Oxidative catabolism of tetrahydropterins

Mixed labelled folic acid was administerd to rats. Exposure to N2O was used to give an insight into the major route of scission within the monoglutamate pool, results suggest that THF formed during transport from the gut lumen to the plasma is the major route of scission within the gut. Peroxides in corn oil and arising as a result of lipid peroxidation and autoxidation increase catabolism of the monoglutamate pool and decrease incorporation of administered folates into the polyglutamate pool. It is suggested that peroxides may oxidise B12 resulting in inhibition of methionine synthetase, this results in diminished polyglutamation and increased urinary excretion of 5 CH3THF. Fats undergo peroxidation within tissues, the resulting peroxides increase catabolism of the polyglutamate pool. It is suggested that the NBT assay may reflect polyglutamate breakdown. Antioxidants such as vitamin E (and DES) decrease catabolism of the monoglutamate pool. Administration of DES resulted in changes similar to those observed during malignancy, it is suggested that these changes may precede the onset of tumour development. Vitamin E elevates brain DHPR activity. Since lowered DHPR levels and disturbed THB metabolism have been observed in aging and Down's syndrome it is proposed that vitamin E therapy may prove beneficial in situations where oxidative stress is increased. Brain DHPR activity was increased on administration of peroxides suggesting that in situations of oxidative stress (which may result in increased catabolism of THB) the salvage pathway may be stimulated and loss of THB minimised. N2O exposure had no effect on THB metabolism suggesting that the stimulatory role of 5 CH3THF is due to its role as a methyl donor.

DNA methylation at cytosine position 5

DNA methylation appears to be involved in the regulation of gene expression. Transcriptionally inactive (silenced) genes normally contain a high proportion of 5-methyl-2'-deoxycytosine residues whereas transcriptionally active genes show much reduced levels. There appears good reason to believe that chemical agents capable of methylating 2'-deoxycytosine might affect gene expression and as a result of hypermethylating promoter regions of cytosine-guanine rich oncogenic sequences, cancer related genes may be silenced. This thesis describes the synthesis of a number of `electrophilic' S-methylsulphonium compounds and assesses their ability to act as molecules capable of methylating cytosine at position 5 and also considers their potential as cytotoxic agents. DNA is methylated in vivo by DNA methyltransferase utilising S-adenoxylmethionine as the methyl donor. This thesis addresses the theory that S-adenoxylmethionine may be replaced as the methyl donor for DNA methytransferase by other sulphonium compounds. S-[3H-methyl]methionine sulphonium iodide was synthesised and experiments to assess the ability of this compounds to transfer methyl groups to cytosine in the presence of DNA methyltransferase were unsuccessful. A proline residue adjacent to a cysteine residue has been identified to a highly conserved feature of the active site region of a large number of prokaryotic DNA methyltransferases. The thesis examines the possibility that short peptides containing the Pro-Cys fragment may be able to facilitate the alkylation of cytosine position 5 by sulphonium compounds. Peptides were synthesised up to 9 amino acids in length but none were shown to exhibit significant activity. Molecular modelling techniques, including Chem-X, Quanta, BIPED and protein structure prediction programs were used to assess any structural similarities that may exist between short peptides containing a Pro-Cys fragment and similar sequences present in proteins. A number of similar structural features were observed.

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.

The Val66Met polymorphism of the BDNF gene influences trigeminal Pain-Related evoked responses

Cortical pain processing is associated with large-scale changes in neuronal connectivity, resulting from neural plasticity phenomena of which brain-derived neurotrophic factor (BDNF) is a central driver. The common single nucleotide polymorphism Val66Met is associated with reduced BDNF activity. Using the trigeminal pain-related evoked potential (tPREP) to repeated electrical painful stimuli, we investigated whether the methionine substitution at codon 66 of the BDNF gene was associated with changes in cortical processing of noxious stimuli. Fifty healthy volunteers were genotyped: 30 were Val/Val and 20 were Met-carriers. tPREPs to 30 stimuli of the right supraorbital nerve using a concentric electrode were recorded. The N2 and P2 component latencies and the N2-P2 amplitude were measured over the 30 stimuli and separately, by dividing the measurements in 3 consecutive blocks of 10 stimuli. The average response to the 30 stimuli did not differ in latency or amplitude between the 2 genotypes. There was a decrease in the N2-P2 amplitude between first and third block in the Val/Val group but not in Met-carriers. BDNF Val66Met is associated with reduced decremental response to repeated electrical stimuli, possibly as a result of ineffective mechanisms of synaptic memory and brain plasticity associated with the polymorphism. PERSPECTIVE: BDNF Val66Met polymorphism affects the tPREP N2-P2 amplitude decrement and influences cortical pain processing through neurotrophin-induced neural plasticity, or through a direct BDNF neurotransmitter-like effect. Our findings suggest that upcoming BDNF central agonists might in the future play a role in pain management.

