RESUMEN
Gymnema sylvestre (GS) is a traditional medicinal plant known for its hypoglycemic and hypolipidemic effects. Gurmarin (hereafter Gur-1) is the only known active peptide in GS. Gur-1 has a suppressive sweet taste effect in rodents but no or only a very weak effect in humans. Here, 8 gurmarin-like peptides (Gur-2 to Gur-9) and their isoforms are reported in the GS transcriptome. The molecular mechanism of sweet taste suppression by Gur-1 is still largely unknown. Therefore, the complete architecture of human and mouse sweet taste receptors T1R2/T1R3 and their interaction with Gur-1 to Gur-9 were predicted by AlphaFold-Multimer (AF-M) and validated. Only Gur-1 and Gur-2 interact with the T1R2/T1R3 receptor. Indeed, Gur-1 and Gur-2 bind to the region of the cysteine-rich domain (CRD) and the transmembrane domain (TMD) of the mouse T1R2 subunit. In contrast, only Gur-2 binds to the TMD of the human T1R2 subunit. This result suggests that Gur-2 may have a suppressive sweet taste effect in humans. Furthermore, AF-M predicted that Gα-gustducin, a protein involved in sweet taste transduction, interacts with the intracellular domain of the T1R2 subunit. These results highlight an unexpected diversity of gurmarin-like peptides in GS and provide the complete predicted architecture of the human and mouse sweet taste receptor with the putative binding sites of Gur-1, Gur-2, and Gα-gustducin. In addition, gurmarin-like peptides may serve as promising drug scaffolds for the development of antidiabetic molecules.
Asunto(s)
Gymnema sylvestre , Receptores Acoplados a Proteínas G , Humanos , Gymnema sylvestre/metabolismo , Gymnema sylvestre/química , Animales , Ratones , Receptores Acoplados a Proteínas G/metabolismo , Péptidos/química , Péptidos/farmacología , Péptidos/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Gusto/fisiología , Unión Proteica , Secuencia de Aminoácidos , Células HEK293RESUMEN
Cecropins form a family of amphipathic α-helical cationic peptides with broad-spectrum antibacterial properties and potent anticancer activity. The emergence of bacteria and cancer cells showing resistance to cationic antimicrobial peptides (CAMPs) has fostered a search for new, more selective and more effective alternatives to CAMPs. With this goal in mind, we looked for cecropin homologs in the genome and transcriptome of the spruce budworm, Choristoneura fumiferana. Not only did we find paralogs of the conventional cationic cecropins (Cfcec+ ), our screening also led to the identification of previously uncharacterized anionic cecropins (Cfcec- ), featuring a poly-l-aspartic acid C-terminus. Comparative peptide analysis indicated that the C-terminal helix of Cfcec- is amphipathic, unlike that of Cfcec+ , which is hydrophobic. Interestingly, molecular dynamics simulations pointed to the lower conformational flexibility of Cfcec- peptides, relative to that of Cfcec+ . Phylogenetic analysis suggests that the evolution of distinct Cfcec+ and Cfcec- peptides may have resulted from an ancient duplication event within the Lepidoptera. Finally, we found that both anionic and cationic cecropins contain a BH3-like motif (G-[KQR]-[HKQNR]-[IV]-[KQR]) that could interact with Bcl-2, a protein involved in apoptosis; this observation is congruent with previous reports indicating that cecropins induce apoptosis. Altogether, our observations suggest that cecropins may provide templates for the development of new anticancer drugs. We also estimated the antibacterial activity of Cfcec-2 and a ∆Cfce-2 peptide as AMPs by testing directly their ability in inhibiting bacterial growth in a disk diffusion assay and their potential for development of novel therapeutics.
