Examining the Roles of Octopamine and Proctolin as Co-Transmitters in Drosophila melanogaster

The nervous system is a highly complex and intricate system that interacts with and controls nearly all the other body systems. The basic functions of nerve cells are conserved across most species and are very similar between vertebrates and invertebrates. Chemical transmitters (neurotransmitters) facilitate communication between nerve cells and their targets. The effects of these signals can be modified by co-transmitters that are released from neurons in conjunction with neurotransmitters, and by neuromodulators that are released as hormones. This thesis examines the effect of two neuromodulators on neuromuscular junctions of the fruit fly, Drosophila melanogaster. Two modulators, proctolin and octopamine, have been identified in motor nerve terminals and are thought to be released as co-transmitters to modify the effects of glutamate, the neurotransmitter that depolarizes muscle cells and triggers contraction. The neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr) was found to increase the amplitude of body wall muscle contractions elicited by glutamate in the absence of nerve stimulation. Thus, proctolin appears to enhance contractions by acting postsynaptically. Previous work reported that increasing neural activity lowers the threshold and EC50 for proctolin’s ability to enhance nerve-evoked contractions by two orders of magnitude. To determine whether such activity-dependence is caused by increased release of glutamate, effects of varying glutamate concentrations on the effectiveness of proctolin are examined here. The threshold for proctolin to increase body wall contractions decreased from 100 nM to 10 nM when glutamate concentration increased from 5 mM to 7 mM, but the threshold increased again to 100 nM for glutamate concentrations of 10-20 nM. Thus, although the effectiveness of proctolin shows some dependence on glutamate concentration, alterations in glutamate levels do not appear to account entirely for the more substantial and more consistent changes in proctolin threshold that occur with increasing neural activity, reported elsewhere. Since octopamine in known to be present in motor neurons innervating most of the body wall muscles of 3rd instar larvae, it was hypothesized that stimulating the motor neurons should release octopamine together with glutamate, and that increasing motor neuron activity should increase the release of both octopamine and glutamate. This hypothesis led to the prediction that an octopamine antagonist, phentolamine, should reduce the amplitude of nerve-evoked contractions, and that the antagonist should be more effective when the motor neurons are stimulated at higher frequencies. Phentolamine, however, did not alter the amplitude of body wall muscle contractions elicited by stimulating the motor axons using impulse bursts with intraburst stimulus frequencies of 5, 32 and 50 Hz. Surprisingly, exogenously applied octopamine did enhance the amplitude of nerve-evoked contractions, and, this effect was antagonized by phentolamine when contractions were elicited by impulse bursts with frequencies of 5 and 50 Hz. At a concentration of 1x10-6 M, octopamine did not induce contractions or alter the amplitude of glutamate-evoked contractions. These results do not support the hypothesis that endogenous octopamine is released onto muscle fibers as a co- transmitter to augment contraction amplitude. One possible explanation for these findings is the octopamine may be released at higher concentrations at neuromuscular synapses, and the effects of octopamine on nerve-evoked contractions are mediated presynaptically, by increasing transmitter release. Overall, the results of this thesis indicate that both octopamine and proctolin modulate muscle contractions in an activity-dependent manner; the level of external nerve-stimulus or exogenous glutamate concentration alter the effectiveness of the contransmitters. However, further work is needed to elucidate the mechanisms of such activity-dependence

Peptide and amine actions on the neurogenic Limulus heart: Biochemical mechanisms of modulation

The biochemical basis underlying the chronotropic and inotropic effects of peptides and amines on the neurogenic heart of the horseshoe crab, Limulus polyphemus, was investigated. This study focused on the role of cyclic nucleotide and phosphatidylinostiol metabolism in specific actions of amines and peptides endogenous to the Limulus nervous system. Biochemical and electrophysiological analyses of amine and peptide actions on specific cellular targets within the neurogenic heart network were performed to characterize the intracellular mechanisms responsible for the excitatory effects of these neuromodulators. Octopamine and the catecholamines dopamine, norepinephrine and epinephrine utilize the second messenger cyclic AMP at multiple cellular sites to increase the rate and strength of heart contractions. These amines increase burst rate in the cardiac ganglion by a cAMP-dependent mechanism. Amines also increase cardiac muscle contractility and enhance cardiac neuromuscular transmission by a cAMP-dependent process. Cyclic GMP does not appear to be involved in any of the excitatory actions of amines, although it may play a role in cardiac inhibition. Several proctolin-like and FMRFamide-like peptides are widely distributed in the Limulus CNS and may play a role in the regulation of cardiac output. The peptide proctolin utilizes the phosphatidylinositol second messenger system in its actions on cardiac muscle contractility and excitability. Dopamine may also activate this system, as this amine has several proctolin-like actions on Limulus cardiac muscle. Finally, the FMRFamide-like cardioexcitatory peptide limadrin, like the amines, appears to produce excitation of heart rate by increasing levels of cAMP in the Limulus cardiac ganglion. These results indicate that peptide and amine neuromodulators share several second messenger systems to produce their characteristic responses on the Limulus heartbeat

