A novel tachykinin-related peptide receptor of Octopus vulgaris--evolutionary aspects of invertebrate tachykinin and tachykinin-related peptide

The tachykinin (TK) and tachykinin-related peptide (TKRP) family represent one of the largest peptide families in the animal kingdom and exert their actions via a subfamily of structurally related G-protein-coupled receptors. In this study, we have identified a novel TKRP receptor from the Octopus heart, oct-TKRPR. oct-TKRPR includes domains and motifs typical of G-protein-coupled receptors. Xenopus oocytes that expressed oct-TKRPR, like TK and TKRP receptors, elicited an induction of membrane chloride currents coupled to the inositol phosphate/calcium pathway in response to Octopus TKRPs (oct-TKRP I-VII) with moderate ligand selectivity. Substance P and Octopus salivary gland-specific TK, oct-TK-I, completely failed to activate oct-TKRPR, whereas a Substance P analog containing a C-terminal Arg-NH2 exhibited equipotent activation of oct-TKRPs. These functional analyses prove that oct-TKRPs, but not oct-TK-I, serve as endogenous functional ligands through oct-TKRPR, although both of the family peptides were identified in a single species, and the importance of C-terminal Arg-NH2 in the specific recognition of TKRPs by TKRPR is conserved through evolutionary lineages of Octopus. Southern blotting of RT-PCR products revealed that the oct-TKRPR mRNA was widely distributed in the central and peripheral nervous systems plus several peripheral tissues. These results suggest multiple physiologic functions of oct-TKRPs as neuropeptides both in the Octopus central nervous system and in peripheral tissues. This is the first report on functional discrimination between invertebrate TKRPs and salivary gland-specific TKs.     

Identification of a tachykinin-related peptide with orexigenic properties in the German cockroach

A number of evidences suggest that tachykinin-related peptides (TRPs) of insects can stimulate food consumption after being released from the midgut to the hemolymph. The idea of the present work has been to test this hypothesis in the anautogenous cockroach Blattella germanica. First, we have identified the peptide LemTRP-1 (APSGFLGVR-NH(2)) from brain extracts, by means of an ELISA developed with a polyclonal antibody against this peptide. ELISA studies have also shown that, whereas brain LemTRP-1 levels were fairly constant, midgut levels increase to a maximum on day 3 after adult emergence, falling thereafter until the end of the gonadotrophic cycle. Interestingly, maximum values of food consumption are concomitant with the decrease of LemTRP-1 immunoreactivity in the midgut. Furthermore, starvation decreases LemTRP-1 immunoreactivity in midgut, whereas in the hemolymph it increases. Finally, injection of synthetic LemTRP-1 to adult females significantly stimulates food consumption. The whole observations suggest that LemTRP-1 is released from the midgut to the hemolymph when sustained food consumption is required to maintain vitellogenesis at the highest levels, and that LemTRP-1 in the hemolymph stimulates food consumption in these days.     

A novel tachykinin-related peptide receptor. Sequence, genomic organization, and functional analysis.

Structurally tachykinin-related peptides have been isolated from various invertebrate species and shown to exhibit their biological activities through a G-protein-coupled receptor (GPCR) for a tachykinin-related peptide. In this paper, we report the identification of a novel tachykinin-related peptide receptor, the urechistachykinin receptor (UTKR) from the echiuroid worm, Urechis unitinctus. The deduced UTKR precursor includes seven transmembrane domains and typical sites for mammalian tachykinin receptors and invertebrate tachykinin-related peptide receptors. A functional analysis of the UTKR expressed in Xenopus oocytes demonstrated that UTKR, like tachykinin receptors and tachykinin-related peptide receptors, activates calcium-dependent signal transduction upon binding to its endogenous ligands, urechistachykinins (Uru-TKs) I-V and VII, which were isolated as Urechis tachykinin-related peptides from the nervous tissue of the Urechis unitinctus in our previous study. UTKR responded to all Uru-TKs equivalently, showing that UTKR possesses no selective affinity with Uru-TKs. In contrast, UTKR was not activated by substance P or an Uru-TK analog containing a C-terminal Met-NH2 instead of Arg-NH2. Furthermore, the genomic analysis revealed that the UTKR gene, like mammalian tachykinin receptor genes, consists of five exons interrupted by four introns, and all the intron-inserted positions are completely compatible with those of mammalian tachykinin receptor genes. These results suggest that mammalian tachykinin receptors and invertebrate tachykinin-related peptide receptors were evolved from a common ancestral GPCR gene. This is the first identification of an invertebrate tachykinin-related peptide receptor from other species than insects and also of the genomic structure of a tachykinin-related peptide receptor gene.     

