RESUMO
Asna1, also known as TRC40, is implicated in the delivery of tail-anchored (TA) proteins into the endoplasmic reticulum (ER), in vesicle-mediated transport, and in chaperoning unfolded proteins during oxidative stress/ATP depletion. Here, we show that Asna1 inactivation in pancreatic progenitor cells leads to redistribution of the Golgi TA SNARE proteins syntaxin 5 and syntaxin 6, Golgi fragmentation, and accumulation of cytosolic p62+ puncta. Asna1-/- multipotent progenitor cells (MPCs) selectively activate integrated stress response signaling and undergo apoptosis, thereby disrupting endocrine and acinar cell differentiation, resulting in pancreatic agenesis. Rescue experiments implicate the Asna1 ATPase activity and a CXXC di-cysteine motif in ensuring Golgi integrity, syntaxin 5 localization and MPC survival. Ex vivo inhibition of retrograde transport reproduces the perturbed Golgi morphology, and syntaxin 5 and syntaxin 6 expression, whereas modulation of p53 activity, using PFT-α and Nutlin-3, prevents or reproduces apoptosis in Asna1-deficient and wild-type MPCs, respectively. These findings support a role for the Asna1 ATPase activity in ensuring the survival of pancreatic MPCs, possibly by counteracting p53-mediated apoptosis.
Assuntos
ATPases Transportadoras de Arsenito/metabolismo , Células-Tronco Multipotentes/metabolismo , Pâncreas/embriologia , Animais , Apoptose/genética , ATPases Transportadoras de Arsenito/genética , Sobrevivência Celular/fisiologia , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Camundongos , Camundongos Knockout , Células-Tronco Multipotentes/citologia , Pâncreas/citologia , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Herpes simplex virus type 1 (HSV1), a member of the alphaherpesvirinae, can cause recurrent facial lesions and encephalitis. Two membrane envelopment processes, one at the inner nuclear membrane and a second at cytoplasmic membranes are crucial for a productive viral infection. Depending on the subfamily, herpesviruses encode more than 11 different transmembrane proteins including members of the tail-anchored protein family. HSV1 encodes three tail-anchored proteins pUL34, pUL56 and pUS9 characterized by a single hydrophobic region positioned at their C-terminal end that needs to be released from the ribosome prior to posttranslational membrane insertion. Asna1/TRC40 is an ATPase that targets tail-anchored proteins to the endoplasmic reticulum in a receptor-dependent manner. Cell biological data point to a critical and general role of Asna1/TRC40 in tail-anchored protein biogenesis. With this study, we aimed to determine the importance of the tail-anchored insertion machinery for HSV1 infection. METHODS: To determine protein-protein interactions, the yeast-two hybrid system was applied. Asna1/TRC40 was depleted using RNA interference. Transient transfection and virus infection experiments followed by indirect immunofluorescence analysis were applied to analyse the localization of viral proteins as well as the impact of Asna1/TRC40 depletion on virus infection. RESULTS: All HSV1 tail-anchored proteins specifically bound to Asna1/TRC40 but independently localized to their target membranes. While non-essential for cell viability, Asna1/TRC40 is required for efficient HSV1 replication. We show that early events of the replication cycle like virion entry and overall viral gene expression were unaffected by depletion of Asna1/TRC40. Furthermore, equal amounts of infectious virions were formed and remained cell-associated. This indicated that both nuclear egress of capsids that requires the essential tail-anchored protein pUL34, and secondary envelopment to form infectious virions were successfully completed. Despite large part of the virus life cycle proceeding normally, viral propagation was more than 10 fold reduced. We show that depletion of Asna1/TRC40 specifically affected a step late in infection during release of infectious virions to the extracellular milieu. CONCLUSIONS: Asna1/TRC40 is required at a late step of herpesviral infection for efficient release of mature virions to the extracellular milieu. This study reveals novel tools to decipher exocytosis of newly formed virions as well as hitherto unknown cellular targets for antiviral therapy.
