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1.
Nat Immunol ; 17(2): 187-95, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26726812

RESUMEN

Studies of repertoires of mouse monoclonal CD4(+) T cells have revealed several mechanisms of self-tolerance; however, which mechanisms operate in normal repertoires is unclear. Here we studied polyclonal CD4(+) T cells specific for green fluorescent protein expressed in various organs, which allowed us to determine the effects of specific expression patterns on the same epitope-specific T cells. Peptides presented uniformly by thymic antigen-presenting cells were tolerated by clonal deletion, whereas peptides excluded from the thymus were ignored. Peptides with limited thymic expression induced partial clonal deletion and impaired effector T cell potential but enhanced regulatory T cell potential. These mechanisms were also active for T cell populations specific for endogenously expressed self antigens. Thus, the immunotolerance of polyclonal CD4(+) T cells was maintained by distinct mechanisms, according to self-peptide expression patterns.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Expresión Génica , Tolerancia Inmunológica , Péptidos/genética , Péptidos/inmunología , Secuencia de Aminoácidos , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Autoantígenos/química , Autoantígenos/genética , Autoantígenos/inmunología , Autoinmunidad , Supresión Clonal/genética , Supresión Clonal/inmunología , Epítopos de Linfocito T/química , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Femenino , Genes Reporteros , Ratones , Ratones Transgénicos , Péptidos/química , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Timo/inmunología , Timo/metabolismo
2.
Immunity ; 48(5): 923-936.e4, 2018 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-29752065

RESUMEN

The development of T cell tolerance in the thymus requires the presentation of host proteins by multiple antigen-presenting cell (APC) types. However, the importance of transferring host antigens from transcription factor AIRE-dependent medullary thymic epithelial cells (mTECs) to bone marrow (BM) APCs is unknown. We report that antigen was primarily transferred from mTECs to CD8α+ dendritic cells (DCs) and showed that CD36, a scavenger receptor selectively expressed on CD8α+ DCs, mediated the transfer of cell-surface, but not cytoplasmic, antigens. The absence of CD8α+ DCs or CD36 altered thymic T cell selection, as evidenced by TCR repertoire analysis and the loss of allo-tolerance in murine allogeneic BM transplantation (allo-BMT) studies. Decreases in these DCs and CD36 expression in peripheral blood of human allo-BMT patients correlated with graft-versus-host disease. Our findings suggest that CD36 facilitates transfer of mTEC-derived cell-surface antigen on CD8α+ DCs to promote tolerance to host antigens during homeostasis and allo-BMT.


Asunto(s)
Antígenos de Superficie/inmunología , Antígenos CD36/inmunología , Tolerancia Inmunológica/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Timo/inmunología , Animales , Antígenos de Superficie/metabolismo , Trasplante de Médula Ósea , Antígenos CD36/genética , Antígenos CD36/metabolismo , Antígenos CD8/inmunología , Antígenos CD8/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Reguladores/metabolismo , Timo/metabolismo , Trasplante Homólogo
3.
Proc Natl Acad Sci U S A ; 121(20): e2320268121, 2024 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-38709934

RESUMEN

Insulin is a central autoantigen in the pathogenesis of T1D, and thymic epithelial cell expression of insulin under the control of the Autoimmune Regulator (Aire) is thought to be a key component of maintaining tolerance to insulin. In spite of this general working model, direct detection of this thymic selection on insulin-specific T cells has been somewhat elusive. Here, we used a combination of highly sensitive T cell receptor transgenic models for detecting thymic selection and sorting and sequencing of Insulin-specific CD4+ T cells from Aire-deficient mice as a strategy to further define their selection. This analysis revealed a number of unique t cell receptor (TCR) clones in Aire-deficient hosts with high affinity for insulin/major histocompatibility complex (MHC) ligands. We then modeled the thymic selection of one of these clones in Aire-deficient versus wild-type hosts and found that this model clone could escape thymic negative selection in the absence of thymic Aire. Together, these results suggest that thymic expression of insulin plays a key role in trimming and removing high-affinity insulin-specific T cells from the repertoire to help promote tolerance.


