Structural and functional properties of the Kunitz-type and C-terminal domains of Amblyomin-X supporting its antitumor activity.

Amblyomin-X is a Kunitz-type FXa inhibitor identified through the transcriptome analysis of the salivary gland from Amblyomma sculptum tick. This protein consists of two domains of equivalent size, triggers apoptosis in different tumor cell lines, and promotes regression of tumor growth, and reduction of metastasis. To study the structural properties and functional roles of the N-terminal (N-ter) and C-terminal (C-ter) domains of Amblyomin-X, we synthesized them by solid-phase peptide synthesis, solved the X-Ray crystallographic structure of the N-ter domain, confirming its Kunitz-type signature, and studied their biological properties. We show here that the C-ter domain is responsible for the uptake of Amblyomin-X by tumor cells and highlight the ability of this domain to deliver intracellular cargo by the strong enhancement of the intracellular detection of molecules with low cellular-uptake efficiency (p15) after their coupling with the C-ter domain. In contrast, the N-ter Kunitz domain of Amblyomin-X is not capable of crossing through the cell membrane but is associated with tumor cell cytotoxicity when it is microinjected into the cells or fused to TAT cell-penetrating peptide. Additionally, we identify the minimum length C-terminal domain named F2C able to enter in the SK-MEL-28 cells and induces dynein chains gene expression modulation, a molecular motor that plays a role in the uptake and intracellular trafficking of Amblyomin-X.

Binding properties to nicotinic acetylcholine receptors can explain differential toxicity of neonicotinoid insecticides in Chironomidae.

Neonicotinoids are neuroactive insecticides commonly detected in freshwater ecosystems. Recent studies have indicated that these compounds are markedly toxic to Chironomidae, a widespread family of ecologically important aquatic insects. However, despite their sensitivity, the pharmacological mechanisms driving neonicotinoid toxicity have yet to be characterized in these insect species. Here, we used a combination of saturation and competition binding studies to characterize neonicotinoid binding properties to nicotinic acetylcholine receptors (nAChR) in two different Chironomidae (Chironomus riparius and Chironomus dilutus) at two different life stages (larval and adult). Using radiolabeled imidacloprid ([3H]-IMI), we characterized and compared receptor density (Bmax), imidacloprid binding affinity (KD), and receptor binding affinity (Ki) to three different neonicotinoid competitors (imidacloprid, clothianidin, and thiamethoxam). We then compared receptor density and binding affinity parameters derived for Chironomidae to data previously generated for other dipterans and agricultural pests. We found that there were limited differences in neonicotinoid binding between C. riparius and C. dilutus, with both organisms demonstrating high affinities for imidacloprid (KD = 0.22-0.87 nM) and high receptor densities (Bmax = 0.92-6.53 pmol/mg). However, there were significant differences between life-stages, with larvae expressing higher densities of nicotinic acetylcholine receptors and higher imidacloprid affinities than adults. Moreover, there were compound-specific differences in receptor affinity, with larval stages displaying relative affinities (Ki) that generally correlated with acute neonicotinoid toxicity (e.g. clothianidin ≥ imidacloprid >>> thiamethoxam). Finally, compared to other dipterans and agricultural pests, Chironomidae display very high densities of high affinity nicotinic acetylcholine receptors, which likely contribute to their sensitivity. Results indicated that receptor-level differences in neonicotinoid binding may be responsible for ecotoxicological differences amongst distinct insect species, and they likely play a role in life stage-, and compound-level toxicity differences previously observed for Chironomidae. Overall, this study highlights the value of understanding the toxicological mechanisms of action of neonicotinoids in sensitive, non-target aquatic insects, to better predict adverse effects associated with unintentional neonicotinoid exposure.

Multiple roles of nicotine on cell proliferation and inhibition of apoptosis: implications on lung carcinogenesis.