The impact of the Val158Met catechol-O-methyltransferase genotype on neural correlates of sad facial affect processing in patients with bipolar disorder and their relatives

Background - The Met allele of the catechol-O-methyltransferase (COMT) valine-to-methionine (Val158Met) polymorphism is known to affect dopamine-dependent affective regulation within amygdala-prefrontal cortical (PFC) networks. It is also thought to increase the risk of a number of disorders characterized by affective morbidity including bipolar disorder (BD), major depressive disorder (MDD) and anxiety disorders. The disease risk conferred is small, suggesting that this polymorphism represents a modifier locus. Therefore our aim was to investigate how the COMT Val158Met may contribute to phenotypic variation in clinical diagnosis using sad facial affect processing as a probe for its neural action. Method - We employed functional magnetic resonance imaging to measure activation in the amygdala, ventromedial PFC (vmPFC) and ventrolateral PFC (vlPFC) during sad facial affect processing in family members with BD (n=40), MDD and anxiety disorders (n=22) or no psychiatric diagnosis (n=25) and 50 healthy controls. Results - Irrespective of clinical phenotype, the Val158 allele was associated with greater amygdala activation and the Met allele with greater signal change in the vmPFC and vlPFC. Signal changes in the amygdala and vmPFC were not associated with disease expression. However, in the right vlPFC the Met158 allele was associated with greater activation in all family members with affective morbidity compared with relatives without a psychiatric diagnosis and healthy controls. Conclusions - Our results suggest that the COMT Val158Met polymorphism has a pleiotropic effect within the neural networks subserving emotional processing. Furthermore the Met158 allele further reduces cortical efficiency in the vlPFC in individuals with affective morbidity. © 2010 Cambridge University Press.

Parental diet, pregnancy outcomes and offspring health:metabolic determinants in developing oocytes and embryos

The periconceptional period, embracing the terminal stages of oocyte growth and post-fertilisation development up to implantation, is sensitive to parental nutrition. Deficiencies or excesses in a range of macro- and micronutrients during this period can lead to impairments in fertility, fetal development and long-term offspring health. Obesity and genotype-related differences in regional adiposity are associated with impaired liver function and insulin resistance, and contribute to fatty acid-mediated impairments in sperm viability and oocyte and embryo quality, all of which are associated with endoplasmic reticulum stress and compromised fertility. Disturbances to maternal protein metabolism can elevate ammonium concentrations in reproductive tissues and disturb embryo and fetal development. Associated with this are disturbances to one-carbon metabolism, which can lead to epigenetic modifications to DNA and associated proteins in offspring that are both insulin resistant and hypertensive. Many enzymes involved in epigenetic gene regulation use metabolic cosubstrates (e.g. acetyl CoA and S-adenosyl methionine) to modify DNA and associated proteins, and so act as 'metabolic sensors' providing a link between parental nutritional status and gene regulation. Separate to their genomic contribution, spermatozoa can also influence embryo development via direct interactions with the egg and by seminal plasma components that act on oviductal and uterine tissues. © IETS 2014.

Thiol redox homeostasis in neurodegenerative disease

This review provides an overview of the biochemistry of thiol redox couples and the significance of thiol redox homeostasis in neurodegenerative disease. The discussion is centred on cysteine/cystine redox balance, the significance of the xc- cystine-glutamate exchanger and the association between protein thiol redox balance and neurodegeneration, with particular reference to Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and glaucoma. The role of thiol disulphide oxidoreductases in providing neuroprotection is also discussed.

The role of DNA methylation in aging, rejuvenation, and age-related disease

DNA methylation is a major control program that modulates gene expression in a plethora of organisms. Gene silencing through methylation occurs through the activity of DNA methyltransferases, enzymes that transfer a methyl group from S-adenosyl-l-methionine to the carbon 5 position of cytosine. DNA methylation patterns are established by the de novo DNA methyltransferases (DNMTs) DNMT3A and DNMT3B and are subsequently maintained by DNMT1. Aging and age-related diseases include defined changes in 5-methylcytosine content and are generally characterized by genome-wide hypomethylation and promoter-specific hypermethylation. These changes in the epigenetic landscape represent potential disease biomarkers and are thought to contribute to age-related pathologies, such as cancer, osteoarthritis, and neurodegeneration. Some diseases, such as a hereditary form of sensory neuropathy accompanied by dementia, are directly caused by methylomic changes. Epigenetic modifications, however, are reversible and are therefore a prime target for therapeutic intervention. Numerous drugs that specifically target DNMTs are being tested in ongoing clinical trials for a variety of cancers, and data from finished trials demonstrate that some, such as 5-azacytidine, may even be superior to standard care. DNMTs, demethylases, and associated partners are dynamically shaping the methylome and demonstrate great promise with regard to rejuvenation. © Copyright 2012, Mary Ann Liebert, Inc. 2012.