Asunto(s)
Proteína AIRE , Insulina , Receptores de Antígenos de Linfocitos T , Timo , Animales , Ratones , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Células Clonales , Tolerancia Inmunológica , Insulina/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Timo/inmunología , Timo/metabolismo , Timo/citología , Factores de Transcripción/metabolismo , Factores de Transcripción/genética
5.
Proc Natl Acad Sci U S A ; 115(20): 5265-5270, 2018 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-29712852

RESUMEN

Regulatory T cells (Tregs) control organ-specific autoimmunity in a tissue antigen-specific manner, yet little is known about their specificity in a natural repertoire. In this study, we used the nonobese diabetic (NOD) mouse model of autoimmune diabetes to investigate the antigen specificity of Tregs present in the inflamed tissue, the islets of Langerhans. Compared with Tregs present in spleen and lymph node, Tregs in the islets showed evidence of antigen stimulation that correlated with higher proliferation and expression of activation markers CD103, ICOS, and TIGIT. T cell receptor (TCR) repertoire profiling demonstrated that islet Treg clonotypes are expanded in the islets, suggesting localized antigen-driven expansion in inflamed islets. To determine their specificity, we captured TCRαß pairs from islet Tregs using single-cell TCR sequencing and found direct evidence that some of these TCRs were specific for islet-derived antigens including insulin B:9-23 and proinsulin. Consistently, insulin B:9-23 tetramers readily detected insulin-specific Tregs in the islets of NOD mice. Lastly, islet Tregs from prediabetic NOD mice were effective at preventing diabetes in Treg-deficient NOD.CD28-/- recipients. These results provide a glimpse into the specificities of Tregs in a natural repertoire that are crucial for opposing the progression of autoimmune diabetes.


Asunto(s)
Enfermedades Autoinmunes/inmunología , Diabetes Mellitus Tipo 1/inmunología , Modelos Animales de Enfermedad , Tolerancia Inmunológica/inmunología , Insulina/inmunología , Trasplante de Islotes Pancreáticos/inmunología , Linfocitos T Reguladores/inmunología , Animales , Autoantígenos/inmunología , Enfermedades Autoinmunes/terapia , Diabetes Mellitus Tipo 1/terapia , Ratones Endogámicos NOD , Ratones SCID
6.
Proc Natl Acad Sci U S A ; 109(15): E898-904, 2012 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-22431588

RESUMEN

Genome-wide studies have identified associations between polymorphisms in the IFN regulatory factor-5 (Irf5) gene and a variety of human autoimmune diseases. Its functional role in disease pathogenesis, however, remains unclear, as studies in Irf5(-/-) mice have reached disparate conclusions regarding the importance of this transcription factor in type I IFN production and antibody responses. We identified a spontaneous genomic duplication and frameshift mutation in the guanine exchange factor dedicator of cytokinesis 2 (Dock2) that has arisen in at least a subset of circulating Irf5(-/-) mice and inadvertently been bred to homozygosity. Retroviral expression of DOCK2, but not IRF-5, rescued defects in plasmacytoid dendritic cell and B-cell development, and Irf5(-/-) mice lacking the mutation in Dock2 exhibited normal plasmacytoid dendritic cell and B-cell development, largely intact type I IFN responses, and relatively normal antibody responses to viral infection. Thus, confirmation of the normal Dock2 genotype in circulating Irf5(-/-) mice is warranted, and our data may partly explain conflicting results in this field.


Asunto(s)
Formación de Anticuerpos/inmunología , Proteínas Activadoras de GTPasa/genética , Factores Reguladores del Interferón/deficiencia , Interferón Tipo I/biosíntesis , Mutación/genética , Secuencia de Aminoácidos , Animales , Linfocitos B/inmunología , Secuencia de Bases , Cromosomas de los Mamíferos/genética , Cruzamientos Genéticos , Células Dendríticas/inmunología , Femenino , Proteínas Activadoras de GTPasa/química , Proteínas Activadoras de GTPasa/metabolismo , Duplicación de Gen/genética , Sitios Genéticos/genética , Factores de Intercambio de Guanina Nucleótido , Humanos , Factores Reguladores del Interferón/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Fenotipo
7.
J Virol ; 82(18): 8956-64, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18632856

