RESUMEN
Administration of vaccines by the nasal route has recently proven to be one of the most efficient ways for inducing both mucosal and systemic antibody responses in experimental animals. Our results demonstrate that P40, a well-defined outer membrane protein A from Klebsiella pneumoniae, is indeed a carrier molecule suitable for nasal immunization. Using fragments from the respiratory syncytial virus subgroup A (RSV-A) G protein as antigen models, it has been shown that P40 is able to induce both systemic and mucosal immunity when fused or coupled to a protein or a peptide and administered intranasally (i.n.) to naive or K. pneumoniae-primed mice. Confocal analyses of nasal mucosa-associated lymphoid tissue after i.n. instillation of P40 showed that this molecule is able to cross the nasal epithelium and target CD11c-positive cells likely to be murine dendritic cells or macrophages. More importantly, this targeting of antigen-presenting cells following i.n. immunization with a subunit of the RSV-A molecule in the absence of any mucosal adjuvant results in both upper and lower respiratory tract protection against RSV-A infection.
Asunto(s)
Adyuvantes Inmunológicos , Células Presentadoras de Antígenos/inmunología , Antígenos Virales/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Klebsiella pneumoniae/inmunología , Infecciones por Virus Sincitial Respiratorio/prevención & control , Vacunas contra Virus Sincitial Respiratorio/inmunología , Vacunas Sintéticas/inmunología , Proteínas Virales/inmunología , Administración Intranasal , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Transporte Biológico , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunidad Mucosa , Tejido Linfoide/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mucosa Nasal/inmunología , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Infecciones por Virus Sincitial Respiratorio/inmunología , Vacunación/métodosRESUMEN
Several cytotoxic T lymphocyte peptide-based vaccines against hepatitis B, human immunodeficiency virus and melanoma were recently studied in clinical trials. One interesting melanoma vaccine candidate alone or in combination with other tumor antigens, is the decapeptide ELA. This peptide is a Melan-A/MART-1 antigen immunodominant peptide analog, with an N-terminal glutamic acid. It has been reported that the amino group and gamma-carboxylic group of glutamic acids, as well as the amino group and gamma-carboxamide group of glutamines, condense easily to form pyroglutamic derivatives. To overcome this stability problem, several peptides of pharmaceutical interest have been developed with a pyroglutamic acid instead of N-terminal glutamine or glutamic acid, without loss of pharmacological properties. Unfortunately compared with ELA, the pyroglutamic acid derivative (PyrELA) and also the N-terminal acetyl-capped derivative (AcELA) failed to elicit cytotoxic T lymphocyte (CTL) activity. Despite the apparent minor modifications introduced in PyrELA and AcELA, these two derivatives probably have lower affinity than ELA for the specific class I major histocompatibility complex. Consequently, in order to conserve full activity of ELA, the formation of PyrELA must be avoided. Furthermore, this stability problem is worse in the case of clinical grade ELA, produced as an acetate salt, like most of the pharmaceutical grade peptides. We report here that the hydrochloride salt, shows higher stability than the acetate salt and may be suitable for use in man. Similar stability data were also obtained for MAGE-3, another N-terminal glutamic acid containing CTL peptide in clinical development, leading us to suggest that all N-terminal glutamic acid and probably glutamine-containing CTL peptide epitopes may be stabilized as hydrochloride salts.
Asunto(s)
Antígenos de Neoplasias , Ácido Glutámico/química , Isoantígenos/metabolismo , Melanoma/inmunología , Fragmentos de Péptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Resinas de Intercambio Aniónico , Vacunas contra el Cáncer/inmunología , Línea Celular/inmunología , Línea Celular/metabolismo , Cromatografía Líquida de Alta Presión , Cromo/metabolismo , Epítopos de Linfocito T , Granulocitos , Humanos , Inmunización , Ratones , Proteínas de Neoplasias/química , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/metabolismo , Péptidos/síntesis química , Péptidos/química , Péptidos/metabolismo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Respiratory syncytial virus (RSV) is a major respiratory pathogen responsible for severe pulmonary disease. We have developed a parenterally administered vaccine, BBG2Na, which is currently in a phase III clinical trial. BBG2Na comprises residues 130--230 of RSV-A G protein (G2Na) fused to the BB carrier protein. In this study, we show that BBG2Na can be delivered by the nasal route and generates both mucosal and systemic antibody responses when co-administered with cholera toxin B or a newly described delivery system, zwittergent 3--14. We found that nasal BBG2Na administration protects against RSV challenge and does not induce lung immunopathology upon subsequent RSV challenge.
