PerR controls Mn-dependent resistance to oxidative stress in Neisseria gonorrhoeae

In previous studies it has been established that resistance to superoxide by Neisseria gonorrhoeae is dependent on the accumulation of Mn(II) ions involving the ABC transporter, MntABC. A mutant strain lacking the periplasmic binding protein component (MntC) of this transport system is hypersensitive to killing by superoxide anion. In this study the mntC mutant was found to be more sensitive to H2O2 killing than the wild-type. Analysis of regulation of MntC expression revealed that it was de-repressed under low Mn(II) conditions. The N. gonorrhoeae mntABC locus lacks the mntR repressor typically found associated with this locus in other organisms. A search for a candidate regulator of mntABC expression revealed a homologue of PerR, a Mn-dependent peroxide-responsive regulator found in Gram-positive organisms. A perR mutant expressed more MntC protein than wild-type, and expression was independent of Mn(II), consistent with a role for PerR as a repressor of mntABC expression. The PerR regulon of N. gonorrhoeae was defined by microarray analysis and includes ribosomal proteins, TonB-dependent receptors and an alcohol dehydrogenase. Both the mntC and perR mutants had reduced intracellular survival in a human cervical epithelial cell model.

Subcellular localization of mammalian type II membrane proteins

Application of a computational membrane organization prediction pipeline, MemO, identified putative type II membrane proteins as proteins predicted to encode a single alpha-helical transmembrane domain (TMD) and no signal peptides. MemO was applied to RIKEN's mouse isoform protein set to identify 1436 non-overlapping genomic regions or transcriptional units (TUs), which encode exclusively type II membrane proteins. Proteins with overlapping predicted InterPro and TMDs were reviewed to discard false positive predictions resulting in a dataset comprised of 1831 transcripts in 1408 TUs. This dataset was used to develop a systematic protocol to document subcellular localization of type II membrane proteins. This approach combines mining of published literature to identify subcellular localization data and a high-throughput, polymerase chain reaction (PCR)-based approach to experimentally characterize subcellular localization. These approaches have provided localization data for 244 and 169 proteins. Type II membrane proteins are localized to all major organelle compartments; however, some biases were observed towards the early secretory pathway and punctate structures. Collectively, this study reports the subcellular localization of 26% of the defined dataset. All reported localization data are presented in the LOCATE database (http://www.locate.imb.uq.edu.au).

Inhibition of transforming growth factor-beta 1 signaling attenuates ataxia telanglectasia mutated activity in response to genotoxic stress

Ionizing radiation causes DNA damage that elicits a cellular program of damage control coordinated by the kinase activity of ataxia telangiectasia mutated protein (ATM). Transforming growth factor beta (TGF beta)-1, which is activated by radiation, is a potent and pleiotropic mediator of physiologic and pathologic processes. Here we show that TGF beta inhibition impedes the canonical cellular DNA damage stress response. Irradiated Tgf beta 1 nail murine epithelial cells or human epithelial cells treated with a small-molecule inhibitor of TGF beta type I receptor kinase exhibit decreased phosphorylation of Chk2, Rad17, and p53; reduced gamma H2AX radiation-induced foci; and increased radiosensitivity compared with TGF beta competent cells. We determined that loss of TGF beta signaling in epithelial cells truncated ATM autophosphorylation and significantly reduced its kinase activity, without affecting protein abundance. Addition of TGF beta restored functional ATM and downstream DNA damage responses. These data reveal a heretofore undetected critical link between the microenvironment and ATM, which directs epithelial cell stress responses, cell fate, and tissue integrity. Thus, Tgf beta 1, in addition to its role in homoeostatic growth control, plays a complex role in regulating responses to genotoxic stress, the failure of which would contribute to the development of cancer; conversely, inhibiting TGF beta may be used to advantage in cancer therapy.

