RESUMEN
As part of a general project aimed at elucidating the initiation of mucin-type O-glycosylation in helminth parasites, we have characterized a novel ppGalNAc-T (UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase) from the cestode Echinococcus granulosus (Eg-ppGalNAc-T1). A full-length cDNA was isolated from a library of the tissue-dwelling larval stage of the parasite, and found to code for a 654-amino-acid protein containing all the structural features of ppGalNAc-Ts. Functional characterization of a recombinant protein lacking the transmembrane domain showed maximal activity at 28 degrees C, in the range 6.5-7.5 pH units and in the presence of Cu2+. In addition, it transferred GalNAc to a broad range of substrate peptides, derived from human mucins and O-glycosylated parasite proteins, including acceptors containing only serine or only threonine residues. Interestingly, the C-terminal region of Eg-ppGalNAc-T1 bears a highly unusual lectin domain, considerably longer than the one from other members of the family, and including only one of the three ricin B repeats generally present in ppGalNAc-Ts. Furthermore, a search for conserved domains within the protein C-terminus identified a fragment showing similarity to a recently defined domain, specialized in the binding of organic phosphates (CYTH). The role of the lectin domain in the determination of the substrate specificity of these enzymes suggests that Eg-ppGalNAc-T1 would be involved in the glycosylation of a special type of substrate. Analysis of the tissue distribution by in situ hybridization and immunohistochemistry revealed that this transferase is expressed in the hydatid cyst wall and the subtegumental region of larval worms. Therefore it could participate in the biosynthesis of O-glycosylated parasite proteins exposed at the interface between E. granulosus and its hosts.
Asunto(s)
Echinococcus granulosus/enzimología , Lectinas/química , N-Acetilgalactosaminiltransferasas/química , Péptidos/química , Secuencia de Aminoácidos , Animales , Células COS/química , Células COS/metabolismo , Bovinos , Enfermedades de los Bovinos/enzimología , Enfermedades de los Bovinos/parasitología , Línea Celular , Chlorocebus aethiops , Clonación Molecular , Cobre/fisiología , ADN Complementario/genética , ADN de Helmintos/genética , Equinococosis/enzimología , Equinococosis/veterinaria , Proteínas del Helminto/biosíntesis , Proteínas del Helminto/química , Proteínas del Helminto/genética , Proteínas del Helminto/fisiología , Concentración de Iones de Hidrógeno , Lectinas/genética , Manganeso/metabolismo , Datos de Secuencia Molecular , N-Acetilgalactosaminiltransferasas/biosíntesis , N-Acetilgalactosaminiltransferasas/genética , N-Acetilgalactosaminiltransferasas/fisiología , Péptidos/genética , Estructura Terciaria de Proteína , Análisis de Secuencia de ADN , Especificidad por Sustrato/genética , Polipéptido N-AcetilgalactosaminiltransferasaRESUMEN
Tumor necrosis factor (TNF)-alpha is produced by cells of the immune system and is a key mediator in immune and inflammatory reactions. Through interaction with widely expressed receptors (TNF receptor 1 and TNF receptor 2), TNF-alpha is able to orchestrate the expression of a range of downstream proinflammatory molecules. Over the past decade novel biologics that inhibit TNF-alpha have been developed as extremely effective treatments for rheumatoid arthritis. Structurally, these biologics are antibodies, or TNF receptors on an antibody backbone that bind TNF-alpha directly and are delivered to patients by repeated injection. Gene therapy offers an improved approach to delivering biologics as a single administration of their encoding genetic material. In the present study we demonstrate the therapeutic effect of a small molecular weight dimeric TNF receptor 2 (dTNFR) constitutively expressed from plasmid DNA, delivered intramuscularly with electroporation, after disease onset in a collagen-induced arthritis model. Regulated promoters that enable the production of a transgene to be controlled are more suited to the application of gene therapy in the clinic. Regulated expression of dTNFR from the plasmid pGTRTT was also therapeutic in the mouse collagen-induced arthritis model when the inducer doxycycline was also administered, whereas no therapeutic effect was observed in the absence of doxycycline. The therapeutic effect of dTNFR expressed from a constitutive or regulated plasmid was dependent on the degree of disease activity at the time of DNA injection. The observations of this study are considered with regard to the disease model, the magnitude of gene regulation, and the path to clinical application.
