RESUMO
Host cell proteins (HCPs) are process-related impurities present in biopharmaceuticals and are generally considered to be critical quality attributes. Changes in a biopharmaceutical production process may result in qualitative shifts in the HCP population. These shifts are not necessarily detectable when overall HCP levels are measured with traditional approaches such as enzyme-linked immunosorbent assays (ELISAs). Thus, the development of techniques that complement the ELISA's functionality is desirable. Here, a mass spectrometry (MS)-based approach for the analysis of HCP populations in biopharmaceuticals is presented. It consists of (i) the generation of exclusion lists that represent the masses of the active pharmaceutical ingredient (API), (ii) the compilation of inclusion lists based on an HCP catalog derived from the analysis of protein A-purified samples, and (iii) the analysis of purified biopharmaceuticals using the generated exclusion and inclusion lists. With this approach, it was possible to increase sensitivity for HCP detection compared with a standard liquid chromatography tandem MS (LC-MS/MS) run. The workflow was successfully implemented in a comparability exercise assessing HCP populations in drug substance samples before and after a process change. Furthermore, the results suggest that size can be an important factor in the copurification of HCPs and API.
Assuntos
Técnicas de Química Analítica/métodos , Proteínas/análise , Espectrometria de Massas em Tandem , Cromatografia Líquida de Alta Pressão , Peso Molecular , Preparações Farmacêuticas/análise , Proteínas/metabolismo , Proteínas S100/análise , Proteínas S100/metabolismo , Tripsina/metabolismoRESUMO
We report the draft genome sequence of the red harvester ant, Pogonomyrmex barbatus. The genome was sequenced using 454 pyrosequencing, and the current assembly and annotation were completed in less than 1 y. Analyses of conserved gene groups (more than 1,200 manually annotated genes to date) suggest a high-quality assembly and annotation comparable to recently sequenced insect genomes using Sanger sequencing. The red harvester ant is a model for studying reproductive division of labor, phenotypic plasticity, and sociogenomics. Although the genome of P. barbatus is similar to other sequenced hymenopterans (Apis mellifera and Nasonia vitripennis) in GC content and compositional organization, and possesses a complete CpG methylation toolkit, its predicted genomic CpG content differs markedly from the other hymenopterans. Gene networks involved in generating key differences between the queen and worker castes (e.g., wings and ovaries) show signatures of increased methylation and suggest that ants and bees may have independently co-opted the same gene regulatory mechanisms for reproductive division of labor. Gene family expansions (e.g., 344 functional odorant receptors) and pseudogene accumulation in chemoreception and P450 genes compared with A. mellifera and N. vitripennis are consistent with major life-history changes during the adaptive radiation of Pogonomyrmex spp., perhaps in parallel with the development of the North American deserts.
Assuntos
Formigas/genética , Redes Reguladoras de Genes/genética , Genoma de Inseto/genética , Genômica/métodos , Filogenia , Animais , Formigas/fisiologia , Sequência de Bases , Clima Desértico , Hierarquia Social , Dados de Sequência Molecular , América do Norte , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Receptores Odorantes/genética , Análise de Sequência de DNARESUMO
BACKGROUND: Gene bodies are the most evolutionarily conserved targets of DNA methylation in eukaryotes. However, the regulatory functions of gene body DNA methylation remain largely unknown. DNA methylation in insects appears to be primarily confined to exons. Two recent studies in Apis mellifera (honeybee) and Nasonia vitripennis (jewel wasp) analyzed transcription and DNA methylation data for one gene in each species to demonstrate that exon-specific DNA methylation may be associated with alternative splicing events. In this study we investigated the relationship between DNA methylation, alternative splicing, and cross-species gene conservation on a genome-wide scale using genome-wide transcription and DNA methylation data. RESULTS: We generated RNA deep sequencing data (RNA-seq) to measure genome-wide mRNA expression at the exon- and gene-level. We produced a de novo transcriptome from this RNA-seq data and computationally predicted splice variants for the honeybee genome. We found that exons that are included in transcription are higher methylated than exons that are skipped during transcription. We detected enrichment for alternative splicing among methylated genes compared to unmethylated genes using fisher's exact test. We performed a statistical analysis to reveal that the presence of DNA methylation or alternative splicing are both factors associated with a longer gene length and a greater number of exons in genes. In concordance with this observation, a conservation analysis using BLAST revealed that each of these factors is also associated with higher cross-species gene conservation. CONCLUSIONS: This study constitutes the first genome-wide analysis exhibiting a positive relationship between exon-level DNA methylation and mRNA expression in the honeybee. Our finding that methylated genes are enriched for alternative splicing suggests that, in invertebrates, exon-level DNA methylation may play a role in the construction of splice variants by positively influencing exon inclusion during transcription. The results from our cross-species homology analysis suggest that DNA methylation and alternative splicing are genetic mechanisms whose utilization could contribute to a longer gene length and a slower rate of gene evolution.