The genotoxic effects of tobacco carcinogens have long been recognized, the contribution of tobacco components to cancerogenesis by cell surface receptor signaling is relatively unexplored. Nicotine, the principal tobacco alkaloid, acts through nicotinic acetylcholine receptor (nAChR). nAChR are functionally present on human lung airway epithelial cells, on lung carcinoma [SCLC and NSCLC] and on mesothelioma and build a part of an autocrine-proliferative network that facilitates the growth of neoplastic cells. Different nAChR subunit gene expression patterns are expressed between NSCLC from smokers and non-smokers. Although there is no evidence that nicotine itself could induce cancer, different studies established that nicotine promotes in vivo the growth of cancer cells and the proliferation of endothelial cells suggesting that nicotine might contribute to the progression of tumors already initiated. These observations led to the hypothesis that nicotine might be playing a direct role in the promotion and progression of human lung cancers. Here, we briefly overview the role and the effects of nicotine on pulmonary cell growth and physiology and its feasible implications in lung carcinogenesis.

Alteration of the in vivo nicotinic receptor density in ADNFLE patients: a PET study.

Nicotinic acetylcholine receptors (nAChRs) are involved in a familial form of frontal lobe epilepsy, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). In several ADNFLE families, mutations were identified in the nAChR alpha4 or beta2 subunit, which together compose the main cerebral nAChR. Electrophysiological assessment using in vitro expression systems indicated a gain of function of the mutant receptors. However the precise mechanisms by which they contribute to the pathogenesis of a focal epilepsy remain obscure, especially since alpha4beta2 nAChRs are known to be widely distributed within the entire brain. PET study using [18F]-F-A-85380, a high affinity agonist at the alpha4beta2 nAChRs, allows the determination of the regional distribution and density of the nAChRs in healthy volunteers and in ADNFLE patients, thus offering a unique opportunity to investigate some in vivo consequences of the molecular defect. We have assessed nAChR distribution in eight non-smoking ADNFLE patients (from five families) bearing an identified mutation in nAChRs and in seven age-matched non-smoking healthy volunteers using PET and [(18)F]-F-A-85380. Parametric images of volume of distribution (Vd) were generated as the ratio of tissue to plasma radioactivities. The images showed a clear difference in the pattern of the nAChR density in the brains of the patients compared to the healthy volunteers. Vd values revealed a significant increase (between 12 and 21%, P < 0.05) in the ADNFLE patients in the mesencephalon, the pons and the cerebellum when compared to control subjects. Statistical parametric mapping (SPM) was then used to better analyse subtle regional differences. This analysis confirmed clear regional differences between patients and controls: patients had increased nAChR density in the epithalamus, ventral mesencephalon and cerebellum, but decreased nAChR density in the right dorsolateral prefrontal region. In five patients who underwent an additional [(18)F]-fluorodeoxyglucose (FDG) PET experiment, hypometabolism was observed in the neighbouring area of the right orbitofrontal cortex. The demonstration of a regional nAChR density decrease in the prefrontal cortex, despite the known distribution of these receptors throughout the cerebral cortex, is consistent with a focal epilepsy involving the frontal lobe. We also propose that the nAChR density increase in mesencephalon is involved in the pathophysiology of ADNFLE through the role of brainstem ascending cholinergic systems in arousal.

Development of novel therapeutic strategies for lung cancer: targeting the cholinergic system.

One of the earliest descriptions of non-neuronal ACh synthesis was by Morris who reported that ACh was synthesized in the placenta [1]; furthermore, Falugi et al. showed the presence of AChE in human fibrosarcoma cells [2]. Afterward, the expression of ACh, AChE, and cholinergic receptors in non-neuronal cells was reported in several studies [3-16]. Indeed, recent data reported that SCLC expresses a cholinergic autocrine loop that can regulate cell growth. Such work demonstrates that SCLC cells have a cholinergic phenotype and that ACh exerts as an autocrine growth factor in human lung tumours [16]. Moreover, it has been recently reported that nicotine in lung adenocarcinoma A549 cells, potently induces Bad phosphorylation at serine (S)112, S136 and S155 in a mechanism involving activation of MAPKs, ERK1/2, PI3K/AKT and PKA through the linking to alpha7-receptors [9]. Bad phosphorylation results in sequestering Bad from mitochondria and subsequently interacting with 14-3-3 in the cytosol [9]. We have recently reported that human malignant pleural mesothelioma expresses a cholinergic system, involved in cell growth regulation. Hence, mesothelioma cells growth is modulated by the cholinergic system in which agonists (i.e. nicotine) have a proliferative effect and antagonists (i.e. curare or alpha-cobratoxin) have an inhibitory effect. Furthermore apoptosis mechanisms are under the control of the cholinergic system (nicotine antiapoptotic via induction of NF-kappaB complexes and phosphorylation of Bad at S112, curare proapoptotic via G0-G1 arrest p21waf-1-dependent, but p53-independent) [16]. The involvement of the non-neuronal cholinergic system in lung cancer and mesothelioma appears reasonable and opens up new translational research strategies.