RESUMEN

West Nile virus (WNV) is a neurotropic flavivirus that has emerged globally as a significant cause of viral encephalitis in humans, especially in immunocompromised individuals. Previous studies have shown essential protective roles for antiviral cytokines (e.g., alpha interferon [IFN-alpha] and IFN-gamma) against WNV in mice. However, studies using cell culture offer conflicting answers regarding whether tumor necrosis factor alpha (TNF-alpha) has an anti-WNV function. To test the biological significance of TNF-alpha against WNV in vivo, experiments were performed with TNF receptor-1 (TNF-R1)-deficient and TNF-alpha-depleted C57BL/6 mice. TNF-R1(-/-) mice had enhanced mortality and decreased survival time after WNV infection compared to congenic wild-type mice. Consistent with this, administration of a neutralizing anti-TNF-alpha monoclonal antibody also decreased survival after WNV infection. Relatively small differences in viral burdens in peripheral tissues of TNF-R1(-/-) mice were observed, and this occurrence correlated with a modest antiviral effect of TNF-alpha on primary macrophages but not dendritic cells. In contrast, the viral titers detected in the central nervous systems of TNF-R1(-/-) mice were significantly increased compared to those of wild-type mice, although TNF-alpha did not have a direct antiviral effect in primary neuron cultures. Whereas no defect in priming of adaptive B- and T-cell responses in TNF-R1(-/-) mice was observed, there were significant reductions in accumulations of CD8+ T cells and macrophages in the brain. Our data are most consistent with a model in which interaction of TNF-alpha with TNF-R1 protects against WNV infection by regulating migration of protective inflammatory cells into the brain during acute infection.


Asunto(s)
Movimiento Celular/inmunología , Sistema Nervioso Central/inmunología , Leucocitos Mononucleares/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/patogenicidad , Animales , Anticuerpos Antivirales/sangre , Encéfalo/inmunología , Encéfalo/virología , Linfocitos T CD8-positivos/inmunología , Sistema Nervioso Central/virología , Humanos , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/genética , Fiebre del Nilo Occidental/mortalidad , Fiebre del Nilo Occidental/virología , Virus del Nilo Occidental/inmunología
8.
J Virol ; 82(22): 10964-74, 2008 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-18786989

RESUMEN

The B-cell response against West Nile virus (WNV), an encephalitic Flavivirus of global concern, is critical to controlling central nervous system dissemination and neurological sequelae, including death. Here, using a well-characterized mouse model of WNV infection, we examine the factors that govern early B-cell activation. Subcutaneous inoculation with a low dose of replicating WNV results in extensive B-cell activation in the draining lymph node (LN) within days of infection as judged by upregulation of the surface markers CD69, class II major histocompatibility complex, and CD86 on CD19(+) cells. B-cell activation in the LN but not the spleen was dependent on signals through the type I alpha/beta interferon (IFN-alpha/beta) receptor. Despite significant activation in the draining LN at day 3 after infection, WNV-specific B cells were not detected by immunoglobulin M enzyme-linked immunospot analysis until day 7. Liposome depletion experiments demonstrate that B-cell activation after WNV infection was not affected by the loss of F4/80(+) or CD169(+) subcapsular macrophages. Nonetheless, LN myeloid cells were essential for control of viral replication and survival from infection. Overall, our data suggest that the massive, early polyclonal B-cell activation occurring in the draining LN after WNV infection is immunoglobulin receptor and macrophage independent but requires sustained signals through the type I IFN-alpha/beta receptor.


Asunto(s)
Linfocitos B/inmunología , Interferón-alfa/inmunología , Interferón beta/inmunología , Activación de Linfocitos , Receptores de Antígenos/inmunología , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/inmunología , Animales , Antígenos CD/biosíntesis , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Linfocitos B/química , Antígeno B7-2/biosíntesis , Antígenos de Histocompatibilidad Clase II/biosíntesis , Lectinas Tipo C , Procedimientos de Reducción del Leucocitos , Ganglios Linfáticos/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Bazo/inmunología , Regulación hacia Arriba
9.
PLoS One ; 14(6): e0218063, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31181113

RESUMEN

[This corrects the article DOI: 10.1371/journal.pone.0200374.].

