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1.
Nature ; 444(7122): 1088-91, 2006 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-17167413

RESUMO

Angiotensin -converting enzyme 2 (ACE2) is a regulator of the renin angiotensin system involved in acute lung failure, cardiovascular functions and severe acute respiratory syndrome (SARS) infections in mammals. A gene encoding a homologue to ACE2, termed collectrin (Tmem27), has been identified in immediate proximity to the ace2 locus. The in vivo function of collectrin was unclear. Here we report that targeted disruption of collectrin in mice results in a severe defect in renal amino acid uptake owing to downregulation of apical amino acid transporters in the kidney. Collectrin associates with multiple apical transporters and defines a novel group of renal amino acid transporters. Expression of collectrin in Xenopus oocytes and Madin-Darby canine kidney (MDCK) cells enhances amino acid transport by the transporter B(0)AT1. These data identify collectrin as a key regulator of renal amino acid uptake.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Rim/metabolismo , Glicoproteínas de Membrana/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Polaridade Celular , Cães , Regulação para Baixo , Feminino , Masculino , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/metabolismo , Tirosina/metabolismo , Xenopus
2.
J Immunol ; 174(3): 1472-8, 2005 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-15661906

RESUMO

The E3 ubiquitin ligase Casitas B cell lymphoma-b (Cbl-b) plays a critical role in the development of autoimmunity and sets the threshold for T cell activation. In the absence of Cbl-b, T cells stimulated via the TCR respond similarly to those that have received a CD28-mediated costimulatory signal, suggesting that the absence of Cbl-b substitutes for CD28-mediated costimulation. In this study, we show that loss of Cbl-b restores Ig class switching and germinal center formation in Vav1 mutant mice in response to an in vivo viral challenge. Genetic inactivation of Cbl-b also rescues impaired antiviral IgG production in CD28-mutant mice. Moreover, loss of CD28 results in disorganization of follicular dendritic cell clusters, which is also rescued by the Cbl-b mutation. Intriguingly, despite restored antiviral in vivo immunity and follicular dendritic cell clusters, loss of Cbl-b did not rescue germinal center formation in CD28-deficient mice. Mechanistically, in vivo vesicular stomatitis virus-induced IL-4 and IFN-gamma production and up-regulation of the inducible costimulatory molecule ICOS were dependent on CD28, and could not be rescued by the loss of Cbl-b. These data provide genetic evidence that CD28-dependent in vivo immune responses and Ig class switching can be genetically uncoupled from germinal center formation and ICOS induction by Cbl-b-Vav1-regulated signaling pathways.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Doenças Autoimunes/imunologia , Antígenos CD28/fisiologia , Proteínas de Ciclo Celular/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Anticorpos Antivirais/biossíntese , Antígenos de Diferenciação de Linfócitos T/biossíntese , Doenças Autoimunes/genética , Doenças Autoimunes/patologia , Antígenos CD28/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Agregação Celular/genética , Agregação Celular/imunologia , Proteínas de Ciclo Celular/genética , Citocinas/antagonistas & inibidores , Citocinas/biossíntese , Células Dendríticas Foliculares/patologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Centro Germinativo/patologia , Switching de Imunoglobulina/genética , Imunoglobulina G/biossíntese , Proteína Coestimuladora de Linfócitos T Induzíveis , Camundongos , Camundongos Knockout , Aglutinina de Amendoim/biossíntese , Fenótipo , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-cbl , Proteínas Proto-Oncogênicas c-vav , Infecções por Rhabdoviridae/genética , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/patologia , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Regulação para Cima/genética , Vírus da Estomatite Vesicular Indiana/imunologia
3.
J Immunol ; 175(6): 3790-9, 2005 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-16148125

RESUMO

Protein kinase B (PKBalpha/Akt1) a PI3K-dependent serine-threonine kinase, promotes T cell viability in response to many stimuli and regulates homeostasis and autoimmune disease in vivo. To dissect the mechanisms by which PKB inhibits apoptosis, we have examined the pathways downstream of PKB that promote survival after cytokine withdrawal vs Fas-mediated death. Our studies show that PKB-mediated survival after cytokine withdrawal is independent of protein synthesis and the induction of NF-kappaB. In contrast, PKB requires de novo gene transcription by NF-kappaB to block apoptosis triggered by the Fas death receptor. Using gene-deficient and transgenic mouse models, we establish that NF-kappaB1, and not c-Rel, is the critical signaling molecule downstream of the PI3K-PTEN-PKB signaling axis that regulates lymphocyte homeostasis.


Assuntos
Homeostase , Linfócitos/fisiologia , NF-kappa B/fisiologia , Transdução de Sinais , Animais , Apoptose , Sobrevivência Celular , Citocinas , Linfócitos/citologia , Camundongos , Camundongos Transgênicos , NF-kappa B/metabolismo , Transcrição Gênica , Receptor fas
4.
Proc Natl Acad Sci U S A ; 100(5): 2783-8, 2003 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-12594341

RESUMO

RNA interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections. Hepatitis C virus (HCV) is a major cause of chronic liver disease and affects >270 million individuals worldwide. The HCV genome is a single-stranded RNA that functions as both a messenger RNA and replication template, making it an attractive target for the study of RNA interference. Double-stranded small interfering RNA (siRNA) molecules designed to target the HCV genome were introduced through electroporation into a human hepatoma cell line (Huh-7) that contained an HCV subgenomic replicon. Two siRNAs dramatically reduced virus-specific protein expression and RNA synthesis to levels that were 90% less than those seen in cells treated with negative control siRNAs. These same siRNAs protected naive Huh-7 cells from challenge with HCV replicon RNA. Treatment of cells with synthetic siRNA was effective >72 h, but the duration of RNA interference could be extended beyond 3 weeks through stable expression of complementary strands of the interfering RNA by using a bicistronic expression vector. These results suggest that a gene-therapeutic approach with siRNA could ultimately be used to treat HCV.