Asunto(s)
Antibacterianos/química , Antineoplásicos/química , Cecropinas/química , Proteínas de Insectos/química , Péptidos/química , Proteínas Proto-Oncogénicas c-bcl-2/química , Secuencia de Aminoácidos , Animales , Antibacterianos/metabolismo , Antibacterianos/farmacología , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Sitios de Unión , Cecropinas/genética , Cecropinas/metabolismo , Cecropinas/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Evolución Molecular , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Proteínas de Insectos/farmacología , Simulación de Dinámica Molecular , Mariposas Nocturnas/química , Mariposas Nocturnas/fisiología , Péptidos/metabolismo , Filogenia , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Electricidad EstáticaRESUMEN
CLEC12A is a myeloid inhibitory receptor that negatively regulates inflammation in mouse models of autoimmune and autoinflammatory arthritis. Reduced CLEC12A expression enhances myeloid cell activation and inflammation in CLEC12A knock-out mice with collagen antibody-induced or gout-like arthritis. Similarly to other C-type lectin receptors, CLEC12A harbours a stalk domain between its ligand binding and transmembrane domains. While it is presumed that the cysteines in the stalk domain have multimerisation properties, their role in CLEC12A expression and/or signaling remain unknown. We thus used site-directed mutagenesis to determine whether the stalk domain cysteines play a role in CLEC12A expression, internalisation, oligomerisation, and/or signaling. Mutation of C118 blocks CLEC12A transport through the secretory pathway diminishing its cell-surface expression. In contrast, mutating C130 does not affect CLEC12A cell-surface expression but increases its oligomerisation, inducing ligand-independent phosphorylation of the receptor. Moreover, we provide evidence that CLEC12A dimerisation is regulated in a redox-dependent manner. We also show that antibody-induced CLEC12A cross-linking induces flotillin oligomerisation in insoluble membrane domains in which CLEC12A signals. Taken together, these data indicate that the stalk cysteines in CLEC12A differentially modulate this inhibitory receptor's expression, oligomerisation and signaling, suggestive of the regulation of CLEC12A in a redox-dependent manner during inflammation.
Asunto(s)
Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Proteínas de la Membrana/metabolismo , Células Mieloides/metabolismo , Multimerización de Proteína/genética , Receptores Mitogénicos/genética , Receptores Mitogénicos/metabolismo , Línea Celular Tumoral , Cisteína/metabolismo , Células HEK293 , Células HeLa , Humanos , Inflamación/genética , Lectinas Tipo C/biosíntesis , Proteínas de la Membrana/genética , Mutagénesis Sitio-Dirigida , Fosforilación , Dominios Proteicos/genética , Transporte de Proteínas/genética , Receptores Mitogénicos/biosíntesis , Transducción de Señal/inmunologíaRESUMEN
KEY MESSAGE: We explored the genetic basis of SNF-related traits through GWAS and identified 40 candidate genes. This study provides fundamental insights into SNF-related traits and will accelerate efforts for SNF breeding. Symbiotic nitrogen fixation (SNF) increases sustainability by supplying biological nitrogen for crops to enhance yields without damaging the ecosystem. A better understanding of this complex biological process is critical for addressing the triple challenges of food security, environmental degradation, and climate change. Soybean plants, the most important legume worldwide, can form a mutualistic interaction with specialized soil bacteria, bradyrhizobia, to fix atmospheric nitrogen. Here we report a comprehensive genome-wide association study of 11 SNF-related traits using 79K GBS-derived SNPs in 297 African soybeans. We identified 25 QTL regions encompassing 40 putative candidate genes for SNF-related traits including 20 genes with no prior known role in SNF. A line with a large deletion (164 kb), encompassing a QTL region containing a strong candidate gene (CASTOR), exhibited a marked decrease in SNF. This study performed on African soybean lines provides fundamental insights into SNF-related traits and yielded a rich catalog of candidate genes for SNF-related traits that might accelerate future efforts aimed at sustainable agriculture.
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Regulación de la Expresión Génica de las Plantas/genética , Glycine max/genética , Fijación del Nitrógeno/genética , Simbiosis/genética , Biomasa , Estudio de Asociación del Genoma Completo , Genotipo , Fijación del Nitrógeno/fisiología , Fenotipo , Filogenia , Fitomejoramiento , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Brotes de la Planta/genética , Brotes de la Planta/crecimiento & desarrollo , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , Nódulos de las Raíces de las Plantas/genética , Glycine max/crecimiento & desarrollo , Glycine max/metabolismo , Simbiosis/fisiologíaRESUMEN
Identifying the molecular changes that lead to ecological specialization during speciation is one of the major goals of molecular evolution. One question that remains to be thoroughly investigated is whether ecological specialization derives strictly from adaptive changes and their associated trade-offs, or from conditionally neutral mutations that accumulate under relaxed selection. We used whole-genome sequencing, genome annotation and computational analyses to identify genes that have rapidly diverged between two incipient species of Saccharomyces paradoxus that occupy different climatic regions along a south-west to north-east gradient. As candidate loci for ecological specialization, we identified genes that show signatures of adaptation and accelerated rates of amino acid substitutions, causing asymmetric evolution between lineages. This set of genes includes a glycyl-tRNA-synthetase, GRS2, which is known to be transcriptionally induced under heat stress in the model and sister species S. cerevisiae. Molecular modelling, expression analysis and fitness assays suggest that the accelerated evolution of this gene in the Northern lineage may be caused by relaxed selection. GRS2 arose during the whole-genome duplication (WGD) that occurred 100 million years ago in the yeast lineage. While its ohnolog GRS1 has been preserved in all post-WGD species, GRS2 has frequently been lost and is evolving rapidly, suggesting that the fate of this ohnolog is still to be resolved. Our results suggest that the asymmetric evolution of GRS2 between the two incipient S. paradoxus species contributes to their restricted climatic distributions and thus that ecological specialization derives at least partly from relaxed selection rather than a molecular trade-off resulting from adaptive evolution.