10.
J Virol ; 81(23): 12816-26, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17881453

RESUMEN

Neutralization of flaviviruses in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Previous studies demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral ridge of domain III (DIII) of the West Nile virus (WNV) E protein strongly protect against infection in animals. Based on X-ray crystallography and sequence analysis, an analogous type-specific neutralizing epitope for individual serotypes of the related flavivirus dengue virus (DENV) was hypothesized. Using yeast surface display of DIII variants, we defined contact residues of a panel of type-specific, subcomplex-specific, and cross-reactive MAbs that recognize DIII of DENV type 2 (DENV-2) and have different neutralizing potentials. Type-specific MAbs with neutralizing activity against DENV-2 localized to a sequence-unique epitope on the lateral ridge of DIII, centered at the FG loop near residues E383 and P384, analogous in position to that observed with WNV-specific strongly neutralizing MAbs. Subcomplex-specific MAbs that bound some but not all DENV serotypes and neutralized DENV-2 infection recognized an adjacent epitope centered on the connecting A strand of DIII at residues K305, K307, and K310. In contrast, several MAbs that had poor neutralizing activity against DENV-2 and cross-reacted with all DENV serotypes and other flaviviruses recognized an epitope with residues in the AB loop of DIII, a conserved region that is predicted to have limited accessibility on the mature virion. Overall, our experiments define adjacent and structurally distinct epitopes on DIII of DENV-2 which elicit type-specific, subcomplex-specific, and cross-reactive antibodies with different neutralizing potentials.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Virus del Dengue/inmunología , Epítopos/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Reacciones Cruzadas , Mapeo Epitopo , Pruebas de Neutralización
11.
PLoS One ; 13(7): e0200374, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30044821

RESUMEN

Cathepsin H is a member of the papain superfamily of lysosomal cysteine proteases. It is the only known aminopeptidase in the family and is reported to be involved in cancer and other major diseases. Like many other proteases, it is synthesized as an inactive proenzyme. Although the crystal structure of mature porcine cathepsin H revealed the binding of the mini-chain and provided structural basis for the aminopeptidase activity, detailed structural and functional information on the inhibition and activation of procathepsin H has been elusive. Here we present the crystal structures of human procathepsin H at 2.00 Å and 1.66 Å resolution. These structures allow us to explore in detail the molecular basis for the inhibition of the mature domain by the prodomain. Comparison with cathepsin H structure reveals how mini-chain reorients upon activation. We further demonstrate that procathepsin H is not auto-activated but can be trans-activated by cathepsin L.


Asunto(s)
Catepsina H/metabolismo , Precursores Enzimáticos/metabolismo , Catepsina H/química , Catepsina H/genética , Catepsina L/química , Catepsina L/metabolismo , Cristalización , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Células HEK293 , Humanos , Simulación de Dinámica Molecular , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
12.
J Exp Med ; 208(13): 2599-606, 2011 Dec 19.
Artículo en Inglés | MEDLINE | ID: mdl-22162833

RESUMEN

Memory B cells (MBCs) and long-lived plasma cells (LLPCs) persist after clearance of infection, yet the specific and nonredundant role MBCs play in subsequent protection is unclear. After resolution of West Nile virus infection in mice, we demonstrate that LLPCs were specific for a single dominant neutralizing epitope, such that immune serum poorly inhibited a variant virus that encoded a mutation at this critical epitope. In contrast, a large fraction of MBC produced antibody that recognized both wild-type (WT) and mutant viral epitopes. Accordingly, antibody produced by the polyclonal pool of MBC neutralized WT and variant viruses equivalently. Remarkably, we also identified MBC clones that recognized the mutant epitope better than the WT protein, despite never having been exposed to the variant virus. The ability of MBCs to respond to variant viruses in vivo was confirmed by experiments in which MBCs were adoptively transferred or depleted before secondary challenge. Our data demonstrate that class-switched MBC can respond to variants of the original pathogen that escape neutralization of antibody produced by LLPC without a requirement for accumulating additional somatic mutations.