Ascorbic acid induces an oxidative stress in CCRF cells activating the transcription factors AP-1 and NFRB

We have previously tested the effects of high dose AA supplements on human volunteers in terms of reducing DNA damage, as a possible mechanism of the vitamin’s proposed protective effect against cancer and detected a transient, pro-oxidant effect at high doses (500 mg/day). Herein, we present evidence of a pro-oxidant effect of the vitamin when added to CCRF cells at extracellular concentrations which mimic those present in human serum in vivo (50–150AM). The activation of the transcription factor AP-1 was optimal at 100 AM AA following 3h exposure at 37jC. A minimum dose of 50 AM of AA activated NFnB but there appeared to be no dose-dependent effect. Increases of 2–3 fold were observed for both transcription factors when cells were exposed to 100 AM AA for 3h, comparing well with the pro-oxidant effect of H2O2 at similar concentrations. In parallel experiments the activation of AP-1 (binding to DNA) was potentiated when cells were pre-incubated with AA prior to exposure with H2O2. Cycloheximide pretreatment (10 Ag/ml for 15min) caused a 50% inhibition of AP-1 binding to DNA suggesting that it was due to a combination of increasing the binding of pre-existing Fos and Jun and an increase in their de novo synthesis. Cellular localisation was confirmed by immunocytochemistry using antibodies specific for c-Fos and c-Jun proteins. These results suggest that extracellular AA can elicit an intracellular stress response resulting in the activation of the oxidative stress-responsive transcription factors AP-1 and NFnB. These transcription factors are involved in the induction of genes associated with an oxidative stress response, cell cycle arrest and DNA repair confirmed by our cDNA microarray analysis (Affymetrix). This may explain the abilty for AA to appear to inhibit 8-oxodG, yet simultaneously generate another oxidative stress biomarker, 8-oxo-dA. These results suggest a completely novel DNA repair action for AA. Whether this action is relevant to our in vivo findings will be the subject of our future research.

Lipoxidation adducts with peptides and proteins:deleterious modifications or signalling mechanisms?

Protein lipoxidation refers to the modification by electrophilic lipid oxidation products to form covalent adducts, which for many years has been considered as a deleterious consequence of oxidative stress. Oxidized lipids or phospholipids containing carbonyl moieties react readily with lysine to form Schiff bases; alternatively, oxidation products containing α,β-unsaturated moieties are susceptible to nucleophilic attack by cysteine, histidine or lysine residues to yield Michael adducts, overall corresponding to a large number of possible protein adducts. The most common detection methods for lipoxidized proteins take advantage of the presence of reactive carbonyl groups to add labels, or use antibodies. These methods have limitations in terms of specificity and identification of the modification site. The latter question is satisfactorily addressed by mass spectrometry, which enables the characterization of the adduct structure. This has allowed the identification of lipoxidized proteins in physiological and pathological situations. While in many cases lipoxidation interferes with protein function, causing inhibition of enzymatic activity and increased immunogenicity, there are a small number of cases where lipoxidation results in gain of function or activity. For certain proteins lipoxidation may represent a form of redox signaling, although more work is required to confirm the physiological relevance and mechanisms of such processes. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. © 2013 Elsevier B.V.

Transcriptional Response of Two Core Photosystem Genes in Symbiodinium spp. Exposed to Thermal Stress

Mutualistic symbioses between scleractinian corals and endosymbiotic dinoflagellates (Symbiodinium spp.) are the foundation of coral reef ecosystems. For many coral-algal symbioses, prolonged episodes of thermal stress damage the symbiont's photosynthetic capability, resulting in its expulsion from the host. Despite the link between photosynthetic competency and symbiont expulsion, little is known about the effect of thermal stress on the expression of photosystem genes in Symbiodinium. This study used real-time PCR to monitor the transcript abundance of two important photosynthetic reaction center genes, psbA(encoding the D1 protein of photosystem II) and psaA (encoding the P700 protein of photosystem I), in four cultured isolates (representing ITS2-types A13, A20, B1, and F2) and two in hospite Symbiodinium spp. within the coral Pocillopora spp. (ITS2-types C1b-c and D1). Both cultured and in hospite Symbiodinium samples were exposed to elevated temperatures (32°C) over a 7-day period and examined for changes in photochemistry and transcript abundance. Symbiodinium A13 and C1b-c (both thermally sensitive) demonstrated significant declines in both psbA and psaA during the thermal stress treatment, whereas the transcript levels of the other Symbiodinium types remained stable. The downregulation of both core photosystem genes could be the result of several different physiological mechanisms, but may ultimately limit repair rates of photosynthetic proteins, rendering some Symbiodinium spp. especially susceptible to thermal stress.