Receptors of peptide hormones in the gut of rice brown planthopper (Nilaparvata lugens) as targets for novel insecticides

The rice brown planthopper, Nilaparvata lugens, is a pest of rice where it can be responsible for single season crop losses of up to 38% (IRRI statistic). Due to the absence of an effective Bacillus thuringiensis (Bt) toxin against these insect pests current control methods are reliant on synthetic insecticides. However, these broad acting insecticides can be harmful to beneficial insects, and resistance to these synthetic insecticides amongst N.lugens populations is now common. Therefore, other novel approaches are needed for the control of this rice pest species, which is addressed in this thesis. This project aims to characterise proteins expressed in the N.lugens gut that could act as potential 'targets' for the design of insecticidal proteins. G protein coupled receptor (GPCR) proteins were identified as 'target' proteins and N.lugens candidates were identified and characterised. A cDNA predicted to encode a diuretic hormone receptor (DHR) in Nilaparvata lugens was cloned using RNA extracted from gut tissue as a template in a PCR based strategy. The coding sequence (639 amino acids, 69.7 kDa) has seven predicted transmembrane domains and is a member of the calcitonin/secretin/corticotropin releasing factor family of G-protein coupled receptors. The protein has greatest similarity to Acheta domesticus DHR (Q16983) with 51% sequence identity. N.lugens DHR has a large N-terminal extracellular domain (amino acids 1-260) putatively involved in hormone binding. This domain was expressed as a recombinant protein in E.coli, purified under denaturing conditions, refolded and used to raise a polyclonal antibody in rabbit. Purified anti-DHR IgG bound specifically to a putative DHR polypeptide extracted from N.lugens gut tissues on western blots. Immunolocalisation experiments using dissected guts showed that anti-DHR antibody bound specifically to the Malpighian tubules. The N- terminal hormone-binding domain is located on the cell surface and is exposed to the haemolymph in vivo. Anti-DHR antibodies delivered to insects via artificial diet showed no binding to gut or tubule tissue, and had no effect on survival. Ingested antibodies were not detected in the haemolymph. Another GPCR protein with high similarity to insect allatostatin receptors (ALSTR) was isolated by PCR using highly degenerate primers, from N.lugens gut specific cDNA. The coding sequence (383 amino acids, M, 42.3 kDa) has seven predicted transmembrane domains and is a member of the SST/galanin/opoid receptor family of G-protein coupled receptors. The putative N.lugens ALSTR has greatest similarity to Periplaneta americana ALSTR (AF336364) with 69% overall sequence identity. Isolation of the full-length N.lugens ALSTR is the first step necessary for the design of novel insecticidal proteins

Modulation of muscle contraction by a FMRFamide-related peptide

A FMRFamide-like neuropeptide with the sequence "DRNFLRF-NH2" was recently isolated from pericardial organs of crayfish (Mercier et aI., Peptides, 14, 137-143, 1993). This neuropeptide, referred to as "DF2'" has already been shown to elicit cardioexcitation and to enhance synaptic transmission at neuromuscular junctions. Possible effects ofDF2 on muscle were investigated using superficial extensor muscles of the abdomen of the crayfish, Procambarus clar/ai. These muscles are of the tonic type and generate slow contractions that affect posture. DF2, at concentrations of 10-8 M or higher, increased muscle tonus and induced spontaneous, rhythmic contractions. These effects were antagonized by 5 rnM Mn2+ but not by lO-7M tetrodotoxin (TTX). Thus, they represent direct actions on muscle cells (rather than effects on motor neurons) and are likely to involve calcium influx. In contrast, deep abdominal extensor muscles, responsible for rapid swimming movements, and superficial flexor muscles do not generate contractions in response to the peptide. 2 Spontaneous contractions were also induced in the superficial extensor muscles by decreasing the temperature to II-13°C. Such contractions were also TTX-insensitive and they were antagonized by adding calcium channel blockers (Mn2+, Cd2+ or Ni2+) or by removing calcium from the bathing solution. This suggests that the spontaneous contractions depend on an influx of calcium from the extracellular solution. N-type and L-type voltage dependent calcium channel blockers did not reduce the effect of the peptide or the spontaneous contractions suggesting that calcium influx is not through N- or L-type calcium channels

