may dictate functional divergence across species during the course of evolution, especially with regards to the evolutionary pressures on obligate intracellular parasites. A bioinformatic analysis of betaCOP amino acid sequences was conducted for 49 eukaryotic species. Cloning and sequence analysis of the Toxoplasma gondii betaCOP homologue revealed several amino acid insertions unique to T. gondii and one C-terminal insertion that is unique to apicomplexan parasites. These findings led us to investigate the possibility that betaCOP experienced functional divergence during the course of its evolution. Bayesian phylogenetic analysis revealed a tree consistent with pan eukaryote distribution and long-branch lengths were observed among the apicomplexans. Further analysis revealed that kinetoplast betaCOP underwent the most amount of change, leading to perhaps an overall change of function. In comparison, T. gondii exhibited subtle yet specific amino acid changes. The amino acid substitutions did not occur in the same places as other lineages, suggesting that TgbetaCOP has a role specific to the apicomplexans. Our work identifies 48 residues that are likely to be functionally important when comparing apicomplexan, kinetoplastid, and fungal betaCOP.