RESUMO
The IFN stimulated gene 15 (ISG15) encodes a 15-kDa ubiquitin-like protein, that is induced by type I IFNs and is conjugated to the bulk of newly synthesized polypeptides at the ribosome. ISG15 functions as an antiviral molecule possibly by being covalently conjugated to viral proteins and disturbing virus particle assembly. Here, we have investigated the effect of ISGylation on degradation and antigen presentation of viral and cellular proteins. ISGylation did not induce proteasomal degradation of bulk ISG15 target proteins neither after overexpressing ISG15 nor after induction by IFN-ß. The MHC class I cell surface expression of splenocytes derived from ISG15-deficient mice or mice lacking the catalytic activity of the major de-ISGylating enzyme USP18 was unaltered as compared to WT mice. Fusion of ubiquitin or FAT10 to the long-lived nucleoprotein (NP) of lymphocytic choriomeningitis virus accelerated the proteasomal degradation of NP while fusion to ISG15 did not detectably speed up NP degradation. Nevertheless, MHC-I restricted presentation of two epitopes of NP were markedly enhanced when it was fused to ISG15 similarly to fusion with ubiquitin or FAT10. Thus, we provide evidence that ISG15 can enhance the presentation of antigens on MHC-I most likely by promoting co-translational antigen processing.
Assuntos
Apresentação de Antígeno/imunologia , Citocinas/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Ubiquitinas/imunologia , Animais , Citocinas/deficiência , Citocinas/genética , Citocinas/metabolismo , Células HEK293 , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Vírus da Coriomeningite Linfocítica/genética , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/imunologia , Proteínas do Nucleocapsídeo/metabolismo , Complexo de Endopeptidases do Proteassoma/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Modificação Traducional de Proteínas/imunologia , Proteólise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Ubiquitina Tiolesterase/deficiência , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/imunologia , Ubiquitinas/deficiência , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
CONTEXT: Thyroid-associated ophthalmopathy (TAO) is the component of Graves' disease characterized by orbital inflammation and connective tissue remodeling. The IGF-1 receptor (IGF-1R) and TSH receptor (TSHR) form a physical and functional complex in orbital fibroblasts. A subset of these fibroblasts is derived from infiltrating CD34(+) fibrocytes. Teprotumumab (RV 001, R1507) is a human monoclonal anti-IGF-1R blocking antibody currently undergoing a phase 2 clinical trial in patients with active TAO. OBJECTIVE: To determine whether teprotumumab inhibits the induction by TSH of IL-6 and IL-8 in fibrocytes. DESIGN: Fibrocytes were treated without or with teprotumumab in combination with IGF-1 or TSH. MAIN OUTCOME MEASURES: IL-6 and IL-8 mRNA expression and protein production were analyzed by real-time PCR and Luminex, respectively. Phosphorylated Akt (S473) levels were analyzed by Western blot. TSHR and IGF-1R display was assessed by flow cytometry. RESULTS: Fibrocyte display of IGF-1R and TSHR was reduced with teprotumumab, as were IGF-1- and TSH-dependent phosphorylated Akt levels. TSH induction of IL-6 and IL-8 mRNA and protein was also reduced by the monoclonal antibody. CONCLUSIONS: Teprotumumab attenuates the actions of both IGF-1 and TSH in fibrocytes. Specifically, it blocks the induction of proinflammatory cytokines by TSH. These results provide, at least in part, the molecular rationale for interrogating the therapeutic efficacy of this antibody in TAO.
Assuntos
Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/farmacologia , Fibroblastos/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Tireotropina/antagonistas & inibidores , Anticorpos Bloqueadores/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Células Cultivadas , Fibroblastos/imunologia , Fibroblastos/metabolismo , Doença de Graves/genética , Doença de Graves/imunologia , Doença de Graves/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Modificação Traducional de Proteínas/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/metabolismo , Tireotropina/farmacologiaRESUMO
The influence of viral envelope glycans is often overlooked, but one should bear in mind that variable glycosylation may affect the properties of viral envelope glycoproteins and potentially alter the course of an infection. Hence, there is a need for simple methods that can be use to identify changes in the glycosylation pattern of viral glycoproteins in a large number of samples. We describe here methods for the analysis of cell-line specific changes in glycosylation of the respiratory syncytial virus (RSV) attachment glycoprotein (G), which involve the use of lectins and anti-carbohydrate antibodies. Given the role of the G glycoprotein in RSV antigenicity, we also describe procedures based on Western blotting to determine the effect of G protein glycosylation changes on reactivity with human sera. We found that glycosylation of the C-terminal domain of the G protein reduces reactivity with human sera, indicating that variable glycosylation may contribute to evasion of the humoral immune response by RSV.