Assuntos
Hepatite C/metabolismo , Fígado/citologia , Interferência de RNA , RNA Interferente Pequeno/fisiologia , RNA Viral/genética , RNA/metabolismo , Replicação Viral/fisiologia , Anticorpos Monoclonais/metabolismo , Northern Blotting , Western Blotting , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Eletroporação , Vetores Genéticos , Humanos , Modelos Genéticos , Mutação , Plasmídeos/metabolismo , RNA Mensageiro/metabolismo , Fatores de Tempo , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas , Replicação Viral/genética
5.
Proc Natl Acad Sci U S A ; 99(17): 11305-10, 2002 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-12172011

RESUMO

The tumor suppressor function of p53 has been attributed to its ability to regulate apoptosis and the cell cycle. In mammals, DNA damage, aberrant growth signals, chemotherapeutic agents, and UV irradiation activate p53, a process that is regulated by several posttranslational modifications. In Drosophila melanogaster, however, the regulation modes of p53 are still unknown. Overexpression of D. melanogaster p53 (Dmp53) in the eye induced apoptosis, resulting in a small eye phenotype. This phenotype was markedly enhanced by coexpression with D. melanogaster Chk2 (DmChk2) and was almost fully rescued by coexpression with a dominant-negative (DN), kinase-dead form of DmChk2. DN DmChk2 also inhibited Dmp53-mediated apoptosis in response to DNA damage, whereas overexpression of Grapes (Grp), the Drosophila Chk1-homolog, and its DN mutant had no effect on Dmp53-induced phenotypes. DmChk2 also activated the Dmp53 transactivation activity in cultured cells. Mutagenesis of Dmp53 amino terminal Ser residues revealed that Ser-4 is critical for its responsiveness toward DmChk2. DmChk2 activates the apoptotic activity of Dmp53 and Ser-4 is required for this effect. Contrary to results in mammals, Grapes, the Drosophila Chk1-homolog, is not involved in regulating Dmp53. Chk2 may be the ancestral regulator of p53 function.


Assuntos
Apoptose/efeitos da radiação , Proteínas de Drosophila , Drosophila melanogaster/efeitos da radiação , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Animais Geneticamente Modificados , Sequência de Bases , Linhagem Celular , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Clonagem Molecular , Primers do DNA/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Citometria de Fluxo , Genes p53/efeitos da radiação , Genoma , Humanos , Rim , Cinética , Larva , Fases de Leitura Aberta , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes/metabolismo , Transfecção , Proteína Supressora de Tumor p53/genética
6.
Immunity ; 21(2): 167-77, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15308098

RESUMO

Antigen-specific immunotolerance limits the expansion of self-reactive T cells involved in autoimmune diseases. Here, we show that the E3 ubiquitin ligase Cbl-b is upregulated in T cells after tolerizing signals. Loss of Cbl-b in mice results in impaired induction of T cell tolerance both in vitro and in vivo. Importantly, rechallenge of Cbl-b mutant mice with the tolerizing antigen results in massive lethality. Moreover, ablation of Cbl-b resulted in exacerbated autoimmunity. Mechanistically, loss of Cbl-b rescues reduced calcium mobilization of anergic T cells, which was attributed to Cbl-b-mediated regulation of PLCgamma-1 phosphorylation. Our results show a critical role for Cbl-b in the regulation of peripheral tolerance and anergy of T cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Anergia Clonal/imunologia , Linfócitos T/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Transferência Adotiva , Animais , Antígenos/imunologia , Anergia Clonal/fisiologia , Enterotoxinas/imunologia , Técnicas In Vitro , Camundongos , Fosfolipase C gama , Proteínas Proto-Oncogênicas c-cbl , Linfócitos T/imunologia , Fosfolipases Tipo C/metabolismo , Ubiquitina-Proteína Ligases/imunologia
7.
Nat Immunol ; 4(9): 899-906, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12925852

RESUMO

We investigated the in vivo function of the B7 family member B7-H3 (also known as B7RP-2) by gene targeting. B7-H3 inhibited T cell proliferation mediated by antibody to T cell receptor or allogeneic antigen-presenting cells. B7-H3-deficient mice developed more severe airway inflammation than did wild-type mice in conditions in which T helper cells differentiated toward type 1 (T(H)1) rather than type 2 (T(H)2). B7-H3 expression was consistently enhanced by interferon-gamma but suppressed by interleukin 4 in dendritic cells. B7-H3-deficient mice developed experimental autoimmune encephalomyelitis several days earlier than their wild-type littermates, and accumulated higher concentrations of autoantibodies to DNA. Thus, B7-H3 is a negative regulator that preferentially affects T(H)1 responses.


Assuntos
Antígeno B7-1/imunologia , Células Th1/imunologia , Animais , Autoanticorpos/imunologia , Autoanticorpos/metabolismo , Antígenos B7 , Antígeno B7-1/biossíntese , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Regulação para Baixo/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Citometria de Fluxo , Interferon gama/imunologia , Interleucina-4/imunologia , Ativação Linfocitária/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Orthomyxoviridae/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia
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