Asunto(s)
Vacunas contra Virus Sincitial Respiratorio/administración & dosificación , Virus Sincitiales Respiratorios/inmunología , Administración Intranasal , Animales , Anticuerpos Antivirales/biosíntesis , Toxina del Cólera/administración & dosificación , Femenino , Proteína HN/inmunología , Humanos , Inmunidad Mucosa , Inyecciones Intramusculares , Ratones , Ratones Endogámicos BALB C , Mucosa Nasal/efectos de los fármacos , Mucosa Nasal/patología , Compuestos de Amonio Cuaternario/administración & dosificación , Compuestos de Amonio Cuaternario/toxicidad , Vacunas de Subunidad/administración & dosificación , Proteínas del Envoltorio ViralRESUMEN
Respiratory syncytial virus (RSV) is an important respiratory pathogen in man, against which no vaccine is available. However, recent evidence suggests that antibodies to the RSV F and G proteins may play an important role in disease prevention. We previously demonstrated that BBG2Na, a subunit vaccine candidate including residues 130-230 of the Long strain G protein, protects rodents against RSV challenge. Using a panel of monoclonal antibodies (MAb) and synthetic peptides, five linear B cell epitopes were identified that mapped to residues 152-163, 165-172, 171-187 (two over-lapping epitopes) and 196-204. Antibody passive transfer and peptide immunisation studies revealed that all were protective. Pepscan analyses of anti-RSV-A and BBG2Na murine polyclonal sera suggested stronger immunogenicity of some protective epitopes (protectopes) in the context of BBG2Na compared with live virus. However, all the identified murine B cell protectopes were conserved in RSV seropositive humans. Should these protectopes correspond with protection in humans, BBG2Na may constitute a very interesting vaccine candidate against RSV.
Asunto(s)
Linfocitos B/inmunología , Virus Sincitiales Respiratorios/inmunología , Proteínas Virales/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Anticuerpos Antivirales , Antígenos Virales/genética , Mapeo Epitopo , Epítopos , Humanos , Inmunización Pasiva , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Virus Sincitiales Respiratorios/genética , Vacunas de Subunidad/genética , Vacunas de Subunidad/farmacología , Proteínas Virales/genética , Vacunas Virales/genética , Vacunas Virales/farmacologíaRESUMEN
The decapeptide ELA (ELAGIGILTV), a Melan-A/MART-1 antigen immunodominant peptide analogue, is an interesting melanoma vaccine candidate alone or in combination with other tumour antigens. P40, the recombinant outer membrane protein A of Klebsiella pneumoniae (kpOmpA), was recently shown to target dendritic cells and to induce peptide-specific CTLs. Here we investigated the adjuvant role of P40 mixed or chemically conjugated to ELA. This compound is an N-terminal glutamic acid-containing peptide. However, it has been reported that the amino group and the gamma-carboxylic group of glutamic acids easily condense to form pyroglutamic derivatives. Usually, to overcome this stability problem, peptides of pharmaceutical interest were developed with a pyroglutamic acid instead of N-terminal glutamic acid, without loss of pharmacological properties. Unfortunately, the pyroglutamic acid derivative (PyrELA) as well as the N-terminal acetyl capped derivative (AcELA) failed to elicit CTL activity when mixed with P40 adjuvant protein. Despite the apparent minor modifications introduced by PyrELA and AcELA, these two derivatives have probably lower affinity than ELA for the class I Major Histocompatibility Complex. Furthermore, this stability problem is worse in the case of clinical grade ELA, produced as an acetate salt, like most of the pharmaceutical grade peptides. We report here that the hydrochloride shows a higher stability than the acetate and may be suitable for use in man.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Melanoma/terapia , Linfocitos T Citotóxicos/inmunología , Animales , Vacunas contra el Cáncer/química , Vacunas contra el Cáncer/uso terapéutico , Cromatografía Líquida de Alta Presión , Melanoma/inmunología , Ratones , Ratones Transgénicos , Modelos MolecularesRESUMEN
