RESUMO
Diversity, a hallmark of G protein-coupled receptor (GPCR) signaling, partly stems from alternative splicing of a single gene generating more than one isoform for a receptor. Additionally, receptor responses to ligands can be attenuated by desensitization upon prolonged or repeated ligand exposure. Both phenomena have been demonstrated and exemplified by the deuterostome tachykinin signaling system, although the role of phosphorylation in desensitization remains a subject of debate. Here, we describe the signaling system for tachykinin-related peptides (TKRPs) in a protostome, mollusk Aplysia. We cloned the Aplysia TKRP precursor, which encodes three TKRPs (apTKRP-1, apTKRP-2a, and apTKRP-2b) containing the FXGXR-amide motif. In situ hybridization and immunohistochemistry showed predominant expression of TKRP mRNA and peptide in the cerebral ganglia. TKRPs and their posttranslational modifications were observed in extracts of central nervous system ganglia using mass spectrometry. We identified two Aplysia TKRP receptors (apTKRPRs), named apTKRPR-A and apTKRPR-B. These receptors are two isoforms generated through alternative splicing of the same gene and differ only in their intracellular C termini. Structure-activity relationship analysis of apTKRP-2b revealed that both C-terminal amidation and conserved residues of the ligand are critical for receptor activation. C-terminal truncates and mutants of apTKRPRs suggested that there is a C-terminal phosphorylation-independent desensitization for both receptors. Moreover, apTKRPR-B also exhibits phosphorylation-dependent desensitization through the phosphorylation of C-terminal Ser/Thr residues. This comprehensive characterization of the Aplysia TKRP signaling system underscores the evolutionary conservation of the TKRP and TK signaling systems, while highlighting the intricacies of receptor regulation through alternative splicing and differential desensitization mechanisms.
RESUMO
The discovery of insulin in the early 1900s ushered in the era of research related to peptides acting as hormones and neuromodulators, among other regulatory roles. These essential gene products are found in all organisms, from the most primitive to the most evolved, and carry important biologic information that coordinates complex physiology and behavior; their misregulation has been implicated in a variety of diseases. The evolutionary origins of at least 30 neuropeptide signaling systems have been traced to the common ancestor of protostomes and deuterostomes. With the use of relevant animal models and modern technologies, we can gain mechanistic insight into orthologous and paralogous endogenous peptides and translate that knowledge into medically relevant insights and new treatments. Groundbreaking advances in medicine and basic science influence how signaling peptides are defined today. The precise mechanistic pathways for over 100 endogenous peptides in mammals are now known and have laid the foundation for multiple drug development pipelines. Peptide biologics have become valuable drugs due to their unique specificity and biologic activity, lack of toxic metabolites, and minimal undesirable interactions. This review outlines modern technologies that enable neuropeptide discovery and characterization, and highlights lessons from nature made possible by neuropeptide research in relevant animal models that is being adopted by the pharmaceutical industry. We conclude with a brief overview of approaches/strategies for effective development of peptides as drugs. SIGNIFICANCE STATEMENT: Neuropeptides, an important class of cell-cell signaling molecules, are involved in maintaining a range of physiological functions. Since the discovery of insulin's activity, over 100 bioactive peptides and peptide analogs have been used as therapeutics. Because these are complex molecules not easily predicted from a genome and their activity can change with subtle chemical modifications, mass spectrometry (MS) has significantly empowered peptide discovery and characterization. This review highlights contributions of MS-based research towards the development of therapeutic peptides.
Assuntos
Insulinas , Neuropeptídeos , Animais , Humanos , Mamíferos/metabolismo , Espectrometria de Massas/métodos , Neuropeptídeos/análise , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Peptídeos , Poder PsicológicoRESUMO
Peptidergic dense-core vesicles are involved in packaging and releasing neuropeptides and peptide hormones-critical processes underlying brain, endocrine and exocrine function. Yet, the heterogeneity within these organelles, even for morphologically defined vesicle types, is not well characterized because of their small volumes. We present image-guided, high-throughput mass spectrometry-based protocols to chemically profile large populations of both dense-core vesicles and lucent vesicles for their lipid and peptide contents, allowing observation of the chemical heterogeneity within and between these two vesicle populations. The proteolytic processing products of four prohormones are observed within the dense-core vesicles, and the mass spectral features corresponding to the specific peptide products suggest three distinct dense-core vesicle populations. Notable differences in the lipid mass range are observed between the dense-core and lucent vesicles. These single-organelle mass spectrometry approaches are adaptable to characterize a range of subcellular structures.
