O2 activation at bioinspired complexes: dinuclear copper systems and mononuclear non-heme iron compounds. Mechanisms and catalytic applications in oxidative transformations

L'activació d'oxigen que té lloc en els éssers vius constitueix una font d'inspiració pel desenvolupament d'alternatives als oxidants tradicionals, considerats altament tòxics i nocius. En aquesta treball s'utilitzen compostos sintètics com a models del centre actiu de proteïnes dinuclears de coure i mononuclears de ferro de tipus no-hemo que participen en l'activació d'oxigen en els éssers vius. Els sistemes dinuclears de coure mostren un centre de tipus coure(III) bis(oxo) que és capaç de dur a terme l'ortho-hidroxilació de fenols de manera similar a la reacció que catalitza la proteïna tirosinasa. Per altra banda, els sistemes de ferro desenvolupats en aquest treball actuen com a models de les dioxigenases de Rieske i poden dur a terme l'hidroxilació estereoespecífica d'alcans i l'epoxidació i cis-dihidroxilació d'olefines utilitzant peròxid d'hidrogen com a agent oxidant. Tot plegat demostra que el desenvolupament de sistemes model constitueix una bona estratègia per l'estudi dels sistemes naturals.

Carotenoids Biosynthesis – a review

As plantas sintetizam uma enorme variedade de metabolitos, que podem ser classificados em dois grupos, de acordo com as suas funções: metabolitos primários, que participam na nutrição e processos metabólicos essenciais no interior da própria planta, e metabolitos secundários (também referidos como produtos naturais), os quais influenciam as interacções ecológicas entre as plantas e o ambiente. Os carotenóides são metabolitos secundários derivados do isopreno. O isopentenil-pirofosfato (IPP) é a unidade básica para a biossíntese dos carotenóides. O esqueleto carbonado dos carotenóides é sintetizado por adição sucessiva das unidades em C5 que vão formar geranilgeranilpirofosfato, intermediário em C20 que por condensação origina a estrutura em C40. Recentemente assumia-se que todos os isoprenóides se sintetizavam a partir do acetil-CoA via ácido mevalónico. Estudos recentes mostraram que o percurso metabólico começa com a síntese do IPP via ácido mevalónico (MVA) e/ou via metileritritol 4-fosfato (MEP). Neste trabalho discutem-se os avanços no conhecimento destas diferentes vias m tabólicas assim como as enzimas e reacções envolvidas na biossíntese dos carotenóides a partir da unidade fundamental (IPP).

Outlining eicosanoid biosynthesis in the crustacean Daphnia

Background: Eicosanoids are biologically active, oxygenated metabolites of three C20 polyunsaturated fatty acids. They act as signalling molecules within the autocrine or paracrine system in both vertebrates and invertebrates mainly functioning as important mediators in reproduction, the immune system and ion transport. The biosynthesis of eicosanoids has been intensively studied in mammals and it is known that they are synthesised from the fatty acid, arachidonic acid, through either the cyclooxygenase (COX) pathway; the lipoxygenase (LOX) pathway; or the cytochrome P450 epoxygenase pathway. However, little is still known about the synthesis and structure of the pathway in invertebrates. Results: Here, we show transcriptomic evidence from Daphnia magna (Crustacea: Branchiopoda) together with a bioinformatic analysis of the D. pulex genome providing insight on the role of eicosanoids in these crustaceans as well as outlining a putative pathway of eicosanoid biosynthesis. Daphnia appear only to have one copy of the gene encoding the key enzyme COX, and phylogenetic analysis reveals that the predicted protein sequence of Daphnia COX clusters with other invertebrates. There is no current evidence of an epoxygenase pathway in Daphnia; however, LOX products are most certainly synthesised in daphnids. Conclusion: We have outlined the structure of eicosanoid biosynthesis in Daphnia, a key genus in freshwater ecosystems. Improved knowledge of the function and synthesis of eicosanoids in Daphnia and other invertebrates could have important implications for several areas within ecology. This provisional overview of daphnid eicosanoid biosynthesis provides a guide on where to focus future research activities in this area.