CD86 is an important costimulatory molecule for the priming and activation of naive and memory T cells, respectively. Here, we show that soluble CD86 is detected in human serum. Soluble CD86 is produced by resting monocytes and results from an alternatively spliced transcript (CD86deltaTM) characterized by deletion of the transmembrane domain. Recombinant CD86deltaTM binds to CD28 and CTLA-4 and induces the activation of T cells after stimulation with anti-CD3 mAb. CD86deltaTM also induces IFNgamma production by virus-specific CD8+ memory human T cells stimulated with the Flu M1 peptide. The concentrations of soluble CD86 found in human serum are sufficient to induce biological activity. Soluble CD86 molecule, therefore, appears to be a functional costimulatory molecule playing a potentially important role in immune surveillance.
Asunto(s)
Antígenos CD/fisiología , Activación de Linfocitos/inmunología , Glicoproteínas de Membrana/fisiología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Antígenos CD/biosíntesis , Antígenos CD/sangre , Antígenos CD/genética , Antígeno B7-2 , Células COS , Epítopos de Linfocito T/inmunología , Humanos , Memoria Inmunológica/genética , Interfase/inmunología , Células Jurkat , Activación de Linfocitos/genética , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/sangre , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Monocitos/inmunología , Monocitos/metabolismo , Empalme del ARN/inmunología , ARN Mensajero/biosíntesis , ARN Mensajero/metabolismo , Proteínas Recombinantes/biosíntesis , Solubilidad , Transcripción Genética/inmunología , Transfección/inmunologíaRESUMEN
The immunogenicity and protective efficacy of BBG2Na, a novel recombinant respiratory syncytial virus subunit vaccine candidate, was assessed in BALB/c mice under various conditions of dose, administration route and number of immunisations. A single intra-peritoneal (i.p.) dose of 2 microg, or two doses of 0.2 microg, were sufficient to induce elevated RSV-A serum antibodies and sterilising lung protective immunity. Serum antibody titres were significantly boosted following second immunisations, but not a third. Of three routes of immunisation, i.p. induced the highest RSV-A antibody titres, followed in efficacy by the intra-muscular (i. m.) and subcutaneous (s.c.) routes. Nonetheless, all three routes induced comparable and sterilising lung protection. In contrast, upper respiratory tract protection was observed only after i.p. vaccination, although significant viral titre reductions were evident following i.m. or s.c. immunisations. Interestingly, Pepscan analyses indicated that antibody epitope usage was highest in i.p. and lowest in i.m. immunised mice, respectively. Nonetheless, all routes resulted in antibody responses to known lung protective epitopes (protectopes). Thus, the prevention of serious lower respiratory tract disease, the principle goal of a RSV vaccine, but not URT infection, is dose dependent but unlikely to be influenced by the route of BBG2Na administration.
Asunto(s)
Antígenos Virales/inmunología , Proteínas Bacterianas/inmunología , Proteína HN , Virus Sincitiales Respiratorios/inmunología , Proteínas del Envoltorio Viral/inmunología , Proteínas Virales de Fusión/inmunología , Proteínas Virales/inmunología , Vacunas Virales/administración & dosificación , Animales , Anticuerpos Antivirales/biosíntesis , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Inyecciones Intraperitoneales , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/inmunología , Virus Sincitiales Respiratorios/aislamiento & purificación , Vacunas Virales/inmunologíaRESUMEN
We analyzed the interaction between a bacterial cell wall protein and dendritic cells (DCs). Outer membrane protein A from Klebsiella pneumoniae (kpOmpA) specifically bound to professional antigen presenting cells and was endocytosed by immature DCs via a receptor-dependent mechanism. kpOmpA signaled through Toll-like receptor 2, induced DCs to produce interleukin 12 and induced maturation of DCs. Whole antigen that was coupled to kpOmpA and injected into mice was taken up by DCs and delivered to the conventional cytosolic MHC class I presentation pathway. kpOmpA also primed antigen-specific CD8+ CTLs in the absence of CD4+ T cell help or adjuvant and elicited therapeutic immunity to antigen-expressing tumors. Thus, OmpA belongs to a class of proteins that are able to elicit CTL responses to exogenous antigen.