Assuntos
Aplysia/citologia , Ensaios de Triagem em Larga Escala/métodos , Aprendizado de Máquina , Organelas/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , AnimaisRESUMO
Exploring the chemical content of individual cells not only reveals underlying cell-to-cell chemical heterogeneity but is also a key component in understanding how cells combine to form emergent properties of cellular networks and tissues. Recent technological advances in many analytical techniques including mass spectrometry (MS) have improved instrumental limits of detection and laser/ion probe dimensions, allowing the analysis of micron and submicron sized areas. In the case of MS, these improvements combined with MS's broad analyte detection capabilities have enabled the rise of single-cell and single-organelle chemical characterization. As the chemical coverage and throughput of single-cell measurements increase, more advanced statistical and data analysis methods have aided in data visualization and interpretation. This review focuses on secondary ion MS and matrix-assisted laser desorption/ionization MS approaches for single-cell and single-organelle characterization, which is followed by advances in mass spectral data visualization and analysis.
RESUMO
Extended periods of bed rest and limb immobilization are required for healing post-injury or disease, yet disuse can result in significant muscle atrophy and decreased quality of life in older adults. Physical rehabilitation is commonly prescribed to recover these deficits, yet accumulation of reactive oxygen species and sustained rates of protein degradation persist during the rehabilitation period that can significantly delay or prevent recovery. Pericytes, considered the primary mesenchymal and vascular stromal cell in skeletal muscle, secrete beneficial factors that maintain baseline muscle mass, yet minimal information exists regarding the pericyte response to disuse and recovery. In the current study, single-cell RNA sequencing and functional assays were performed to demonstrate that pericytes in mouse skeletal muscle lose the capacity to synthesize antioxidants during disuse and recovery. This information was used to guide the design of a strategy in which healthy donor pericytes were stimulated with hydrogen peroxide (H2 O2 ) to produce small extracellular vesicles (sEVs) that effectively restored myofibre size in adult and aged muscle after disuse. Proteomic assessment detected 11 differentially regulated proteins in primed sEVs that may account for recovery of muscle, including proteins associated with extracellular matrix composition and anti-inflammatory and antioxidant processes. This study demonstrates that healthy H2 O2 -primed pericyte-derived sEVs effectively improve skeletal muscle recovery after immobilization, presenting a novel acellular approach to rebuild muscle mass in older adults after a period of disuse. KEY POINTS: Previous studies suggest that prolonged oxidative stress is a barrier to skeletal muscle recovery after a period of immobilization. In this study we demonstrate that muscle-resident perivascular stromal cells (pericytes) become dysfunctional and lack the capacity to mount an antioxidant defence after disuse in mice. Hydrogen peroxide treatment of healthy pericytes in vitro simulates the release of small extracellular vesicles (sEVs) that effectively recover skeletal muscle fibre size and extracellular matrix remodelling in young adult and aged mice after disuse. Pericyte-derived sEVs present a novel acellular strategy to recover skeletal muscle after disuse.
Assuntos
Peróxido de Hidrogênio , Qualidade de Vida , Camundongos , Animais , Peróxido de Hidrogênio/metabolismo , Antioxidantes/metabolismo , Proteômica , Músculo Esquelético/fisiologia , Atrofia Muscular/metabolismoRESUMO
Neuropeptides are a chemically diverse class of cell-to-cell signaling molecules that are widely expressed throughout the central nervous system, often in a cell-specific manner. While cell-to-cell differences in neuropeptides is expected, it is often unclear how exactly neuropeptide expression varies among neurons. Here we created a microscopy-guided, high-throughput single cell matrix-assisted laser desorption/ionization mass spectrometry approach to investigate the neuropeptide heterogeneity of individual neurons in the central nervous system of the neurobiological model Aplysia californica, the California sea hare. In all, we analyzed more than 26,000 neurons from 18 animals and assigned 866 peptides from 66 prohormones by mass matching against an in silico peptide library generated from known Aplysia prohormones retrieved from the UniProt database. Louvain-Jaccard (LJ) clustering of mass spectra from individual neurons revealed 40 unique neuronal populations, or LJ clusters, each with a distinct neuropeptide profile. Prohormones and their related peptides were generally found in single cells from ganglia consistent with the prohormones' previously known ganglion localizations. Several LJ clusters also revealed the cellular colocalization of behaviorally related prohormones, such as an LJ cluster exhibiting achatin and neuropeptide Y, which are involved in feeding, and another cluster characterized by urotensin II, small cardiac peptide, sensorin A, and FRFa, which have shown activity in the feeding network or are present in the feeding musculature. This mass spectrometry-based approach enables the robust categorization of large cell populations based on single cell neuropeptide content and is readily adaptable to the study of a range of animals and tissue types.
