Functional and biophysical analysis of the C-terminus of the CGRP-receptor; a family B GPCR

G-protein coupled receptors (GPCRs) typically have a functionally important C-terminus which, in the largest subfamily (family A), includes a membrane-parallel eighth helix. Mutations of this region are associated with several diseases. There are few C-terminal studies on the family B GPCRs and no data supporting the existence of a similar eighth helix in this second major subfamily, which has little or no sequence homology to family A GPCRs. Here we show that the C-terminus of a family B GPCR (CLR) has a disparate region from N400 to C436 required for CGRP-mediated internalization, and a proximal region of twelve residues (from G388 to W399), in a similar position to the family A eighth helix, required for receptor localization at the cell surface. A combination of circular and linear dichroism, fluorescence and modified waterLOGSY NMR spectroscopy (SALMON) demonstrated that a peptide mimetic of this domain readily forms a membrane-parallel helix anchored to the liposome by an interfacial tryptophan residue. The study reveals two key functions held within the C-terminus of a family B GPCR and presents support for an eighth helical region with striking topological similarity to the nonhomologous family A receptor. This helix structure appears to be found in most other family B GPCRs.

Biochemical aspects of Tourette's Syndrome

Kynurenine (KYN) is the first stable metabolite of the kynurenine pathway, the major route of tryptophan. (TRP) metabolIsm. In the liver, cortisol-inducible tIyptophan-2,3-dioxygenase (TDO) is the first enzyme and rate limiting step. In extrahepatic tissues, it is superceded by indoleamine-2,3-dioxygenase (IDO), an enzyme with a wider substrate specificity. Earlier work in this research group has found substantial elevations in plasma KYN in fasting Tourette's Syndrome (TS) patients with normal TRP and neopterin. The aim of our initial pilot study was to confirm this increase in KYN in fasting human TS patients compared with normal controls, and to see how changes in diet :ay influence certain kynurenine pathway variables. However, we failed to detect a change in plasma KYN, TRP, kynurenic acid (KYNA), neopterin or cortisol between the fasting TS and control groups. Moreover, none of the variables was affected by dietary status, and thus candidates selected for the larger cross-sectional study were permitted to eat and drink freely on the day that blood samples were submitted, but were requested to avoid products containing caffeine, aspirin or nicotine. In the cross-sectional study, TS patients exhibited significantly higher plasma KYN concentrations than controls, although the magnitude of the change was much smaller than originally found. This may be due to differences in detection procedure and the seasonal fluctuation of some biochemical variables, notably cortisol. The generalised increase in neopterin in the TS subject group, suggests a difference in the activity of cytokine-inducible IDO as a likely source for this elevated KYN. Other kynurenine pathway metabolites, specifIcally TRP, 3-hydroxykynurenine (HKY), 3-hydroxyanthranilic acid (HAA) and KYNA were unchanged. In view of recent speculation of the potential therapeutic effects of nicotine in TS, the lower KYN concentrations observed in TS smokers, compared with non-smoking TS patients, was another interesting finding. Tic-like movements, such as head-shakes (HS), which occur in rodents both spontaneously and following diverse drug treatments, closely resemble tic behaviours in humans. The animal tic model was used to examine what effects nicotine may have on shaking behaviours and on selected TRP metabolites. Acute systemic administration of nicotine to mice, produced a dose-dependent reduction in HS frequency (induced by the 5-HT2A/2C agonist DOl), which appeared to be mediated via central nicotinic cholinergic receptors, since mecamylamine pretreatment abolished this effect. Conversely, twice daily subcutaneous injections of nicotine for 7 days, led to an increase in spontaneous and DOI-induced HS. Chronic nicotine also caused a significant elevation m plasma and whole brain KYN concentrations, but plasma TRP, HKY, HAA and KYNA were unaltered. In addition, no change in brain 5-HT or 5-HIAA concentrations or 5-HT turnover, was found. Despite contrasting results from human and animal studIes, a role for nicotine in the mediation of tic-like movements is indicated. The relevance of the kynurenine pathway to TS and the potential role played by nicotine in modifying tic-like behaviours is discussed.