Asunto(s)
Presentación de Antígeno , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Células Dendríticas/citología , Células Dendríticas/inmunología , Proteínas de Drosophila , Animales , Células Presentadoras de Antígenos/citología , Células Presentadoras de Antígenos/inmunología , Diferenciación Celular , Línea Celular , Endocitosis , Femenino , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Klebsiella pneumoniae/inmunología , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/terapia , Ovalbúmina/inmunología , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Linfocitos T Citotóxicos/inmunología , Receptor Toll-Like 2 , Receptores Toll-LikeAsunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/inmunología , Klebsiella pneumoniae/química , Klebsiella pneumoniae/inmunología , Péptidos/química , Péptidos/inmunología , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/inmunología , Vacunas Conjugadas/química , Vacunas Conjugadas/inmunología , Animales , Proteínas Portadoras/química , Fenómenos Químicos , Química Física , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/metabolismo , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/química , Ratones , Ratones Endogámicos BALB CRESUMEN
We have recently cloned a new protein, recombinant P40 (rP40). When tested in vivo after conjugation to a B-cell epitope, rP40 induces an important antibody response without the need for adjuvant. To characterize its potency, this carrier protein was coupled to a peptide derived from respiratory syncytial virus attachment G protein (G1'). After immunization of mice with the rP40-G1' conjugate, strong antipeptide antibodies were detected, whereas peptide alone was not immunogenic. To emphasize the carrier properties of rP40, a polysaccharide derived from Haemophilus influenzae type b (Hib) was coupled to it. Immunoglobulin G responses against the Hib polysaccharide were observed after coupling to rP40. Interestingly, an antipeptide antibody response was observed despite preexisting anti-rP40 antibodies generated by preimmunization with rP40. In addition, rP40 compares well with the reference carrier protein, tetanus toxoid (TT), since antibody responses of equal intensity were observed when a peptide or a polysaccharide was coupled to TT and rP40. Moreover, rP40 had advantages compared to TT; e.g., it induced a mixed Th1/Th2 response, whereas TT induced only a Th2 profile. Together, the results indicate that rP40 is a novel carrier protein with potential for use as an alternative carrier for human vaccination.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Portadoras/inmunología , Proteína HN , Klebsiella pneumoniae/química , Animales , Anticuerpos Antibacterianos/sangre , Cápsulas Bacterianas , Femenino , Vacunas contra Haemophilus/inmunología , Inmunización , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Polisacáridos Bacterianos/inmunología , Proteínas Recombinantes/inmunología , Toxina Tetánica/inmunología , Proteínas del Envoltorio Viral , Proteínas Virales/inmunologíaRESUMEN
A recombinant fusion protein (BBG2Na) comprising the central conserved domain of the respiratory syncytial virus subgroup A (RSV-A) (Long) G protein (residues 130 to 230) and an albumin binding domain of streptococcal protein G was shown previously to protect mouse upper (URT) and lower (LRT) respiratory tracts against intranasal RSV challenge (U. F. Power, H. Plotnicky-Gilquin, T. Huss, A. Robert, M. Trudel, S. Stahl, M. Uhlén, T. N. Nguyen, and H. Binz, Virology 230:155-166, 1997). Panels of monoclonal antibodies (MAbs) and synthetic peptides were generated to facilitate dissection of the structural elements of this domain implicated in protective efficacy. All MAbs recognized native RSV-A antigens, and five linear B-cell epitopes were identified; these mapped to residues 152 to 163, 165 to 172, 171 to 187 (two overlapping epitopes), and 196 to 204, thereby covering the highly conserved cysteine noose domain. Antibody passive-transfer and peptide immunization studies revealed that all epitopes were implicated in protection of the LRT, but not likely the URT, against RSV-A challenge. Pepscan analyses of anti-RSV-A and anti-BBG2Na murine polyclonal sera revealed lower-level epitope usage within the central conserved region in the former, suggesting diminished immunogenicity of the implicated epitopes in the context of the whole virus. However, Pepscan analyses of RSV-seropositive human sera revealed that all of the murine B-cell protective epitopes (protectopes) that mapped to the central conserved domain were recognized in man. Should these murine protectopes also be implicated in human LRT protection, their clustering around the highly conserved cysteine noose region will have important implications for the development of RSV vaccines.