Asunto(s)
Epítopos de Linfocito B/inmunología , Memoria Inmunológica , Mutación , Células Plasmáticas/inmunología , Proteínas Virales/inmunología , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Formación de Anticuerpos , Epítopos de Linfocito B/genética , Humanos , Ratones , Células Plasmáticas/metabolismo , Proteínas Virales/genética , Fiebre del Nilo Occidental/genética , Virus del Nilo Occidental/genética
13.
J Clin Invest ; 119(11): 3266-77, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19855131

RESUMEN

West Nile virus (WNV) causes asymptomatic infection in most humans, but for undefined reasons, approximately 20% of immunocompetent individuals develop West Nile fever, a potentially debilitating febrile illness, and approximately 1% develop neuroinvasive disease syndromes. Notably, since its emergence in 1999, WNV has become the leading cause of epidemic viral encephalitis in North America. We hypothesized that CD4+ Tregs might be differentially regulated in subjects with symptomatic compared with those with asymptomatic WNV infection. Here, we show that in 32 blood donors with acute WNV infection, Tregs expanded significantly in the 3 months after index (RNA+) donations in all subjects. Symptomatic donors exhibited lower Treg frequencies from 2 weeks through 1 year after index donation yet did not show differences in systemic T cell or generalized inflammatory responses. In parallel prospective experimental studies, symptomatic WNV-infected mice also developed lower Treg frequencies compared with asymptomatic mice at 2 weeks after infection. Moreover, Treg-deficient mice developed lethal WNV infection at a higher rate than controls. Together, these results suggest that higher levels of peripheral Tregs after infection protect against severe WNV disease in immunocompetent animals and humans.


Asunto(s)
Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Fiebre del Nilo Occidental/inmunología , Fiebre del Nilo Occidental/patología , Adulto , Anciano , Animales , Donantes de Sangre , Proliferación Celular , Femenino , Humanos , Inmunidad Innata , Recuento de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Fenotipo , ARN Viral/sangre , Factores de Tiempo , Fiebre del Nilo Occidental/mortalidad , Fiebre del Nilo Occidental/fisiopatología , Virus del Nilo Occidental/fisiología , Adulto Joven
14.
Science ; 322(5904): 1097-100, 2008 Nov 14.
Artículo en Inglés | MEDLINE | ID: mdl-19008445

RESUMEN

Although in vitro observations suggest that cross-presentation of antigens is mediated primarily by CD8alpha+ dendritic cells, in vivo analysis has been hampered by the lack of systems that selectively eliminate this cell lineage. We show that deletion of the transcription factor Batf3 ablated development of CD8alpha+ dendritic cells, allowing us to examine their role in immunity in vivo. Dendritic cells from Batf3-/- mice were defective in cross-presentation, and Batf3-/- mice lacked virus-specific CD8+ T cell responses to West Nile virus. Importantly, rejection of highly immunogenic syngeneic tumors was impaired in Batf3-/- mice. These results suggest an important role for CD8alpha+ dendritic cells and cross-presentation in responses to viruses and in tumor rejection.


Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/fisiología , Antígenos CD8/análisis , Reactividad Cruzada , Citotoxicidad Inmunológica , Células Dendríticas/inmunología , Proteínas Represoras/fisiología , Linfocitos T Citotóxicos/inmunología , Traslado Adoptivo , Animales , Anticuerpos Antivirales/sangre , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/deficiencia , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Linfocitos T CD4-Positivos/inmunología , Células Dendríticas/trasplante , Femenino , Fibrosarcoma/inmunología , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Represoras/genética , Bazo/inmunología , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/inmunología
15.
Eur J Immunol ; 37(7): 1845-54, 2007 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-17559174

RESUMEN

Infection with West Nile virus (WNV) causes fatal encephalitis in immunocompromised animals. Previous studies in mice have established that T cell protection is required for clearance of WNV infection from tissues and preventing viral persistence. The current study assessed whether specific WNV peptide epitopes could elicit a cytotoxic T lymphocyte (CTL) response capable of protecting against virus infection. Hidden Markov model analysis was used to identify WNV-encoded peptides that bound the MHC class I proteins K(b) or D(b). Of the 35 peptides predicted to bind MHC class I molecules, one immunodominant CTL recognition peptide was identified in each of the envelope and non-structural protein 4B genes. Addition of these but not control peptides to CD8(+) T cells from WNV-infected mice induced IFN-gamma production. CTL clones that were generated ex vivo lysed peptide-pulsed or WNV-infected target cells in an antigen-specific manner. Finally, adoptive transfer of a mixture of envelope- and non-structural protein 4B-specific CTL to recipient mice protected against lethal WNV challenge. Based on this, we conclude that CTL responses against immundominant WNV epitopes confer protective immunity and thus should be targets for inclusion in new vaccines.