Mapping nitro-tyrosine modifications in fibrinogen by mass spectrometry as a biomarker for inflammatory disease

There is a growing awareness that inflammatory diseases have an oxidative pathology, which can result in specific oxidation of amino acids within proteins. It is known that patients with inflammatory disease have higher levels of plasma protein nitro-tyrosine than healthy controls. Fibrinogen is an abundant plasma protein, highly susceptible to such oxidative modifications, and is therefore a potential marker for oxidative protein damage. The aim of this study was to map tyrosine nitration in fibrinogen under oxidative conditions and identify susceptible residues. Fibrinogen was oxidised with 0.25mM and 1mM SIN-1, a peroxynitrite generator, and methionine was used to quench excess oxidant in the samples. The carbonyl assay was used to confirm oxidation in the samples. The carbonyl levels were 2.3, 8.72 and 11.5nmol/mg protein in 0, 0.25mM and 1mM SIN-1 samples respectively. The samples were run on a SDS-PAGE gel and tryptically digested before analysis by HPLC MS-MS. All 3 chains of fibrinogen were observed for all treatment conditions. The overall sequence coverage for fibrinogen determined by Mascot was between 60-75%. The oxidised samples showed increases in oxidative modifications in both alpha and beta chains, commonly methionine sulfoxide and tyrosine nitration, correlating with increasing SIN-1 treatment. Tyrosines that were most susceptible were Tyr135 (tryptic peptide YLQEIYNSNNQK) and Tyr277 (tryptic peptide GGSTSYGTGSETESPR), but several other nitrated tyrosines were also identified with high confidence. Identification of these susceptible peptides will allow design of sequences-specific biomarkers of oxidative and nitrative damage to plasma protein in inflammatory conditions.

Residue-specific investigation of the relationship between oxidation and activity of a dual specificity phosphatase by mass spectrometry

Redox regulation of signalling pathways is critical in proliferation and apoptosis; redox imbalance can lead to pathologies such as inflammation and cancer. Vaccinia H1-related protein (VHR; DUSP3) is a dual-specificity phosphatase important in controlling MAP kinase activity during cell cycle. the active-site motif contains a cysteine that acts as a nucleophile during catalysis. We used VHR to investigate the effect of oxidation in vitro on phosphatase activity, with the aim of determining how the profile of site-specific modification related to catalytic activity. Recombinant human VHR was expressed in E. coli and purified using a GST-tag. Protein was subjected to oxidation with various concentrations of SIN-1 or tetranitromethane (TNM) as nitrating agents, or HOCl. the activity was assayed using either 3-O-methylfluorescein phosphate with fluorescence detection or PIP3 by phosphate release with malachite green. the sites of oxidation were mapped using HPLC coupled to tandem mass spectrometry on an ABSciex 5600TripleTOF following in-gel digestion. More than 25 different concentration-dependent oxidative modifications to the protein were detected, including oxidations of methionine, cysteine, histidine, lysine, proline and tyrosine, and the % oxidized peptide (versus unmodified peptide) was determined from the extracted ion chromatograms. Unsurprisingly, methionine residues were very susceptible to oxidation, but there was a significant different in the extent of their oxidation. Similarly, tyrosine residues varied greatly in their modifications: Y85 and Y138 were readily nitrated, whereas Y38, Y78 and Y101 showed little modification. Y138 must be phosphorylated for MAPK phosphatase activity, so this susceptibility impacts on signalling pathways. Di- and tri- oxidations of cysteine residues were observed, but did not correlate directly with loss of activity. Overall, the catalytic activity did not correlate with redox state of any individual residue, but the total oxidative load correlated with treatment concentration and activity. This study provides the first comprehensive analysis of oxidation modifications of VHR, and demonstrates both heterogenous oxidant effects and differential residue susceptibility in a signalling phosphatase.

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.

The role of protein oxidation in ageing & disease

Ageing is a natural phenomenon of the human lifecycle, yet it is still not understood what causes the deterioration of the human body near the end of the lifespan. One popular theory is the Free Radical Theory of Ageing, which proposes that oxidative damage to biomolecules causes ageing of tissues. The ageing population is affected by many chronic diseases. This study focused on sarcopenia (muscle loss in ageing) and obesity as two models for comparison of oxidative damage in muscle proteins in mice. The aim of the study was to develop advanced mass spectrometry methods to detect specific oxidative modifications to mouse muscle proteins, including oxidation, nitration, chlorination, and carbonyl group formation, but western blotting was also used to provide complementary information on the oxidative state of proteins from aged and obese muscle. Mass spectrometry proved to be a powerful tool, enabling identification of the types of modifications present, the sites at which they were present and percentage of the peptide populations that were modified. Targeted and semi-targeted mass spectrometry methods were optimised for the identification and quantitation of the oxidised residues in muscle proteins. The development of the quantitative methods enabled comparisons of mass spectrometry instruments. Both the Time of Flight and QTRAP systems showed advantages of using the different mass analysers to quantify oxidative modifications. Several oxidised residues were characterised and quantified in both the obese and sarcopenic models, and higher levels of oxidation were found compared to their control counterparts. Residues found to be oxidised were oxidation of proline, tyrosine and tryptophan, dioxidation of methionine, allysine and nitration of tyrosine. However quantification was performed on methionine dioxidation and cysteine trioxidation containing residues in SERCA. The combination of measuring residue susceptibility and functional studies could contribute to understanding the overall role of oxidation in ageing and obesity.