Redox Regulation of Ras Proteins in Dictyostelium discoideum

Reactive oxygen species are a normal consequence of life in an aerobic environment. However when they deviate from the narrow permissible range in cells, oxidative damage can occur. Dictyostelium discoideum is a model organism ideal for the study of cell signaling events such as those affected by oxidative stress. It was previously shown that Ras signaling in Dictyostelium is affected by genetic inactivation of the antioxidant enzyme Superoxide dismutase C (SodC) and in vitro data suggests that the NKCD motif of Ras is the redox target of superoxide. The main objective of this project was to determine the mechanism of superoxide mediated Ras regulation in vivo. To accomplish the main objective, we cloned, and in some cases, mutated different Ras proteins and later determined their activity in wild type and sodC- cells. RasC and RasD showed normal activation in sodC- cells, however RasG and RasS displayed high Ras activity. These last two Ras proteins contain the NKC118D motif inside the nucleotide binding region. A mutation of cysteine118 to alanine in RasG rendered the protein less active in sodC- than the wild type RasG protein and a mutation alanine118 to cysteine in RasD conferred redox sensitivity to this small GTPase. Additionally, the propensity of RasG to be targeted by superoxide was evident when the environment of wild type cells was manipulated to induce the internal generation of superoxide through changes in the extracellular ion levels mainly magnesium. Lack of magnesium ions increased the intracellular level of superoxide and severely hampered directional cell migration. Chemotaxis of cells expressing RasG was negatively impacted by the absence of magnesium ions; however rasG- cells did not seem to be affected in their ability to perform chemotaxis. The last experiment implies that RasG is an important mediator of cell signaling during oxidative stress, responsible for preventing cells from continuing their developmental program. Our study suggests that the cysteine residue in the NKCD motif is essential for mediating the redox sensitivity of Ras proteins in Dictyostelium and that RasG is an essential mediator of the response to oxidative stress in this organism.

Health-promoting properties of bioactive peptides derived from milk proteins in infant food : a review

Peer reviewed

Proteomic and phosphoproteomic analysis of polyethylene glycol-induced osmotic stress in root tips of common bean (Phaseolus vulgaris L.)

Previous studies have shown that polyethylene glycol (PEG)-induced osmotic stress (OS) reduces cell-wall (CW) porosity and limits aluminium (Al) uptake by root tips of common bean (Phaseolus vulgaris L.). A subsequent transcriptomic study suggested that genes related to CW processes are involved in adjustment to OS. In this study, a proteomic and phosphoproteomic approach was applied to identify OS-induced protein regulation to further improve our understanding of how OS affects Al accumulation. Analysis of total soluble proteins in root tips indicated that, in total, 22 proteins were differentially regulated by OS; these proteins were functionally categorized. Seventy-seven per- cent of the total expressed proteins were involved in metabolic pathways, particularly of carbohydrate and amino acid metabolism. An analysis of the apoplastic proteome revealed that OS reduced the level of five proteins and increased that of seven proteins. Investigation of the total soluble phosphoproteome suggested that dehydrin responded to OS with an enhanced phosphorylation state without a change in abundance. A cellular immunolocalization analysis indicated that dehydrin was localized mainly in the CW. This suggests that dehydrin may play a major protective role in the OS-induced physical breakdown of the CW structure and thus maintenance of the reversibility of CW extensibility during recovery from OS. The proteomic and phosphoproteomic analyses provided novel insights into the complex mechanisms of OS-induced reduction of Al accumulation in the root tips of common bean and highlight a key role for modification of CW structure.