Asunto(s)
Especiación Genética , Filogeografía/métodos , Saccharomyces/genética , Ecología , Evolución Molecular , Duplicación de Gen/genética , Genes Fúngicos/genética , Genoma Fúngico/genética , Filogenia , Saccharomyces cerevisiae/genética , Especificidad de la EspecieRESUMEN
Using massively parallel sequencing data from two species with different life history traits, American lobster (Homarus americanus) and Arctic Char (Salvelinus alpinus), we highlight how an unbalanced sex ratio in the samples and a few sex-linked markers may lead to false interpretations of population structure and thus to potentially erroneous management recommendations. Here, multivariate analyses revealed two genetic clusters separating samples by sex instead of by expected spatial variation: inshore and offshore locations in lobster, or east and west locations in Arctic Char. To further investigate this, we created several subsamples artificially varying the sex ratio in the inshore/offshore and east/west groups and then demonstrated that significant genetic differentiation could be observed despite panmixia in lobster, and that FST values were overestimated in Arctic Char. This pattern was due to 12 and 94 sex-linked markers driving differentiation for lobster and Arctic Char, respectively. Removing sex-linked markers led to nonsignificant genetic structure in lobster and a more accurate estimation of FST in Arctic Char. The locations of these markers and putative identities of genes containing or nearby the markers were determined using available transcriptomic and genomic data, and this provided new information related to sex determination in both species. Given that only 9.6% of all marine/diadromous population genomic studies to date have reported sex information, we urge researchers to collect and consider individual sex information. Sex information is therefore relevant for avoiding unexpected biases due to sex-linked markers as well as for improving our knowledge of sex determination systems in nonmodel species.
Asunto(s)
Genética de Población , Secuenciación de Nucleótidos de Alto Rendimiento , Nephropidae/genética , Razón de Masculinidad , Trucha/genética , Animales , Femenino , Marcadores Genéticos , Masculino , Análisis Multivariante , Polimorfismo de Nucleótido Simple , Sesgo de SelecciónRESUMEN
Periodic outbreaks of spruce budworm (SBW) affect large areas of ecologically and economically important conifer forests in North America, causing tree mortality and reduced forest productivity. Host resistance against SBW has been linked to growth phenology and the chemical composition of foliage, but the underlying molecular mechanisms and population variation are largely unknown. Using a genomics approach, we discovered a ß-glucosidase gene, Pgßglu-1, whose expression levels and function underpin natural resistance to SBW in mature white spruce (Picea glauca) trees. In phenotypically resistant trees, Pgßglu-1 transcripts were up to 1000 times more abundant than in non-resistant trees and were highly enriched in foliage. The encoded PgßGLU-1 enzyme catalysed the cleavage of acetophenone sugar conjugates to release the aglycons piceol and pungenol. These aglycons were previously shown to be active against SBW. Levels of Pgßglu-1 transcripts and biologically active acetophenone aglycons were substantially different between resistant and non-resistant trees over time, were positively correlated with each other and were highly variable in a natural white spruce population. These results suggest that expression of Pgßglu-1 and accumulation of acetophenone aglycons is a constitutive defence mechanism in white spruce. The progeny of resistant trees had higher Pgßglu-1 gene expression than non-resistant progeny, indicating that the trait is heritable. With reported increases in the intensity of SBW outbreaks, influenced by climate, variation of Pgßglu-1 transcript expression, PgßGLU-1 enzyme activity and acetophenone accumulation may serve as resistance markers to better predict impacts of SBW in both managed and wild spruce populations.
