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[
WormBook,
2008]
The role of neuropeptides in modulating behavior is slowly being elucidated. With the sequencing of the C. elegans genome, the extent of the neuropeptide genes in C. elegans can be determined. To date, 113 neuropeptide genes encoding over 250 distinct neuropeptides have been identified. Of these, 40 genes encode insulin-like peptides, 31 genes encode FMRFamide-related peptides, and 42 genes encode non-insulin, non-FMRFamide-related neuropeptides. As in other systems, C. elegans neuropeptides are derived from precursor molecules that must be post-translationally processed to yield the active peptides. These precursor molecules contain a single peptide, multiple copies of a single peptide, multiple distinct peptides, or any combination thereof. The neuropeptide genes are expressed extensively throughout the nervous system, including in sensory, motor, and interneurons. In addition, some of the genes are also expressed in non-neuronal tissues, such as the somatic gonad, intestine, and vulval hypodermis. To address the effects of neuropeptides on C. elegans behavior, animals in which the different neuropeptide genes are inactivated or overexpressed are being isolated. In a complementary approach the receptors to which the neuropeptides bind are also being identified and examined. Among the knockout animals analyzed thus far, defects in locomotion, dauer formation, egg laying, ethanol response, and social behavior have been reported. These data suggest that neuropeptides have a modulatory role in many, if not all, behaviors in C. elegans.
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[
Adv Exp Med Biol,
2010]
Nematode neuropeptide systems comprise an exceptionally complex array of approximately 250 peptidic signaling molecules that operate within a structurally simple nervous system of approximately 300 neurons. A relatively complete picture of the neuropeptide complement is available for Caenorhabditis elegans, with 30 flp, 38 ins and 43 nlp genes having been documented; accumulating evidence indicates similar complexity in parasitic nematodes from clades I, III, IV and V. In contrast, the picture for parasitic platyhelminths is less clear, with the limited peptide sequence data available providing concrete evidence for only FMRFamide-like peptide (FLP) and neuropeptide F (NPF) signaling systems, each of which only comprises one or two peptides. With the completion of the Schmidtea meditteranea and Schistosoma mansoni genome projects and expressed sequence tag datasets for other flatworm parasites becoming available, the time is ripe for a detailed reanalysis ofneuropeptide signalingin flatworms. Although the actual neuropeptides provide limited obvious value as targets for chemotherapeutic-based control strategies, they do highlight the signaling systems present in these helminths and provide tools for the discovery of more amenable targets such as neuropeptide receptors or neuropeptide processing enzymes. Also, they offer opportunities to evaluate the potential of their associated signaling pathways as targets through RNA interference (RNAi)-based, target validation strategies. Currently, within both helminth phyla, theflp signaling systems appear to merit further investigation as they are intrinsically linked with motor function, a proven target for successful anti-parasitics; it is clear that some nematode NLPs also play a role in motor function and could have similar appeal. At this time, it is unclear if flatworm NPF and nematode INS peptides operate in pathways that have utility for parasite control. Clearly, RNAi-based validation could be a starting point for scoring potential target pathways within neuropeptide signaling for parasiticide discovery programs. Also, recent successes in the application of in planta-based RNAi control strategies for plant parasitic nematodes reveal a strategy whereby neuropeptide encoding genes could become targets for parasite control. The possibility of developing these approaches for the control of animal and human parasites is intriguing, but will require significant advances in the delivery of RNAi-triggers.
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[
WormBook,
2006]
In Drosophila and vertebrates, Hedgehog (Hh) signalling is mediated by a cascade of genes, which play essential roles in cell proliferation and survival, and in patterning of the embryo, limb buds and organs. In C. elegans, this pathway has undergone considerable evolutionary divergence; genes encoding homologues of key pathway members, including Hh, Smoothened, Cos2, Fused and Suppressor of Fused, are absent. Surprisingly, over sixty proteins (i.e. WRT, GRD, GRL, and QUA), encoded by a set of genes collectively referred to as the Hh-related genes, and two co-orthologs ( PTC-1 ,-3) of fly Patched, a Hh receptor, are present in C. elegans. Several of the Hh-related proteins are bipartite and all can potentially generate peptides with signalling activity, although none of these peptides shares obvious sequence similarity with Hh. In addition, the ptc -related ( ptr ) genes, which are present in a single copy in Drosophila and vertebrates and encode proteins closely related to Patched, have undergone an expansion in number in nematodes. A number of functions, including roles in molting, have been attributed to the C. elegans Hh-related, PTC and PTR proteins; most of these functions involve processes that are associated with the trafficking of proteins, sterols or sterol-modified proteins. Genes encoding other components of the Hh signalling pathway are also found in C. elegans, but their functions remain to be elucidated.