Asunto(s)
Antígenos Virales/inmunología , Epítopos de Linfocito B/inmunología , Proteína HN , Infecciones por Virus Sincitial Respiratorio/inmunología , Virus Sincitial Respiratorio Humano/inmunología , Proteínas Virales/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Sitios de Unión , Modelos Animales de Enfermedad , Mapeo Epitopo , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Péptidos/síntesis química , Péptidos/inmunología , Proteínas Recombinantes de Fusión/inmunología , Infecciones por Virus Sincitial Respiratorio/prevención & control , Proteínas del Envoltorio ViralRESUMEN
Using phage-display technology, a novel binding protein (Z-affibody) showing selective binding to the RSV (Long strain) G protein was selected from a combinatorial library of a small alpha-helical protein domain (Z), derived from staphylococcal protein A (SPA). Biopanning of the Z-library against a recombinant fusion protein comprising amino acids 130-230 of the G protein from RSV-subgroup A, resulted in the selection of a Z-affibody (Z(RSV1)) which showed G protein specific binding. Using biosensor technology, the affinity (K(D)) between Z(RSV1) and the recombinant protein was determined to be in the micromolar range (10(-6) M). Interestingly, the Z(RSV1) affibody was demonstrated to also recognize the partially (54%) homologous G protein of RSV subgroup B with similar affinity. Using different recombinant RSV G protein derived fragments, the binding was found to be dependent on the presence of the cysteinyl residues proposed to be involved in the formation of an intramolecular disulfide-constrained loop structure, indicating a conformation-dependent binding. Results from epitope mapping studies, employing a panel of monoclonal antibodies directed to different RSV G protein subfragments, suggest that the Z(RSV1) affibody binding site is located within the region of amino acids 164-186 of the G protein. This region contains a 13 amino acid residue sequence which is totally conserved between subgroups A and B of RSV and extends into the cystein loop region (amino acids 173-186). The potential use of the RSV G protein-specific Z(RSV1) affibody in diagnostic and therapeutic applications is discussed.
Asunto(s)
Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Proteína HN , Péptidos/química , Péptidos/inmunología , Virus Sincitial Respiratorio Humano/inmunología , Proteínas Virales/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Bacteriófagos/genética , Técnicas Biosensibles , Mapeo Epitopo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/inmunología , Virus Sincitial Respiratorio Humano/química , Proteína Estafilocócica A/química , Proteína Estafilocócica A/inmunología , Proteínas del Envoltorio ViralRESUMEN
Cyclin-dependent kinase (Cdk) mutations that prevent entry into the mitotic cell cycle of budding yeast fail to block meiotic DNA replication, suggesting there may be fundamental differences between these pathways. However, S phase in meiosis was found to depend on the same B-type cyclins (Clb5 and Clb6) as it does in mitosis. Meiosis differs instead in the mechanism that controls removal of the Cdk inhibitor Sic1. Destruction of Sic1 and activation of a Clb5-dependent kinase in meiotic cells required the action of the meiosis-specific protein kinase Ime2, thereby coupling early meiotic gene expression to control of DNA replication for meiosis.