Asunto(s)
Resistencia a la Enfermedad/genética , Picea/fisiología , Proteínas de Plantas/fisiología , beta-Glucosidasa/fisiología , Acetofenonas/metabolismo , Animales , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Herbivoria , Larva/fisiología , Modelos Moleculares , Datos de Secuencia Molecular , Mariposas Nocturnas/fisiología , Picea/enzimología , Picea/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estructura Terciaria de Proteína , beta-Glucosidasa/química , beta-Glucosidasa/genética , beta-Glucosidasa/metabolismoRESUMEN
UNLABELLED: HIV-1 acquires an impressive number of foreign components during its formation. Despite all previous efforts spent studying the nature and functionality of virus-anchored host molecules, the exact mechanism(s) through which such constituents are acquired by HIV-1 is still unknown. However, in the case of ICAM-1, one of the most extensively studied transmembrane proteins found associated with mature virions, the Pr55(Gag) precursor polyprotein appears to be a potential interaction partner. We investigated and characterized at the molecular level the process of ICAM-1 incorporation using initially a Pr55(Gag)-based virus-like particle (VLP) model. Substitution of various domains of Pr55(Gag), such as the nucleocapsid, SP2, or p6, had no effect on the acquisition of ICAM-1. We found that the structural matrix protein (MA) is mandatory for ICAM-1 incorporation within VLPs, and we confirmed this novel observation with the replication-competent HIV-1 molecular clone NL4.3. Additional studies suggest that the C-terminal two-thirds of MA, and especially 13 amino acids positioned inside the fifth α-helix, are important. Moreover, based on three-dimensional (3D) modeling of protein-protein interactions (i.e., protein-protein docking) and further validation by a virus capture assay, we found that a series of acidic residues in the MA domain interact with basic amino acids located in the ICAM-1 cytoplasmic tail. Our findings provide new insight into the molecular mechanism governing the acquisition of ICAM-1, a host molecule known to enhance HIV-1 infectivity in a significant manner. Altogether, these observations offer a new avenue for the development of antiviral therapeutics that are directed at a target of host origin. IMPORTANCE: Intercellular adhesion molecule 1 (ICAM-1) is a cell surface host component known to be efficiently inserted within emerging HIV-1 particles. It has been demonstrated that host-derived ICAM-1 molecules act as a strong attachment factor and increase HIV-1 infectivity substantially. Despite previous efforts spent studying virus-associated host molecules, the precise mechanism(s) through which such constituents are inserted within emerging HIV-1 particles still remains obscure. Previous data suggest that the Pr55(Gag) precursor polyprotein appears as a potential interaction partner with ICAM-1. In the present study, we demonstrate that the HIV-1 matrix domain plays a key role in the ICAM-1 incorporation process. Some observations were confirmed with whole-virus preparations amplified in primary human cells, thereby providing physiological significance to our data.
Asunto(s)
Antígenos VIH/metabolismo , VIH-1/fisiología , Interacciones Huésped-Patógeno , Molécula 1 de Adhesión Intercelular/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Ensamble de VirusRESUMEN
Investigating how environmental features shape the genetic structure of populations is crucial for understanding how they are potentially adapted to their habitats, as well as for sound management. In this study, we assessed the relative importance of spatial distribution, ocean currents and sea surface temperature (SST) on patterns of putatively neutral and adaptive genetic variation among American lobster from 19 locations using population differentiation (PD) approaches combined with environmental association (EA) analyses. First, PD approaches (using bayescan, arlequin and outflank) found 28 outlier SNPs putatively under divergent selection and 9770 neutral SNPs in common. Redundancy analysis revealed that spatial distribution, ocean current-mediated larval connectivity and SST explained 31.7% of the neutral genetic differentiation, with ocean currents driving the majority of this relationship (21.0%). After removing the influence of spatial distribution, no SST were significant for putatively neutral genetic variation whereas minimum annual SST still had a significant impact and explained 8.1% of the putatively adaptive genetic variation. Second, EA analyses (using Pearson correlation tests, bayescenv and lfmm) jointly identified seven SNPs as candidates for thermal adaptation. Covariation at these SNPs was assessed with a spatial multivariate analysis that highlighted a significant temperature association, after accounting for the influence of spatial distribution. Among the 505 candidate SNPs detected by at least one of the three approaches, we discovered three polymorphisms located in genes previously shown to play a role in thermal adaptation. Our results have implications for the management of the American lobster and provide a foundation on which to predict how this species will cope with climate change.
