RESUMEN
Tissue factor (TF) is the major physiological initiator of the coagulation cascade and has an important function in the morbidity and mortality associated with many disease states, including cancer-associated thrombosis and atherosclerosis. TF normally exists in a partially encrypted state and its de-encryption on circulating monocytes, platelets or endothelial cells by inflammatory mediators can lead to thrombosis. Furthermore, many cancer cells express large amounts of TF and these cells communicate readily with the circulation through the fenestrated tumor endothelium. To assess agents or conditions that modulate the encryption state of TF, we developed a continuous assay for the determination of TF procoagulant activity (PCA) in a cell-based system. We have shown the use of this assay at detecting agents that de-encrypt TF thereby leading to an increase in TF PCA in three cancer cell lines, namely, T24/83 bladder carcinoma cells and PC-3 and DU145 prostate cancer cells. Further, through use of this assay, we have shown that the endoplasmic reticulum calcium pump inhibitor, thapsigargin, stimulates the de-encryption of TF. The continuous assay for the determination of TF PCA proved to have inherently less intra- and inter-assay variability than the widely used discontinuous assay and is considerably less labor intensive. Further, the continuous assay produced progress curves that were compatible with curve fitting to allow for the determination of the nature of reaction as well as rate constants for the underlying enzymes, TF/FVIIa and FXa. The continuous assay for the assessment of TF PCA on intact cells is applicable for high-throughput screening to allow for the determination of compounds that modulate TF PCA.
Asunto(s)
Fenómenos Fisiológicos Celulares , Tromboplastina/metabolismo , Biomarcadores de Tumor/análisis , Carcinoma/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/fisiología , Inhibidores Enzimáticos/farmacología , Humanos , Immunoblotting , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/metabolismo , Tapsigargina/farmacología , Tromboplastina/análisis , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
The p68-derived peptide, QIVDVCHDV, was identified by a reverse immunology approach as capable of reconstituting an epitope recognized by the melanoma-reactive cytotoxic T lymphocyte (CTL) line VMM5. The peptide has not been demonstrated definitively on the cell surface by mass spectrometry; thus, it is not yet considered appropriate for use in human melanoma vaccines. Interestingly, however, the antigenicity of this peptide was affected by spontaneous modifications at two distinct residues. Spontaneous modification of the QIVDVCHDV peptide can occur at the cysteine residue at position 6 or at the N-terminal glutamine residue, and both modifications dramatically affect CTL recognition. Avoidance of an acidic environment prevents the conversion of the N-terminal glutamine residue to pyroglutamic acid, a conversion that inhibits binding of the peptide to HLA-A2 and diminishes recognition by CTLs. Substitution of asparagine for the N-terminal glutamine and substitution of serine for the cysteine were shown to enhance the binding of the peptide to HLA-A2 and to enhance the recognition of the peptide by CTLs. These findings suggest general strategies for enhancing the antigenicity of other peptides containing similar amino acids in their sequence.
Asunto(s)
Vacunas contra el Cáncer , Cisteína/química , Glutamina/química , Antígeno HLA-A2/química , Inmunoterapia/métodos , Péptidos/química , Antígenos de Neoplasias/química , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , ARN Helicasas DEAD-box , Bases de Datos como Asunto , Epítopos/química , Antígenos de Histocompatibilidad Clase I/química , Humanos , Espectrometría de Masas , Melanoma/inmunología , Melanoma/metabolismo , Modelos Químicos , Proteínas Quinasas/química , Estructura Terciaria de Proteína , ARN Helicasas/química , Linfocitos T Citotóxicos/metabolismoRESUMEN
The Nek family of protein kinases in humans is composed of 11 members that share an amino-terminal catalytic domain related to NIMA, an Aspergillus kinase involved in the control of several aspects of mitosis, and divergent carboxyl-terminal tails of varying length. Nek6 (314AA) and Nek7 (303AA), 76% identical, have little noncatalytic sequence but bind to the carboxyl-terminal noncatalytic tail of Nercc1/Nek9, a NIMA family protein kinase that is activated in mitosis. Microinjection of anti-Nercc1 antibodies leads to spindle abnormalities and prometaphase arrest or chromosome missegregation. Herein we show that Nek6 is increased in abundance and activity during mitosis; activation requires the phosphorylation of Ser206 on the Nek6 activation loop. This phosphorylation and the activity of recombinant Nek6 is stimulated by coexpression with an activated mutant of Nercc1. Moreover, Nercc1 catalyzes the direct phosphorylation of prokaryotic recombinant Nek6 at Ser206 in vitro concomitant with 20-25-fold activation of Nek6 activity; Nercc1 activates Nek7 in vitro in a similar manner. Nercc1/Nek9 is likely to be responsible for the activation of Nek6 during mitosis and probably participates in the regulation of Nek7 as well. These findings support the conclusion that Nercc1/Nek9 and Nek6 represent a novel cascade of mitotic NIMA family protein kinases whose combined function is important for mitotic progression.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Activación Enzimática , Humanos , Cinética , Ratones , Mitosis , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Quinasa 1 Relacionada con NIMA , Quinasas Relacionadas con NIMA , Fosforilación , Reacción en Cadena de la Polimerasa , Proteínas Serina-Treonina Quinasas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Type 1 diabetes is an autoimmune disease in which autoreactive T cells attack and destroy the insulin-producing pancreatic beta cells. CD8+ T cells are essential for this beta cell destruction, yet their specific antigenic targets are largely unknown. Here, we reveal that the autoantigen targeted by a prevalent population of pathogenic CD8+ T cells in nonobese diabetic mice is islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP). Through tetramer technology, IGRP-reactive T cells are readily detected in islets and peripheral blood directly ex vivo. The human IGRP gene maps to a diabetes susceptibility locus, suggesting that IGRP also may be an antigen for pathogenic T cells in human type 1 diabetes and, thus, a new, potential target for diagnostic and therapeutic approaches.