Insulin production and signaling in renal tubules of Drosophila is under control of tachykinin-related peptide and regulates stress resistance

The insulin-signaling pathway is evolutionarily conserved in animals and regulates growth, reproduction, metabolic homeostasis, stress resistance and life span. In Drosophila seven insulin-like peptides (DILP1-7) are known, some of which are produced in the brain, others in fat body or intestine. Here we show that DILP5 is expressed in principal cells of the renal tubules of Drosophila and affects survival at stress. Renal (Malpighian) tubules regulate water and ion homeostasis, but also play roles in immune responses and oxidative stress. We investigated the control of DILP5 signaling in the renal tubules by Drosophila tachykinin peptide (DTK) and its receptor DTKR during desiccative, nutritional and oxidative stress. The DILP5 levels in principal cells of the tubules are affected by stress and manipulations of DTKR expression in the same cells. Targeted knockdown of DTKR, DILP5 and the insulin receptor dInR in principal cells or mutation of Dilp5 resulted in increased survival at either stress, whereas over-expression of these components produced the opposite phenotype. Thus, stress seems to induce hormonal release of DTK that acts on the renal tubules to regulate DILP5 signaling. Manipulations of S6 kinase and superoxide dismutase (SOD2) in principal cells also affect survival at stress, suggesting that DILP5 acts locally on tubules, possibly in oxidative stress regulation. Our findings are the first to demonstrate DILP signaling originating in the renal tubules and that this signaling is under control of stress-induced release of peptide hormone.     

Isolation of a unique peptide inhibitor of hormone-responsive adenylate cyclase.

We have perfused isolated rat livers with hypocalcemic (4.4 mg 100 ml) Krebs-Ringer bicarbonate albumin buffer. After 15 min of perfusion, a substance appeared in the perfusate which decreased rat renal adenylate cyclase activation by parathyroid hormone (PTH). The material in the perfusate was purified greater than 50,000-fold by Bio-Gel P-10 chromatography. The purified antagonist decreased the activation of rat renal cortical adenylate cyclase by PTH, glucagon, and epinephrine 75 to 100%. Concentration response curves for each of the hormones indicated a noncompetitive interaction of the inhibitor with the hormone. The inhibition was not species-specific, as the activation of the parathyroid hormone-responsive adenylate cyclase in cat renal cortex was also abolished by the inhibitor from the perfused rat liver. The inhibitor is a peptide, Mr equal to similar to 1000, which is heat-stable, acid-stable, alkai-labile, and is destroyed by trypsin, leucine aminopeptidase, and elastase. It is not destroyed by phosphodiesterase, 5'-nucleotidase, alkaline phosphatase, neuraminidase, RNase, or phospholipase A. The inhibitor is not produced by isolated rat livers perfused with normocalcemic perfusion media. It is unclear whether the peptide is synthesized by the liver or whether it is a breakdown product of a larger peptide or protein in the liver. This is the first reported peptide inhibitor of adenylate cyclase.     

Inactivation of a tachykinin-related peptide: identification of four neuropeptide-degrading enzymes in neuronal membranes of insects from four different orders