Assuntos
Processamento Alternativo , Abelhas/genética , Metilação de DNA , Genes de Insetos , Transcriptoma , Vespas/genética , Animais , Sequência de Bases , Sequência Conservada , Ilhas de CpG , Evolução Molecular , Éxons/genética , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Dados de Sequência Molecular , Análise de Sequência de RNARESUMO
Vitellogenin (Vg) is an egg-yolk precursor protein in most oviparous species. In honeybee (Apis mellifera), the protein (AmVg) also affects social behavior and life-span plasticity. Despite its manifold functions, the AmVg molecule remains poorly understood. The subject of our structure-oriented AmVg study is its polyserine tract - a little-investigated repetitive protein segment mostly found in insects. We previously reported that AmVg is tissue specifically cleaved in the vicinity of this tract. Here, we show that, despite its potential for an open, disordered structure, AmVg is unexpectedly resistant to trypsin/chymotrypsin digestion at the tract. Our findings suggest that multiple phosphorylation plays a role in this resilience. Sequence variation is highly pronounced at the polyserine region in insect Vgs. We demonstrate that sequence differences in this region can lead to structural variation, as NMR and circular dichroism (CD) evidence assign different conformational propensities to polyserine peptides from the honeybee and the jewel wasp Nasonia vitripennis; the former is extended and disordered and the latter more compact and helical. CD analysis of the polyserine region of bumblebee Bombus ignitus and wasp Pimpla nipponica supports a random coil structure in these species. The spectroscopic results strengthen our model of the AmVg polyserine tract as a flexible domain linker shielded by phosphorylation.
Assuntos
Vitelogeninas/química , Sequência de Aminoácidos , Animais , Abelhas/genética , Abelhas/metabolismo , Sítios de Ligação , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Peptídeos/química , Fosforilação , Conformação Proteica , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Espectrometria de Massas em Tandem , Vitelogeninas/genética , Vitelogeninas/metabolismo , Vespas/genética , Vespas/metabolismoRESUMO
Regardless of genetic makeup, a female honey bee becomes a queen or worker depending on the food she receives as a larva. For decades, it has been known that nutrition and juvenile hormone (JH) signaling determine the caste fate of the individual bee. However, it is still largely unclear how these factors are connected. To address this question, we suppressed nutrient sensing by RNA interference (RNAi)-mediated gene knockdown of IRS (insulin receptor substrate) and TOR (target of rapamycin) in larvae reared on queen diet. The treatments affected several layers of organismal organization that could play a role in the response to differential nutrition between castes. These include transcript profiles, proteomic patterns, lipid levels, DNA methylation response and morphological features. Most importantly, gene knockdown abolished a JH peak that signals queen development and resulted in a worker phenotype. Application of JH rescued the queen phenotype in either knockdown, which demonstrates that the larval response to JH remains intact and can drive normal developmental plasticity even when IRS or TOR transcript levels are reduced. We discuss our results in the context of other recent findings on honey bee caste and development and propose that IRS is an alternative substrate for the Egfr (epidermal growth factor receptor) in honey bees. Overall, our study describes how the interplay of nutritional and hormonal signals affects many levels of organismal organization to build different phenotypes from identical genotypes.