The characterization, functional expression, and localization of the first arthropod myokinin receptor from the southern cattle tick, Boophilus microplus (Acari: ixodidae)

Myokinins are invertebrate neuropeptides with myotropic and diuretic activity. The lymnokinin receptor from the snail Lymnaea stagnalis was the only previously identified myokinin receptor. A cDNA encoding a neuropeptide receptor was cloned from the southern cattle tick, Boophilus microplus. The deduced amino acid sequence was 40 % identical to the lymnokinin receptor. The receptor transcript is present in all tick life stages as determined by semiquantitative RT-PCR. When expressed in mammalian CHO-K1 cells, myokinins at nanomolar concentrations induced increases in intracellular calcium as measured by fluorescent cytometry. The rank order of potency for peptides tested was FFFSWS-NH2≥FFFSWG-NH2≥FFSWG-NH2>FYSWG-NH2>muscakinin>lymnokinin>>APTGFFGVR-NH2. The receptor coupled to a pertussis toxin insensitive G protein. Absence of extracellular calcium did not inhibit the calcium response, indicating the release of Ca2+ from intracellular stores. Receptor transcript was detected by RT-PCR in the dissected synganglia, ovaries, salivary glands, guts and Malpighian tubules of partially engorged adult female ticks. It is concluded that the B. microplus receptor is the first myokinin receptor cloned from an arthropod, and the first neuropeptide receptor known from the Acari. The presence of this receptor transcript in multiple tissues and all life stages suggests a multifunctional role in ticks

Mode of action of FMRFamide-like peptide on drosophila body wall muscle conractions

Neuropeptides are the largest group of signalling chemicals that can convey the information from the brain to the cells of all tissues. DPKQDFMRFamide, a member of one of the largest families of neuropeptides, FMRFamide-like peptides, has modulatory effects on nerve-evoked contractions of Drosophila body wall muscles (Hewes et aI.,1998) which are at least in part mediated by the ability of the peptide to enhance neurotransmitter release from the presynaptic terminal (Hewes et aI., 1998, Dunn & Mercier., 2005). However, DPKQDFMRFamide is also able to act directly on Drosophila body wall muscles by inducing contractions which require the influx of extracellular Ca 2+ (Clark et aI., 2008). The present study was aimed at identifying which proteins, including the membrane-bound receptor and second messenger molecules, are involved in mechanisms mediating this myotropic effect of the peptide. DPKQDFMRFamide induced contractions were reduced by 70% and 90%, respectively, in larvae in which FMRFamide G-protein coupled receptor gene (CG2114) was silenced either ubiquitously or specifically in muscle tissue, when compared to the response of the control larvae in which the expression of the same gene was not manipulated. Using an enzyme immunoassay (EIA) method, it was determined that at concentrations of 1 ~M- 0.01 ~M, the peptide failed to increase cAMP and cGMP levels in Drosophila body wall muscles. In addition, the physiological effect of DPKQDFMRFamide at a threshold dose was not potentiated by 3-lsobutyl-1-methylxanthine, a phosphodiesterase inhibitor, nor was the response to 1 ~M peptide blocked or reduced by inhibitors of cAMP-dependent or cGMP-dependent protein kinases. The response to DPKQDFMRFamide was not affected in the mutants of the phosholipase C-~ (PLC~) gene (norpA larvae) or IP3 receptor mutants, which suggested that the PLC-IP3 pathway is not involved in mediat ing the peptide's effects. Alatransgenic flies lacking activity of calcium/calmodul in-dependent protein kinase (CamKII showed an increase in muscle tonus following the application of 1 JlM DPKQDFMRFamide similar to the control larvae. Heat shock treatment potentiated the response to DPKQDFMRFamide in both ala1 and control flies by approximately 150 and 100 % from a non heat-shocked larvae, respectively. Furthermore, a CaMKII inhibitor, KN-93, did not affect the ability of peptide to increase muscle tonus. Thus, al though DPKQDFMRFamide acts through a G-protein coupled FMRFamide receptor, it does not appear to act via cAMP, cGMP, IP3, PLC or CaMKl1. The mechanism through which the FMRFamide receptor acts remains to be determined