Oxidative stress and its haemodynamic consequences in chronic kidney disease

Chronic kidney disease (CKD) is associated with increased cardiovascular risk in comparison with the general population. This can be observed even in the early stages of CKD, and rises in proportion to the degree of renal impairment. Not only is cardiovascular disease (CVD) more prevalent in CKD, but its nature differs too, with an excess of morbidity and mortality associated with congestive cardiac failure, arrhythmia and sudden death, as well as the accelerated atherosclerosis which is also observed. Conventional cardiovascular risk factors such as hypertension, dyslipidaemia, obesity, glycaemia and smoking, are highly prevalent amongst patients with CKD, although in many of these examples the interaction between risk factor and disease differs from that which exists in normal renal function. Nevertheless, the extent of CVD cannot be fully explained by these conventional risk factors, and non-conventional factors specific to CKD are now recognised to contribute to the burden of CVD. Oxidative stress is a state characterised by excessive production of reactive oxygen species (ROS) and other radical species, a reduction in the capacity of antioxidant systems, and disturbance in normal redox homeostasis with depletion of protective vascular signalling molecules such as nitric oxide (NO). This results in oxidative damage to macromolecules such as lipids, proteins and DNA which can alter their functionality. Moreover, many enzymes are sensitive to redox regulation such that oxidative modification to cysteine thiol groups results in activation of signalling cascades which result in adverse cardiovascular effects such as vascular and endothelial dysfunction. Endothelial dysfunction and oxidative stress are present in association with many conventional cardiovascular risk factors, and can be observed even prior to the development of overt, clinical, vascular pathology, suggesting that these phenomena represent the earliest stages of CVD. In the presence of CKD, there is increased ROS production due to upregulated NADPH oxidase (NOX), increase in a circulating asymmetric dimethylarginine (ADMA), uncoupling of endothelial nitric oxide synthase (eNOS) as well as other mechanisms. There is also depletion in exogenous antioxidants such as ascorbic acid and tocopherol, and a reduction in activity of endogenous antioxidant systems regulated by the master gene regulator Nrf-2. In previous studies, circulating markers of oxidative stress have been shown to be increased in CKD, together with a reduction in endothelial function in a stepwise fashion relating to the severity of renal impairment. Not only is CVD linked to oxidative stress, but the progression of CKD itself is also in part dependent on redox sensitive mechanisms. For example, administration of the ROS scavenger tempol attenuates renal injury and reduces renal fibrosis seen on biopsy in a mouse model of CKD, whilst conversely, supplementation with the NOS inhibitor L-NAME causes proteinuria and renal impairment. Previous human studies examining the effect of antioxidant administration on vascular and renal function have been conflicting however. The work contained in this thesis therefore examines the effect of antioxidant administration on vascular and endothelial function in CKD. Firstly, 30 patients with CKD stages 3 – 5, and 20 matched hypertensive controls were recruited. Participants with CKD had lower ascorbic acid, higher TAP and ADMA, together with higher augmentation index and pulse wave velocity. There was no difference in baseline flow mediated dilatation (FMD) between groups. Intravenous ascorbic acid increased TAP and O2-, and reduced central BP and augmentation index in both groups, and lowered ADMA in the CKD group only. No effect on FMD was observed. The effects of ascorbic acid on kidney function was then investigated, however this was hindered by the inherent drawbacks of existing methods of non-invasively measuring kidney function. Arterial spin labelling MRI is an emerging imaging technique which allows measurement of renal perfusion without administration of an exogenous contrast agent. The technique relies upon application of an inversion pulse to blood within the vasculature proximal to the kidneys, which magnetically labels protons allowing measurement upon transit to the kidney. At the outset of this project local experience using ASL MRI was limited and there ensued a prolonged pre-clinical phase of testing with the aim of optimising imaging strategy. A study was then designed to investigate the repeatability of ASL MRI in a group of 12 healthy volunteers with normal renal function. The measured T1 longitudinal relaxation times and ASL MRI perfusion values were in keeping with those found in the literature; T1 time was 1376 ms in the cortex and 1491 ms in the whole kidney ROI, whilst perfusion was 321 mL/min/100g in the cortex, and 228 mL/min/100g in the whole kidney ROI. There was good reproducibility demonstrated on Bland Altman analysis, with a CVws was 9.2% for cortical perfusion and 7.1% for whole kidney perfusion. Subsequently, in a study of 17 patients with CKD and 24 healthy volunteers, the effects of ascorbic acid on renal perfusion was investigated. Although no change in renal perfusion was found following ascorbic acid, it was found that ASL MRI demonstrated significant differences between those with normal renal function and participants with CKD stages 3 – 5, with increased cortical and whole kidney T1, and reduced cortical and whole kidney perfusion. Interestingly, absolute perfusion showed a weak but significant correlation with progression of kidney disease over the preceding year. Ascorbic acid was therefore shown to have a significant effect on vascular biology both in CKD and in those with normal renal function, and to reduce ADMA only in patients with CKD. ASL MRI has shown promise as a non-invasive investigation of renal function and as a biomarker to identify individuals at high risk of progressive renal impairment.