Tachykinin-related peptides (TRP) are widely distributed in the CNS of insects, where they are likely to function as transmitters/modulators. Metabolic inactivation by membrane ecto-peptidases is one mechanism by which peptide signalling is terminated in the CNS. Using locustatachykinin-1 (LomTK-1, GPSGFYGVRamide) as a substrate and several selective peptidase inhibitors, we have compared the types of membrane associated peptidases present in the CNS of four insects, Locusta migratoria, Leucophaea maderae, Drosophila melanogaster and Lacanobia oleracea. A neprilysin (NEP)-like activity cleaving the G-F peptide bond was the major LomTK-1-degrading peptidase detected in locust brain membranes. NEP activity was also found in Leucophaea brain membranes, but the major peptidase was an angiotensin converting enzyme (ACE), cleaving the G-V peptide bond. Drosophila adult head and larval neuronal membranes cleaved the G-F and G-V peptide bonds. Phosphoramidon inhibited both these cleavages, but with markedly different potencies, indicating the presence in the fly brain of two NEP-like enzymes with different substrate and inhibitor specificity. In Drosophila, membrane ACE did not make a significant contribution to the cleavage of the G-V bond. In contrast, ACE was an important membrane peptidase in Lacanobia brain, whereas very little neuronal NEP could be detected. A dipeptidyl peptidase IV (DPP IV) that removed the GP dipeptide from the N-terminus of LomTK-1 was also found in Lacanobia neuronal membranes. This peptidase was a minor contributor to LomTK-1 metabolism by neuronal membranes from all four insect species. In Lacanobia, LomTK-1 was also a substrate for a deamidase that converted LomTK-1 to the free acid form. However, the deamidase was not an integral membrane protein and could be a lysosomal contaminant. It appears that insects from different orders can have different complements of neuropeptide-degrading enzymes. NEP, ACE and the deamidase are likely to be more efficient than the common DPP IV activity at terminating neuropeptide signalling since they cleave close to the C-terminus of the tachykinin, a region essential for maintaining biological activity.     

Sarcolipin, the "proteolipid" of skeletal muscle sarcoplasmic reticulum, is a unique, amphipathic, 31-residue peptide.

The sarcoplasmic reticulum of rabbit skeletal muscle contains a small "proteolipid," i.e., a protein which is soluble in acidic CHCl3/CH3OH. We propose the name sarcolipin for this small protein, to signify its lipid-like solubility and association with the sarcoplasmic reticulum. We have determined the following amino acid sequence for sarcolipin, using protein chemistry methods: M E R S T R E L C L N F T V V L I T V I L I W L L V R S Y Q Y. This 31-residue sequence includes a 19-residue hydrophobic segment which probably spans the sarcoplasmic reticulum membrane. The molecular weight calculated from the sequence, 3733, agrees with that measured by fast atom bombardment mass spectrometry, showing that sarcolipin contains no attached fatty acyl or other prosthetic groups.     

Human is a unique species among primates in terms of telomere length.

TRF (terminal restriction fragments) length in various tissues of non-human primates such as Macaca mulatta (rhesus monkey), Macaca fuscata (Japanese monkey), Macaca fascicularis (crab-eating monkey), Pan troglodytes (common chimpanzee), and Pongo pygmaeus (orangutan) was at least 23 kb without exception, which was quite different from that of human somatic tissues (smaller than 10 kb). The distribution pattern of telomerase activity among tissues was similar between human and non-human primates, while the activity level showed some differences such as that strong telomerase activity was observed in gastrointestinal and lymphocytic tissues from non-human primates. The human appears to be a unique species among primates in terms of telomere length.     

Heparin is a unique marker of progenitors in the glial cell lineage.

The oligodendrocyte-type-2 astrocyte progenitor cells (precursors of oligodendrocytes and type-2 astrocytes) are an excellent system in which to study differentiation as they can be manipulated in vitro. Maintenance of oligodendrocyte-type-2 astrocyte progenitor cells requires basic fibroblast growth factor, a growth factor whose action normally depends on a heparan sulfate coreceptor. Biochemical analysis revealed a most surprising result: that the oligodendrocyte-type-2 astrocyte progenitors did not synthesize heparan sulfate, the near ubiquitous N-sulfated cell surface polysaccharide, but the chemically related heparin in a form that was almost completely N- and O-sulfated. The heparin was detected in the pericellular fraction of the cells and the culture medium. In contrast the differentiated glial subpopulations (oligodendrocytes and type-2 astrocytes) synthesized typical heparan sulfate but with distinctive fine structural features for each cell type. Thus heparin is a unique differentiation marker in the glial lineage. Previously heparin has been found only in a subset of mature mast cells called the connective tissue mast cells. Its presence within the developing nervous system on a precise population of progenitors may confer specific and essential recognition properties on those cells in relation to binding soluble growth and/or differentiation factors and the extracellular matrix.     