Assuntos
Aplysia , Neurônios , Neuropeptídeos , Animais , Aplysia/fisiologia , Sistema Nervoso Central/metabolismo , Neurônios/química , Neurônios/metabolismo , Neuropeptídeos/química , Neuropeptídeos/metabolismo , Análise de Célula Única , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Single cell analysis strives to probe molecular heterogeneity in morphologically similar cell populations through quantitative or qualitative measurements of genetic, proteomic, or metabolic products. Here, we applied mass analysis of single neurons to investigate cell-cell signaling peptides. The multiplicity of endogenous cell-cell signaling peptides is a common source of chemical diversity among cell populations. Certain peptides can undergo post-translational isomerization of select residues, which has important physiological consequences. The limited number of single cell analysis techniques that are sensitive to peptide stereochemistry make it challenging to study isomerization at the individual cell level. We performed capillary electrophoresis (CE) with mass spectrometry (MS) detection to characterize the peptide content of single cells. Using complementary trapped ion mobility spectrometry (TIMS) separations, we measured the stereochemical configurations of three neuropeptide gene products derived from the pleurin precursor in individual neurons (N = 3) isolated from the central nervous system of Aplysia californica. An analysis of the resultant mobility profiles indicated >98% of the detectable pleurin-derived peptides exist as the nonisomerized, all-l forms in individual neuron cell bodies. However, we observed 44% of the Plrn2 peptide from the pleurin precursor was present as the isomerized, d-residue-containing form in the nerve tissue. These findings demonstrate an unusual distribution of isomerized peptides in A. californica and establish CE-TIMS MS as a powerful analytical tool for investigating peptide stereochemistry at the single cell level.
Assuntos
Espectrometria de Mobilidade Iônica , Proteômica , Eletroforese Capilar , Espectrometria de Massas , PeptídeosRESUMO
Chronic use of opioids can produce opioid-induced hyperalgesia (OIH), and when used to treat migraine, these drugs can result in increased pain and headache chronicity. We hypothesized that overlapping mechanisms between OIH and chronic migraine occur through neuropeptide dysregulation. Using label-free, non-biased liquid chromatography-mass spectrometry to identify and measure changes in more than 1500 neuropeptides under these two conditions, we observed only 16 neuropeptides that were altered between the two conditions. The known pro-migraine molecule, calcitonin-gene related peptide, was among seven peptides associated with chronic migraine, with several pain-processing neuropeptides among the nine other peptides affected in OIH. Further, composite peptide complements Pituitary adenylate cyclase-activating polypeptide (PACAP), Vasoactive intestinal peptide (VIP) and Secretogranin (SCG) showed significant changes in both chronic migraine and OIH. In a follow-up pharmacological study, we confirmed the role of PACAP in models of these two disorders, validating the effectiveness of our peptidomic approach, and identifying PACAP as a mechanistic link between chronic migraine and OIH. Data are available via ProteomeXchange with identifier PXD013362.