Biomarkers of Nitrosative Stress: Development and validation of a new analytical method for 3-Nitrotyrosine quantification

Background: The nitration of tyrosine residues in proteins is associated with nitrosative stress, resulting in the formation of 3-nitrotyrosine (3-NT). 3-NT levels in biological samples have been associated with numerous physiological and pathological conditions. For this reason, several attempts have been made in order to develop methods that accurately quantify 3-NT in biological samples. Regarding chromatographic methods, they seem to be very accurate, showing very good sensibility and specificity. However, accurate quantification of this molecule, which is present at very low concentrations both at physiological and pathological states, is always a complex task and a target of intense research. Objectives: We aimed to develop a simple, rapid, low-cost and sensitive 3-NT quantification method for use in medical laboratories as an additional tool for diagnosis and/or treatment monitoring of a wide range of pathologies. We also aimed to evaluate the performance of the HPLC-based method developed here in a wide range of biological matrices. Material and methods: All experiments were performed on a Hitachi LaChrom Elite® HPLC system and separation was carried out using a Lichrocart® 250-4 Lichrospher 100 RP-18 (5μm) column. The method was further validated according to ICH guidelines. The biological matrices tested were serum, whole blood, urine, B16 F-10 melanoma cell line, growth medium conditioned with the same cell line, bacterial and yeast suspensions. Results: From all the protocols tested, the best results were obtained using 0.5% CH3COOH:MeOH:H2O (15:15:70) as the mobile phase, with detection at wavelengths 215, 276 and 356 nm, at 25ºC, and using a flow rate of 1 mL/min. By using this protocol, it was possible to obtain a linear calibration curve (correlation coefficient = 1), limits of detection and quantification in the order of ng/mL, and a short analysis time (<15 minutes per sample). Additionally, the developed protocol allowed the successful detection and quantification of 3-NT in all biological matrices tested, with detection at 356 nm. Conclusion: The method described in this study, which was successfully developed and validated for 3-NT quantification, is simple, cheap and fast, rendering it suitable for analysis in a wide range of biological matrices.

Improving protein yield from mammalian cells by manipulation of stress response pathways

Monoclonal antibodies are a class of therapeutic that is an expanding area of the lucrative biopharmaceutical industry. These complex proteins are predominantly produced from large cultures of mammalian cells; the industry standard cell line being Chinese Hamster Ovary (CHO) cells. A number of optimisation strategies have led to antibody titres from CHO cells increasing by a hundred-fold, and it has been proposed that a further bottleneck in biosynthesis is in protein folding and assembly within the secretory pathway. To alleviate this bottleneck, a CHO-derived host cell line was generated by researchers at the pharmaceutical company UCB that stably overexpressed two critical genes: XBP1, a transcription factor capable of expanding the endoplasmic reticulum and upregulating protein chaperones; and Ero1α, an oxidase that replenishes the machinery of disulphide bond formation. This host cell line, named CHO-S XE, was confirmed to have a high yield of secreted antibody. The work presented in this thesis further characterises CHO-S XE, with the aim of using the information gained to lead the generation of novel host cell lines with more optimal characteristics than CHO-S XE. In addition to antibodies, it was found that CHO-S XE had improved production of two other secreted proteins: one with a simple tertiary structure and one complex multi-domain protein; and higher levels of a number of endogenous protein chaperones. As a more controlled system of gene expression to unravel the specific roles of XBP1 and Ero1α in the secretory properties of CHO-S XE, CHO cells with inducible overexpression of XBP1, Ero1α, or a third gene involved in the Unfolded Protein Response, GADD34, were generated. From these cell lines, it was shown that more antibody was secreted by cells with induced overexpression of XBP1; however, Ero1α and GADD34 overexpression did not improve antibody yield. Further investigation revealed that endogenous XBP1 splicing was downregulated in the presence of an abundance of the active form of XBP1. This result indicated a novel aspect of the regulation of the activity of IRE1, the stress-induced endoribonuclease responsible for XBP1 splicing. Overall, the work described in this thesis confirms that the overexpression of XBP1 has an enhancing effect on the secretory properties of CHO cells; information which could contribute to the development of host cells with a greater capacity for antibody production.