Asunto(s)
Aclimatación/genética , Genética de Población , Nephropidae/genética , Polimorfismo de Nucleótido Simple , Temperatura , Animales , Femenino , Frecuencia de los Genes , Genómica , Modelos Genéticos , Análisis Multivariante , América del Norte , Análisis Espacial , Movimientos del AguaRESUMEN
BACKGROUND: Glycoside hydrolase family 32 (GH32) enzymes cleave the glycosidic bond between two monosaccharides or between a carbohydrate and an aglycone moiety. GH32 enzymes have been studied in prokaryotes and in eukaryotes but not in viruses. FINDINGS: This is the first analysis of GH32 enzymes in Bacillus subtilis phage SP10, ÏNIT1 and SPG24. Phylogenetic analysis, molecular docking and secretability predictions suggest that phage GH32 enzymes function as levan (fructose homopolysaccharide) fructotransferase. CONCLUSIONS: We showed that viruses also contain GH32 enzymes and that our analyses in silico strongly suggest that these enzymes function as levan fructotransferase.
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Fagos de Bacillus/enzimología , Fagos de Bacillus/genética , Bacillus subtilis/virología , Glicósido Hidrolasas/genética , Secuencia de Aminoácidos , Análisis por Conglomerados , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Filogenia , Conformación Proteica , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de SecuenciaRESUMEN
Major histocompatibility (MHC) immune system genes may evolve in response to pathogens in the environment. Because they also may affect mate choice, they are candidates for having great importance in ecological speciation. Here, we use next-generation sequencing to test the general hypothesis of parallelism in patterns of MHCIIß diversity and bacterial infections among five dwarf and normal whitefish sympatric pairs. A second objective was to assess the functional relationships between specific MHCIIß alleles and pathogens in natural conditions. Each individual had between one and four alleles, indicating two paralogous loci. In Cliff Lake, the dwarf ecotype was monomorphic for the most common allele. In Webster Lake, the skew in the allelic distribution was towards the same allele but in the normal ecotype, underscoring the nonparallel divergence among lakes. Our signal of balancing selection matched putative peptide binding region residues in some cases, but not in others, supporting other recent findings of substantial functional differences in fish MHCIIß compared with mammals. Individuals with fewer alleles were less likely to be infected; thus, we found no evidence for the heterozygote advantage hypothesis. MHCIIß alleles and pathogenic bacteria formed distinct clusters in multivariate analyses, and clusters of certain alleles were associated with clusters of pathogens, or sometimes the absence of pathogens, indicating functional relationships at the individual level. Given that patterns of MHCIIß and bacteria were nonparallel among dwarf and normal whitefish pairs, we conclude that pathogens driving MHCIIß evolution did not play a direct role in their parallel phenotypic evolution.
Asunto(s)
Evolución Molecular , Variación Genética , Complejo Mayor de Histocompatibilidad/genética , Salmonidae/genética , Adaptación Biológica , Animales , Ambiente , Perfilación de la Expresión Génica , Genética de Población , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , LagosRESUMEN
BACKGROUND: We identified two families with Paget's disease of bone (PDB) linked to the p.Pro392Leu mutation within the SQSTM1 gene displaying a possible digenism. This study aimed at identifying this second genetic variant cosegregating with the p.Pro392Leu mutation and at characterizing its impact on the clinical and cellular phenotypes of PDB. METHODS: Whole exome sequencing was performed in one patient per family and two healthy controls. We compared clinical characteristics of PDB in 14 relatives from the two families. The osteoclastic phenotype was compared in in vitro differentiated osteoclasts from 31 participants carrying the DOCK6 and/or SQSTM1 variants. Tridimensional models of SQSTM1 and DOCK6 proteins were generated to evaluate the impact of these variants on their stability and flexibility. Statistical analyses were performed with Graphpad prism. RESULTS: Whole-exome sequencing allowed us to identify the p.Val45Ile missense variant in the DOCK6 gene in patients. In both families, the mean age at PDB diagnosis was delayed in pagetic patients carrier of the p.Val45Ile variant alone compared to those carrying the p.Pro392Leu mutation alone (67 vs. 44 years, P = 0.03). Although both p.Val45Ile and p.Pro392Leu variants gave rise to a pagetic phenotype of osteoclast versus healthy controls, the p.Val45Ile variant was found to attenuate the severity of the osteoclastic phenotype of PDB caused by the p.Pro392Leu mutation when both variants were present. The DOCK6 mRNA expression was higher in carriers of the p.Val45Ile variant than in pagetic patients without any mutations and healthy controls. Structural bioinformatics analyses suggested that the p.Pro392Leu mutation might rigidify the UBA domain and thus decrease its possible intramolecular interaction with a novel domain, the serum response factor-transcription factor (SRF-TF)-like domain, whereas the p.Val45Ile variant may decrease SRF-TF-like activity. CONCLUSION: The p.Val45Ile variant may attenuate the severity of the clinical phenotype of PDB in patient carriers of both variants. In vitro, the rare variant of the DOCK6 may have a modifier effect on the p.Pro392Leu mutation, possibly via its effect on the SRF-TF-like.