Investigation into the biochemical changes in Tourette Syndrome with a potential for pharmacological manipulation

Kynurenine (KYN) is the first stable metabolite of the kynurenine pathway, which accounts for over 95% of tryptophan metabolism. Two previous studies by this research group reported elevated plasma KYN in Tourette syndrome (TS) patients when compared with age and sex matched controls and another study showed that KYN potentiated 5-HT2A-mediated head-shakes (HS) in rodents. These movements have been suggested to model tics in TS. This raised the questions how KYN acts in eliciting this response and whether it is an action of its own or of a further metabolite along the kynurenine pathway. In the liver, where most of the kynurenine pathway metabolism takes place under physiological conditions, the first and the rate limiting enzyme is tryptophan-dioxygenase (TDO) which can be induced by cortisol. In extrahepatic tissues the same step of the pathway is catalyzed by indoleamine-dioxygenase (IDO), which is induced by cytokines, predominantly interferon-y (INF-y). Plasma neopterin, which shows parallel increase with KYN following immune stimulation, was also found elevated in one of these studies positively correlating with KYN. In the present work animal studies suggested that KYN potentiates and quinolinic acid (QUINA) dose dependently inhibits the 5-HT2A-mediated HS response in mice. The potentiating effect seen with KYN was suggested to be an effect of KYN itself. Radioligand binding and phosphoinositide (PI) hydrolysis studies were done to explore the mechanisms by which kynurenine pathway metabolites could alter a 5-HT2A-receptor mediated response. None of the kynurenine pathway metabolites tested showed direct binding to 5-HT2A-receptors. PI hydrolysis studies with KYN and QUINA showed that KYN did not have any effect while QUINA inhibited 5-HT2A-mediated PI hydrolysis. Plasma cortisol determination in TS patients with elevated plasma KYN did not show elevated plasma cortisol levels, suggesting that the increase of plasma KYN in these TS patients is unlikely to be due to an increased TDO activity induced by increased cortisol. Attention deficit hyperactivity disorder (ADHD) is commonly associated with TS. Salivary cortisol detected in a group of children primarily affected with ADHD showed significantly lower salivary cortisol levels when compared with age and sex matched controls. Plasma tryptophan, KYN, neopterin, INF-y and KYN/tryptophan ratio and night-time urinary 6-sulphatoxymelatonin (aMT6s) excretion measured in a group of TS patients did not show any difference in their levels when compared with age and sex matched controls, but TS patients failed to show the expected positive correlation seen between plasma INF-y, neopterin and KYN and the negative correlation seen between plasma KYN and night-time urinary aMT6s excretion seen in healthy controls. The relevance of the kynurenine pathway, melatonin secretion and cortisol to Tourette Syndrome and associated conditions and the mechanism by which KYN and QUINA alter the 5-HT2A-receptor mediated HS response are discussed.

Combinatorial approach to multi-substituted 1,4-Benzodiazepines as novel non-peptide CCK-antagonists

For the drug discovery process, a library of 168 multisubstituted 1,4-benzodiazepines were prepared by a 5-step solid phase combinatorial approach. Substituents were varied in the 3,5, 7 and 8-position on the benzodiazepine scaffold. The combinatorial library was evaluated in a CCK radiolabelled binding assay and CCKA (alimentary) and CCKB (brain) selective lead structures were discovered. The template of CCKA selective 1,4-benzodiazepin-2-ones bearing the tryptophan moiety was chemically modified by selective alkylation and acylation reactions. These studies provided a series of Asperlicin naturally analogues. The fully optimised Asperlicin related compound possessed a similar CCKA activity as the natural occuring compound. 3-Alkylated 1,4-benzodiazepines with selectivity towards the CCKB receptor subtype were optimised on A) the lipophilic side chain and B) the 2-aminophenyl-ketone moiety, together with some stereochemical changes. A C3 unit in the 3-position of 1,4-benzodiazepines possessed a CCKB activity within the nanomolar range. Further SAR optimisation on the N1-position by selective alkylation resulted in an improved CCKB binding with potentially decreased activity on the GABAA/benzodiazepine receptor complex. The in vivo studies revealed two N1-alkylated compounds containing unsaturated alkyl groups with anxiolytic properties. Alternative chemical approaches have been developed, including a route that is suitable for scale up of the desired target molecule in order to provide sufficient quantities for further in vivo evaluation.