Induction of heme oxygenase 1 by moderately oxidized low-density lipoproteins in human vascular smooth muscle cells: role of mitogen-activated protein kinases and Nrf2

Oxidized low-density lipoproteins (LDL) play a central role in atherogenesis and induce expression of the antioxidant stress protein heme oxygenase 1 (HO-1). In the present study we investigated induction of HO-1 and adaptive increases in reduced glutathione (GSH) in human aortic smooth muscle cells (SMC) in response to moderately oxidized LDL (moxLDL, 100 mu g protein/ml, 24 h), a species containing high levels of lipid hydroperoxides. Expression and activity of HO-1 and GSH levels were elevated to a greater extent by moxLDL than highly oxidized LDL but unaffected by native or acetylated LDL. Inhibitors of protein kinase C (PKC) or mitogen-activated protein kinases (MAPK) p38(MAPK) and MEK or c-jun-NH2-terminal kinase (JNK) significantly attenuated induction of HO-1. Phosphorylation of p38(MAPK), extracellular signal-regulated kinase (ERK1/2), or JNK and nuclear translocation of the transcription factor Nrf2 were enhanced following acute exposure of SMC to rnoxLDL (100 mu g proteiri/ml, 1-2 h). Pretreatment of SMC with the antioxidant vitamin C (100 mu M, 24 h) attenuated the induction of HO-1 by moxLDL. Native and oxidized LDL did not alter basal levels of intracellular ATP, mitochondrial dehydrogenase activity, or expression of the lectin-like oxidized LDL receptor (LOX-1) in SMC. These findings demonstrate for the first time that activation of PKC, p38(MAPK), JNK, ERK1/2, and Nrf2 by oxidized LDL in human SMC leads to HO-1 induction, constituting an adaptive response against oxidative injury that can be ameliorated by vitamin C. (C) 2005 Elsevier Inc. All rights reserved.

Effects of apoE genotype on macrophage inflammation and heme oxygenase-1 expression

In order to gain a more comprehensive understanding of the aetiology of apolipoprotein E4 genotype-cardiovascular disease (CVD) associations, the impact of the apoE genotype on the macrophage inflammatory response was examined. The murine monocyte-macrophage cell line (RAW 264.7) stably transfected to produce equal amounts of human apoE3 or apoE4 was used. Following LPS stimulation, apoE4-macrophages showed higher and lower concentrations of tumour necrosis factor alpha (pro-inflammatory) and interleukin 10 (anti-inflammatory), respectively, both at mRNA and protein levels. In addition, increased expression of heme oxygenase-1 (a stress-induced anti-inflammatory protein) was observed in the apoE4-cells. Furthermore, in apoE4-macrophages, an enhanced transactivation of the key redox sensitive transcription factor NF-kappa B was shown. Current data indicate that apoE4 macrophages have an altered inflammatory response, which may contribute to the higher CVD risk observed in apoE4 carriers. (c) 2007 Elsevier Inc. All rights reserved.

The Biosynthesis of Artemisinin (Qinghaosu) and the Phytochemistry of Artemisia annua L. (Qinghao)