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Osteítis Deformante , Proteínas Adaptadoras Transductoras de Señales/genética , Adulto , Anciano , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Mutación , Osteítis Deformante/genética , Osteoclastos , Fenotipo , Proteína Sequestosoma-1/genética , Factores de Transcripción/genéticaRESUMEN
Insects have developed various adaptations to survive harsh winter conditions. Among freeze-intolerant species, some produce "antifreeze proteins" (AFPs) that bind to nascent ice crystals and inhibit further ice growth. Such is the case of the spruce budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae), a destructive North American conifer pest that can withstand temperatures below -30°C. Despite the potential importance of AFPs in the adaptive diversification of Choristoneura, genomic tools to explore their origins have until now been limited. Here we present a chromosome-scale genome assembly for C. fumiferana, which we used to conduct comparative genomic analyses aimed at reconstructing the evolutionary history of tortricid AFPs. The budworm genome features 16 genes homologous to previously reported C. fumiferana AFPs (CfAFPs), 15 of which map to a single region on chromosome 18. Fourteen of these were also detected in five congeneric species, indicating Choristoneura AFP diversification occurred before the speciation event that led to C. fumiferana. Although budworm AFPs were previously considered unique to the genus Choristoneura, a search for homologs targeting recently sequenced tortricid genomes identified seven CfAFP-like genes in the distantly related Notocelia uddmanniana. High structural similarity between Notocelia and Choristoneura AFPs suggests a common origin, despite the absence of homologs in three related tortricids. Interestingly, one Notocelia AFP formed the C-terminus of a "zonadhesin-like" protein, possibly representing the ancestral condition from which tortricid AFPs evolved. Future work should clarify the evolutionary path of AFPs between Notocelia and Choristoneura and assess the role of the "zonadhesin-like" protein as precursor of tortricid AFPs.
RESUMEN
BACKGROUND: Manipulative parasites are thought to liberate molecules in their external environment, acting as manipulation factors with biological functions implicated in their host's physiological and behavioural alterations. These manipulation factors are part of a complex mixture called the secretome. While the secretomes of various parasites have been described, there is very little data for a putative manipulative parasite. It is necessary to study the molecular interaction between a manipulative parasite and its host to better understand how such alterations evolve. METHODS: Here, we used proteomics to characterize the secretome of a model cestode with a complex life cycle based on trophic transmission. We studied Schistocephalus solidus during the life stage in which behavioural changes take place in its obligatory intermediate fish host, the threespine stickleback (Gasterosteus aculeatus). We produced a novel genome sequence and assembly of S. solidus to improve protein coding gene prediction and annotation for this parasite. We then described the whole worm's proteome and its secretome during fish host infection using LC-MS/MS. RESULTS: A total of 2290 proteins were detected in the proteome of S. solidus, and 30 additional proteins were detected specifically in the secretome. We found that the secretome contains proteases, proteins with neural and immune functions, as well as proteins involved in cell communication. We detected receptor-type tyrosine-protein phosphatases, which were reported in other parasitic systems to be manipulation factors. We also detected 12 S. solidus-specific proteins in the secretome that may play important roles in host-parasite interactions. CONCLUSIONS: Our results suggest that S. solidus liberates molecules with putative host manipulation functions in the host and that many of them are species-specific.