Assuntos
Analgésicos Opioides/efeitos adversos , Hiperalgesia/induzido quimicamente , Transtornos de Enxaqueca/tratamento farmacológico , Neuropeptídeos/metabolismo , Neurotransmissores/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Analgésicos Opioides/uso terapêutico , Animais , Comportamento Animal , Cromatografia Líquida , Modelos Animais de Doenças , Transtornos da Cefaleia/complicações , Transtornos da Cefaleia/tratamento farmacológico , Hiperalgesia/complicações , Hiperalgesia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transtornos de Enxaqueca/complicações , Transtornos de Enxaqueca/metabolismo , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Flax (Linum usitatissimum L.) is grown for fiber and seed in many countries. Flax cultivars differ in the oil composition and, depending on the ratio of fatty acids, are used in pharmaceutical, food, or paint industries. It is known that genes of SAD (stearoyl-ACP desaturase) and FAD (fatty acid desaturase) families play a key role in the synthesis of fatty acids, and some alleles of these genes are associated with a certain composition of flax oil. However, data on genetic polymorphism of these genes are still insufficient. RESULTS: On the basis of the collection of the Institute for Flax (Torzhok, Russia), we formed a representative set of 84 cultivars and lines reflecting the diversity of fatty acid composition of flax oil. An approach for the determination of full-length sequences of SAD1, SAD2, FAD2A, FAD2B, FAD3A, and FAD3B genes using the Illumina platform was developed and deep sequencing of the 6 genes in 84 flax samples was performed on MiSeq. The obtained high coverage (about 400x on average) enabled accurate assessment of polymorphisms in SAD1, SAD2, FAD2A, FAD2B, FAD3A, and FAD3B genes and evaluation of cultivar/line heterogeneity. The highest level of genetic diversity was observed for FAD3A and FAD3B genes - 91 and 62 polymorphisms respectively. Correlation analysis revealed associations between particular variants in SAD and FAD genes and predominantly those fatty acids whose conversion they catalyze: SAD - stearic and oleic acids, FAD2 - oleic and linoleic acids, FAD3 - linoleic and linolenic acids. All except one low-linolenic flax cultivars/lines contained both the substitution of tryptophan to stop codon in the FAD3A gene and histidine to tyrosine substitution in the FAD3B gene, while samples with only one of these polymorphisms had medium content of linolenic acid and cultivars/lines without them were high-linolenic. CONCLUSIONS: Genetic polymorphism of SAD and FAD genes was evaluated in the collection of flax cultivars and lines with diverse oil composition, and associations between particular polymorphisms and the ratio of fatty acids were revealed. The achieved results are the basis for the development of marker-assisted selection and DNA-based certification of flax cultivars.
Assuntos
Ácidos Graxos Dessaturases/genética , Ácidos Graxos/metabolismo , Linho/genética , Variação Genética , Oxigenases de Função Mista/genética , Substituição de Aminoácidos , DNA de Plantas , Linho/enzimologia , Linho/metabolismo , Genes de Plantas , Heterogeneidade Genética , Oxigenases de Função Mista/metabolismo , Análise de Sequência de DNA , Ácido alfa-Linolênico/metabolismoRESUMO
The evolution of tRNA genes in mitochondrial (mt) genomes is a complex process that includes duplications, degenerations, and transpositions, as well as a specific process of identity change through mutations in the anticodon (tRNA gene remolding or tRNA gene recruitment). Using amphipod-specific tRNA models for annotation, we show that tRNA duplications are more common in the mt genomes of amphipods than what was revealed by previous annotations. Seventeen cases of tRNA gene duplications were detected in the mt genomes of amphipods, and ten of them were tRNA genes that underwent remolding. The additional tRNA gene findings were verified using phylogenetic analysis and genetic distance analysis. The majority of remolded tRNA genes (seven out of ten cases) were found in the mt genomes of endemic amphipod species from Lake Baikal. All additional mt tRNA genes arose independently in the Baikalian amphipods, indicating the unusual plasticity of tRNA gene evolution in these species assemblages. The possible reasons for the unusual abundance of additional tRNA genes in the mt genomes of Baikalian amphipods are discussed. The amphipod-specific tRNA models developed for MiTFi refine existing predictions of tRNA genes in amphipods and reveal additional cases of duplicated tRNA genes overlooked by using less specific Metazoa-wide models. The application of these models for mt tRNA gene prediction will be useful for the correct annotation of mt genomes of amphipods and probably other crustaceans.
Assuntos
Anfípodes/classificação , Anfípodes/genética , Duplicação Gênica , Genoma Mitocondrial/genética , RNA de Transferência/genética , Animais , Evolução Molecular , Genes Mitocondriais/fisiologia , Especiação Genética , Lagos , Mutação , Filogenia , Filogeografia , SibériaRESUMO
Cocaine addiction afflicts nearly 1 million adults in the United States, and to date, there are no known treatments approved for this psychiatric condition. Women are particularly vulnerable to developing a cocaine use disorder and suffer from more serious cardiac consequences than men when using cocaine. Estrogen is one biological factor contributing to the increased risk for females to develop problematic cocaine use. Animal studies have demonstrated that estrogen (17ß-estradiol or E2) enhances the rewarding properties of cocaine. Although E2 affects the dopamine system, the molecular and cellular mechanisms of E2-enhanced cocaine reward have not been characterized. In this study, quantitative top-down proteomics was used to measure intact proteins in specific regions of the female mouse brain after mice were trained for cocaine-conditioned place preference, a behavioral test of cocaine reward. Several proteoform changes occurred in the ventral tegmental area after combined cocaine and E2 treatments, with the most numerous proteoform alterations on myelin basic protein, indicating possible changes in white matter structure. There were also changes in histone H4, protein phosphatase inhibitors, cholecystokinin, and calmodulin proteoforms. These observations provide insight into estrogen signaling in the brain and may guide new approaches to treating women with cocaine use disorder.
