RESUMEN
The 2,5-Diketopiperazines (DKPs) constitute a large family of natural products with important biological activities. Bicyclomycin is a clinically-relevant DKP antibiotic that is the first and only member in a class known to target the bacterial transcription termination factor Rho. It derives from cyclo-(L-isoleucyl-L-leucyl) and has an unusual and highly oxidized bicyclic structure that is formed by an ether bridge between the hydroxylated terminal carbon atom of the isoleucine lateral chain and the alpha carbon of the leucine in the diketopiperazine ring. Here, we paired in vivo and in vitro studies to complete the characterization of the bicyclomycin biosynthetic gene cluster. The construction of in-frame deletion mutants in the biosynthetic gene cluster allowed for the accumulation and identification of biosynthetic intermediates. The identity of the intermediates, which were reproduced in vitro using purified enzymes, allowed us to characterize the pathway and corroborate previous reports. Finally, we show that the putative antibiotic transporter was dispensable for the producing strain.
Asunto(s)
Antibacterianos/biosíntesis , Vías Biosintéticas/genética , Genes Bacterianos/genética , Familia de Multigenes , Streptomyces/genética , Antibacterianos/química , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Compuestos Bicíclicos Heterocíclicos con Puentes/metabolismo , Dicetopiperazinas/química , Hidroxilación , Modelos Químicos , Estructura Molecular , Mutación , Streptomyces/metabolismoRESUMEN
OBJECTIVE: We previously showed that mycobacterial Hsp70-derived peptide B29 induced B29-specific Treg cells that suppressed experimental arthritis in mice via cross-recognition of their mammalian Hsp70 homologs. The aim of the current study was to characterize B29 binding and specific CD4+ T cell responses in the context of human major histocompatibility complex (MHC) molecules. METHODS: Competitive binding assays were performed to examine binding of peptide B29 and its mammalian homologs to HLA molecules. The effect of B29 immunization in HLA-DQ8-transgenic mice with proteoglycan-induced arthritis was assessed, followed by ex vivo restimulation with B29 to examine the T cell response. Human peripheral blood mononuclear cells were used to investigate the presence of B29-specific T cells with immunoregulatory potential. RESULTS: The binding affinity of the B29 peptide was high to moderate for multiple HLA-DR and HLA-DQ molecules, including those highly associated with rheumatoid arthritis. This binding was considered to be functional, because B29 immunization resulted in the suppression of arthritis and T cell responses in HLA-DQ8-transgenic mice. In humans, we demonstrated the presence and expansion of B29-specific CD4+ T cells, which were cross-reactive with the mammalian homologs. Using HLA-DR4+ tetramers specific for B29 or the mammalian homolog mB29b, we showed expansion of cross-reactive T cells, especially the human FoxP3+ CD4+CD25+ T cell population, after in vitro stimulation with B29. CONCLUSION: These results demonstrated a conserved fine specificity and functionality of B29-induced Treg cell responses in the context of the human MHC. Based on these findings, a path for translation of the experimental findings for B29 into a clinical immunomodulatory therapeutic approach is within reach.
Asunto(s)
Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Linfocitos T CD4-Positivos/inmunología , Epítopos de Linfocito T/inmunología , Antígenos HLA-DQ/inmunología , Complejo Mayor de Histocompatibilidad/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T/inmunología , Animales , Unión Competitiva , Separación Celular , Células Cultivadas , Reacciones Cruzadas , Encefalinas/inmunología , Femenino , Factores de Transcripción Forkhead/inmunología , Humanos , Técnicas In Vitro , Integrina beta1/inmunología , Subunidad alfa del Receptor de Interleucina-2/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Precursores de Proteínas/inmunologíaRESUMEN
Cyclodipeptide synthases (CDPSs) constitute a family of peptide bond-forming enzymes that use aminoacyl-tRNAs for the synthesis of cyclodipeptides. Here, we describe the activity of 41 new CDPSs. We also show that CDPSs can be classified into two main phylogenetically distinct subfamilies characterized by specific functional subsequence signatures, named NYH and XYP. All 11 previously characterized CDPSs belong to the NYH subfamily, suggesting that further special features may be yet to be discovered in the other subfamily. CDPSs synthesize a large diversity of cyclodipeptides made up of 17 proteinogenic amino acids. The identification of several CDPSs having the same specificity led us to determine specificity sequence motifs that, in combination with the phylogenetic distribution of CDPSs, provide a first step toward being able to predict the cyclodipeptides synthesized by newly discovered CDPSs. The determination of the activity of ten more CDPSs with predicted functions constitutes a first experimental validation of this predictive approach.