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Cestodos/química , Cestodos/genética , Enfermedades de los Peces/parasitología , Peces/parasitología , Proteínas del Helminto/genética , Interacciones Huésped-Parásitos , Animales , Cestodos/clasificación , Cestodos/patogenicidad , Proteínas del Helminto/biosíntesis , Proteínas del Helminto/metabolismo , Estadios del Ciclo de Vida , Proteómica/métodos , SecretomaRESUMEN
The enzyme kinetics of the amide ligase MurE, a cell wall biosynthesis enzyme, from Pseudomonas aeruginosa were determined using the synthesized nucleotide substrate UDP-MurNAc-Ala-Glu (uridine 5'-diphosphoryl N-acetylmuramoyl-L-alanyl-D-glutamate). When coupled to a competitive bio-panning technique using a M13 phage display library encoding approximately 2.7 x 10(9) random peptide permutations and the specific substrates meso-A2pm (meso-diaminopimelic acid) and ATP, a peptide inhibitor of MurE was identified. The MurEp1 dodecamer selected and synthesized inhibited MurE ATPase activity with an IC(50) value of 500 microM. The inhibition was shown to be time-dependent and was reversed by the addition of meso-A2pm or UDP-MurNAc-Ala-Glu during the pre-incubation step. Kinetic analysis defined MurEp1 as a mixed inhibitor against both substrates with K(i) values of 160 and 80 microM respectively. MurEp1 was found to interfere in meso-A2pm and UDP-MurNAc-Ala-Glu binding necessary for amide bond formation. Modelling of Ps. aeruginosa MurE and docking of MurEp1 on the Ps. aeruginosa MurE surface indicated that MurEp1 binds at the juxtaposition of both meso-A2pm- and UDP-MurNAc-Ala-Glu-binding sites in the closed conformational state of the enzyme. Identification of the MurEp1 residues involved in MurE binding and inhibition will allow the development of a novel class of inhibitors having a novel mode of action against MurE.
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Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Oligopéptidos/química , Péptido Sintasas/antagonistas & inhibidores , Péptido Sintasas/química , Péptidos/química , Pseudomonas aeruginosa/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Sitios de Unión , Dipéptidos/química , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Oligopéptidos/metabolismo , Péptido Sintasas/metabolismo , Péptidos/metabolismo , Conformación Proteica , Pseudomonas aeruginosa/metabolismo , Uridina Difosfato Ácido N-Acetilmurámico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurámico/químicaRESUMEN
Valosin-containing protein (VCP)/p97/Cdc48 is an AAA + ATPase associated with many ubiquitin-dependent cellular pathways that are central to protein quality control. VCP binds various cofactors, which determine pathway selectivity and substrate processing. Here, we used co-immunoprecipitation and mass spectrometry studies coupled to in silico analyses to identify the Leishmania infantum VCP (LiVCP) interactome and to predict molecular interactions between LiVCP and its major cofactors. Our data support a largely conserved VCP protein network in Leishmania including known but also novel interaction partners. Network proteomics analysis confirmed LiVCP-cofactor interactions and provided novel insights into cofactor-specific partners and the diversity of LiVCP complexes, including the well-characterized VCP-UFD1-NPL4 complex. Gene Ontology analysis coupled with digitonin fractionation and immunofluorescence studies support cofactor subcellular compartmentalization with either cytoplasmic or organellar or vacuolar localization. Furthermore, in silico models based on 3D homology modeling and protein-protein docking indicated that the conserved binding modules of LiVCP cofactors, except for NPL4, interact with specific binding sites in the hexameric LiVCP protein, similarly to their eukaryotic orthologs. Altogether, these results allowed us to build the first VCP protein interaction network in parasitic protozoa through the identification of known and novel interacting partners potentially associated with distinct VCP complexes.
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Simulación por Computador , Leishmania infantum/química , Complejos Multiproteicos/química , Proteínas Protozoarias/química , Proteína que Contiene Valosina/química , Leishmania infantum/metabolismo , Complejos Multiproteicos/metabolismo , Estructura Cuaternaria de Proteína , Proteínas Protozoarias/metabolismo , Proteína que Contiene Valosina/metabolismoRESUMEN
The small Hsp DmHsp27 from Drosophila melanogaster is one of the few small heat shock proteins (sHsps) found within the nucleus. We report that its dimerization is independent of disulfide bond formation and seems to rely on salt bridges. Unlike metazoan sHsps, DmHsp27 forms two populations of oligomers not in equilibrium. Mutations at highly conserved arginine residues in mammalian sHsps have been reported to be associated with protein conformational defects and intracellular aggregation. Independent mutation of three highly conserved arginines (R122, R131, and R135) to glycine in DmHsp27 results in only one population of higher molecular weight form. In vitro, the chaperone-like activity of wild-type DmHsp27 was comparable with that of its two isolated populations and to the single population of the R122G, R131G, and R135G using luciferase as substrate. However, using insulin, the chaperone-like activity of wild-type DmHsp27 was lower than that of R122G and R131G mutants. Altogether, the results characterize wild-type DmHsp27 and its alpha-crystallin domain (ACD) arginine mutants and may give insight into protection mechanism of sHsps.