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[
1982]
Much of this meeting is devoted to the study of multi-gene families and the differential expression of various members during muscle development. Structural analysis of myosin and then other muscle proteins by peptide mapping and amino acid sequencing first suggested that these isoforms are the products of different genes. The use of antibodies specific to distinct structural gene products has permitted detailed investigations of myosin structure, biosynthesis and degradation, and cellular location as muscle development proceeds. The small nematode, Caenorhabditis elegans, is a laboratory animal which offers genetic dissection and manipulation as tools in deciphering of gene regulation in terms of specific protein synthesis during muscle development. The examination of specific mutants by protein chemistry and immunochemistry has already proved a powerful comination in many fields.
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[
1977]
Native myosin purified from the wild-type, N2, and a body-wall defective mutant, E675, of the nematode contains two myosins, each homogeneous for different heavy chains. These myosins can be resolved from one another on hydroxyapatite and, when cleavaged with CNBr, they yield different peptide-fragments. In E190, one of the homogeneous myosins is absent.
e190 and
e675 are alleles of the same gene,
unc-54. The myosin lacking in E190 is the same one affected in E675. This suggests that
unc-54 is the structural gene for a myosin heavy chain. In order to determine the role of these different myosins, we plan to use antibodies to locate the myosins on thick filaments from body-wall muscle. Additionally, we are studying the patterns of synthesis and degradation of the two myosins in the wild-type and muscle-defective mutants in order to discover how the observed stoichiometry is maintained.
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[
Adv Exp Med Biol,
2010]
Neuropeptides are short sequences ofamino acids that function in all multicellular organisms to communicate information between cells. The first sequence ofa neuropeptide was reported in 1970' and the number of identified neuropeptides remained relatively small until the 1990s when the DNA sequence of multiple genomes revealed treasure troves ofinformation. Byblasting away at the genome, gene families, the sizes ofwhich were previously unknown, could now be determined. This information has led to an exponential increase in the number of putative neuropeptides and their respective gene families. The molecular biology age greatly benefited the neuropeptide field in the nematode Caenorhabditis elegans. Its genome was among the first to be sequenced and this allowed us the opportunity to screen the genome for neuropeptide genes. Initially, the screeningwas slow, as the Genefinder and BLAST programs had difficulty identifying small genes and peptides. However, as the bioinformatics programs improved, the extent of the neuropeptide gene families in C. elegans gradually emerged.
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[
Methods Mol Biol,
2011]
Quantitative proteomics aims to identify and quantify proteins in cells or organisms that have been obtained from different biological origin (e.g., "healthy vs. diseased"), that have received different treatments, or that have different genetic backgrounds. Protein expression levels can be quantified by labeling proteins with stable isotopes, followed by mass spectrometric analysis. Stable isotopes can be introduced in vitro by reacting proteins or peptides with isotope-coded reagents (e.g., iTRAQ, reductive methylation). A preferred way, however, is the metabolic incorporation of heavy isotopes into cells or organisms by providing the label, in the form of amino acids (such as in SILAC) or salts, in the growth media. The advantage of in vivo labeling is that it does not suffer from side reactions or incomplete labeling that might occur in chemical derivatization. In addition, metabolic labeling occurs at the earliest possible moment in the sample preparation process, thereby minimizing the error in quantitation. Labeling with the heavy stable isotope of nitrogen (i.e., (15)N) provides an efficient way for accurate protein quantitation. Where the application of SILAC is mostly restricted to cell culture, (15)N labeling can be used for micro-organisms as well as a number of higher (multicellular) organisms. The most prominent examples of the latter are Caenorhabditis elegans and Drosophila (fruit fly), two important model organisms for a range of regulatory processes underlying developmental biology. Here we describe in detail the labeling with (15)N atoms, with a particular focus on fruit flies and C. elegans. We also describe methods for the identification and quantitation of (15)N-labeled proteins by mass spectrometry and bioinformatic analysis.