Assuntos
Encéfalo/efeitos dos fármacos , Cocaína/farmacologia , Estradiol/farmacologia , Proteoma/metabolismo , Proteômica/métodos , Animais , Encéfalo/metabolismo , Condicionamento Clássico/efeitos dos fármacos , Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Estrogênios/farmacologia , Feminino , Camundongos Endogâmicos C57BL , Ovariectomia , Recompensa , Área Tegmentar Ventral/efeitos dos fármacos , Área Tegmentar Ventral/metabolismoRESUMO
A better understanding of neuromodulation in a behavioral system requires identification of active modulatory transmitters. Here, we used identifiable neurons in a neurobiological model system, the mollusc Aplysia, to study neuropeptides, a diverse class of neuromodulators. We took advantage of two types of feeding neurons, B48 and B1/B2, in the Aplysia buccal ganglion that might contain different neuropeptides. We performed a representational difference analysis (RDA) by subtraction of mRNAs in B48 versus mRNAs in B1/B2. The RDA identified an unusually long (2025 amino acids) peptide precursor encoding Aplysia leucokinin-like peptides (ALKs; e.g. ALK-1 and ALK-2). Northern blot analysis revealed that, compared with other ganglia (e.g. the pedal-pleural ganglion), ALK mRNA is predominantly present in the buccal ganglion, which controls feeding behavior. We then used in situ hybridization and immunohistochemistry to localize ALKs to specific neurons, including B48. MALDI-TOF MS on single buccal neurons revealed expression of 40 ALK precursor-derived peptides. Among these, ALK-1 and ALK-2 are active in the feeding network; they shortened the radula protraction phase of feeding motor programs triggered by a command-like neuron. We also found that this effect may be mediated by the ALK-stimulated enhancement of activity of an interneuron, which has previously been shown to terminate protraction. We conclude that our multipronged approach is effective for determining the structure and defining the diverse functions of leucokinin-like peptides. Notably, the ALK precursor is the first verified nonarthropod precursor for leucokinin-like peptides with a novel, marked modulatory effect on a specific parameter (protraction duration) of feeding motor programs.
Assuntos
Aplysia/fisiologia , Gânglios dos Invertebrados/fisiologia , Neuropeptídeos/metabolismo , Animais , Aplysia/química , Aplysia/citologia , Aplysia/genética , Comportamento Alimentar , Gânglios dos Invertebrados/química , Gânglios dos Invertebrados/metabolismo , Neurônios/química , Neurônios/citologia , Neurônios/metabolismo , Neuropeptídeos/análise , Neuropeptídeos/genética , Processamento de Proteína Pós-Traducional , RNA Mensageiro/análise , RNA Mensageiro/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Over the past decade, advances in mass spectrometry-based proteomics have accelerated brain proteome research aimed at studying the expression, dynamic modification, interaction and function of proteins in the nervous system that are associated with physiological and behavioral processes. With the latest hardware and software improvements in top-down mass spectrometry, the technology has expanded from mere protein profiling to high-throughput identification and quantification of intact proteoforms. Murine systems are broadly used as models to study human diseases. Neuroscientists specifically study the mouse brain from inbred strains to help understand how strain-specific genotype and phenotype affect development, functioning, and disease progression. This work describes the first application of label-free quantitative top-down proteomics to the analysis of the mouse brain proteome. Operating in discovery mode, we determined physiochemical differences in brain tissue from four healthy inbred strains, C57BL/6J, DBA/2J, FVB/NJ, and BALB/cByJ, after probing their intact proteome in the 3.5-30 kDa mass range. We also disseminate these findings using a new tool for top-down proteomics, TDViewer and cataloged them in a newly established Mouse Brain Proteoform Atlas. The analysis of brain tissues from the four strains identified 131 gene products leading to the full characterization of 343 of the 593 proteoforms identified. Within the results, singly and doubly phosphorylated ARPP-21 proteoforms, known to inhibit calmodulin, were differentially expressed across the four strains. Gene ontology (GO) analysis for detected differentially expressed proteoforms also helps to illuminate the similarities and dissimilarities in phenotypes among these inbred strains.