The synthesis and evaluation of small organic molecules as cholecystokinin antagonists

Cholecystokinin (CCK) is a peptide hormone, present in the alimentary and the CNS. It is the most abundant peptide in the brain. CCK has been implicated in a number of disorders. The link between CCK and anxiety was the basis for this research. A comprehensive discussion on the many types of CCK receptor antagonists is included. For the drug discovery process, a number of synthetic approaches have been investigated and alternative chemical approaches developed. 1,4-Benzodiazepine analogues were prepared, with substitutents In the 1,2 & 3- position of the benzodiazepine scaffold varied, and substituted 3-anilino benzodiazepines exhibited the greatest in vitro activity towards the CCKA receptor subtype. Through extensive screening, pyrazolinone-ureido derivatives were identified, optimised, SAR studied and re-screened. A comprehensive in vivo study on the most active analogue is included, which has a number of common structural features with L-36S, 260 including activity. Pyrazolinone-amide derivatives, bearing the tryptophan moiety were equally active. A number of existing and novel furan- 2(SH)-one building blocks were prepared, from which a selected mini-library of 4- amino-substituted furan-2(SH)-ones were prepared and evaluated. All synthesised compounds were evaluated in a CCK radiolabelled binding assay (CCKA & CCKB), with compounds demonstrating receptor selectivity and lead structures being discovered. The work in this thesis has identified a number of highly active prime structures, from which further investigations are essential in providing more in vitro & in vivo data and the need to prepare more analogues.

Growth, sporulation and spore properties of bacillus stearothemophilus produced in chemically defined media

The nutritional requirements for the vegetative growth of B. stearothermophilus strains NCIB 8919, NCTC lO,OO3 (wild) were found to be DL-methionine, biotin, nicotinic acid, thiamin, glucose and mineral salts. Strains NCIB 8920 required in addition L-tryptophan. B. stearothermophilus NCTC lO,OO3 (mutant) grew in a medium containing only glucose and mineral salts. Separate chemically defined media for the growth of Bacillus stearothermophilus strains NCIB 8919, 8920, NCTC lO,OO3 (wild) and NCTC lO,OO3 (mutant) were developed. Optimally aerated culture of B. stearothermonhilus NCTC lO,OO3(mutant) required 1.0 x 10-4 M. Mn2+ and 2.4 x 10-3 M. glutamic acid for optimal sporulation. Specific nutrient depletion of growth affected percentage sporulation. Spore suspensions of B. stearothermophilus NCTC 10,003 (mutant) were prepared from media in which sulphate (SO4-), nitrogen (N-),phosphate (Po4-), carbon (C-), magnesium-carbon simultaneously (Ng-C-) depleted growth. The heat resistance, dormancy and chemistry of these spores varied considerably. B. stearothermophilus NCTC 10,003 10,00310,00(mutant) spores prepared from carbon depleted cultures containing high and low concentrations of calcium, iron or manganese showed variations in heat resistance,dormancy and chemical composition. Progressive increase in the concentration of medium calciumfrom 1.0 X 10-5  M to 1.4 X 10-4 M. progressively increased theheat resistance of B. stearothermophilus NCTC 10,003 (mutant) spores prepared from nitrogen depleted cultures (N-). The thermodynamic functions for germination rate, magnesium and manganese release of N- and SO4- spores were within the range expected of enzymic reactions. The thermodynamic functions for the breaking of dormancy in SO4- spores and that for the release of D.P.A. were identical. Sublethal heating of SO4- spores (96.5°C and below) induced dormancy in these spores, whereas heating above 96.5°C gave rise to heat activation. Pooled results of the chemical analyses of all spore types studied showed that the concentration of D.P.A. and calcium were positively related to heat resistance whereas magnesium concentration and Mg/Ca molar ratio were inversely proportional to heat resistance.