Single crystal x-ray diffraction studies on small, medium and large molecules

Chapter 1. Production of crystals for diffraction analysis would be assisted by the devising of a set of rules which, given molecular formula, could predict crystal formation conditions. By studying trends in structural properties of a group of closely related simple molecules, deductions could be drawn which could then be applied more generally. Chalcone derivatives with minor substituent differences were recrystallised. X-ray diffraction data collected and the structures solved and refined. Additionally, NMR and UV studies were performed, investigating an observed dimerisation reaction. Chapter 2. Discovery of peptide hormones and neurotransmitters has stimulated the study of structure-activity relationships, although the structure of these molecules is often poorly defined. Proctolin, a linear pentapeptide, is a neurotransmitter in insects. Crystallisation was attempted, with the aim of deducing the active conformation structure, thereby assisting in design of small molecule analogues for use as non-cholinergic pesticides. No diffraction was observed from the crystals produced. Chapter 3. Glucosamine 6-phosphate synthase is an N-terminal nucleophile amidotransferase catalysing the first step in the hexosamine pathway, from which all amino-sugar containing macromolecules are derived. Structure determination of each of two subdomains was attempted. In one case, pseudo-symmetry appeared to obstruct structure solution. The symmetry has subsequently been understood and the structure obtained. Crystals of the second domain are rotationally disordered. Chapters 4 and 5. Recent advances in macromolecular crystallographic techniques have facilitated the collection of an increasing number of high quality, atomic resolution data sets. Methods for refinement, previously limited to small molecule structures, have increasing relevance for proteins. Atomic resolution refinements using these evolving protocols have been performed on two small proteins, rubredoxin from Desulfovibrio vulgaris and the protein G immunoglobulin-binding domain. Appropriate treatment of the solvent structure in a protein crystal and the benefit to be gained by using sharpened density maps during refinement were investigated

Characterization of the gut peptidome and the function of brain-gut peptides with regard to food intake and metabolism in Drosophila melanogaster and the agricultural pest Delia radicum

Regulatory peptides, which comprise neuropeptides and peptide hormones, are cell-cell signaling molecules that control a variety of biological processes in insects and other metazoans. The insect midgut, like the mammalian digestive system, contains numerous peptide-producing endocrine cells, and the diversity of insect enteroendocrine peptides gives a hint at their relevance for metabolism, energy balance and feeding behavior. In the first study, we characterized the midgut peptidome of adult and larval Drosophila melanogaster by extraction of peptides from midgut tissue and subsequent LC-MS/MS analysis of peptide structures. By this means we identified 24 peptides originating from 9 different peptide precursors. All gut peptides were found in identical form within the CNS and thus represent brain-gut peptides. Processing of Drosophila neuropeptide hormones was previously shown to require the subtilisin-like proprotein convertase 2 (dPC2, AMON). Our results suggest a general need of AMON for gut peptide production as well. In the second study, we could expand the knowledge on peptide structures of relevant insects. We investigated the peptidome of the cabbage maggot (i.e., the larva of the cabbage root fly Delia radicum), which causes substantial agricultural damage by feeding on plant roots. By mass spectrometric analysis of CNS, neurohemal organ and gut tissue, we could characterize 38 peptides belonging to diverse insect peptide families. Moreover, we identified a new peptide with sequence similarity to the eclosion hormone precursor of several Drosophila species. Immunocytochemical characterization of peptide-producing neurons and enteroendocrine cells in cabbage maggots showed that peptide distribution was largely similar to Drosophila larvae. The observed similarities in the peptidergic systems of both species suggest that Drosophila can serve as a genetically accessible pest species model in terms of peptidergic regulation of, e.g., metabolism. In the future, our results could be of use for the development of a targeted, peptide-based management of cabbage root flies. In the third study, we analyzed the role of allatostatin A (AstA), a peptide family commonly occurring in insects (and other arthropods). Previous studies had already demonstrated a role for AstA in metabolic regulation and nutritional homeostasis of Drosophila. We addressed the question whether specific effects were connected to the activity of certain subsets of the numerous AstA-producing cells found in adult fruit flies. AstA neurons are located in different regions of the CNS. The thorax also contains a few peripheral AstA neurons. The hindgut and the posteriormost portion of the midgut are innervated by central AstA neurons. In addition, a large number of enteroendocrine AstA cells are scattered across the epithelium of the posterior midgut. Thermogenetic activation of certain AstA cells significantly reduced food intake of flies, and also considerably diminished their locomotor activity. The combination of our results with findings of a previous study suggested that two pairs of AstA-producing posterior lateral protocerebrum neurons function to promote satiety, while enteroendocrine AstA cells seem to regulate locomotor activity. In addition, our findings indicated that AstA cells might directly and indirectly influence defecation behavior, while no effect on water and ion homeostasis could be observed. Furthermore, we tested the effect of synthetic AstA peptides on isolated midguts in vitro and observed a dose-dependent inhibition of midgut motility. Downregulation of AstA receptor mRNA in the gut musculature via RNAi showed that the DAR-2 receptor mediates the myoinhibitory effect of AstA peptides. Altogether, by influencing satiety, locomotion, gut peristalsis and possibly also defecation, AstA cells appear to affect different levels of metabolism and different tissues, seemingly promoting several interrelated processes connected to food intake