Asunto(s)
Proteínas de Ciclo Celular , Ciclina B , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Proteínas Fúngicas/metabolismo , Meiosis , Proteínas Quinasas/metabolismo , Fase S , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citología , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Replicación del ADN , Inhibidores Enzimáticos/metabolismo , Proteínas Fúngicas/genética , Genes Fúngicos , Péptidos y Proteínas de Señalización Intracelular , Mutación , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Surface display of recombinant proteins on bacteria and phages has become an important tool in bioscience. To evaluate the various host systems, a great need exists for quantitative methods to determine the densities of displayed proteins and peptides on the bacteria and phage surfaces. Here we describe how a method previously applied for quantification of surface proteins on mammalian cells has been adapted for quantification of chimeric receptors surface-displayed on bacteria; in this study, the bacteria being recombinant staphylococci. The presented method takes advantage of fluorescence-activated cell sorting (FACS) technology and a new type of nonfluorescent plastic beads, similar in size (2 microns in diameter) to bacterial cells, and thus suitable for generation of calibration curves from which the number of chimeric receptors can be obtained. The method was used to estimate the number of antigenic sites on two types of recombinant staphylococci, both carrying heterologous chimeric receptors, and it was found that the recombinant Staphylococcus carnosus cells carried approximately 10(4) surface-displayed antigenic sites, while recombinant Staphylococcus xylosus exposed approximately 3 x 10(3) sites per cell. The use of the deviced method for different applications is discussed.
Asunto(s)
Citometría de Flujo , Proteínas Recombinantes/análisis , Staphylococcus/química , Animales , Anticuerpos Monoclonales , Proteínas Bacterianas/genética , Membrana Celular/química , Femenino , Expresión Génica , Vectores Genéticos , Ratones , Ratones Endogámicos BALB C , Microesferas , Receptores de Albúmina/análisis , Receptores de Albúmina/genética , Albúmina Sérica/metabolismoRESUMEN
Regulation of cell cycle progression occurs in part through the targeted degradation of both activating and inhibitory subunits of the cyclin-dependent kinases. During G1, CDC4, encoding a WD-40 repeat protein, and CDC34, encoding a ubiquitin-conjugating enzyme, are involved in the destruction of these regulators. Here we describe evidence indicating that CDC53 also is involved in this process. Mutations in CDC53 cause a phenotype indistinguishable from those of cdc4 and cdc34 mutations, numerous genetic interactions are seen between these genes, and the encoded proteins are found physically associated in vivo. Cdc53p defines a large family of proteins found in yeasts, nematodes, and humans whose molecular functions are uncharacterized. These results suggest a role for this family of proteins in regulating cell cycle proliferation through protein degradation.
Asunto(s)
Proteína Quinasa CDC2/genética , Proteínas de Ciclo Celular/genética , Ciclo Celular , Proteínas F-Box , Fase G1/genética , Fase S/genética , Saccharomyces cerevisiae/genética , Proteína p53 Supresora de Tumor/genética , Ubiquitina-Proteína Ligasas , Secuencia de Aminoácidos , Secuencia de Bases , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Mutación , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae , Alineación de SecuenciaRESUMEN
Previously we demonstrated that calmodulin binds to the carboxy terminus of Spc110p, an essential component of the Saccharomyces cerevisiae spindle pole body (SPB), and that this interaction is required for chromosome segregation. Immunoelectron microscopy presented here shows that calmodulin and thus the carboxy terminus of Spc110p localize to the central plaque. We created temperature-sensitive SPC110 mutations by combining PCR mutagenesis with a plasmid shuffle strategy. The temperature-sensitive allele spc110-220 differs from wild type at two sites. The cysteine 911 to arginine mutation resides in the calmodulin-binding site and alone confers a temperature-sensitive phenotype. Calmodulin overproduction suppresses the temperature sensitivity of spc110-220. Furthermore, calmodulin levels at the SPB decrease in the mutant cells at the restrictive temperature. Thus, calmodulin binding to Spc110-220p is defective at the nonpermissive temperature. Synchronized mutant cells incubated at the nonpermissive temperature arrest as large budded cells with a G2 content of DNA and suffer considerable lethality. Immunofluorescent staining demonstrates failure of nuclear DNA segregation and breakage of many spindles. Electron microscopy reveals an aberrant nuclear structure, the intranuclear microtubule organizer (IMO), that differs from a SPB but serves as a center of microtubule organization. The IMO appears during nascent SPB formation and disappears after SPB separation. The IMO contains both the 90-kD and the mutant 110-kD SPB components. Our results suggest that disruption of the calmodulin Spc110p interaction leads to the aberrant assembly of SPB components into the IMO, which in turn perturbs spindle formation.
