RESUMO
BACKGROUND: Acyclovir-resistant herpes simplex viruses (HSV) are commonly recovered from immunocompromised individuals who exhibit chronic and/or disseminated herpetic lesions. OBJECTIVES AND STUDY DESIGN: The virulence and response to topical acyclovir therapy were evaluated in a mouse model of zosteriform cutaneous HSV infection using two HSV-1 isolates from the same immunocompromised patient (one susceptible and one resistant to acyclovir). RESULTS: The acyclovir-resistant virus, with a Thr63Ile change in the ATP-binding site of the thymidine kinase gene, produced almost as many skin lesions as the wild-type susceptible virus. As expected from in vitro susceptibility data, the herpetic lesions of the mice infected with the drug-resistant virus did not respond to topical acyclovir therapy. CONCLUSIONS: Some thymidine kinase HSV mutants associated with drug resistance may retain their pathogenicity at least in the mouse model described in this study.
Assuntos
Aciclovir/farmacologia , Antivirais/farmacologia , Herpes Simples/tratamento farmacológico , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/patogenicidade , Aciclovir/administração & dosagem , Administração Tópica , Substituição de Aminoácidos , Animais , Antivirais/administração & dosagem , Modelos Animais de Doenças , Farmacorresistência Viral , Feminino , Herpes Simples/virologia , Herpesvirus Humano 1/isolamento & purificação , Humanos , Camundongos , Camundongos Pelados , Mutação de Sentido Incorreto , Timidina Quinase/genética , VirulênciaRESUMO
Proprotein convertases (PCs) have been proposed to play a role in tumor necrosis factor-alpha converting enzyme (TACE) processing/activation. Using the furin-deficient LoVo cells, as well as the furin-proficient synoviocytes and HT1080 cells expressing the furin inhibitor alpha(1)-PDX, we demonstrate that furin activity alone is not sufficient for effective maturation and activation of the TACE enzyme. Data from in vitro and in vivo cleavage assays indicate that PACE-4, PC5/PC6, PC1 and PC2 can directly cleave the TACE protein and/or peptide. PC inhibition in macrophages reduced the release of soluble TNF-alpha from transmembrane pro-TNF-alpha. We therefore conclude that furin, in addition to other candidate PCs, is involved in TACE maturation and activation.
Assuntos
Precursores Enzimáticos/metabolismo , Metaloendopeptidases/metabolismo , Pró-Proteína Convertases/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas ADAM , Proteína ADAM17 , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Precursores Enzimáticos/química , Furina/antagonistas & inibidores , Furina/deficiência , Furina/genética , Furina/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Células Jurkat , Metaloendopeptidases/química , Pró-Proteína Convertases/antagonistas & inibidores , Pró-Proteína Convertases/genética , Inibidores de Proteases/farmacologia , Ratos , Receptores do Fator de Necrose Tumoral/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Membrana Sinovial/citologia , Transfecção , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/farmacologiaRESUMO
G protein-coupled receptors (GPCRs) represent a vast family of transmembrane proteins involved in the regulation of several physiological responses. The thromboxane A2 receptor (present as two isoforms: TP alpha and TP beta) is a GPCR displaying diverse pharmacological effects. As seen for many other GPCRs, TP beta is regulated by agonist-induced internalization. In the present study, we report the identification by yeast two-hybrid screening of Nm23-H2, a nucleoside diphosphate kinase, as a new interacting molecular partner with the C-terminal tail of TP beta. This interaction was confirmed in a cellular context when Nm23-H2 was co-immunoprecipitated with TP beta in HEK293 cells, a process dependent on agonist stimulation of the receptor. We observed that agonist-induced internalization of TP beta was regulated by Nm23-H2 through modulation of Rac1 signaling. Immunofluorescence microscopy in HEK293 cells revealed that Nm23-H2 had a cytoplasmic and nuclear localization but was induced to translocate to the plasma membrane upon stimulation of TP beta to show extensive co-localization with the receptor. Our findings represent the first demonstration of an interaction of an Nm23 protein with a membrane receptor and constitute a novel molecular regulatory mechanism of GPCR endocytosis.