Assuntos
Abelhas/enzimologia , Hierarquia Social , Mel , Proteínas Substratos do Receptor de Insulina/metabolismo , Hormônios Juvenis/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Animais , Abelhas/genética , Metilação de DNA/genética , Sistema Endócrino/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Proteínas de Insetos/metabolismo , Proteínas Substratos do Receptor de Insulina/genética , Larva/enzimologia , Larva/genética , Metabolismo dos Lipídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genéticaRESUMO
The insulin/insulin-like signalling (IIS) network is conserved among animals and is central to growth and development. In eusocial honeybees (Apis mellifera), IIS is hypothesized to shape female caste fate. We tested this hypothesis via RNA interference (RNAi) knockdown of the insulin receptor substrate (IRS) homologue, a key adaptor protein in IIS. Female larvae naturally develop into queens (reproductives) or workers (helpers) after being fed rich versus limited diets, respectively. Feeding larvae a rich diet mixed with dsRNA (double stranded RNA) targeting irs gene transcript decreased irs mRNA abundance and caused development of worker morphology. Controls receiving rich larval diet and control dsRNA developed queen morphology. Whole-body mass spectrometry profiling of larvae collected 72, 96 and 120 h after dsRNA treatments revealed proteomic differences between irs gene knockdowns and controls, including levels of hexamerin 110, a storage protein connected to natural caste differences.
Assuntos
Abelhas/metabolismo , Comportamento Animal/fisiologia , Proteínas Substratos do Receptor de Insulina/metabolismo , Animais , Feminino , Regulação da Expressão Gênica/fisiologia , Interferência de RNA , Comportamento SocialRESUMO
SARS-CoV-2 enters host cells after binding through its spike glycoprotein to the angiotensin-converting enzyme 2 (ACE2) receptor. Soluble ACE2 ectodomains bind and neutralize the virus, yet their short in vivo half-live limits their therapeutic use. This limitation can be overcome by fusing the fragment crystallizable (Fc) part of human immunoglobulin G (IgG) to the ACE2 ectodomain, but this bears the risk of Fc-receptor activation and antibody-dependent cellular cytotoxicity. Here, we describe optimized ACE2-IgG4-Fc fusion constructs that avoid Fc-receptor activation, preserve the desired ACE2 enzymatic activity and show promising pharmaceutical properties. The engineered ACE2-IgG4-Fc fusion proteins neutralize the original SARS-CoV, pandemic SARS-CoV-2 as well as the rapidly spreading SARS-CoV-2 alpha, beta and delta variants of concern. Importantly, these variants of concern are inhibited at picomolar concentrations proving that ACE2-IgG4 maintains - in contrast to therapeutic antibodies - its full antiviral potential. Thus, ACE2-IgG4-Fc fusion proteins are promising candidate anti-antivirals to combat the current and future pandemics.
Assuntos
Enzima de Conversão de Angiotensina 2 , Antivirais/síntese química , Tratamento Farmacológico da COVID-19 , Imunoglobulina G , Internalização do Vírus/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/uso terapêutico , Antivirais/uso terapêutico , Humanos , Ligação Proteica , SARS-CoV-2/efeitos dos fármacosRESUMO
Statistical mining and integration of complex molecular data including metabolites, proteins, and transcripts is one of the critical goals of systems biology (Ideker, T., Galitski, T., and Hood, L. (2001) A new approach to decoding life: systems biology. Annu. Rev. Genomics Hum. Genet. 2, 343-372). A number of studies have demonstrated the parallel analysis of metabolites and large scale transcript expression. Protein analysis has been ignored in these studies, although a clear correlation between transcript and protein levels is shown only in rare cases, necessitating that actual protein levels have to be determined for protein function analysis. Here, we present an approach to investigate the combined covariance structure of metabolite and protein dynamics in a systemic response to abiotic temperature stress in Arabidopsis thaliana wild-type and a corresponding starch-deficient mutant (phosphoglucomutase-deficient). Independent component analysis revealed phenotype classification resolving genotype-dependent response effects to temperature treatment and genotype-independent general temperature compensation mechanisms. An observation is the stress-induced increase of raffinose-family-oligosaccharide levels in the absence of transitory starch storage/mobilization in temperature-treated phosphoglucomutase plants indicating that sucrose synthesis and storage in these mutant plants is sufficient to bypass the typical starch storage/mobilization pathways under abiotic stress. Eventually, sample pattern recognition and correlation network topology analysis allowed for the detection of specific metabolite-protein co-regulation and assignment of a circadian output regulated RNA-binding protein to these processes. The whole concept of high-dimensional profiling data integration from many replicates, subsequent multivariate statistics for dimensionality reduction, and covariance structure analysis is proposed to be a major strategy for revealing central responses of the biological system under study.
Assuntos
Proteínas de Arabidopsis/análise , Arabidopsis/metabolismo , Temperatura Baixa , Temperatura Alta , Proteômica/métodos , Rafinose/metabolismo , Amido/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cromatografia Gasosa-Espectrometria de Massas/métodos , Fenótipo , Análise Serial de ProteínasRESUMO
N-glycosylation is a common post-translational modification of biopharmaceutical products. Certain types of N-glycans have been shown to influence important properties of monoclonal antibody products including pharmacokinetics and effector functions. Complex biopharmaceuticals e.g. Fc fusion proteins may contain several N- and O-glycosylation sites. Domain specific characterization of two Fc fusion proteins showed an Fc N-glycosylation pattern comparable to IgG molecules. The receptor N-glycosylation was found to contain some larger and more complex N-glycans compared to the Fc part. Analyses of samples from non-clinical studies of the two studied fusion proteins indicate that their N-glycans impact pharmacokinetic properties. Interestingly, besides the type of N-glycan this influence on the pharmacokinetics depends also on the glycosylation site and thus the accessibility on the protein. The same type of N-glycan can influence the clearance of fusion proteins when located at the receptor part, but not if located at the Fc part. In this study, it is shown that N-glycans with terminal galactose or N-acetylglucosamine residues have a negative impact on serum half-life when located at the receptor part. Terminal sialylation of galactose residues prevents this faster clearance even when only one sialic acid is present. O-acetylation, a modification of sialic acids does not impact pharmacokinetics. Thus, type and accessibility of N-glycan moieties of fusion proteins both play an important role in pharmacokinetics. Finally, detailed site specific analysis is critical in the development of biopharmaceuticals.
Assuntos
Anticorpos Monoclonais/farmacocinética , Produtos Biológicos/farmacocinética , Fragmentos Fc das Imunoglobulinas/farmacologia , Polissacarídeos/química , Proteínas Recombinantes de Fusão/farmacocinética , Acetilglucosamina/química , Anticorpos Monoclonais/química , Produtos Biológicos/química , Glicosilação , Meia-Vida , Fragmentos Fc das Imunoglobulinas/química , Proteínas Recombinantes de Fusão/químicaRESUMO
BACKGROUND: Honey bee (Apis mellifera) workers are characterized by complex social behavior. Their life-history is dominated by a period of within-nest activity followed by a phase of long-distance flights and foraging. General insights into insect metabolism imply that foraging onset is associated with fundamental metabolic changes, and theory on social evolution suggests metabolic adaptations that are advantageous for the colony as a whole. RESULTS: Here we address the life-history characteristics of workers with LC-MS/MS based relative quantification of major proteins. Our approach includes: i. Calculation of a false positive rate for the identifications, ii. Support of relative protein quantification results obtained from spectral count by non-parametric statistics, and iii. Correction for Type 1 error inflation using a bootstrap iteration analysis. Our data are consistent with the use of glucose as the main fuel for honey bee flight. Moreover, the data delivers information on the expression of ATPsynthases/ATPases, and provide new insights into nurse- and forager-specific patterns of protection against oxidative stress. CONCLUSION: The results show the suitability of this approach to investigate fundamental biochemical changes in an insect, and provide new evidence for metabolic specializations that occur during the social ontogeny of worker honey bees.
RESUMO
Methods such as mRNA expression profiling have provided a vast amount of genomic and transcriptomic information about plants and other organisms. However, there is explicit indication that considerable metabolic control is executed on the metabolite and on the protein level including protein modifications, thereby constituting the phenotypic plasticity. Consequently, the analysis of the molecular phenotype demands the step toward mass spectrometry (MS)-based postgenomic techniques such as metabolomics and proteomics. This chapter describes a straightforward protocol for simultaneously extracting metabolites and proteins from the same biological sample in preparation for MS analysis. Furthermore, protocols for profiling polar metabolites using gas chromatography time-of-flight MS and for shotgun proteomics using liquid chromatography-MS are discussed. A practical course is laid out that outlines all the basic steps, from harvesting to data analysis. These steps enable the correlative study of metabolite and protein dynamics with minimal technical variation. Biological variability of independent samples is exploited for variance analysis and pattern recognition.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Perfilação da Expressão Gênica/métodos , Folhas de Planta/metabolismo , Proteômica/métodos , Arabidopsis/química , Proteínas de Arabidopsis/isolamento & purificação , Cromatografia Líquida/instrumentação , Cromatografia Líquida/métodos , Cromatografia Gasosa-Espectrometria de Massas/instrumentação , Cromatografia Gasosa-Espectrometria de Massas/métodos , Folhas de Planta/químicaRESUMO
Methylation of DNA is an epigenetic mechanism that influences patterns of gene expression. DNA methylation marks contribute to adaptive phenotypic variation but are erased during development. The role of DNA methylation in adaptive evolution is therefore unclear. We propose that environmentally-induced DNA methylation causes phenotypic heterogeneity that provides a substrate for selection via forces that act on the epigenetic machinery. For example, selection can alter environmentally-induced methylation of DNA by acting on the molecular mechanisms used for the genomic targeting of DNA methylation. Another possibility is that specific methylation marks that are environmentally-induced, yet non-heritable, could influence preferential survival and lead to consistent methylation of the same genomic regions over time. As methylation of DNA is known to increase the likelihood of cytosine-to-thymine transitions, non-heritable adaptive methylation marks can drive an increased likelihood of mutations targeted to regions that are consistently marked across several generations. Some of these mutations could capture, genetically, the phenotypic advantage of the epigenetic mark. Thereby, selectively favored transitory alterations in the genome invoked by DNA methylation could ultimately become selectable genetic variation through mutation. We provide evidence for these concepts using examples from different taxa, but focus on experimental data on large-scale DNA sequencing that expose between-group genetic variation after bidirectional selection on honeybees, Apis mellifera.
Assuntos
Adaptação Fisiológica/genética , Abelhas/genética , Abelhas/fisiologia , Metilação de DNA/genética , Meio Ambiente , Genoma de Inseto/genética , Animais , Sequência de Bases/genética , Evolução Molecular , Variação Genética , Mutação/genética , FenótipoRESUMO
Many animals are characterized by declining brain function at advanced ages, including honeybees (Apis mellifera). Variation in honeybee social development, moreover, results in individual differences in the progression of aging that may be accelerated, delayed, and sometimes reversed by changes in behavior. Here, we combine manipulations of social development with a measurement of sensory sensitivity, Pavlovian (associative) learning, and a proteomic technique to study the brain of aged honeybees. First, we confirm that sensory sensitivity can remain intact during aging, and that age-associated learning deficits are specific to bees that forage, a behavior typically expressed after a period of nursing activity. These initial data go beyond previous findings by showing how foragers age in social groups of different age compositions and sizes. Thereafter, we establish that learning ability can recover in aged foragers that revert to nursing tasks. Finally, we use liquid chromatography coupled to tandem mass spectrometry (LC-MS(2)) to describe proteomic differences between central brains, from reverted former foragers that varied in recovery of learning performance, and from nurse bees that varied in learning ability but never foraged. We find that recovery is positively associated with levels of stress response/cellular maintenance proteins in the central brain, while variation in learning before aging is negatively associated with the amounts of metabolic enzymes in the brain tissue. Our work provides the strongest evidence, thus far, for reversibility of learning deficits in aged honeybees, and indicates that recovery-related brain plasticity is connected to cellular stress resilience, maintenance and repair processes.
Assuntos
Envelhecimento/fisiologia , Abelhas/fisiologia , Aprendizagem por Discriminação/fisiologia , Análise de Variância , Animais , Comportamento Animal/fisiologia , Química Encefálica , Condicionamento Psicológico/fisiologia , Proteoma , Recuperação de Função Fisiológica/fisiologia , Olfato/fisiologia , Percepção Gustatória/fisiologiaRESUMO
In honeybee societies, distinct caste phenotypes are created from the same genotype, suggesting a role for epigenetics in deriving these behaviorally different phenotypes. We found no differences in DNA methylation between irreversible worker and queen castes, but substantial differences between nurses and forager subcastes. Reverting foragers back to nurses reestablished methylation levels for a majority of genes and provides, to the best of our knowledge, the first evidence in any organism of reversible epigenetic changes associated with behavior.
Assuntos
Comportamento Animal/fisiologia , Metilação de DNA/fisiologia , Epigenômica , Animais , Abelhas/genética , Metilação de DNA/genética , Proteínas de Insetos/genética , Proteínas de Insetos/fisiologia , FenótipoRESUMO
Honeybee workers are essentially sterile female helpers that make up the majority of individuals in a colony. Workers display a marked change in physiology when they transition from in-nest tasks to foraging. Recent technological advances have made it possible to unravel the metabolic modifications associated with this transition. Previous studies have revealed extensive remodeling of brain, thorax, and hypopharyngeal gland biochemistry. However, data on changes in the abdomen is scarce. To narrow this gap we investigated the proteomic composition of abdominal tissue in the days typically preceding the onset of foraging in honeybee workers. In order to get a broader representation of possible protein dynamics, we used workers of two genotypes with differences in the age at which they initiate foraging. This approach was combined with RNA interference-mediated downregulation of an insulin/insulin-like signaling component that is central to foraging behavior, the insulin receptor substrate (irs), and with measurements of glucose and lipid levels. Our data provide new insight into the molecular underpinnings of phenotypic plasticity in the honeybee, invoke parallels with vertebrate metabolism, and support an integrated and irs-dependent association of carbohydrate and lipid metabolism with the transition from in-nest tasks to foraging.
Assuntos
Abelhas/genética , Abelhas/fisiologia , Regulação da Expressão Gênica , Proteômica/métodos , Abdome/fisiologia , Animais , Carboidratos/química , Regulação para Baixo , Feminino , Genótipo , Glucose/metabolismo , Insulina/metabolismo , Metabolismo dos Lipídeos , Lipídeos/química , Modelos Genéticos , Fenótipo , Proteoma , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/metabolismoRESUMO
Polistes paper wasps are models for understanding conditions that may have characterized the origin of worker and queen castes and, therefore, the origin of paper wasp sociality. Polistes is "primitively eusocial" by virtue of having context-dependent caste determination and no morphological differences between castes. Even so, Polistes colonies have a temporal pattern in which most female larvae reared by the foundress become workers, and most reared by workers become future-reproductive gynes. This pattern is hypothesized to reflect development onto two pathways, which may utilize mechanisms that regulate diapause in other insects. Using expressed sequence tags (ESTs) for Polistes metricus we selected candidate genes differentially expressed in other insects in three categories: 1) diapause vs. non-diapause phenotypes and/or worker vs. queen differentiation, 2) behavioral subcastes of worker honey bees, and 3) no a priori expectation of a role in worker/gyne development. We also used a non-targeted proteomics screen to test for peptide/protein abundance differences that could reflect larval developmental divergence. We found that foundress-reared larvae (putative worker-destined) and worker-reared larvae (putative gyne-destined) differed in quantitative expression of sixteen genes, twelve of which were associated with caste and/or diapause in other insects, and they also differed in abundance of nine peptides/proteins. Some differentially-expressed genes are involved in diapause regulation in other insects, and other differentially-expressed genes and proteins are involved in the insulin signaling pathway, nutrient metabolism, and caste determination in highly social bees. Differential expression of a gene and a peptide encoding hexameric storage proteins is especially noteworthy. Although not conclusive, our results support hypotheses of 1) larval developmental pathway divergence that can lead to caste bias in adults and 2) nutritional differences as the foundation of the pathway divergence. Finally, the differential expression in Polistes larvae of genes and proteins also differentially expressed during queen vs. worker caste development in honey bees may indicate that regulatory mechanisms of caste outcomes share similarities between primitively eusocial and advanced eusocial Hymenoptera.
Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Proteínas de Insetos/genética , Predomínio Social , Vespas/genética , Animais , Viés , Cromatografia Líquida , Análise Discriminante , Genes de Insetos/genética , Proteínas de Insetos/metabolismo , Larva/genética , Espectrometria de Massas , Peptídeos/genética , Peptídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Processos de Determinação SexualRESUMO
Insect diapause is an alternative life-history strategy used to increase longevity and survival in harsh environmental conditions. Even though some aspects of diapause are well investigated, broader scale studies that elucidate the global metabolic adjustments required for this remarkable trait, are rare. In order to better understand the metabolic changes during early insect diapause, we used a shotgun proteomics approach on early diapausing and non-diapausing larvae of the recently sequenced hymenopteran model organism Nasonia vitripennis. Our results deliver insights into the molecular underpinnings of diapause in Nasonia and corroborate previously reported diapause-associated features for invertebrates, such as a diapause-dependent abundance change for heat shock and storage proteins. Furthermore, we observed a diapause-dependent switch in enzymes involved in glycerol synthesis and a vastly changed capacity for protein synthesis and degradation. The abundance of structural proteins and proteins involved in protein synthesis decreased with increasing diapause duration, while the abundance of proteins likely involved in diapause maintenance (e.g. ferritins) increased. Only few potentially diapause-specific proteins were identified suggesting that diapause in Nasonia relies to a large extent on a modulation of pre-existing pathways. Studying a diapause syndrome on a proteomic level rather than isolated pathways or physiological networks, has proven to be an efficient and successful avenue to understand molecular mechanisms involved in diapause.
Assuntos
Proteínas de Insetos/química , Insetos/fisiologia , Proteômica , Animais , Cromatografia Líquida , Estágios do Ciclo de Vida , Espectrometria de Massas em TandemRESUMO
With age, worker honey bees normally proceed from performing activities inside the nest to foraging in the field, creating an age-related division of labor. We previously established that the whole-body protein profiles of nest workers and foragers are different, and proposed that this proteomic divergence in part is explained by a shift in metabolic requirements as worker bees initiate intense flight. The unique plasticity of honey bee worker ontogeny, however, provides further opportunities to investigate if such changes in the proteome are dynamic or, alternatively, are permanently induced. Through manipulation of the social structure of colonies, foragers can be forced to revert to nest tasks, and in the current study we investigate how protein profiles respond to such reverse development. By using a quantitative LC-MS/MS-based approach in conjunction with robust statistical validation we show that after reversal from foraging to nest activities, subsets of proteins are detected at relative concentrations that characterize nest bees, whereas other proteins remain unchanged at relative concentrations normally found in foragers. In all, we quantified the levels of 81 proteins, and for 22 of these we found significant differences between worker groups before and after reversion. We interpret these patterns as examples of plasticity and robustness at the proteome level that are linked to characteristics of behavior and aging in Apis mellifera.
Assuntos
Abelhas/fisiologia , Proteínas de Insetos/metabolismo , Proteoma/metabolismo , Animais , Anexinas/metabolismo , Abelhas/metabolismo , Comportamento Animal/fisiologia , Cromatografia Líquida , Análise por Conglomerados , Citocromos c/metabolismo , Feminino , Proteínas de Insetos/análise , Malato Desidrogenase/metabolismo , Proteoma/análise , Receptores Odorantes/metabolismo , Espectrometria de Massas em Tandem , Fatores de Tempo , alfa-Glucosidases/metabolismoRESUMO
Many essential cellular functions such as growth rate, motility, and metabolic activity are linked to reversible protein phosphorylation, since they are controlled by signaling cascades based mainly on phosphorylation/dephosphorylation events. Quantification of global or site-specific protein phosphorylation is not straightforward with standard proteomic techniques. The coupling of capillary liquid chromatography (microLC) with ICP-MS (inductively coupled plasma-mass spectrometry) is a method which allows a quantitative screening of protein extracts for their phosphorus and sulfur content, and thus provides access to the protein phosphorylation degree. In extension of a recent pilot study, we analyzed protein extracts from the model organisms Arabidopsis thaliana and Chlamydomonas reinhardtii as representatives for multicellular and unicellular green photosynthetically active organisms. The results indicate that the average protein phosphorylation level of the algae C. reinhardtii is higher than that of A. thaliana. Both the average phosphorylation levels were found to be between the extreme values determined so far for prokaryotes (C. glutamicum, lowest levels) and eukaryotes (Mus musculus, highest levels). Tissue samples of A. thaliana representing different stages of plant development showed varying levels of protein phosphorylation indicating a different adjustment of the kinase/phosphatase system. We also utilized the microLC-ICP-MS technology to estimate the efficiency of a novel phosphoprotein enrichment method based on aluminum hydroxide, since the enrichment of phosphorylated species is often an essential step for their molecular characterization.
Assuntos
Fosfoproteínas/metabolismo , Proteínas de Plantas/metabolismo , Animais , Arabidopsis/metabolismo , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Arabidopsis/metabolismo , Células Cultivadas , Chlamydomonas reinhardtii/isolamento & purificação , Chlamydomonas reinhardtii/metabolismo , Cromatografia Líquida , Espectrometria de Massas , Camundongos , Fragmentos de Peptídeos/isolamento & purificação , Fosfoproteínas/isolamento & purificação , Fósforo/análise , Fosforilação , Proteômica , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Enxofre/análiseRESUMO
The standard strategy for analysis by tandem mass spectrometry of protein phosphorylation at serine or threonine utilizes the neutral loss of H3PO4 (= 97.977/z) from proteolytic peptide molecular ions as marker fragmentation. Manual control of automatically performed neutral loss-based phosphopeptide identifications is strongly recommended, since these data may contain false-positive results. These are connected to the experimental neutral loss m/z error, to competing peptide fragmentation pathways, to limitations in data interpretation software, and to the general growth of protein sequence databases. The fragmentation-related limitations of the neutral loss approach cover (i) the occurrence of abundant 'close-to-98/z' neutral loss fragmentations, (ii) the erroneous assignment of a neutral loss other than loss of H3PO4 due to charge state mix-up, and (iii) the accidental occurrence of any fragment ion in the m/z windows of interest in combination with a charge-state mix-up. The 'close-to-98/z' losses comprise loss of proline (97.053/z), valine (99.068/z), threonine (101.048/z), or cysteine (103.009/z) preferably from peptides with N-terminal sequences PP, VP, TP, or CP, and loss of 105.025/z from alkylated methionine. Confusion with other neutral losses may occur, when their m/z window coincides with a 98/z window as result of a charge state mix-up. Neutral loss of sulfenic acid from oxidized methionine originating from a doubly charged precursor (63.998/2 = 31.999) may thus mimic the loss of phosphoric acid from a triply charged phosphopeptide (97.977/3 = 32.659). As a consequence of the large complexity of proteomes, peptide sequence ions may occur in one of the mass windows of H3PO4 loss around 97.977/z. Practical examples for false-positive annotations of phosphopeptides are given for the first two groups of error. The majority of these can be readily recognized using the guidelines presented in this study.