Asunto(s)
Proteínas Bacterianas/química , Dipéptidos/química , Proteínas Fúngicas/química , Péptido Sintasas/química , Péptidos Cíclicos/química , Secuencias de Aminoácidos , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Biología Computacional , Ciclización , Bases de Datos Genéticas , Dipéptidos/biosíntesis , Dipéptidos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/clasificación , Proteínas Fúngicas/genética , Expresión Génica , Datos de Secuencia Molecular , Biosíntesis de Péptidos Independientes de Ácidos Nucleicos , Péptido Sintasas/biosíntesis , Péptido Sintasas/genética , Péptidos Cíclicos/biosíntesis , Péptidos Cíclicos/genética , Filogenia , Estructura Terciaria de Proteína , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidad por SustratoRESUMEN
Cyclin B1 (CCNB1) is considered as a potential target for a cancer vaccine, as it is overexpressed in many malignant cells, while being transiently expressed in normal cells. To evaluate the CD4 T cell response to CCNB1, we derived T cell lines by multiple weekly rounds of stimulation with recombinant CCNB1 of T cells collected in healthy donors (long-term T cell assays). T cell lines were specific for 15 immunodominant peptides and derived preferentially from naive T cells. From 74 overlapping peptides, 20 peptides were selected for their broad specificity of binding to HLA class II molecules and included most of the immunodominant epitopes. They primed in vitro a large number of specific CD4 T cell lines in all the donors. Immunodominant epitopes were the most efficacious in long-term T cell assays, both in terms of number of specific T cell lines and number of responding donors. The 20 peptides were also submitted to short-term T cell assays using cells collected in healthy and cancer patients with the aim to evaluate the memory response. The recognized peptides differed from the immunodominant peptides and were part of the best promiscuous peptides. We also observed pre-existing CCNB1-specifc IgG Abs in both healthy and cancer donors. Long- and short-term T cell assays revealed that CCNB1 contained many CD4 T cell epitopes, which are differentially recognized by pre-existing naive and memory CD4 T cells. These observations are of value for the design of cancer vaccines.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Ciclina B1/inmunología , Epítopos de Linfocito T/inmunología , Memoria Inmunológica , Neoplasias/inmunología , Anticuerpos/inmunología , Estudios de Casos y Controles , Línea Celular , Epítopos de Linfocito T/metabolismo , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Epítopos Inmunodominantes/inmunología , Inmunoglobulina G/inmunología , Péptidos/inmunología , Péptidos/metabolismo , Unión Proteica , Especificidad del Receptor de Antígeno de Linfocitos T/inmunologíaRESUMEN
Burkholderia pseudomallei is the causative agent of melioidosis characterized by pneumonia and fatal septicemia and prevalent in Southeast Asia. Related Burkholderia species are strong risk factors of mortality in cystic fibrosis (CF). The B. pseudomallei flagellar protein FliC is strongly seroreactive and vaccination protects challenged mice. We assessed B. pseudomallei FliC peptide binding affinity to multiple HLA class II alleles and then assessed CD4 T cell immunity in HLA class II transgenic mice and in seropositive individuals in Thailand. T cell hybridomas were generated to investigate cross-reactivity between B. pseudomallei and the related Burkholderia species associated with Cepacia Complex CF. B. pseudomallei FliC contained several peptide sequences with ability to bind multiple HLA class II alleles. Several peptides were shown to encompass strong CD4 T cell epitopes in B. pseudomallei-exposed individuals and in HLA transgenic mice. In particular, the p38 epitope is robustly recognized by CD4 T cells of seropositive donors across diverse HLA haplotypes. T cell hybridomas against an immunogenic B. pseudomallei FliC epitope also cross-reacted with orthologous FliC sequences from Burkholderia multivorans and Burkholderia cenocepacia, important pathogens in CF. Epitopes within FliC were accessible for processing and presentation from live or heat-killed bacteria, demonstrating that flagellin enters the HLA class II Ag presentation pathway during infection of macrophages with B. cenocepacia. Collectively, the data support the possibility of incorporating FliC T cell epitopes into vaccination programs targeting both at-risk individuals in B. pseudomallei endemic regions as well as CF patients.