Sch9 regulates intracellular protein ubiquitination by controlling stress responses

Protein ubiquitination and the subsequent degradation are important means by which aberrant proteins are removed from cells, a key requirement for long-term survival. In this study, we found that the overall level of ubiquitinated proteins dramatically decreased as yeast cell grew from log to stationary phase. Deletion of SCH9, a gene encoding a key protein kinase for longevity control, decreased the level of ubiquitinated proteins in log phase and this effect could be reversed by restoring Sch9 function. We demonstrate here that the decrease of ubiquitinated proteins in sch9Δ cells in log phase is not caused by changes in ubiquitin expression, proteasome activity, or autophagy, but by enhanced expression of stress response factors and a decreased level of oxidative stress. Our results revealed for the first time how Sch9 regulates the level of ubiquitinated proteins and provides new insight into how Sch9 controls longevity.

Antioxidative peptides derived from food proteins

The search for natural antioxidants is an ongoing endeavour as an aid to combat the harmful effects of free radicals. Research advances in the past few decades have shown that, by controlled enzymatic hydrolysis, natural antioxidants can be produced from food proteins. In this chapter, the role of certain antioxidative peptides derived from food proteins is discussed in relation to their prospect in the prevention of oxidative stress. The molecular diversity of these food peptides is described together with their pharmacological effects and mechanisms of action in relation to antioxidation. The production of these peptides and the elucidation of their antioxidative peptides are also presented. Owing to their therapeutic potential, antioxidative peptides derived from food proteins can be incorporated as ingredients in functional foods, nutraceuticals and pharmaceuticals, where their biological activities may inhibit product oxidation or assist in the control and prevention of diseases induced by free radicals. However, further insightful research is needed to overcome certain scientific challenges and thereby increase and promote consumer acceptance of these natural antioxidants.

Redox regulation of Ras proteins in Dictyostelium discoideum

Reactive oxygen species are a normal consequence of life in an aerobic environment. However when they deviate from the narrow permissible range in cells, oxidative damage can occur. Dictyostelium discoideum is a model organism ideal for the study of cell signaling events such as those affected by oxidative stress. It was previously shown that Ras signaling in Dictyostelium is affected by genetic inactivation of the antioxidant enzyme Superoxide dismutase C (SodC) and in vitro data suggests that the NKCD motif of Ras is the redox target of superoxide.^ The main objective of this project was to determine the mechanism of superoxide mediated Ras regulation in vivo. To accomplish the main objective, we cloned, and in some cases, mutated different Ras proteins and later determined their activity in wild type and sodC- cells. RasC and RasD showed normal activation in sodC- cells, however RasG and RasS displayed high Ras activity. These last two Ras proteins contain the NKC118D motif inside the nucleotide binding region. A mutation of cysteine 118 to alanine in RasG rendered the protein less active in sodC- than the wild type RasG protein and a mutation alanine118 to cysteine in RasD conferred redox sensitivity to this small GTPase. Additionally, the propensity of RasG to be targeted by superoxide was evident when the environment of wild type cells was manipulated to induce the internal generation of superoxide through changes in the extracellular ion levels mainly magnesium. Lack of magnesium ions increased the intracellular level of superoxide and severely hampered directional cell migration. Chemotaxis of cells expressing RasG was negatively impacted by the absence of magnesium ions; however rasG- cells did not seem to be affected in their ability to perform chemotaxis. The last experiment implies that RasG is an important mediator of cell signaling during oxidative stress, responsible for preventing cells from continuing their developmental program. Our study suggests that the cysteine residue in the NKCD motif is essential for mediating the redox sensitivity of Ras proteins in Dictyostelium and that RasG is an essential mediator of the response to oxidative stress in this organism.^