Asunto(s)
Autoantígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Glucosa-6-Fosfatasa , Islotes Pancreáticos/inmunología , Proteínas/inmunología , Animales , Autoantígenos/genética , Células COS , Chlorocebus aethiops , Cromatografía de Afinidad , Células Clonales/inmunología , Modelos Animales de Enfermedad , Genes Codificadores de la Cadena alfa de los Receptores de Linfocito T , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T , Antígenos H-2/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Fragmentos de Péptidos/inmunología , Proteínas/genética , Organismos Libres de Patógenos Específicos , TransfecciónRESUMEN
Minor histocompatibility (H) antigens crucially affect the outcome of human leukocyte antigen (HLA)-identical allogeneic stem cell transplantation (SCT). To understand the basis of alloimmune responses against minor H antigens, identification of minor H peptides and their antigenicity-determining mechanisms is essential. Here we report the identification of HA-3 and its encoding gene. The HA-3 peptide, VTEPGTAQY (HA-3T), is encoded by the lymphoid blast crisis (Lbc) oncogene. We thus show for the first time that a leukemia-associated oncogene can give rise to immunogenic T-cell epitopes that may have participated in antihost and antileukemic alloimmune responses. Genotypic analysis of HA-3- individuals revealed the allelic counterpart VMEPGTAQY (HA-3M). Despite the lack of T-cell recognition of HA-3- cells, the Thr-->Met substitution had only a modest effect on peptide binding to HLA-A1 and a minimal impact on recognition by T cells when added exogenously to target cells. This substitution did not influence transporter associated with antigen processing (TAP) transport, but, in contrast to the HA-3T peptide, HA-3M is destroyed by proteasome-mediated digestion. Thus, the immunogenicity of minor H antigens can result from proteasome-mediated destruction of the negative allelic peptide.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Epítopos de Linfocito T/genética , Complejos Multienzimáticos/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas/genética , Proteínas de Anclaje a la Quinasa A , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Transportadoras de Casetes de Unión a ATP/metabolismo , Enfermedad Aguda , Proteínas Adaptadoras Transductoras de Señales , Alelos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Presentación de Antígeno , Linfocitos T CD8-positivos/inmunología , Células Clonales/inmunología , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Femenino , Genotipo , Antígeno HLA-A1/metabolismo , Humanos , Leucemia Mieloide/inmunología , Leucemia Mieloide/terapia , Masculino , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Linaje , Trasplante de Células Madre de Sangre Periférica , Polimorfismo Genético , Complejo de la Endopetidasa Proteasomal , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The fundamental unit of eukaryotic chromatin, the nucleosome, consists of genomic DNA wrapped around the conserved histone proteins H3, H2B, H2A and H4, all of which are variously modified at their amino- and carboxy-terminal tails to influence the dynamics of chromatin structure and function -- for example, conjugation of histone H2B with ubiquitin controls the outcome of methylation at a specific lysine residue (Lys 4) on histone H3, which regulates gene silencing in the yeast Saccharomyces cerevisiae. Here we show that ubiquitination of H2B is also necessary for the methylation of Lys 79 in H3, the only modification known to occur away from the histone tails, but that not all methylated lysines in H3 are regulated by this 'trans-histone' pathway because the methylation of Lys 36 in H3 is unaffected. Given that gene silencing is regulated by the methylation of Lys 4 and Lys 79 in histone H3, we suggest that H2B ubiquitination acts as a master switch that controls the site-selective histone methylation patterns responsible for this silencing.
Asunto(s)
Cromatina/metabolismo , Regulación Fúngica de la Expresión Génica , Silenciador del Gen , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Cromatina/química , N-Metiltransferasa de Histona-Lisina , Histonas/química , Ligasas/metabolismo , Metilación , Modelos Biológicos , Proteínas Nucleares/metabolismo , Enzimas Ubiquitina-ConjugadorasRESUMEN
Recent studies of histone methylation have yielded fundamental new insights pertaining to the role of this modification in gene activation as well as in gene silencing. While a number of methylation sites are known to occur on histones, only limited information exists regarding the relevant enzymes that mediate these methylation events. We thus sought to identify native histone methyltransferase (HMT) activities from Saccharomyces cerevisiae. Here, we describe the biochemical purification and characterization of Set2, a novel HMT that is site-specific for lysine 36 (Lys36) of the H3 tail. Using an antiserum directed against Lys36 methylation in H3, we show that Set2, via its SET domain, is responsible for methylation at this site in vivo. Tethering of Set2 to a heterologous promoter reveals that Set2 represses transcription, and part of this repression is mediated through the HMT activity of the SET domain. These results suggest that Set2 and methylation at H3 Lys36 play a role in the repression of gene transcription.