Asunto(s)
Antígenos Virales/inmunología , Linfocitos T Citotóxicos/inmunología , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/inmunología , Traslado Adoptivo , Algoritmos , Secuencia de Aminoácidos , Animales , Antígenos Virales/genética , Antígenos Virales/aislamiento & purificación , Antígenos de Histocompatibilidad Clase I/inmunología , Epítopos Inmunodominantes/genética , Epítopos Inmunodominantes/inmunología , Epítopos Inmunodominantes/aislamiento & purificación , Cadenas de Markov , Ratones , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/aislamiento & purificación , Fiebre del Nilo Occidental/prevención & control
16.
J Am Chem Soc ; 127(38): 13124-5, 2005 Sep 28.
Artículo en Inglés | MEDLINE | ID: mdl-16173722

RESUMEN

An elusive goal for nucleic acid enzymology has been deoxyribozymes that ligate RNA rapidly, sequence-generally, with formation of native 3'-5' linkages, and in preparatively useful yield. Using in vitro selection, we have identified Mg2+- and Zn2+-dependent deoxyribozymes that simultaneously fulfill all four of these criteria. The new deoxyribozymes operate under practical incubation conditions and have modest RNA substrate sequence requirements, specifically D downward arrowRA for 9DB1 and A downward arrowR for 7DE5 (D = A, G, or U; R = A or G). These requirements are comparable to those of deoxyribozymes such as 10-23 and 8-17, which are already widely used as biochemical tools for RNA cleavage. We anticipate that the 9DB1 and 7DE5 deoxyribozymes will find immediate practical application for RNA ligation.


Asunto(s)
ADN Catalítico/química , ARN/síntesis química , Catálisis , Factores de Tiempo
17.
Biochemistry ; 44(25): 9217-31, 2005 Jun 28.
Artículo en Inglés | MEDLINE | ID: mdl-15966746

RESUMEN

We report Zn(2+)-dependent deoxyribozymes that ligate RNA. The DNA enzymes were identified by in vitro selection and ligate RNA with k(obs) up to 0.5 min(-)(1) at 1 mM Zn(2+) and 23 degrees C, pH 7.9, which is substantially faster than our previously reported Mg(2+)-dependent deoxyribozymes. Each new Zn(2+)-dependent deoxyribozyme mediates the reaction of a specific nucleophile on one RNA substrate with a 2',3'-cyclic phosphate on a second RNA substrate. Some of the Zn(2+)-dependent deoxyribozymes create native 3'-5' RNA linkages (with k(obs) up to 0.02 min(-)(1)), whereas all of our previous Mg(2+)-dependent deoxyribozymes that use a 2',3'-cyclic phosphate create non-native 2'-5' RNA linkages. On this basis, Zn(2+)-dependent deoxyribozymes have promise for synthesis of native 3'-5'-linked RNA using 2',3'-cyclic phosphate RNA substrates, although these particular Zn(2+)-dependent deoxyribozymes are likely not useful for this practical application. Some of the new Zn(2+)-dependent deoxyribozymes instead create non-native 2'-5' linkages, just like their Mg(2+) counterparts. Unexpectedly, other Zn(2+)-dependent deoxyribozymes synthesize one of three unnatural linkages that are formed upon the reaction of an RNA nucleophile other than a 5'-hydroxyl group. Two of these unnatural linkages are the 3'-2' and 2'-2' linear junctions created when the 2'-hydroxyl of the 5'-terminal guanosine of one RNA substrate attacks the 2',3'-cyclic phosphate of the second RNA substrate. The third unnatural linkage is a branched RNA that results from attack of a specific internal 2'-hydroxyl of one RNA substrate at the 2',3'-cyclic phosphate. When compared with the consistent creation of 2'-5' linkages by Mg(2+)-dependent ligation, formation of this variety of RNA ligation products by Zn(2+)-dependent deoxyribozymes highlights the versatility of transition metals such as Zn(2+) for mediating nucleic acid catalysis.


Asunto(s)
ADN Catalítico/metabolismo , ARN/química , ARN/metabolismo , Zinc/farmacología , Secuencia de Bases , Hidrólisis/efectos de los fármacos , Datos de Secuencia Molecular , Estructura Molecular , ARN/genética , Ribonucleasa T1/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Zinc/metabolismo
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