Asunto(s)
Calmodulina/metabolismo , Proteínas Fúngicas/metabolismo , Microtúbulos/química , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Huso Acromático/química , Sitios de Unión , Calmodulina/análisis , Calmodulina/biosíntesis , Proteínas de Unión a Calmodulina , Ciclo Celular/fisiología , Núcleo Celular/química , Cromosomas Fúngicos , Proteínas del Citoesqueleto , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Microtúbulos/ultraestructura , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrollo , Huso Acromático/ultraestructura , Supresión Genética , TemperaturaRESUMEN
The Saccharomyces cerevisiae CDC3, CDC10, CDC11, and CDC12 genes encode a family of related proteins, the septins, which are involved in cell division and the organization of the cell surface during vegetative growth. A search for additional S. cerevisiae septin genes using the polymerase chain reaction identified SPR3, a gene that had been identified previously on the basis of its sporulation-specific expression. The predicted SPR3 product shows 25-40% identity in amino acid sequence to the previously known septins from S. cerevisiae and other organisms. Immunoblots confirmed the sporulation-specific expression of Spr3p and showed that other septins are also present at substantial levels in sporulating cells. Consistent with the expression data, deletion of SPR3 in either of two genetic backgrounds had no detectable effect on exponentially growing cells. In one genetic background, deletion of SPR3 produced a threefold reduction in sporulation efficiency, although meiosis appeared to be completed normally. In this background, deletion of CDC10 had no detectable effect on sporulation. In the other genetic background tested, the consequences of the two deletions were reversed. Immunofluorescence observations suggest that Spr3p, Cdc3p, and Cdc11p are localized to the leading edges of the membrane sacs that form near the spindle-pole bodies and gradually extend to engulf the nuclear lobes that contain the haploid chromosome sets, thus forming the spores. Deletion of SPR3 does not prevent the localization of Cdc3p and Cdc11p, but these proteins appear to be less well organized, and the intensity of their staining is reduced. Taken together, the results suggest that the septins play important but partially redundant roles during the process of spore formation.
Asunto(s)
Proteínas de Ciclo Celular/biosíntesis , Proteínas del Citoesqueleto , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas de Ciclo Celular/análisis , Proteínas de Ciclo Celular/aislamiento & purificación , División Celular , Cartilla de ADN , Proteínas Fúngicas/biosíntesis , GTP Fosfohidrolasas , Genotipo , Proteínas de la Membrana , Datos de Secuencia Molecular , Familia de Multigenes , Mutagénesis , Reacción en Cadena de la Polimerasa , Profilinas , Proteínas Recombinantes/biosíntesis , Mapeo Restrictivo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Schizosaccharomyces pombe , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Esporas Fúngicas , Factores de TranscripciónRESUMEN
The transition from G1 to S phase of the cell cycle in Saccharomyces cerevisiae requires the activity of the Ubc3 (Cdc34) ubiquitin-conjugating enzyme. S. cerevisiae cells lacking a functional UBC3 (CDC34) gene are able to execute the Start function that initiates the cell cycle but fail to form a mitotic spindle or enter S phase. The Ubc3 (Cdc34) enzyme has previously been shown to catalyze the attachment of multiple ubiquitin molecules to model substrates, suggesting that the role of this enzyme in cell cycle progression depends on its targeting an endogenous protein(s) for degradation. In this report, we demonstrate that the Ubc3 (Cdc34) protein is itself a substrate for both ubiquitination and phosphorylation. Immunochemical localization of the gene product to the nucleus renders it likely that the relevant substrates similarly reside within the nucleus.