Structural and Kinetic Studies of Drug-Resistant Mutants of HIV-1 Protease

The employment of HIV-1 protease (PR) inhibitors (PIs) in antiviral therapy has been successful in reducing mortality of HIV/AIDS patients. However, the long-term efficacy of PIs is challenged by the rapid emergence of drug-resistant mutants of PR. To understand the underlying mechanism of drug resistance, structures and activities of HIV-1 PR and its drug resistant mutants have been extensively studied. Here, PR mutants PRR8Q, PRD30N, PRI47V, PRI50V, PRI54M, PRV82A, and PRN88D/S bearing single substitutions have been investigated by crystallography and kinetics. GRL-0519 is a potent new antiviral inhibitor of HIV-1 PR that possesses tris-tetrahydrofuran (tris-THF) as the P2 ligand. The crystal structures of GRL-0519 were determined at resolutions of 1.06-1.49 Å in complex with the mutants PRR8Q, PRD30N, PRI50V, PRI54M, and PRV82A. I50V lost its interaction with inhibitor while V82Aand I54M compensated for the mutation through the main chain shift and flexibility of 80’s loop (residues 78-82), respectively. The structural changes may account for the worst inhibition of GRL-0519 for PRI50V (60-fold decrease relative to wild-type enzyme)and moderate inhibition for PRI54M and PRV82A (6-7-fold decrease). The large tris-THF group at P2 provides a good fit in the S2 subsite and may be effective against resistant virus with mutations of residues in this subsite. SQV and DRV are two clinical inhibitors that were designed to target the wild type PR and its drug resistant mutants, respectively. The crystal structures of PR mutants PRI47V, PRN88D/s in complex with DRV and mutants PRI47V and PRN88D in complex with SQV with resolutions of 1.13-1.72 Å were also analyzed. Mutation I47V gained more hydrophobic interactions with DRV and SQV. Interestingly, the structural changes did not affect the inhibition of both inhibitors for PRI47V (relative Ki is 0.7 and 1 for DRV and SQV, respectively). DRV and SQV showed 8-fold increase in Ki for PRN88D and only very subtle local changes have been observed on the structures. DRV induced 0.3 fold reduction in Ki for PRN88S and the distal structural changes have been transferred to the active site. This study provided fundamental information for understanding drug resistance and future design of potential antiviral drugs

Role of sulfakinin signaling in feeding of the red flour beetle Tribolium castaneum

Understanding the regulation of feeding is a prerequisite for the development of more effective and environmentally safe control methods of pest insects. Insect sulfakinins (SKs) are neuropeptides associated with the regulation of feeding. However, not much is known about SK and SK signaling in insects such as the model insect Tribolium castaneum. Therefore, the function and mechanism of the SK signaling on the regulation of feeding in T. castaneum was examined in this PhD study. First, the SK signaling inhibits feeding in T. castaneum: food intake was increased when SK signaling was reduced due to the silencing of SK or SK receptor (SKR) gene; food intake was decreased when SK signaling was enhanced via administration of exogenous SK. Second, the ligand-specificity of the two SKRs was examined in a cell-based assay. The two SKRs both have higher affinity to sulfated SK than nonsulfated SK and they respond to sulfated SK in a dose-dependent manner. Third, the three-dimensional structures of two SKRs were predicted by molecular modeling. The structural difference between two SKRs is proposed to contribute to their ligand-specificity. Further studies can provide more details on the mechanism of SK signaling and its potential in the development of new pest control methods