RESUMO
Upon antigen recognition by the T cell receptor, lymphocyte function-associated antigen 1 (LFA-1) physically associates with the leukocyte adhesion molecule CD226 (DNAM-1) and the protein tyrosine kinase Fyn. We show that lentiviral vector-mediated mutant (Y-F322) CD226 transferred into naive CD4+ helper T cells (Ths) inhibited interleukin (IL)-12-independent Th1 development initiated by CD3 and LFA-1 ligations. Moreover, proliferation induced by LFA-1 costimulatory signal was suppressed in mutant (Y-F322) CD226-transduced naive CD4+ and CD8+ T cells in the absence of IL-2. These results suggest that CD226 is involved in LFA-1-mediated costimulatory signals for triggering naive T cell differentiation and proliferation. We also demonstrate that although LFA-1, CD226, and Fyn are polarized at the immunological synapse upon stimulation with anti-CD3 in CD4+ and CD8+ T cells, lipid rafts are polarized in CD4+, but not CD8+, T cells. Moreover, proliferation initiated by LFA-1 costimulatory signal is suppressed by lipid raft disruption in CD4+, but not CD8+, T cells, suggesting that the LFA-1 costimulatory signal is independent of lipid rafts in CD8+ T cells.
Assuntos
Antígenos de Diferenciação de Linfócitos T/fisiologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Linfócitos T/fisiologia , Diferenciação Celular , Divisão Celular , Transferência Genética Horizontal , Vetores Genéticos , Humanos , Interleucina-12/fisiologia , Interleucina-2/farmacologia , Lentivirus/genética , Ativação Linfocitária , Microdomínios da Membrana/fisiologia , Células Th1/fisiologiaRESUMO
Eukaryotic 20S proteasomes are composed of two alpha-rings and two beta-rings, which form an alphabetabetaalpha stacked structure. Here we describe a proteasome-specific chaperone complex, designated Dmp1-Dmp2, in budding yeast. Dmp1-Dmp2 directly bound to the alpha5 subunit to facilitate alpha-ring formation. In Deltadmp1 cells, alpha-rings lacking alpha4 and decreased formation of 20S proteasomes were observed. Dmp1-Dmp2 interacted with proteasome precursors early during proteasome assembly and dissociated from the precursors before the formation of half-proteasomes. Notably, the crystallographic structures of Dmp1 and Dmp2 closely resemble that of PAC3-a mammalian proteasome-assembling chaperone; nonetheless, neither Dmp1 nor Dmp2 showed obvious sequence similarity to PAC3. The structure of the Dmp1-Dmp2-alpha5 complex reveals how this chaperone functions in proteasome assembly and why it dissociates from proteasome precursors before the beta-rings are assembled.
Assuntos
Chaperonas Moleculares/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Cristalografia por Raios X , Precursores Enzimáticos/metabolismo , Chaperonas Moleculares/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/isolamento & purificação , Complexos Multienzimáticos/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/isolamento & purificação , Proteínas Mutantes/metabolismo , Mutação/genética , Complexo de Endopeptidases do Proteassoma/química , Ligação Proteica , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia Estrutural de ProteínaRESUMO
Collagen-induced arthritis (CIA) can be induced even in CIA-resistant H-2(b) background of C57BL/6 mice when these mice are immunized with type II collagen (CII) emulsified in complete Freund's adjuvant (CFA) containing high, but not low, dose of Mycobacterium tuberculosis. Here, we investigated the pathogenesis of CIA in C57BL/6 mice induced by the immunizing protocol. We examined expressions of cytokines, costimulatory molecules and major histocompatibility complex (MHC) class II in draining lymph nodes (DLN) in CIA-induced C57BL/6 mice by quantitative reverse transcription-polymerase chain reaction. We also examined an effect of M. tuberculosis on the expression of these molecules on dendritic cells (DC) in vitro by flow cytometry. We finally examined an effect of M. tuberculosis in CFA used for immunization with CII antigen on priming of CD4+ helper T cells specific to CII in DLN of CIA-induced C57BL/6 mice. The expression of interferon-gamma (IFN-gamma), Interleukin-12p40 (IL-12p40), costimulatory molecules CD40, CD80 and CD86 and MHC class II were up-regulated in DLN of CIA-induced C57BL/6 mice. Expressions of these costimulatory molecules were also up-regulated on DC after stimulation with high, but not low, dose of M. tuberculosis in vitro. Furthermore, priming of CD4+ helper T cells specific to CII antigen in DLN required immunization with CII using CFA containing high, but not low, dose of M. tuberculosis. These results suggested that high dose of M. tuberculosis were required for maturation of DC enough to prime CD4+ helper T cells specific to CII antigen in DLN of H-2b background of C57BL/6 mice.