Tetrahydrobiopterin Metabolism

Tetrahydrobiopterin is the cofactor for the hydroxylation of phenylalanine, tyrosine and tryptophan and is therefore essential for the production of monoamine neurotransmitters. Neopterin, a biosynthetic precusor of tetrahydrobiopterin, and biopterin appear in urine. In normal subjects the urinary neopterin to biopterin ratio has been found to be about 1.00. In patients suffering from Alzheimer's disease, Down's syndrome and depression the urinary neopterin to biopterin ratio has been found to be elevated. In some Alzheimer's and depressed patients the increased urinary neopterin to biopterin ratio is proportional to the severity of the disease. Folates were found not to increase tetrahydrobiopterin biosynthesis in the rat as previously thought. Methotrexate was found to reduce liver biopterin levels and increas_ urinary biopterin levels in the rat. Methotrexate also reduced brain pterin levels but had no influence on liver pterin. Urinary isoxanthopterin, found in some patients, was found to be derived from biopterin and neopterin in the rat. Isoxanthopterin is proposed as an indicator of the levels of tetrahydrobiopterin turnover.

Biological barriers to the nasal delivery of peptide drugs

The nasal absorption of larger peptide and protein drugs is generally low. The importance of the mucus layer and enzymic degradation in reducing absorption were investigated. Reversed-phase high-performance liquid chromatographic (HPLC) methods were developed to assay a variety of compounds. Pig gastric mucus (PGM) was selected to investigate the importance of the mucus layer. A method of treating and storing PGM was developed and evaluated which was representative of the gel in vivo. The nature of the mucus barrier was evaluated in vitro with three-compartment diffusion cells and a series of compounds with differing physicochemical properties. Mucus retarded the diffusion of all the compounds with molecular weight and charge exerting a marked effect. Binding to mucus was investigated by a centrifugation method. All of the compounds tested were found to bind to mucus with the exception of the negatively charged molecule benzoic acid. The small peptides did not demonstrate greater binding to mucus than any of the other compounds evaluated. The effect of some absorption enhancers upon the rate of diffusion of tryptophan through mucus was determined in vi tro. At the concentrations employed the enhancers EDTA, N-acetylcysteine and taurodeoxycholic acid exerted no effect, whilst taurocholic acid and cholic acid, were found to slightly reduce the rate of diffusion. The intracellular and luminal proteolytic activity of the nose was investigated in the sheep animal model with a nasal mucosal homogenate and a nasal wash preparation respectively and a series of chemically similar peptides. Hydrolysis was also investigated with the proteolytic enzymes carboxypeptidase A, cytosolic leucine aminopeptidase and microsomal leucine aminopeptidase. Sheep nasal mucosa possesses significant peptide hydrolase activity capable of degrading all the substrates tested. Considerable variation in susceptibility was observed. Degradation occurred excl us i ve ly at the pept ide bond between the aromatic amino ac id and glycine, indicating some specificity for aromatic amino acids. Hydrolysis profiles indicated the presence of both aminopeptidase and carboxypeptidase enzymes. The specific activity of the microsomal fraction was found to be greater than the cytosolic fraction. Hydrolysis in the nasal wash indicated the presence of either luminal or loosely-bound proteases, which can degrade peptide substrates. The same specificity for aromatic amino acids was observed and aminopeptidase activity demonstrated. The specific activity of the nasal wash was smaller than that of the homogenate.

Development of novel mass spectrometric methods for identifying HOCl-induced modifications to proteins

Protein oxidation is thought to contribute to a number of inflammatory diseases, hence the development of sensitive and specific analytical techniques to detect oxidative PTMs (oxPTMs) in biological samples is highly desirable. Precursor ion scanning for fragment ions of oxidized amino acid residues was investigated as a label-free MS approach to mapping specific oxPTMs in a complex mixture of proteins. Using HOCl-oxidized lysozyme as a model system, it was found that the immonium ions of oxidized tyrosine and tryptophan formed in MS(2) analysis could not be used as diagnostic ions, owing to the occurrence of isobaric fragment ions from unmodified peptides. Using a double quadrupole linear ion trap mass spectrometer, precursor ion scanning was combined with detection of MS(3) fragment ions from the immonium ions and collisionally-activated decomposition peptide sequencing to achieve selectivity for the oxPTMs. For chlorotyrosine, the immonium ion at 170.1 m/z fragmented to yield diagnostic ions at 153.1, 134.1, and 125.1 m/z, and the hydroxytyrosine immonium ion at 152.1 m/z gave diagnostic ions at 135.1 and 107.1 m/z. Selective MS(3) fragment ions were also identified for 2-hydroxytryptophan and 5-hydroxytryptophan. The method was used successfully to map these oxPTMs in a mixture of nine proteins that had been treated with HOCl, thereby demonstrating its potential for application to complex biological samples.