Asunto(s)
Ciclo Celular/genética , Ligasas/biosíntesis , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Complejos de Ubiquitina-Proteína Ligasa , Ubiquitinas/metabolismo , Ciclosoma-Complejo Promotor de la Anafase , Western Blotting , Núcleo Celular/enzimología , Núcleo Celular/ultraestructura , Proteínas Fúngicas/biosíntesis , Fase G1 , Genes Fúngicos , Ligasas/análisis , Ligasas/genética , Fosforilación , Plásmidos , Mapeo Restrictivo , Fase S , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae , Eliminación de Secuencia , Enzimas Ubiquitina-Conjugadoras , Ubiquitina-Proteína LigasasRESUMEN
The function of the essential MIF2 gene in the Saccharomyces cerevisiae cell cycle was examined by overepressing or creating a deficit of MIF2 gene product. When MIF2 was overexpressed, chromosomes missegregated during mitosis and cells accumulated in the G2 and M phases of the cell cycle. Temperature sensitive mutants isolated by in vitro mutagenesis delayed cell cycle progression when grown at the restrictive temperature, accumulated as large budded cells that had completed DNA replication but not chromosome segregation, and lost viability as they passed through mitosis. Mutant cells also showed increased levels of mitotic chromosome loss, supersensitivity to the microtubule destabilizing drug MBC, and morphologically aberrant spindles. mif2 mutant spindles arrested development immediately before anaphase spindle elongation, and then frequently broke apart into two disconnected short half spindles with misoriented spindle pole bodies. These findings indicate that MIF2 is required for structural integrity of the spindle during anaphase spindle elongation. The deduced Mif2 protein sequence shared no extensive homologies with previously identified proteins but did contain a short region of homology to a motif involved in binding AT rich DNA by the Drosophila D1 and mammalian HMGI chromosomal proteins.
Asunto(s)
Anafase , Carbamatos , Proteínas de Unión al ADN , Proteínas Fúngicas/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Huso Acromático/fisiología , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bencimidazoles/farmacología , Northern Blotting , Ciclo Celular , Mapeo Cromosómico , Cromosomas/química , Cromosomas/fisiología , Cromosomas/ultraestructura , Clonación Molecular , ADN/análisis , ADN/genética , Citometría de Flujo , Proteínas Fúngicas/análisis , Proteínas Fúngicas/genética , Expresión Génica/genética , Microtúbulos/efectos de los fármacos , Datos de Secuencia Molecular , Mutágenos/farmacología , Mutación/genética , Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/ultraestructura , Huso Acromático/química , Huso Acromático/ultraestructura , TemperaturaRESUMEN
The spindle pole body (SPB) of Saccharomyces cerevisiae serves as the centrosome in this organism, undergoing duplication early in the cell cycle to generate the two poles of the mitotic spindle. The conditional lethal mutation ndc1-1 has previously been shown to cause asymmetric segregation, wherein all the chromosomes go to one pole of the mitotic spindle (Thomas, J. H., and D. Botstein. 1986. Cell. 44:65-76). Examination by electron microscopy of mutant cells subjected to the nonpermissive temperature reveals a defect in SPB duplication. Although duplication is seen to occur, the nascent SPB fails to undergo insertion into the nuclear envelope. The parental SPB remains functional, organizing a monopolar spindle to which all the chromosomes are presumably attached. Order-of-function experiments reveal that the NDC1 function is required in G1 after alpha-factor arrest but before the arrest caused by cdc34. Molecular analysis shows that the NDC1 gene is essential and that it encodes a 656 amino acid protein (74 kD) with six or seven putative transmembrane domains. This evidence for membrane association is further supported by immunofluorescent localization of the NDC1 product to the vicinity of the nuclear envelope. These findings suggest that the NDC1 protein acts within the nuclear envelope to mediate insertion of the nascent SPB.