Asunto(s)
Proteínas Bacterianas/inmunología , Infecciones por Burkholderia/inmunología , Burkholderia/inmunología , Linfocitos T CD4-Positivos/inmunología , Epítopos de Linfocito T/inmunología , Alelos , Animales , Proteínas Bacterianas/química , Vacunas Bacterianas/inmunología , Infecciones por Burkholderia/genética , Burkholderia pseudomallei/inmunología , Reacciones Cruzadas/inmunología , Fibrosis Quística/prevención & control , Epítopos de Linfocito T/química , Antígenos HLA-DR/genética , Antígenos HLA-DR/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Inmunización , Interferón gamma/biosíntesis , Melioidosis/prevención & control , Ratones , Ratones Transgénicos , Péptidos/inmunología , Péptidos/metabolismo , Unión Proteica , Especificidad del Receptor de Antígeno de Linfocitos T/inmunologíaRESUMEN
Cyclodipeptide synthases form cyclodipeptides from two aminoacyl transfer RNAs. They use a ping-pong mechanism that begins with transfer of the aminoacyl moiety of the first aminoacyl tRNA onto a conserved serine, yielding an aminoacyl enzyme. Combining X-ray crystallography, site-directed mutagenesis and affinity labelling of the cyclodipeptide synthase AlbC, we demonstrate that the covalent intermediate reacts with the aminoacyl moiety of the second aminoacyl tRNA, forming a dipeptidyl enzyme, and identify the aminoacyl-binding sites of the aminoacyl tRNAs.
Asunto(s)
Proteínas Bacterianas/química , Biosíntesis de Péptidos , Péptido Sintasas/química , Ribosomas/química , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Conformación Molecular , Mutagénesis Sitio-Dirigida , ARN de Transferencia/química , Serina/química , Streptomyces/químicaRESUMEN
Cyclodipeptide synthases (CDPSs) use two aminoacyl-tRNA substrates in a sequential ping-pong mechanism to form a cyclodipeptide. The crystal structures of three CDPSs have been determined and all show a Rossmann-fold domain similar to the catalytic domain of class-I aminoacyl-tRNA synthetases (aaRSs). Structural features and mutational analyses however suggest that CDPSs and aaRSs interact differently with their tRNA substrates. We used AlbC from Streptomyces noursei that mainly produces cyclo(l-Phe-l-Leu) to investigate the interaction of a CDPS with its substrates. We demonstrate that Phe-tRNA(Phe) is the first substrate accommodated by AlbC. Its binding to AlbC is dependent on basic residues located in the helix α4 that form a basic patch at the surface of the protein. AlbC does not use all of the Leu-tRNA(Leu) isoacceptors as a second substrate. We show that the G(1)-C(72) pair of the acceptor stem is essential for the recognition of the second substrate. Substitution of D163 located in the loop α6-α7 or D205 located in the loop ß6-α8 affected Leu-tRNA(Leu) isoacceptors specificity, suggesting the involvement of these residues in the binding of the second substrate. This is the first demonstration that the two substrates of CDPSs are accommodated in different binding sites.
Asunto(s)
Proteínas Bacterianas/metabolismo , Péptido Sintasas/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , Streptomyces/enzimología , Proteínas Bacterianas/química , Sitios de Unión , Péptido Sintasas/química , Aminoacil-ARN de Transferencia/química , ARN de Transferencia de Leucina/química , ARN de Transferencia de Leucina/metabolismo , ARN de Transferencia de Fenilalanina/química , ARN de Transferencia de Fenilalanina/metabolismo , Especificidad por SustratoRESUMEN
BACKGROUND: The CD4 T cell response to the tumor antigen Midkine was unknown. RESULTS: Most of the T cell response to Midkine relies on T cell epitopes contained in its signal peptide. CONCLUSION: The signal peptide of Midkine is accessible to HLA class II pathway for CD4 T cell presentation. SIGNIFICANCE: It is a new function for signal peptides to contribute to tumor-specific CD4 T cell response. Because of the key role of CD4 T cell response in immunity to tumors, we investigated the CD4(+) T cell response to the recently identified tumor antigen Midkine (MDK). By weekly stimulations of T lymphocytes harvested from seven HLA-DR-typed healthy donors, we derived CD4(+) T cell lines specific for eight MDK peptides. Most of the T cell lines reacted with the peptides 9-23 and 14-28, located in and overlapping the MDK signal peptide, respectively. Accordingly, the MDK signal peptide appeared to be rich in good binders to common HLA-DR molecules. The peptide 9-23-specific T cell lines were specifically stimulated by autologous dendritic cells loaded with lysates of MDK-transfected cells or with lysates of tumor cells naturally expressing the MDK protein. One T cell line was stimulated by HLA-compatible MDK-transfected tumor cells. By contrast, the peptide 14-28-specific T cell lines were not stimulated in any of these conditions. Our data demonstrate that CD4(+) T cell epitopes present in the signal peptide can be accessible to recognition by CD4(+) T cells and may therefore contribute to tumor immunity, whereas a peptide overlapping the junction between the signal peptide and the mature protein is not.