Assuntos
Química Encefálica , Espectrometria de Massas/métodos , Camundongos Endogâmicos , Proteoma/análise , Proteômica/métodos , Animais , Encéfalo/metabolismo , Cromatografia Líquida/métodos , Feminino , Camundongos Endogâmicos BALB C/metabolismo , Camundongos Endogâmicos C57BL/metabolismo , Camundongos Endogâmicos DBA/metabolismo , Camundongos Endogâmicos/metabolismo , Proteoma/metabolismo , SoftwareRESUMO
Tissue regeneration is a complex process that involves a mosaic of molecules that vary spatially and temporally. Insights into the chemical signaling underlying this process can be achieved with a multiplex and untargeted chemical imaging method such as mass spectrometry imaging (MSI), which can enablede novostudies of nervous system regeneration. A combination of MSI and multivariate statistics was used to differentiate peptide dynamics in the freshwater planarian flatwormSchmidtea mediterraneaat different time points during cephalic ganglia regeneration. A protocol was developed to makeS. mediterraneatissues amenable for MSI. MS ion images of planarian tissue sections allow changes in peptides and unknown compounds to be followed as a function of cephalic ganglia regeneration. In conjunction with fluorescence imaging, our results suggest that even though the cephalic ganglia structure is visible after 6 days of regeneration, the original chemical composition of these regenerated structures is regained only after 12 days. Differences were observed in many peptides, such as those derived from secreted peptide 4 and EYE53-1. Peptidomic analysis further identified multiple peptides from various known prohormones, histone proteins, and DNA- and RNA-binding proteins as being associated with the regeneration process. Mass spectrometry data also facilitated the identification of a new prohormone, which we have named secreted peptide prohormone 20 (SPP-20), and is up-regulated during regeneration in planarians.
Assuntos
Regeneração Nervosa , Neuropeptídeos/análise , Neuropeptídeos/metabolismo , Planárias/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Gânglios/química , Gânglios/fisiologia , Gânglios/ultraestrutura , Regulação da Expressão Gênica , Neurogênese , Neuropeptídeos/genética , Imagem Óptica , Planárias/química , Planárias/genéticaRESUMO
Measurement, identification, and quantitation of endogenous peptides in tissue samples by mass spectrometry (MS) contribute to our understanding of the complex molecular mechanisms of numerous biological phenomena. For accurate results, it is essential to arrest the postmortem degradation of ubiquitous proteins in samples prior to performing peptidomic measurements. Doing so ensures that the detection of endogenous peptides, typically present at relatively low levels of abundance, is not overwhelmed by protein degradation products. Heat stabilization has been shown to inactivate the enzymes in tissue samples and minimize the presence of protein degradation products in the subsequent peptide extracts. However, the efficacy of different heat treatments to preserve the integrity of full-length endogenous peptides has not been well documented; prior peptidomic studies of heat stabilization methods have not distinguished between the full-length (mature) and numerous truncated (possible artifacts of sampling) forms of endogenous peptides. We show that thermal sample treatment via rapid conductive heat transfer is effective for detection of mature endogenous peptides in fresh and frozen rodent brain tissues. Freshly isolated tissue processing with the commercial Stabilizor T1 heat stabilization system resulted in the confident identification of 65% more full-length mature neuropeptides compared to widely used sample treatment in a hot water bath. This finding was validated by a follow-up quantitative multiple reaction monitoring MS analysis of select neuropeptides. The rapid conductive heating in partial vacuum provided by the Stabilizor T1 effectively reduces protein degradation and decreases the chemical complexity of the sample, as assessed by determining total protein content. This system enabled the detection, identification, and quantitation of neuropeptides related to 22 prohormones expressed in individual rat hypothalami and suprachiasmatic nuclei.
Assuntos
Hipotálamo/química , Neuropeptídeos/análise , Sequência de Aminoácidos , Animais , Cromatografia Líquida , Temperatura Alta , Masculino , Proteólise , Proteoma , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Cichlid fishes have evolved remarkably diverse reproductive, social, and feeding behaviors. Cell-to-cell signaling molecules, notably neuropeptides and peptide hormones, are known to regulate these behaviors across vertebrates. This class of signaling molecules derives from prohormone genes that have undergone multiple duplications and losses in fishes. Whether and how subfunctionalization, neofunctionalization, or losses of neuropeptides and peptide hormones have contributed to fish behavioral diversity is largely unknown. Information on fish prohormones has been limited and is complicated by the whole genome duplication of the teleost ancestor. We combined bioinformatics, mass spectrometry-enabled peptidomics, and molecular techniques to identify the suite of neuropeptide prohormones and pituitary peptide products in Astatotilapia burtoni, a well-studied member of the diverse African cichlid clade. RESULTS: Utilizing the A. burtoni genome, we identified 148 prohormone genes, with 21 identified as a single copy and 39 with at least 2 duplicated copies. Retention of prohormone duplicates was therefore 41 %, which is markedly above previous reports for the genome-wide average in teleosts. Beyond the expected whole genome duplication, differences between cichlids and mammals can be attributed to gene loss in tetrapods and additional duplication after divergence. Mass spectrometric analysis of the pituitary identified 620 unique peptide sequences that were matched to 120 unique proteins. Finally, we used in situ hybridization to localize the expression of galanin, a prohormone with exceptional sequence divergence in cichlids, as well as the expression of a proopiomelanocortin, prohormone that has undergone an additional duplication in some bony fish lineages. CONCLUSION: We characterized the A. burtoni prohormone complement. Two thirds of prohormone families contain duplications either from the teleost whole genome duplication or a more recent duplication. Our bioinformatic and mass spectrometric findings provide information on a major vertebrate clade that will further our understanding of the functional ramifications of these prohormone losses, duplications, and sequence changes across vertebrate evolution. In the context of the cichlid radiation, these findings will also facilitate the exploration of neuropeptide and peptide hormone function in behavioral diversity both within A. burtoni and across cichlid and other fish species.
Assuntos
Ciclídeos/genética , Hormônios/genética , Neuropeptídeos/genética , Sequência de Aminoácidos , Animais , Biologia Computacional , Evolução Molecular , Duplicação Gênica , Perfilação da Expressão Gênica , Genoma , Genômica/métodos , Hormônios/química , Família Multigênica , Neuropeptídeos/química , Hipófise/metabolismoRESUMO
BACKGROUND: Amphipods (Crustacea) of Lake Baikal are a very numerous and diverse group of invertebrates generally believed to have originated by adaptive radiation. The evolutionary history and phylogenetic relationships in Baikalian amphipods still remain poorly understood. Sequencing of mitochondrial genomes is a relatively feasible way for obtaining a set of gene sequences suitable for robust phylogenetic inferences. The architecture of mitochondrial genomes also may provide additional information on the mechanisms of evolution of amphipods in Lake Baikal. RESULTS: Three complete and four nearly complete mitochondrial genomes of Baikalian amphipods were obtained by high-throughput sequencing using the Illumina platform. A phylogenetic inference based on the nucleotide sequences of all mitochondrial protein coding genes revealed the Baikalian species to be a monophyletic group relative to the nearest non-Baikalian species with a completely sequenced mitochondrial genome - Gammarus duebeni. The phylogeny of Baikalian amphipods also suggests that the shallow-water species Eulimnogammarus has likely evolved from a deep-water ancestor, however many other species have to be added to the analysis to test this hypothesis. The gene order in all mitochondrial genomes of studied Baikalian amphipods differs from the pancrustacean ground pattern. Mitochondrial genomes of four species possess 23 tRNA genes, and in three genomes the extra tRNA gene copies have likely undergone remolding. Widely varying lengths of putative control regions and other intergenic spacers are typical for the mitochondrial genomes of Baikalian amphipods. CONCLUSIONS: The mitochondrial genomes of Baikalian amphipods display varying organization suggesting an intense rearrangement process during their evolution. Comparison of complete mitochondrial genomes is a potent approach for studying the amphipod evolution in Lake Baikal.
Assuntos
Anfípodes/genética , Evolução Molecular , Genoma Mitocondrial , Anfípodes/classificação , Animais , Composição de Bases , Códon , Ordem dos Genes , Variação Genética , Tamanho do Genoma , Genômica/métodos , Fases de Leitura Aberta , FilogeniaRESUMO
A receptor binding class of d-amino acid-containing peptides (DAACPs) is formed in animals from an enzymatically mediated post-translational modification of ribosomally translated all-l-amino acid peptides. Although this modification can be required for biological actions, detecting it is challenging because DAACPs have the same mass as their all-l-amino acid counterparts. We developed a suite of mass spectrometry (MS) protocols for the nontargeted discovery of DAACPs and validated their effectiveness using neurons from Aplysia californica. The approach involves the following three steps, with each confirming and refining the hits found in the prior step. The first step is screening for peptides resistant to digestion by aminopeptidase M. The second verifies the presence of a chiral amino acid via acid hydrolysis in deuterium chloride, labeling with Marfey's reagent, and liquid chromatography-mass spectrometry to determine the chirality of each amino acid. The third involves synthesizing the putative DAACPs and comparing them to the endogenous standards. Advantages of the method, the d-amino acid-containing neuropeptide discovery funnel, are that it is capable of detecting the d-form of any common chiral amino acid, and the first two steps do not require peptide standards. Using these protocols, we report that two peptides from the Aplysia achatin-like neuropeptide precursor exist as GdYFD and SdYADSKDEESNAALSDFA. Interestingly, GdYFD was bioactive in the Aplysia feeding and locomotor circuits but SdYADSKDEESNAALSDFA was not. The discovery funnel provides an effective means to characterize DAACPs in the nervous systems of animals in a nontargeted manner.
Assuntos
Aminoácidos/análise , Aplysia/química , Neuropeptídeos/análise , Aminoácidos/metabolismo , Animais , Aplysia/citologia , Aplysia/metabolismo , Antígenos CD13/metabolismo , Espectrometria de Massas , Neurônios/química , Neurônios/citologia , Neurônios/metabolismo , Neuropeptídeos/metabolismoRESUMO
Neurochemical differences in the hypothalamic-pituitary axis between individuals and between ages may contribute to differential susceptibility to cocaine abuse. This study measured peptide levels in the pituitary gland (Pit) and lateral hypothalamus (LH) in adolescent (age 30 days) and adult (age 65 days) mice from four standard inbred strains, FVB/NJ, DBA/2J, C57BL/6J, and BALB/cByJ, which have previously been characterized for acute locomotor responses to cocaine. Individual peptide profiles were analyzed using mass spectrometric profiling and principal component analysis. Sequences of assigned peptides were verified by tandem mass spectrometry. Principal component analysis classified all strains according to their distinct peptide profiles in Pit samples from adolescent mice, but not adults. Select pro-opiomelanocortin-derived peptides were significantly higher in adolescent BALB/cByJ and DBA/2J mice than in FVB/NJ or C57BL/6J mice. A subset of peptides in the LH, but not in the Pit, was altered by cocaine in adolescents. A 15 mg/kg dose of cocaine induced greater peptide alterations than a 30 mg/kg dose, particularly in FVB/NJ animals, with larger differences in adolescents than adults. Neuropeptides in the LH affected by acute cocaine administration included pro-opiomelanocortin-, myelin basic protein-, and glutamate transporter-derived peptides. The observed peptide differences could contribute to differential behavioral sensitivity to cocaine among strains and ages. Peptides were measured using mass spectrometry (MALDI-TOF) in individual lateral hypothalamus and pituitary samples from four strains and two ages of inbred mice in response to acute cocaine administration. Principal component analyses (PCA) classified the strains according to their peptide profiles from adolescent mice, and a subset of peptides in the lateral hypothalamus was altered by cocaine in adolescents.
Assuntos
Envelhecimento , Cocaína/administração & dosagem , Inibidores da Captação de Dopamina/administração & dosagem , Região Hipotalâmica Lateral , Peptídeos/metabolismo , Hipófise , Animais , Animais Recém-Nascidos , Cromatografia Líquida , Região Hipotalâmica Lateral/efeitos dos fármacos , Região Hipotalâmica Lateral/crescimento & desenvolvimento , Região Hipotalâmica Lateral/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos/metabolismo , Peptídeos/análise , Hipófise/efeitos dos fármacos , Hipófise/crescimento & desenvolvimento , Hipófise/metabolismo , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Cell-to-cell variability and functional heterogeneity are integral features of multicellular organisms. Chemical classification of cells into cell type is important for understanding cellular specialization as well as organismal function and organization. Assays to elucidate these chemical variations are best performed with single cell samples because tissue homogenates average the biochemical composition of many different cells and oftentimes include extracellular components. Several single cell microanalysis techniques have been developed but tend to be low throughput or require preselection of molecular probes that limit the information obtained. Mass spectrometry (MS) is an untargeted, multiplexed, and sensitive analytical method that is well-suited for studying chemically complex individual cells that have low analyte content. In this work, populations of cells from the rat pituitary, the rat pancreatic islets of Langerhans, and from the Aplysia californica nervous system, are classified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI) MS by their peptide content. Cells were dispersed onto a microscope slide to generate a sample where hundreds to thousands of cells were separately located. Optical imaging was used to determine the cell coordinates on the slide, and these locations were used to automate the MS measurements to targeted cells. Principal component analysis was used to classify cellular subpopulations. The method was modified to focus on the signals described by the lower principal components to explore rare cells having a unique peptide content. This approach efficiently uncovers and classifies cellular subtypes as well as discovers rare cells from large cellular populations.