Redox signalling and detection of protein oxidation by mass spectrometry

It is now recognised that redox control of proteins plays an important role in many signalling pathways both in health and disease. Proteins can undergo a wide variety of oxidative post-translational modifications (oxPTM); while the reversible modifications are thought to be most important in physiological processes, non-reversible oxPTM may contribute to pathological situations and disease. The oxidant is also important in determining the type of oxPTM (chlorination, nitration, etc.), and the susceptibilities of residues vary depending on their structural location. The best characterized oxPTMs involved in signalling modulation are partial oxidations of cysteine to the disulfide, glutathionylated or sulfenic acid forms, but there is increasing evidence that specific oxidations of methionine and tyrosine may have some biological roles. Well understood examples of oxidative regulation include protein tyrosine phosphatases, e.g. PTP1B/C, and members of the MAPK pathways such as MEKK1 and ASK1. Transcription factors such as NFkB and Nrf-2 are also regulated by redox-active cysteines. Improved methods for analysing specific oxPTMs in biological samples are critical for understanding the physiological and pathological roles of these changes, and tandem or MS3 mass spectrometry techniques interfaced with nano-LC separation are being now used. MS3 fragmentation markers for a variety of oxidized residues including tyrosine, tryptophan and proline have been identified, and a precursor ion scanning method that allows the selective identification of these oxPTMs in complex samples has been developed. Such advances in technology offer potential for biomarker development, disease diagnosis and understanding pathology.

Modulation of SERCA in the chronic phase of adjuvant arthritis as a possible adaptation mechanism of redox imbalance

Adjuvant arthritis (AA) is a condition that involves systemic oxidative stress. Unexpectedly, it was found that sarcoplasmic reticulum Ca2 +-ATPase (SERCA) activity was elevated in muscles of rats with AA compared to controls, suggesting possible conformational changes in the enzyme. There was no alteration in the nucleotide binding site but rather in the transmembrane domain according to the tryptophan polar/non-polar fluorescence ratio. Higher relative expression of SERCA, higher content of nitrotyrosine but no increase in phospholipid oxidation in AA SR was found. In vitro treatments of SR with HOCl showed that in AA animals SERCA activity was more susceptible to oxidative stress, but SR phospholipids were more resistant and SERCA could also be activated by phosphatidic acid. It was concluded that increased SERCA activity in AA was due to increased levels of SERCA protein and structural changes to the protein, probably induced by direct and specific oxidation involving reactive nitrogen species.

Mechanism of serca modulation from rats with adjuvant arthritis

We studied the structural and functional alterations of SERCA in rats suffering from adjuvant arthritis (AA). AA was induced by intradermal administration of Mycobacterium butyricum (MB) to the base of the tail of Lewis rats. Injury of SERCA from skeletal muscles of AA rats was analyzed on days 7, 14, 21 and 28 after MB injection. Neither fragmentation, aggregation of SERCA protein, alterations in SH groups, nor oxidation of phosphatidylcholines and phosphatidylethanolamines in SR vesicles were observed in animals with AA. The only ROS/RNS modification was increased formation of nitrotyrosine. The activity of SERCA from AA animals decreased on day 21 after MB injection and was associated with a significant increase of protein carbonyls in sarcoplasmic reticulum (SR). In contrast, on day 28 an increase of SERCA activity was observed and protein carbonyl level reversed to control level. Concerning kinetic parameters, maximum reaction velocity (Vmax) decrease and increase was observed with respect to both substrates (Ca, ATP) on days 21 and 28, respectively, suggesting possible conformational changes of the enzyme. These changes were not associated with alterations in nucleotide binding site situated in cytosol, but rather with tryptophan fluorescence intensity ratio (cytosol/membrane) related to the transmembrane domain of SERCA. Elevated SERCA activity on day 28 was caused by its higher expression. Acidic phospholipids (PA), probably present in SR of AA rats, may contribute to the elevation of Ca-ATPase activity, as PA administration in vitro increased this activity.