Bleomycin hydrolase is a unique thiol aminopeptidase

Bleomycin hydrolase, which hydrolyzes the carboxamide bond in the pyrimidoblamic acid moiety of the bleomycin molecule, also cleaved several p-nitroanilide substrates with a neutral or basic amino acid residue and dipeptide substrates such as L-leucyl-glycine. The activity of bleomycin hydrolase was inhibited by two thiol protease inhibitors, E-64 and leupeptin, as well as by N-ethylmaleimide. These results suggest that bleomycin hydrolase is a thiol aminopeptidase. Magnesium ion, sodium chloride, ethylenediaminetetraacetic acid and 1,2-dihydroxybenzene-3,5-disulfonic acid specifically activated the enzymatic hydrolysis of L-arginine-p-nitroanilide, but did not that of L-leucine-p-nitroanilide. Lineweaver-Burk plots showed that Km values of the enzymatic activity for L-arginine-p-nitroanilide were altered by these reagents, although Vmax values were almost unaltered.     

A tryptic peptide containing a unique serine phosphate residue in rabbit phosphoglucomutase

³²P-labelled phosphoglucomutase was digested with trypsin after denaturation and two peptides were isolated that contained the bulk of the radioactivity bound to peptides. Both peptides appeared to derive from an identical section of the molecule. Peptic and subtilisin digests of the tryptic peptides were prepared. The resulting radioactive peptides were purified and their sequences studied. The presence of a single serine [³²P]phosphate residue was clearly established. Difficulties in purification and low yields, especially of the tryptic peptide, prevented exhaustive sequence studies, but a tentative sequence is proposed as:Ala-Ile-Gly-Gly-Ile-Ile-Leu-Thr-Ala-SerP-His-Asx-Pro-Gly-Gly-Pro-(Asx₂,Gly)-Phe-Gly-Ile-Lys(where SerP represents serine phosphate and Asx represents aspartic acid or asparagine). The results do not support the presence of two serine phosphate residues in the denatured enzyme, but confirm previous results of a unique sequence around a single serine phosphate residue.     

Red pigment-concentrating hormone is not limited to crustaceans

A peptide that was previously assumed to occur exclusively in crustaceans is found in the corpora cardiaca of the stinkbug, Nezara viridula. The sequence of the peptide was deduced from the multiple MS(N) electrospray mass data as that of an octapeptide: pGlu-Ile/Leu-Asn-Phe-Ser-Pro-Gly-Trp amide. This peptide with Leu at position 2 is known as crustacean red pigment-concentrating hormone and code-named Panbo-RPCH. The ambiguity about the amino acid at position 2, Leu or Ile, was solved by isolating the peptide in a single-step by reversed-phase HPLC and establishing co-elution with authentic Panbo-RPCH but not with the Ile(2)-analog. When injected into stinkbugs, synthetic Panbo-RPCH elicited an increase of lipids in the haemolymph. Thus, it is assumed that Panbo-RPCH functions in the stinkbug as a lipid-mobilizing hormone.     

Gastrin-releasing peptide is a mitogen and a morphogen in murine colon cancer.

Little is known about the factors involved in regulating the appearance, or differentiation, of solid tumors including those arising from the colon. We herein demonstrate that the mitogen gastrin-releasing peptide (GRP) is a morphogen, critically important in regulating the differentiation of murine colon cancer. Although epithelial cells lining the mouse colon do not normally express GRP and its receptor (GRP-R), both are aberrantly expressed by all better differentiated cancers in wild-type C57BL/6J mice treated with the carcinogen azoxymethane. Whereas small tumors in both wild-type and GRP-R-deficient (i.e., GRP-R-/-) mice are histologically similar, larger tumors become better differentiated in the former but degenerate into more poorly differentiated mucinous adenocarcinomas in the latter. This alteration in phenotype is attributable to GRP increasing focal adhesion kinase expression in GRP-R-expressing tumors. Consistent with GRP acting as a mitogen, GRP/GRP-R coexpressing tumors in wild-type animals also contain more proliferating cells than those occurring in GRP-R-/- mice. Yet tumors are similarly sized in animals of either genotype receiving azoxymethane for identical times, a finding attributable to the significantly higher number of apoptotic cells detected in GRP/GRP-R coexpressing cancers. Thus, these findings indicate that although GRP is a mitogen, aberrant expression does not result in increased tumor growth. Rather, the mitogenic properties of GRP are subordinate to it acting as a morphogen, where it and its receptor are critically involved in regulating colon cancer histological progression by promoting a well-differentiated phenotype.     