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Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , alfa-Cristalinas/química , Secuencia de Aminoácidos , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/química , Drosophila melanogaster/genética , Proteínas de Choque Térmico/genética , Modelos Moleculares , Mutación Puntual , Conformación Proteica , Dominios Proteicos , Multimerización de ProteínaRESUMEN
The gypsy moth, Lymantria dispar L., is one of the most destructive forest pests in the world. While the subspecies established in North America is the European gypsy moth (L. dispar dispar), whose females are flightless, the two Asian subspecies, L. dispar asiatica and L. dispar japonica, have flight-capable females, enhancing their invasiveness and warranting precautionary measures to prevent their permanent establishment in North America. Various molecular tools have been developed to help distinguish European from Asian subspecies, several of which are based on the mitochondrial barcode region. In an effort to identify additional informative markers, we undertook the sequencing and analysis of the mitogenomes of 10 geographic variants of L. dispar, including two or more variants of each subspecies, plus the closely related L. umbrosa as outgroup. Several regions of the gypsy moth mitogenomes displayed nucleotide substitutions with potential usefulness for the identification of subspecies and/or geographic origins. Interestingly, the mitogenome of one geographic variant displayed significant divergence relative to the remaining variants, raising questions about its taxonomic status. Phylogenetic analyses placed this population from northern Iran as basal to the L. dispar clades. The present findings will help improve diagnostic tests aimed at limiting risks of AGM invasions.
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Genoma Mitocondrial/genética , Genotipo , Geografía , Mariposas Nocturnas/genética , Animales , Femenino , Variación Genética , FilogeniaRESUMEN
In Helicobacter pylori, the heterotrimeric tRNA-dependent amidotransferase (GatCAB) is essential for protein biosynthesis because it catalyzes the conversion of misacylated Glu-tRNA(Gln) and Asp-tRNA(Asn) into Gln-tRNA(Gln) and Asn-tRNA(Asn), respectively. In this study, we used a phage library to identify peptide inhibitors of GatCAB. A library displaying loop-constrained heptapeptides was used to screen for phages binding to the purified GatCAB. To optimize the probability of obtaining competitive inhibitors of GatCAB with respect to its substrate Glu-tRNA(Gln), we used that purified substrate in the biopanning process of the phage-display technique to elute phages bound to GatCAB at the third round of the biopanning process. Among the eluted phages, we identified several that encode cyclic peptides rich in Trp and Pro that inhibit H. pylori GatCAB in vitro. Peptides P10 and P9 were shown to be competitive inhibitors of GatCAB with respect to its substrate Glu-tRNA(Gln), with Ki values of 126 and 392µM, respectively. The docking models revealed that the Trp residues of these peptides form π-π stacking interactions with Tyr81 of the synthetase active site, as does the 3'-terminal A76 of tRNA, supporting their competitive behavior with respect to Glu-tRNA(Gln) in the transamidation reaction. These peptides can be used as scaffolds in the search for novel antibiotics against the pathogenic bacteria that require GatCAB for Gln-tRNA(Gln) and/or Asn-tRNA(Asn) formation.
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Antibacterianos/química , Proteínas Bacterianas/antagonistas & inhibidores , Helicobacter pylori/enzimología , Transferasas de Grupos Nitrogenados/antagonistas & inhibidores , Péptidos Cíclicos/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Dominio Catalítico , Cinética , Modelos Moleculares , Transferasas de Grupos Nitrogenados/química , Transferasas de Grupos Nitrogenados/aislamiento & purificación , Unión ProteicaRESUMEN
Glutaminyl-tRNAGln in Helicobacter pylori is formed by an indirect route requiring a noncanonical glutamyl-tRNA synthetase and a tRNA-dependent heterotrimeric amidotransferase (AdT) GatCAB. Widespread use of this pathway among prominent human pathogens, and its absence in the mammalian cytoplasm, identify AdT as a target for the development of antimicrobial agents. We present here the inhibitory properties of three dipeptide-like sulfone-containing compounds analogous to the transamidation intermediates, which are competitive inhibitors of AdT with respect to Glu-tRNAGln . Molecular docking revealed that AdT inhibition by these compounds depends on π-π stacking interactions between their aromatic groups and Tyr81 of the GatB subunit. The properties of these inhibitors indicate that the 3'-terminal adenine of Glu-tRNAGln plays a major role in binding to the AdT transamidation active site.