Oxidative impairment of plasma and skeletal muscle sarcoplasmic reticulum in rats with adjuvant arthritis - effects of pyridoindole antioxidants

OBJECTIVES: To study possible oxidation of proteins and lipids in plasma and sarcoplasmic reticulum (SR) from skeletal muscles and to assess the effects of pyridoindole antioxidants in rats with adjuvant arthritis (AA) and to analyze modulation of Ca-ATPase activity from SR (SERCA). METHODS: SR was isolated by ultracentrifugation, protein carbonyls in plasma and SR were determined by ELISA. Lipid peroxidation was analyzed by TBARS determination and by mass spectrometry. ATPase activity of SERCA was measured by NADH-coupled enzyme assay. Tryptophan fluorescence was used to analyze conformational alterations. RESULTS: Increase of protein carbonyls and lipid peroxidation was observed in plasma of rats with adjuvant arthritis. Pyridoindole antioxidant stobadine and its methylated derivative SMe1 decreased protein carbonyl formation in plasma, effect of stobadine was significant. Lipid peroxidation of plasma was without any effect of pyridoindole derivatives. Neither protein oxidation nor lipid peroxidation was identified in SR from AA rats. SERCA activity from AA rats increased significantly, stobadine and SMe1 diminished enzyme activity. Ratio of tryptophan fluorescence intensity in SR of AA rats increased and was not influenced by antioxidants. CONCLUSION: Plasma proteins and lipids were oxidatively injured in rats with AA; antioxidants exerted protection only with respect to proteins. In SR, SERCA activity was altered, apparently induced by its conformational changes, as supported by study of tryptophan fluorescence. Stobadine and SMe1 induced a decrease of SERCA activity, elevated in AA rats, but they did not affect conformational changes associated with tryptophan fluorescence.

Use of narrow mass-window, high-resolution extracted product ion chromatograms for the sensitive and selective identification of protein modifications

Protein modifications, including oxidative modifications, glycosylations, and oxidized lipid-protein adducts, are becoming increasingly important as biomarkers and in understanding disease etiology. There has been a great deal of interest in mapping these on Apo B100 from low density lipoprotein (LDL). We have used extracted ion chromatograms of product ions generated using a very narrow mass window from high-resolution tandem mass spectrometric data collected on a rapid scanning quadrupole time-of-flight (QTOF) instrument, to selectively and sensitively detect modified peptides and identify the site and nature of a number of protein modifications in parallel. We have demonstrated the utility of this method by characterizing for the first time oxidized phospholipid adducts to LDL and human serum albumin and for the detection of glycosylation and kynurenin formation from the oxidation of tryptophan residues in LDL. © 2013 American Chemical Society.

Detection and quantification of protein oxidation in sarcopenic models:a mass spectrometry study

Oxidised biomolecules in aged tissue could potentially be used as biomarkers for age-related diseases; however, it is still unclear whether they causatively contribute to ageing or are consequences of the ageing process. To assess the potential of using protein oxidation as markers of ageing, mass spectrometry (MS) was employed for the identification and quantification of oxidative modifications in obese (ob/ob) mice. Lean muscle mass and strength is reduced in obesity, representing a sarcopenic model in which the levels of oxidation can be evaluated for different muscular systems including calcium homeostasis, metabolism and contractility. Several oxidised residues were identified by tandem MS (MS/MS) in both muscle homogenate and isolated sarcoplasmic reticulum (SR), an organelle that regulates intracellular calcium levels in muscle. These modifications include oxidation of methionine, cysteine, tyrosine, and tryptophan in several proteins such as sarcoplasmic reticulum calcium ATPase (SERCA), glycogen phosphorylase, and myosin. Once modifications had been identified, multiple reaction monitoring MS (MRM) was used to quantify the percentage modification of oxidised residues within the samples. Preliminary data suggests proteins in ob/ob mice are more oxidised than the controls. For example SERCA, which constitutes 60-70% of the SR, had approximately a 2-fold increase in cysteine trioxidation of Cys561 in the obese model when compared to the control. Other obese muscle proteins have also shown a similar increase in oxidation for various residues. Further analysis with complex protein mixtures will determine the potential diagnostic use of MRM experiments for analysing protein oxidation in small biological samples such as muscle needle biopsies.