Type 1 diabetes-associated HLA-DQ8 transdimer accommodates a unique peptide repertoire

HLA-DQ2 and HLA-DQ8 are strongly predisposing haplotypes for type 1 diabetes (T1D). Yet HLA-DQ2/8 heterozygous individuals have a synergistically increased risk compared with HLA-DQ2 or HLA-DQ8 homozygote subjects that may result from the presence of a transdimer formed between the α-chain of HLA-DQ2 (DQA1*05:01) and the β-chain of HLA-DQ8 (DQB1*03:02). We generated cells exclusively expressing this transdimer (HLA-DQ8trans), characterized its peptide binding repertoire, and defined a unique transdimer-specific peptide binding motif that was found to be distinct from those of HLA-DQ2 and HLA-DQ8. This motif predicts an array of peptides of islet autoantigens as candidate T cell epitopes, many of which selectively bind to the HLA transdimer, whereas others bind to both HLA-DQ8 and transdimer with similar affinity. Our findings provide a molecular basis for the association between HLA-DQ transdimers and T1D and set the stage for rational testing of potential diabetogenic peptide epitopes.     

Sigpep: calculating unique peptide signature transition sets in a complete proteome background

We have created a new software platform called sigpep that analyzes transition redundancy in selected reaction monitoring assays. Building on this platform, we also created a web application to generate transition sets with unique signatures for targeted peptides. The platform has been made available under the permissive Apache 2.0 open-source license, and the web application can be accessed from http://iomics.ugent.be/sigpep.     

Role of a tachykinin-related peptide and its receptor in modulating the olfactory sensitivity in the oriental fruit fly,Bactrocera dorsalis (Hendel)

Insect tachykinin-related peptide (TRP), an ortholog of tachykinin in vertebrates, has been linked with regulation of diverse physiological processes, such as olfactory perception, locomotion, aggression, lipid metabolism and myotropic activity. In this study, we investigated the function of TRP (BdTRP) and its receptor (BdTRPR) in an important agricultural pest, the oriental fruit fly Bactrocera dorsalis. BdTRPR is a typical G-protein coupled-receptor (GPCR), and it could be activated by the putative BdTRP mature peptides with the effective concentrations (EC₅₀) at the nanomolar range when expressed in Chinese hamster ovary cells. Consistent with its role as a neuromodulator, expression of BdTRP was detected in the central nervous system (CNS) of B. dorsalis, specifically in the local interneurons with cell bodies lateral to the antennal lobe. BdTRPR was found in the CNS, midgut and hindgut, but interestingly also in the antennae. To investigate the role of BdTRP and BdTRPR in olfaction behavior, adult flies were subjected to RNA interference, which led to a reduction in the antennal electrophysiological response and sensitivity to ethyl acetate in the Y-tube assay. Taken together, we demonstrate the impact of TRP/TRPR signaling on the modulation of the olfactory sensitivity in B. dorsalis. The result improve our understanding of olfactory processing in this agriculturally important pest insect.     

A peptide triggers allostery in tet repressor by binding to a unique site

Regulatory proteins often communicate with each other to manage various cellular processes. Such interactions mostly rely on the recognition of small peptide motifs. The activity of other regulatory proteins depends on small molecular weight effectors and allostery. We demonstrate the in vivo regulation of the tetracycline-dependent Tet repressor by an oligopeptide fused to the N or C terminus of thioredoxin A. The binding site of the peptide overlaps but is not identical with the tetracycline binding site. Several TetR mutants that are non-inducible by tetracycline also respond to the peptide. This demonstrates for the first time the conversion of a small molecular weight effector-dependent regulator to a protein-protein contact-dependent potential member of designed signaling chains.