Assuntos
ATPases Transportadoras de Arsenito/metabolismo , Herpesvirus Humano 1/fisiologia , Interações Hospedeiro-Patógeno , Liberação de Vírus , Animais , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Microscopia de Fluorescência , Mapeamento de Interação de Proteínas , Técnicas do Sistema de Duplo-HíbridoRESUMO
There is no cross-resistance between arsenic trioxide and conventional chemotherapeutics. Classical multi-drug resistant (MDR) cells remain sensitive to arsenic trioxide, which may even reverse the drug resistance. Arsenic trioxide is also effective in leukemias/tumors that persist despite conventional cytotoxic or targeted drugs. We obtained a highly arsenic-resistant MDR leukemic cell line, HL-60/RS, by exposing leukemic HL-60 cells to adriamycin selection. We compared the arsenic sensitivity, and the expression and responses to arsenic of the arsenic-related transporters, MRP1, MRP2, and ASNA1, in paired parent/arsenic-resistant HL-60/RS/HL-60 and arsenic-sensitive/parental K562/ADM/K562 cells. Expression levels of MRP1, MRP2, and ASNA1 were negatively correlated with cell sensitivities to arsenic trioxide, and ASNA1 expression notably was highest in HL-60/RS cells and lowest in K562/ADM cells. Expression levels of MRP1, MRP2, and ASNA1 were significantly enhanced in HL-60/RS cells and inhibited in K562/ADM cells by arsenic trioxide treatment, compared with their parental sensitive cells, in accord with the high-resistance of HL-60/RS cells and high-sensitivity of K562/ADM cells. In conclusion, the cross-resistance of conventional chemotherapeutics-resistant leukemic cells to arsenic trioxide is determined by both levels of MRP1, MRP2, and ASNA1, and also by the responses of these transporters to arsenic stress.
Assuntos
Arsenicais/farmacologia , ATPases Transportadoras de Arsenito/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Leucemia/patologia , Óxidos/farmacologia , Antineoplásicos/farmacologia , Arsênio/farmacologia , Trióxido de Arsênio , ATPases Transportadoras de Arsenito/análise , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Células K562 , Leucemia/tratamento farmacológico , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/análise , Proteínas Associadas à Resistência a Múltiplos Medicamentos/efeitos dos fármacosRESUMO
Cisplatin is an essential chemotherapeutic drug in the treatment of many cancers. Its use, however, is limited by the development of resistance in many tumors. The ability to re-sensitize resistant tumors could significantly strengthen cisplatin therapy in patients. Caenorhabditis elegans is a suitable model for studying the cytoplasmic role of cisplatin in tumor cells. We have previously shown that the ATPase ASNA-1 has similar roles as a factor governing cisplatin sensitivity in mammalian tumor cells and C. elegans. Here we study the endoplasmic reticulum (ER) resident chaperone ENPL-1/GRP94 and find that its depletion makes worms sensitive to cisplatin. Elevated ER stress levels in enpl-1 mutants is the likely cause of this sensitivity because a correlation can be made between cisplatin sensitivity and the high ER stress levels. We also find that asna-1 mutants have elevated unfolded protein response (UPR) activity and that the intrinsically cisplatin resistant wild-type worms become sensitive when ER stress is high. We conclude that enpl-1 is a cisplatin sensitizing factor and suggest that manipulation of its levels or of UPR activity will enhance the effects of cisplatin based cancer therapy.
RESUMO
Proteins destined for the endomembrane system of eukaryotic cells are typically translocated into or across the membrane of the endoplasmic reticulum and this process is normally closely coupled to protein synthesis. However, it is becoming increasingly apparent that a significant proportion of proteins are targeted to and inserted into the ER membrane post-translationally, that is after their synthesis is complete. These proteins must be efficiently captured and delivered to the target membrane, and indeed a failure to do so may even disrupt proteostasis resulting in cellular dysfunction and disease. In this review, we discuss the mechanisms by which various protein precursors can be targeted to the ER and either inserted into or translocated across the membrane post-translationally. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum.