RESUMO
We report identification of a beta-type pigment-dispersing hormone (PDH) identical in two water flea species, Daphnia magna and Daphnia pulex. It has been identified by cloning of precursors, chromatographic isolation from tissue extracts followed by immunoassays and de novo-mass spectrometric sequencing. The peptide is restricted to a complex system of distinct interneurons in the brain and visual ganglia, but does not occur in neurosecretory cells projecting to neurohemal organs as in decapod crustaceans. Thirteen neuron types individually identified and reconstructed by immunohistochemistry were almost identical in terms of positions and projection patterns in both species. Several neurons invade and form plexuses in visual ganglia and major brain neuropils including the central body. Five neuron types show contralateral pathways and form plexuses in the lateral, dorsal, or postlateral brain neuropils. Others are local interneurons, and a tritocerebral neuron connects the protocerebrum with the neuropil of the locomotory second antenna. Two visual ganglia neuron types lateral to the medulla closely resemble insect medulla lateral circadian clock neurons containing pigment-dispersing factor based upon positional and projectional criteria. Experiments under 12:12 h light/dark cycles and constant light or darkness conditions showed significant circadian changes in numbers and activities of one type of medulla lateral PDH neuron with an acrophase in the evening. This simple PDH system shows striking homologies to PDH systems in decapod crustaceans and well-known clock neurons in several insects, which suggests evolutionary conservation of an ancient peptidergic interneuronal system that is part of biological clocks.
Assuntos
Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Daphnia/fisiologia , Neurônios/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/imunologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
We report 43 novel genes in the water flea Daphnia pulex encoding 73 predicted neuropeptide and protein hormones as partly confirmed by RT-PCR. MALDI-TOF mass spectrometry identified 40 neuropeptides by mass matches and 30 neuropeptides by fragmentation sequencing. Single genes encode adipokinetic hormone, allatostatin-A, allatostatin-B, allatotropin, Ala(7)-CCAP, CCHamide, Arg(7)-corazonin, DENamides, CRF-like (DH52) and calcitonin-like (DH31) diuretic hormones, two ecdysis-triggering hormones, two FIRFamides, one insulin, two alternative splice forms of ion transport peptide (ITP), myosuppressin, neuroparsin, two neuropeptide-F splice forms, three periviscerokinins (but no pyrokinins), pigment dispersing hormone, proctolin, Met(4)-proctolin, short neuropeptide-F, three RYamides, SIFamide, two sulfakinins, and three tachykinins. There are two genes for a preprohormone containing orcomyotropin-like peptides and orcokinins, two genes for N-terminally elongated ITPs, two genes (clustered) for eclosion hormones, two genes (clustered) for bursicons alpha, beta, and two genes (clustered) for glycoproteins GPA2, GPB5, three genes for different allatostatins-C (two of them clustered) and three genes for IGF-related peptides. Detailed comparisons of genes or their products with those from insects and decapod crustaceans revealed that the D. pulex peptides are often closer related to their insect than to their decapod crustacean homologues, confirming that branchiopods, to which Daphnia belongs, are the ancestor group of insects.
Assuntos
Genômica , Peptídeos/química , Transcriptoma , Adipocinas/metabolismo , Sequência de Aminoácidos , Animais , Biologia Computacional/métodos , Daphnia , Etiquetas de Sequências Expressas , Feminino , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Neuropeptídeos/química , Proteínas/química , Proteômica/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Corazonin (Crz) is an 11 amino acid C-terminally amidated neuropeptide that has been identified in most arthropods examined with the notable exception of beetles and an aphid. The Crz-receptor shares sequence similarity to the GnRH-AKH receptor family thus suggesting an ancestral function related to the control of reproduction and metabolism. In 1989, Crz was purified and identified as a potent cardioaccelerating agent in cockroaches (hence the Crz name based on "corazon", the Spanish word for "heart"). Since the initial assignment as a cardioacceleratory peptide, additional functions have been discovered, ranging from pigment migration in the integument of crustaceans and in the eye of locusts, melanization of the locust cuticle, ecdysis initiation and in various aspects of gregarization in locusts. The high degree of structural conservation of Crz, its well-conserved (immuno)-localization, mainly in specific neurosecretory cells in the pars lateralis, and its many functions, suggest that Crz is vital. Yet, Crz-deficient insects develop normally. Upon reexamining all known effects of Crz, a hypothesis was developed that the evolutionary ancient function of Crz may have been "to prepare animals for coping with the environmental stressors of the day". This function would then complement the role of pigment-dispersing factor (PDF), the prime hormonal effector of the clock, which is thought "to set a coping mechanism for the night".
Assuntos
Artrópodes/química , Proteínas de Insetos/fisiologia , Neuropeptídeos/fisiologia , Estresse Fisiológico/fisiologia , Sequência de Aminoácidos , Animais , Artrópodes/fisiologia , Ritmo Circadiano , Evolução Molecular , Feminino , Coração/fisiologia , Imuno-Histoquímica , Proteínas de Insetos/análise , Proteínas de Insetos/química , Masculino , Melaninas/metabolismo , Muda , Neuropeptídeos/análise , Neuropeptídeos/química , Pigmentos Biológicos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiologia , Receptores LHRH/química , Receptores de Neuropeptídeos/química , Receptores de Neuropeptídeos/fisiologiaRESUMO
This review is focussed on SIFamide. This neuropeptide was discovered as a result of an extensive purification process, typical for 20th century physiology, of an extract of 350,000 flesh flies. Our knowledge of SIFamide greatly expanded since the first publication in 1996. Describing the minor and major findings on this peptide is our lead to summarise a number of innovations that recently became common in research on Arthropods. Mass spectrometry, nanoLC, whole mount immunocytochemistry, genome sequencing, deorphanizing receptors and functional gene knock downs are aspects that dramatically improved and changed peptide research. Some of the techniques mentioned in this review were of course applied before 1996, but they were not widespread. Although the focus of the review is on insects we incorporated the data of SIFamide in Crustaceans as well. SIFamide illustrates that crustaceans and insects might have more in common than was previously anticipated. Today, six isoforms of SIFamide are discovered in many crustaceans, several insects and a tick. The sequence of SIFamide is extremely conserved among these species. Deorphanizing its receptor in Drosophila, learned that both the ligand and receptor are impressively conserved, pointing at a crucial function. Immunohistochemistry and mass spectrometry data reveal that SIFamide is present in the crustacean brain and gut, but restricted to four neurons in the insect pars intercerebralis. The immunoreactive patterns in the brain refer to a neuromodulatory role in combining visual, tactile and olfactory input. Eventually, targeted cell ablation and RNAi revealed that SIFamide modulates sexual behaviour in fruit flies.
Assuntos
Artrópodes/metabolismo , Proteínas de Insetos/fisiologia , Neuropeptídeos/química , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Bases de Dados Genéticas , Imuno-Histoquímica , Proteínas de Insetos/química , Proteínas de Insetos/isolamento & purificação , Mucosa Intestinal/metabolismo , Ligantes , Espectrometria de Massas , Dados de Sequência Molecular , Neuropeptídeos/isolamento & purificação , Neuropeptídeos/fisiologia , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/fisiologia , Alinhamento de Sequência , Análise de Sequência de ProteínaRESUMO
A major unresolved issue in insect endocrinology concerns the question of whether or not insects have sex hormones. Conclusive evidence in favor of the presence of such hormones awaits the establishment of appropriate bioassays in males. The cuticle of sexually mature males of the desert locust Schistocerca gregaria turns yellow in gregarious conditions only. Neither females nor isolated males ever turn yellow. The yellowing is due to the deposition in the cuticle of a male-specific Yellow Protein (YP), of which the amino acid sequence is known. In this paper, we describe the partial cloning of the cDNA encoding this Yellow Protein. The tissue distribution and temporal expression of the YP-mRNA is studied in detail using RT-PCR. Furthermore, an RT-PCR based bioassay was developed, which may serve as a reliable tool to help identify the hormones controlling the yellowing process. In addition to juvenile hormone, we have shown that a factor present in the brain-corpora cardiaca is involved in the yellow coloration, as injection of an extract induces the expression of YP-mRNA in isolated gregarious males.
Assuntos
Gafanhotos/genética , Proteínas de Insetos/genética , Reação em Cadeia da Polimerase/métodos , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/genética , Regulação da Expressão Gênica no Desenvolvimento , Gafanhotos/crescimento & desenvolvimento , Proteínas de Insetos/isolamento & purificação , Masculino , Dados de Sequência Molecular , RNA Mensageiro/genética , Transcrição GênicaRESUMO
BACKGROUND: For holometabolous insects there has been an explosion of proteomic and peptidomic information thanks to large genome sequencing projects. Heterometabolous insects, although comprising many important species, have been far less studied. The migratory locust Locusta migratoria, a heterometabolous insect, is one of the most infamous agricultural pests. They undergo a well-known and profound phase transition from the relatively harmless solitary form to a ferocious gregarious form. The underlying regulatory mechanisms of this phase transition are not fully understood, but it is undoubtedly that neuropeptides are involved. However, neuropeptide research in locusts is hampered by the absence of genomic information. RESULTS: Recently, EST (Expressed Sequence Tag) databases from Locusta migratoria were constructed. Using bioinformatical tools, we searched these EST databases specifically for neuropeptide precursors. Based on known locust neuropeptide sequences, we confirmed the sequence of several previously identified neuropeptide precursors (i.e. pacifastin-related peptides), which consolidated our method. In addition, we found two novel neuroparsin precursors and annotated the hitherto unknown tachykinin precursor. Besides one of the known tachykinin peptides, this EST contained an additional tachykinin-like sequence. Using neuropeptide precursors from Drosophila melanogaster as a query, we succeeded in annotating the Locusta neuropeptide F, allatostatin-C and ecdysis-triggering hormone precursor, which until now had not been identified in locusts or in any other heterometabolous insect. For the tachykinin precursor, the ecdysis-triggering hormone precursor and the allatostatin-C precursor, translation of the predicted neuropeptides in neural tissues was confirmed with mass spectrometric techniques. CONCLUSION: In this study we describe the annotation of 6 novel neuropeptide precursors and the neuropeptides they encode from the migratory locust, Locusta migratoria. By combining the manual annotation of neuropeptides with experimental evidence provided by mass spectrometry, we demonstrate that the genes are not only transcribed but also translated into precursor proteins. In addition, we show which neuropeptides are cleaved from these precursor proteins and how they are post-translationally modified.
Assuntos
Etiquetas de Sequências Expressas , Hormônios de Inseto/genética , Locusta migratoria/genética , Neuropeptídeos/genética , Precursores de Proteínas/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Animais , Genoma de Inseto/genética , Hormônios de Inseto/análise , Locusta migratoria/química , Dados de Sequência Molecular , Neuropeptídeos/análise , Precursores de Proteínas/análise , Proteínas/análise , Proteínas/genética , Homologia de Sequência de Aminoácidos , Taquicininas/análise , Taquicininas/genéticaRESUMO
Degradation of the neuropeptide [His7]-corazonin, a key hormone in phase transition in locusts was studied using [3H][His7]-corazonin, RP-HPLC and mass spectrometry. After 4h incubation, 50 and 75% of [His7]-corazonin could still be found in hemolymph of gregarious and solitarious Schistocerca gregaria, respectively. Under in vivo conditions the half-life was 30 min. These results are in contrast to many other neuropeptides that usually have half lives of a few minutes. The peptide is cleaved first by an endopeptidase, either just before or after the Tyr residue at position 5. Next, the C-terminal degradation fragments are further degraded by a dipeptidyl-peptidase, whereas the N-terminal fragments are further broken down one amino acid at a time. In addition, [Dopa5][His7]-corazonin was detected. Upon synthesis, this unexpected molecular modification turned out to be biologically active in bringing about cuticular melanization.
Assuntos
Gafanhotos/metabolismo , Hemolinfa/metabolismo , Proteínas de Insetos/metabolismo , Neuropeptídeos/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Di-Hidroxifenilalanina/análogos & derivados , Di-Hidroxifenilalanina/farmacologia , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Endopeptidases/metabolismo , Proteínas de Insetos/farmacologia , Neuropeptídeos/farmacologia , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/metabolismo , Pigmentação/efeitos dos fármacos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The precursor of the insect hormone corazonin has been cloned from the honey bee Apis mellifera. The precursor predicts a novel isoform of corazonin, pQTFTYSHGWTNamide, which was confirmed by tandem mass spectrometry. Although Apis corazonin differs only by a glutamine/threonine substitution from [His7]-corazonin, it is considerably less active in the dark color inducing assay on albino locusts. Whole mount fluorescence immunohistochemistry of the central nervous system of the honey bee showed a pattern similar to the ones described for other insects. Four neurons of the lateral protocerebrum project axons towards the retrocerebral complex. It is unlikely that Apis corazonin is present in all hymenopteran species since the presence of this peptide could not be demonstrated by means of mass spectrometry in the retrocerebral complex of the red wood ant Formica rufa and the wasp Vespula saxonica. Instead, we found masses corresponding with [Arg7]- and [His7]-corazonin respectively, suggesting that some of the corazonin isoforms originated late during evolution in different insect orders.
Assuntos
Proteínas de Insetos/genética , Neuropeptídeos/genética , Isoformas de Proteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Abelhas/química , Clonagem Molecular , Imuno-Histoquímica , Proteínas de Insetos/química , Proteínas de Insetos/farmacologia , Locusta migratoria/efeitos dos fármacos , Dados de Sequência Molecular , Neuropeptídeos/química , Neuropeptídeos/farmacologia , Pigmentação/efeitos dos fármacos , Isoformas de Proteínas/química , Isoformas de Proteínas/farmacologia , Precursores de Proteínas/química , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
A brain-retrocerebral complex-subesophageal ganglion acidified methanolic extract of 100 larval Galleria mellonella (greater wax moth) was prepared for the isolation and identification of (neuro)peptides. To reduce sample complexity, the isolated peptides were roughly separated using a single, conventional chromatographic separation step. Subsequently, screening of these fractions with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in combination with nanoflow electrospray ionization quadrupole time-of-flight tandem mass spectrometry resulted in the identification of 12 lepidopteran peptides. None of these had been previously isolated or characterized within this species. VIFTPKLamide encoded by the diapause hormone-pheromone biosynthesis activating neuropeptide precursor was for the first time isolated and biochemically identified in a tissue extract, providing irrefutable evidence of its expression in larval nervous tissue. Another pentapeptide, AMVRFamide, with no resemblance to other lepidopteran peptides, was de novo sequenced and is most related to the neuropeptide F peptide family.
Assuntos
Gânglios dos Invertebrados/metabolismo , Proteínas de Insetos/análise , Mariposas/metabolismo , Neuropeptídeos/análise , Sequência de Aminoácidos , Animais , Química Encefálica , Cromatografia Líquida de Alta Pressão , Gânglios dos Invertebrados/química , Genes de Insetos , Cabeça , Proteínas de Insetos/genética , Cininas/análise , Larva/química , Larva/metabolismo , Dados de Sequência Molecular , Mariposas/química , Mariposas/genética , Neuropeptídeos/genética , Oligopeptídeos/análise , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
A mass spectrometric study of the peptidome of the neurohemal part of the corpora cardiaca of Locusta migratoria and Schistocerca gregaria shows that it contains several unknown peptides. We were able to identify the sequence of one of these peptides as pQSDLFLLSPK. This sequence is identical to the part of the Locusta insulin-related peptide (IRP) precursor that is situated between the signal peptide and the B-chain. We designated this peptide as IRP copeptide. This IRP copeptide is also present in the pars intercerebralis, which is likely to be the site of synthesis. It is identical in both L. migratoria and S. gregaria. It shows no effect on the hemolymph lipid concentration in vivo or muscle contraction in vitro. The IRP copeptide is able to cause a decreased phosphorylase activity in locust fat body in vitro, opposite to the effect of the adipokinetic hormones and therefore possibly represents a glycogenolysis-inhibiting peptide.
Assuntos
Glicogênio/metabolismo , Gafanhotos , Neuropeptídeos/análise , Sistemas Neurossecretores/química , Sequência de Aminoácidos , Animais , Inibidores Enzimáticos/farmacologia , Glicogênio Fosforilase/antagonistas & inibidores , Hormônios de Inseto/farmacologia , Espectrometria de Massas , Dados de Sequência Molecular , Neuropeptídeos/química , Neuropeptídeos/farmacologia , Precursores de Proteínas/química , Análise de Sequência de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Variability in behavior is often put in an unfavorable light as a marker of lack of skill. Here, we provide evidence that increased variability during preferred patterns of coordination is associated with higher flexibility in adopting new patterns. Twelve right-handed subjects performed cyclical bimanual flexion and extension patterns with four homologous and six heterologous joint combinations involving shoulder, elbow, wrist, and finger movements. Preferred (isofrequency) as well as less preferred (multifrequency) coordination patterns were studied. The findings revealed less accurate and less stable 1:1 coordination patterns during heterologous as compared to homologous limb segment combinations. Conversely, coordination patterns with a 2:1 frequency ratio were performed more accurately and more consistently during heterologous as compared to homologous conditions. Accordingly, a lower degree of coupling between effectors during performance of preferred coordination patterns was associated with more successful performance of less familiar patterns. This suggests that variability may promote the creative exploration of new performance modes.
Assuntos
Articulações/fisiologia , Movimento/fisiologia , Desempenho Psicomotor/fisiologia , Adulto , Análise de Variância , Fenômenos Biomecânicos , Lateralidade Funcional , Humanos , Maleabilidade , Fatores de TempoRESUMO
Peptides occur in the whole animal kingdom, from the least evolved phyla with a very simple nervous system (coelenterates) to the highest vertebrates and are involved in most, if not all, physiological processes in animals. Knowing the amino acid sequence of peptide hormones or neurotransmitters is important since this allows for synthesis of large quantities of peptides to perform further functional analysis. Immunocytochemistry, radioimmunoassays (RIA), enzyme-linked immunosorbant assays (ELISA) and mass spectrometry can then provide information on the temporal and spatial distribution and quantification of the (neuro)peptide. Ever since the 1970s, a wealth of peptides has been discovered and investigated and this flow seems to be far from over. This is partially due to the use of new approaches mainly based on chromatographical purifications as well as molecular biological techniques. Surprisingly, peptides have so far been neglected in most proteomic studies. The finalization of the genome projects has opened new opportunities for rapid identification and functional analysis of (neuro)peptides as well. In analogy with the proteomics technology, where all proteins expressed in a cell or tissue are analyzed, the peptidomic approach aims at the simultaneous visualization and identification of the whole peptidome of a cell or tissue, i.e. all expressed peptides with their post-translational modifications (PTMs). This technology provides us with a fast and efficient tool to analyze the peptides from any tissue. This paper reviews the approaches that have been used so far to achieve this.
Assuntos
Peptídeos/química , Proteômica , Sequência de Aminoácidos , Dados de Sequência Molecular , Peptídeos/isolamento & purificaçãoRESUMO
BACKGROUND: The desert locust (Schistocerca gregaria) displays a fascinating type of phenotypic plasticity, designated as 'phase polyphenism'. Depending on environmental conditions, one genome can be translated into two highly divergent phenotypes, termed the solitarious and gregarious (swarming) phase. Although many of the underlying molecular events remain elusive, the central nervous system (CNS) is expected to play a crucial role in the phase transition process. Locusts have also proven to be interesting model organisms in a physiological and neurobiological research context. However, molecular studies in locusts are hampered by the fact that genome/transcriptome sequence information available for this branch of insects is still limited. METHODOLOGY: We have generated 34,672 raw expressed sequence tags (EST) from the CNS of desert locusts in both phases. These ESTs were assembled in 12,709 unique transcript sequences and nearly 4,000 sequences were functionally annotated. Moreover, the obtained S. gregaria EST information is highly complementary to the existing orthopteran transcriptomic data. Since many novel transcripts encode neuronal signaling and signal transduction components, this paper includes an overview of these sequences. Furthermore, several transcripts being differentially represented in solitarious and gregarious locusts were retrieved from this EST database. The findings highlight the involvement of the CNS in the phase transition process and indicate that this novel annotated database may also add to the emerging knowledge of concomitant neuronal signaling and neuroplasticity events. CONCLUSIONS: In summary, we met the need for novel sequence data from desert locust CNS. To our knowledge, we hereby also present the first insect EST database that is derived from the complete CNS. The obtained S. gregaria EST data constitute an important new source of information that will be instrumental in further unraveling the molecular principles of phase polyphenism, in further establishing locusts as valuable research model organisms and in molecular evolutionary and comparative entomology.
Assuntos
Bases de Dados Genéticas , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Gafanhotos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , Regulação da Expressão Gênica , Dados de Sequência Molecular , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
Changes in the color of the cuticle, days after the completion of hardening, are rare in adult insects. Even more so when such changes are specific to one sexual form and coincide with sexual maturation. Adult males of the desert locust Schistocerca gregaria deposit a well characterized 'yellow protein' in their cuticle about 10 days after the adult molt, but only if they live under crowded (gregarious) conditions. Isolated-reared (solitarious) males do not turn yellow, neither do the females. Upon regrouping, yellowing is quickly induced, but again, only in the males. Juvenile hormone (JH) is involved, but its sex- and phase-specific effect suggests that other factors are also involved. We analyzed the recent and classical literature to find out what should be added or changed to the classical way of thinking on sex differentiation in insects so that a comprehensive conceptual framework could emerge. Undervalued and/or new data on male accessory glands as a possible second site of JH synthesis, on ecdysteroids as possible sex steroids, on the transcription factor fruitless in insects and on the evolutionarily highly conserved transcription factor Foxl2 that, when ablated in mice is responsible for the transdifferentiation of the ovaries into testes, are considered.
Assuntos
Gafanhotos/fisiologia , Modelos Animais , Modelos Biológicos , Pigmentação/fisiologia , Diferenciação Sexual/fisiologia , Animais , Ecdisteroides/metabolismo , Hormônios Juvenis/biossíntese , Hormônios Juvenis/metabolismo , Masculino , Neuroendocrinologia , Densidade Demográfica , Fatores de Transcrição/metabolismoRESUMO
Since the early days of cloning the first adipokinetic hormone (AKH) gene, researchers recognized that this gene also codes for a joining region and for a second peptide called adipokinetic hormone precursor related peptide (APRP). In species with more than one AKH gene, such as locusts, APRPs can form both homodimers and heterodimers. Database analysis showed that APRPs might belong to the ancient family of growth hormone releasing factor but they still are functionally orphan. We investigated whether some of the APRP forms play a role in control of reproduction or/and growth via stimulation of ecdysteroidogenesis.
Assuntos
Evolução Molecular , Hormônios de Inseto/metabolismo , Oligopeptídeos/metabolismo , Peptídeos/metabolismo , Precursores de Proteínas/metabolismo , Ácido Pirrolidonocarboxílico/análogos & derivados , Animais , Hormônios de Inseto/genética , Oligopeptídeos/genética , Peptídeos/genética , Precursores de Proteínas/genética , Ácido Pirrolidonocarboxílico/metabolismoRESUMO
MS is currently one of the most important analytical techniques in biological and medical research. ESI and MALDI launched the field of MS into biology. The performance of mass spectrometers increased tremendously over the past decades. Other technological advances increased the analytical power of biological MS even more. First, the advent of the genome projects allowed an automated analysis of mass spectrometric data. Second, improved separation techniques, like nanoscale HPLC, are essential for MS analysis of biomolecules. The recent progress in bioinformatics is the third factor that accelerated the biochemical analysis of macromolecules. The first part of this review will introduce the basics of these techniques. The field that integrates all these techniques to identify endogenous peptides is called peptidomics and will be discussed in the last section. This integrated approach aims at identifying all the present peptides in a cell, organ or organism (the peptidome). Today, peptidomics is used by several fields of research. Special emphasis will be given to the identification of neuropeptides, a class of short proteins that fulfil several important intercellular signalling functions in every animal. MS imaging techniques and biomarker discovery will also be discussed briefly.
Assuntos
Biologia Computacional/métodos , Espectrometria de Massas/métodos , Neuropeptídeos/análise , Proteômica/métodos , Animais , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Biologia Computacional/instrumentação , Humanos , Espectrometria de Massas/instrumentação , Proteômica/instrumentação , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Members of the insulin superfamily are not restricted to vertebrates, but have also been identified in invertebrate species. In the current report, we present the characterization of Scg-insulin-related peptide (IRP), an insulin-related peptide in the desert locust, Schistocerca gregaria. This peptide was isolated from corpora cardiaca (CC) extracts by means of a high-performance liquid chromatography (HPLC)-based purification strategy. Subsequent cloning and sequencing of the corresponding cDNA revealed that the encoded Scg-IRP precursor displays the structural organization that is typical for members of the insulin superfamily. Moreover, immunocytochemistry on brain tissue sections demonstrated the presence of Scg-IRP in median neurosecretory cells of the pars intercerebralis and their projections towards the storage part of the CC. Quantitative real-time RT-PCR studies revealed the presence of Scg-IRP transcripts in a variety of tissues, including nervous tissue and fat body. Furthermore, these transcripts showed a tissue- and phase-dependent, temporal regulation during the reproductive cycle of adult males and females. Finally, we demonstrated that Scg-IRP interacts in vitro with a recombinant neuroparsin, a locust protein displaying sequence similarity with vertebrate IGF binding proteins.
Assuntos
DNA Complementar/metabolismo , Gafanhotos/metabolismo , Hormônios de Inseto/metabolismo , Proteínas de Insetos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Feminino , Hormônios de Inseto/genética , Insulina/metabolismo , Masculino , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Especificidade de Órgãos , Peptídeos/genéticaRESUMO
To fully understand neuronal network operation, the influence of all inputs onto that network must be characterized. As in most systems, many neuronal and hormonal pathways influence the multifunctional motor circuits of the crustacean stomatogastric ganglion (STG), but the actions of only some of them are known. Therefore, we characterized the influence of the kinin peptide family on the gastric mill (chewing) and pyloric (filtering of chewed food) motor circuits in the STG of the crab Cancer borealis. The kinins are myoactive in arthropods and they occur within the arthropod central nervous system (CNS), but their CNS actions are not well characterized in any species. The pevkinins were first identified in the shrimp Penaeus vannamei, but they have yet to be studied in the STG of any species. We identified kinin-like immunolabeling (KLI) in the pericardial organs (POs) in C. borealis, but there was no KLI within the STG. The POs are a major source of hormonal influence on the STG. Pevkinin peptides activated the pyloric circuit and they caused a modest increase in the speed of ongoing pyloric rhythms. This modest influence on cycle speed resulted in part from pevkinin excitation of the lateral pyloric neuron, whose strengthened inhibitory synapse onto the pyloric pacemaker neurons limited the pevkinin-mediated increase in cycle speed. The pevkinin excitation of the pyloric rhythm was not strong enough to interfere with the previously documented, gastric mill rhythm-mediated weakening of the pyloric rhythm. Pevkinin also had little influence on the gastric mill rhythm. These results indicate that the kinin peptides have distinct and selective modulatory actions on the pyloric rhythm.
Assuntos
Braquiúros/metabolismo , Gânglios dos Invertebrados/efeitos dos fármacos , Cininas/farmacologia , Animais , Braquiúros/fisiologia , Condutividade Elétrica , Gânglios dos Invertebrados/metabolismo , Imuno-Histoquímica , Técnicas In Vitro , Cininas/análise , Masculino , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neuropeptídeos/análise , Oligopeptídeos/análise , Piloro/citologia , Piloro/efeitos dos fármacos , Piloro/fisiologiaRESUMO
Neuropeptides, critical brain peptides that modulate animal behavior by affecting the activity of almost every neuronal circuit, are inherently difficult to predict directly from a nascent genome sequence because of extensive posttranslational processing. The combination of bioinformatics and proteomics allows unprecedented neuropeptide discovery from an unannotated genome. Within the Apis mellifera genome, we have inferred more than 200 neuropeptides and have confirmed the sequences of 100 peptides. This study lays the groundwork for future molecular studies of Apis neuropeptides with the identification of 36 genes, 33 of which were previously unreported.
Assuntos
Abelhas/química , Abelhas/genética , Genes de Insetos , Proteínas de Insetos/química , Proteínas de Insetos/genética , Neuropeptídeos/química , Neuropeptídeos/genética , Algoritmos , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Química Encefálica , Códon , Biologia Computacional , Genoma de Inseto , Espectrometria de Massas , Dados de Sequência Molecular , Precursores de Proteínas/química , Precursores de Proteínas/genética , ProteomaRESUMO
An extract of head ganglia and retrocerebral complexes of nondiapausing and diapausing Leptinotarsa decemlineata was prepared to characterize regulatory neuropeptides involved in adult diapause by using a differential peptidomics approach. To reduce sample complexity, both extracts were roughly separated by means of an identical chromatographic step. MALDI-TOF MS led to the identification of proctolin, an adipokinetic hormone, and short neuropeptide F I and II in the extract of nondiapausing beetles. In combination with nano-ESI-Q-TOF MS(2) evidence was found for the presence of three pyrokinins, the first to be identified in a coleopteran species. Pyrokinins, involved in the induction of embryonic diapause in Bombyx mori, were present in both physiological conditions suggesting that they are of minor importance in the regulation of adult diapause in the Colorado potato beetle. A striking difference, detected by the differential peptidomics approach, between both neuropeptide profiles was the absence of ions corresponding to the short neuropeptide F (sNPF) related peptides, also known as Led-NPF-I and -II, in the extract of diapausing animals. Therefore, we postulate that "short NPFs" are involved in the regulation of adult diapause, displayed by the Colorado potato beetle.