Asunto(s)
Antígenos de Neoplasias/inmunología , Citocinas/inmunología , Epítopos de Linfocito T/inmunología , Fragmentos de Péptidos/inmunología , Señales de Clasificación de Proteína/fisiología , Secuencia de Aminoácidos , Presentación de Antígeno , Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Linfocitos T CD4-Positivos , Células Cultivadas , Citocinas/química , Citocinas/metabolismo , Células Dendríticas , Epítopos de Linfocito T/química , Epítopos de Linfocito T/metabolismo , Antígenos HLA-DR/química , Antígenos HLA-DR/metabolismo , Células Hep G2 , Humanos , Midkina , Datos de Secuencia Molecular , Neoplasias/inmunología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión ProteicaRESUMEN
Due to the high degree of sequence identity between Orthopoxvirus species, the specific B and T cell responses raised against these viruses are largely cross-reactive and poorly selective. We therefore searched for CD4 T cell epitopes present in the conserved parts of the Vaccinia genome (VACV) but absent from Variola viruses (VARV), with a view to identifying immunogenic sequences selective for VACV. We identified three long peptide fragments from the B7R, B10R and E7R proteins by in silico comparisons of the poxvirus genomes, and evaluated the recognition of these fragments by VACV-specific T cell lines derived from healthy donors. For the 12 CD4 T cell epitopes identified, we assessed their binding to common HLA-DR allotypes and their capacity to induce peptide-specific CD4 T-cell lines. Four peptides from B7R and B10R displayed a broad binding specificity for HLA-DR molecules and induced multiple T cell lines from healthy donors. Besides their absence from VARV, the two B10R peptide sequences were mutated in the Cowpox virus and completely absent from the Monkeypox genome. This work contributes to the development of differential diagnosis of poxvirus infections.
Asunto(s)
Linfocitos T CD4-Positivos/química , Epítopos de Linfocito T/química , Genoma Viral , Antígenos HLA-DR/química , Virus Vaccinia/genética , Virus de la Viruela/genética , Secuencia de Aminoácidos , Linfocitos T CD4-Positivos/inmunología , Línea Celular , Simulación por Computador , Epítopos , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Antígenos HLA-DR/genética , Antígenos HLA-DR/inmunología , Humanos , Datos de Secuencia Molecular , Unión Proteica , Virus Vaccinia/inmunología , Virus de la Viruela/inmunologíaRESUMEN
We examine here the mechanisms ensuring the fidelity of RNA synthesis by RNA polymerase III (Pol III). Misincorporation could only be observed by using variants of Pol III deficient in the intrinsic RNA cleavage activity. Determination of relative rates of the reactions producing correct and erroneous transcripts at a specific position on a tRNA gene, combined with computational methods, demonstrated that Pol III has a highly efficient proofreading activity increasing its transcriptional fidelity by a factor of 10(3) over the error rate determined solely by selectivity (1.8 x 10(-4)). We show that Pol III slows down synthesis past a misincorporation to achieve efficient proofreading. We discuss our findings in the context of transcriptional fidelity studies performed on RNA Pols, proposing that the fidelity of transcription is more crucial for Pol III than Pol II.
Asunto(s)
ARN Polimerasa III/química , ARN Polimerasa III/metabolismo , ARN/biosíntesis , Transcripción Genética , Secuencia de Bases , Biología Computacional , Variación Genética , Cinética , Modelos Biológicos , Subunidades de Proteína/química , Subunidades de Proteína/aislamiento & purificación , Subunidades de Proteína/metabolismo , ARN Polimerasa III/genética , ARN Polimerasa III/aislamiento & purificación , ARN de Transferencia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Moldes Genéticos , Factores de Transcripción/clasificación , Factores de Transcripción/aislamiento & purificación , Factores de Transcripción/metabolismoRESUMEN
Regulation of ribosome biogenesis is a key element of cell biology, not only because ribosomes are directly required for growth, but also because ribosome production monopolizes nearly 80% of the global transcriptional activity in rapidly growing yeast cells. These observations underscore the need for a tight regulation of ribosome synthesis in response to environmental conditions. In eukaryotic cells, ribosome synthesis involves the activities of the three nuclear RNA polymerases (Pol). Although postulated, there is no clear evidence indicating whether the maintenance of an equimolar supply of ribosomal components reflects communication between the nuclear transcriptional machineries. Here, by constructing a yeast strain expressing a Pol I that remains constitutively competent for the initiation of transcription under stress conditions, we demonstrate that derepression of Pol I transcription leads to a derepression of Pol II transcription that is restricted to the genes encoding ribosomal proteins. Furthermore, we show that the level of 5S rRNA, synthesized by Pol III, is deregulated concomitantly with Pol I transcription. Altogether, these results indicate that a partial derepression of Pol I activity drives an abnormal accumulation of all ribosomal components, highlighting the critical role of the regulation of Pol I activity within the control of ribosome biogenesis.