Asunto(s)
Artritis Experimental/inducido químicamente , Artritis Experimental/terapia , Colágeno Tipo II/inmunología , Proteínas de Neoplasias/uso terapéutico , Plásmidos/efectos de los fármacos , Plásmidos/genética , Animales , Artritis Experimental/sangre , Artritis Reumatoide/terapia , Células COS/química , Células COS/metabolismo , Bovinos , Chlorocebus aethiops , Modelos Animales de Enfermedad , Doxiciclina/metabolismo , Electroporación/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Inyecciones Intramusculares , Luciferasas/genética , Ganglios Linfáticos/citología , Ratones , Ratones Endogámicos DBA , Proteínas de Neoplasias/genética , Plásmidos/administración & dosificación , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Receptores Tipo II del Factor de Necrosis Tumoral/uso terapéutico , Receptores Señuelo del Factor de Necrosis TumoralRESUMEN
Eukaryotes respond to the presence of unfolded protein in the endoplasmic reticulum (ER) by up-regulating the transcription of genes encoding ER protein chaperones, such as BiP. We have isolated a novel human cDNA encoding a homolog to Saccharomyces cerevisiae Ire1p, a proximal sensor for this signal transduction pathway in yeast. The gene product hIre1p is a type 1 transmembrane protein containing a cytoplasmic domain that is highly conserved to the yeast counterpart having a Ser/Thr protein kinase domain and a domain homologous to RNase L. However, the luminal domain has extensively diverged from the yeast gene product. hIre1p expressed in mammalian cells displayed intrinsic autophosphorylation activity and an endoribonuclease activity that cleaved the 5' splice site of yeast HAC1 mRNA, a substrate for the endoribonuclease activity of yeast Ire1p. Overexpressed hIre1p was localized to the ER with particular concentration around the nuclear envelope and some colocalization with the nuclear pore complex. Expression of Ire1p mRNA was autoregulated through a process that required a functional hIre1p kinase activity. Finally, overexpression of wild-type hIre1p constitutively activated a reporter gene under transcriptional control of the rat BiP promoter, whereas expression of a catalytically inactive hIre1p acted in a trans-dominant-negative manner to prevent transcriptional activation of the BiP promoter in response to ER stress induced by inhibition of N-linked glycosylation. These results demonstrate that hIre1p is an essential proximal sensor of the unfolded protein response pathway in mammalian cells.
Asunto(s)
Núcleo Celular/fisiología , Retículo Endoplásmico/fisiología , Endorribonucleasas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Estrés Fisiológico/fisiopatología , Factores de Transcripción , Alanina/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Células COS/química , Células COS/enzimología , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Endorribonucleasas/genética , Expresión Génica/genética , Regulación Enzimológica de la Expresión Génica , Humanos , Membranas Intracelulares/química , Membranas Intracelulares/enzimología , Lisina/genética , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Mutación , Membrana Nuclear/química , Membrana Nuclear/enzimología , Pliegue de Proteína , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Especificidad por Sustrato , Distribución TisularRESUMEN
Viral envelope (Env)-receptor interactions have been implicated in the cell death associated with infection by subgroups B and D avian leukosis-sarcoma viruses (ALVs). A chicken protein, CAR1, was identified that permitted infection of mammalian cells by these viral subgroups. CAR1 bound to a viral Env fusion protein, comprising an ALV-B surface Env protein and the Fc region of an immunoglobulin, indicating that it is a specific viral receptor. CAR1 contains two extracellular cysteine-rich domains characteristic of the TNFR family and a cytoplasmic region strikingly similar to the death domain of TNFR1 and Fas, implicating this receptor in cell killing. Chicken embryo fibroblasts susceptible to ALV-B infection and transfected quail QT6 cells expressing CAR1 underwent apoptosis in response to the Env-Ig fusion protein, demonstrating that this cytopathic ALV receptor can mediate cell death.