The Chinese medicinal plant Artemisia annua L. (Qinghao) is the only known source of the sesquiterpene artemisinin (Qinghaosu), which is used in the treatment of malaria. Artemisinin is a highly oxygenated sesquiterpene, containing a unique 1,2,4-trioxane ring structure, which is responsible for the antimalarial activity of this natural product. The phytochemistry of A. annua is dominated by both sesquiterpenoids and flavonoids, as is the case for many other plants in the Asteraceae family. However, A. annua is distinguished from the other members of the family both by the very large number of natural products which have been characterised to date (almost six hundred in total, including around fifty amorphane and cadinane sesquiterpenes), and by the highly oxygenated nature of many of the terpenoidal secondary metabolites. In addition, this species also contains an unusually large number of terpene allylic hydroperoxides and endoperoxides. This observation forms the basis of a proposal that the biogenesis of many of the highly oxygenated terpene metabolites from A. annua - including artemisinin itself may proceed by spontaneous oxidation reactions of terpene precursors, which involve these highly reactive allyllic hydroperoxides as intermediates. Although several studies of the biosynthesis of artemisinin have been reported in the literature from the 1980s and early 1990s, the collective results from these studies were rather confusing because they implied that an unfeasibly large number of different sesquiterpenes could all function as direct precursors to artemisinin (and some of the experiments also appeared to contradict one another). As a result, the complete biosynthetic pathway to artemisinin could not be stated conclusively at the time. Fortunately, studies which have been published in the last decade are now providing a clearer picture of the biosynthetic pathways in A. annua. By synthesising some of the sesquiterpene natural products which have been proposed as biogenetic precursors to artemisinin in such a way that they incorporate a stable isotopic label, and then feeding these precursors to intact A. annua plants, it has now been possible to demonstrate that dihydroartemisinic acid is a late-stage precursor to artemisinin and that the closely related secondary metabolite, artemisinic acid, is not (this approach differs from all the previous studies, which used radio-isotopically labelled precursors that were fed to a plant homogenate or a cell-free preparation). Quite remarkably, feeding experiments with labeled dihydroartemisinic acid and artemisinic acid have resulted in incorporation of label into roughly half of all the amorphane and cadinane sesquiterpenes which were already known from phytochemical studies of A. annua. These findings strongly support the hypothesis that many of the highly oxygenated sesquiterpenoids from this species arise by oxidation reactions involving allylic hydroperoxides, which seem to be such a defining feature of the chemistry of A. annua. In the particular case of artemisinin, these in vivo results are also supported by in vitro studies, demonstrating explicitly that the biosynthesis of artemisinin proceeds via the tertiary allylic hydroperoxide, which is derived from oxidation of dihydroartemisinic acid. There is some evidence that the autoxidation of dihydroartemisinic acid to this tertiary allylic hydroperoxide is a non-enzymatic process within the plant, requiring only the presence of light; and, furthermore, that the series of spontaneous rearrangement reactions which then convert thi allylic hydroperoxide to the 1,2,4-trioxane ring of artemisinin are also non-enzymatic in nature.

A new role for heme, facilitating release of iron from the bacterioferritin iron biomineral

Bacterioferritin (BFR) from Escherichia coli is a member of the ferritin family of iron storage proteins and has the capacity to store very large amounts of iron as an Fe(3+) mineral inside its central cavity. The ability of organisms to tap into their cellular stores in times of iron deprivation requires that iron must be released from ferritin mineral stores. Currently, relatively little is known about the mechanisms by which this occurs, particularly in prokaryotic ferritins. Here we show that the bis-Met-coordinated heme groups of E. coli BFR, which are not found in other members of the ferritin family, play an important role in iron release from the BFR iron biomineral: kinetic iron release experiments revealed that the transfer of electrons into the internal cavity is the rate-limiting step of the release reaction and that the rate and extent of iron release were significantly increased in the presence of heme. Despite previous reports that a high affinity Fe(2+) chelator is required for iron release, we show that a large proportion of BFR core iron is released in the absence of such a chelator and further that chelators are not passive participants in iron release reactions. Finally, we show that the catalytic ferroxidase center, which is central to the mechanism of mineralization, is not involved in iron release; thus, core mineralization and release processes utilize distinct pathways.

Carbon monoxide mediates the anti-apoptotic effects of heme oxygenase-1 in medulloblastoma DAOY cells via K+ channel inhibition

Tumor cell survival and proliferation is attributable in part to suppression of apoptotic pathways, yet the mechanisms by which cancer cells resist apoptosis are not fully understood. Many cancer cells constitutively express heme oxygenase-1 (HO-1), which catabolizes heme to generate biliverdin, Fe(2+), and carbon monoxide (CO). These breakdown products may play a role in the ability of cancer cells to suppress apoptotic signals. K(+) channels also play a crucial role in apoptosis, permitting K(+) efflux which is required to initiate caspase activation. Here, we demonstrate that HO-1 is constitutively expressed in human medulloblastoma tissue, and can be induced in the medulloblastoma cell line DAOY either chemically or by hypoxia. Induction of HO-1 markedly increases the resistance of DAOY cells to oxidant-induced apoptosis. This effect was mimicked by exogenous application of the heme degradation product CO. Furthermore we demonstrate the presence of the pro-apoptotic K(+) channel, Kv2.1, in both human medulloblastoma tissue and DAOY cells. CO inhibited the voltage-gated K(+) currents in DAOY cells, and largely reversed the oxidant-induced increase in K(+) channel activity. p38 MAPK inhibition prevented the oxidant-induced increase of K(+) channel activity in DAOY cells, and enhanced their resistance to apoptosis. Our findings suggest that CO-mediated inhibition of K(+) channels represents an important mechanism by which HO-1 can increase the resistance to apoptosis of medulloblastoma cells, and support the idea that HO-1 inhibition may enhance the effectiveness of current chemo- and radiotherapies.

Functional γ-aminobutyrate shunt in Listeria monocytogenes: role in acid tolerance and succinate biosynthesis

Listeria monocytogenes, the causative agent of human listeriosis, is known for its ability to withstand severe environmental stresses. The glutamate decarboxylase (GAD) system is one of the principal systems utilized by the bacterium to cope with acid stress, a reaction that produces γ-aminobutyrate (GABA) from glutamate. Recently, we have shown that GABA can accumulate intracellularly under acidic conditions, even under conditions where no extracellular glutamate-GABA exchange is detectable. The GABA shunt, a pathway that metabolizes GABA to succinate, has been described for several other bacterial genera, and the present study sought to determine whether L. monocytogenes has this metabolic capacity, which, if present, could provide a possible route for succinate biosynthesis in L. monocytogenes. Using crude protein extracts from L. monocytogenes EGD-e, we show that this strain exhibits activity for the two main enzyme reactions in the GABA shunt, GABA aminotransferase (GABA-AT) and succinic semialdehyde dehydrogenase (SSDH). Two genes were identified as candidates for encoding these enzyme activities, argD (GABA-AT) and lmo0913 (SSDH). Crude protein extracts prepared from a mutant lacking a functional argD gene significantly reduced GABA-AT activity, while an lmo0913 mutant lost all detectable SSDH activity. The deletion of lmo0913 increased the acid tolerance of EGD-e and showed an increased accumulation of intracellular GABA, suggesting that this pathway plays a significant role in the survival of this pathogen under acidic conditions. This is the first report of such a pathway in the genus Listeria, which highlights an important link between metabolism and acid tolerance and also presents a possible compensatory pathway to partially overcome the incomplete tricarboxylic acid cycle of Listeria.

Heme-induced biomarkers associated with red meat promotion of colon cancer are not modulated by the intake of nitrite

Red and processed meat consumption is associated with the risk of colorectal cancer. Three hypotheses are proposed to explain this association, via heme-induced oxidation of fat, heterocyclic amines, or N-nitroso compounds. Rats have often been used to study these hypotheses, but the lack of enterosalivary cycle of nitrate in rats casts doubt on the relevance of this animal model to predict nitroso- and heme-associated human colon carcinogenesis. The present study was thus designed to clarify whether a nitrite intake that mimics the enterosalivary cycle can modulate hemeinduced nitrosation and fat peroxidation. This study shows that, in contrast with the starting hypothesis, drinking water added with nitrite to mimic the salivary nitrite content did not change the effect of hemoglobin on biochemicalmarkers linked to colon carcinogenesis, notably lipid peroxidation and cytotoxic activity in the colon of rat. However, ingested sodium nitrite increased fecal nitrosocompounds level, but their fecal concentration and their nature (iron-nitrosyl) would probably not be associated with an increased risk of cancer.We thus suggest that the rat model could be relevant for study the effect of red meat on colon carcinogenesis, in spite of the lack of nitrite in the saliva of rats.

A mathematical model of the sterol regulatory element binding protein 2 cholesterol biosynthesis pathway

Cholesterol is one of the key constituents for maintaining the cellular membrane and thus the integrity of the cell itself. In contrast high levels of cholesterol in the blood are known to be a major risk factor in the development of cardiovascular disease. We formulate a deterministic nonlinear ordinary differential equation model of the sterol regulatory element binding protein 2 (SREBP-2) cholesterol genetic regulatory pathway in an hepatocyte. The mathematical model includes a description of genetic transcription by SREBP-2 which is subsequently translated to mRNA leading to the formation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), a main precursor of cholesterol synthesis. Cholesterol synthesis subsequently leads to the regulation of SREBP-2 via a negative feedback formulation. Parameterised with data from the literature, the model is used to understand how SREBP-2 transcription and regulation affects cellular cholesterol concentration. Model stability analysis shows that the only positive steady-state of the system exhibits purely oscillatory, damped oscillatory or monotic behaviour under certain parameter conditions. In light of our findings we postulate how cholesterol homestasis is maintained within the cell and the advantages of our model formulation are discussed with respect to other models of genetic regulation within the literature.