Nitroglycerin metabolism by Phanerochaete chrysosporium: evidence for nitric oxide and nitrite formation.

We have demonstrated that a filamentous fungus Phanerochaete chrysosporium converts glyceryl trinitrate (GTN) into its di- and mononitrate derivatives concurrently with the formation of nitric oxide detected by electron paramagnetic resonance (EPR), and the formation of nitrite. The metabolisms of nitrite and nitrate by the fungus are evaluated and taken into account when considering GTN degradation. Lack of evidence for nitrate formation from GTN suggests that an esterase-type activity is not involved. Furthermore, the kinetics of appearance of the hemoprotein-NO and non-heme protein-NO (FeS-NO) complexes indicate that an enzymatic process producing NO directly from GTN may be involved concurrently with a glutathione transferase-like system.

Site-directed disulfide reduction using an affinity reagent: application on the nicotinic acetylcholine receptor.

The aim of this study was to present a new concept of site-directed reduction of disulfide bonds based upon the use of an affinity ligand harbouring a readily oxidizable dithiol. The cysteine bond involved in the acetylcholine binding site of the AChoR was specifically reduced by a carbamylcholine analogue. The ligand, in its oxidized form, was characterized by an affinity constant of 20 microM for the agonist binding site. In its dithiol form, it specifically reduced the disulfide between Cys-192 and Cys-193 on the alpha-subunits of the nicotinic acetylcholine receptor. This reduction needed 10 times lower concentration when carried out with site-directed reducing agent (ARA) than with DTT, and was highly specific for the alpha-subunits. The contribution of the carbamylcholine moiety of the site-directed reducing agent was clearly demonstrated in kinetic studies where reduction abilities of ARA, DTT and the methylated analogue of ARA (MeRA) were compared. At the same concentration (20 microM), DTT and MeRA had a 25 times lower initial rate of reduction than ARA. With 200 microM of DTT this initial reduction was still 4 times lower. Furthermore, the use of a maleimido undecagold cluster which specifically labeled the reduced nicotinic receptor opens the way to structural analysis of the agonist binding site by electron microscopy. These results demonstrate the potency of this kind of site-directed reducing agent for structural study of receptors or enzymes involving a disulfide bond in their active site.

Interaction of protein ligands with receptor fragments. On the residues of curaremimetic toxins that recognize fragments 128-142 and 185-199 of the alpha-subunit of the nicotinic acetylcholine receptor.

Using a solid-phase assay, we found that 3H-labeled alpha Cobtx from Naja naja siamensis, a long-chain curaremimetic toxin, and 3H-labelled toxin alpha from Naja nigricollis, a short-chain toxin both bind specifically but with substantially different affinities (Kd = 4 x 10(-7) M and 50 x 10(-6) M) to fragment 185-199 (T alpha 185-199) of the alpha-subunit of the acetylcholine receptor (AcChoR) from Torpedo marmorata. Then we show that monoderivatizations of residues common to both long-chain and short-chain toxins (Tyr-25, Lys-27, Trp-29, and Lys-53) or to long-chain toxins only (Cys-30 and Cys-34) do not affect the binding of the toxins to T alpha 185-199, suggesting that none of these invariant residues in implicated in the recognition of this AcChoR region. alpha Cobtx and toxin alpha bind to the fragment 128-142 (T alpha 128-142) with more similar affinities (Kd = 3 x 10(-7) M and 1.4 x 10(-6) M) and their binding is dramatically affected by the single abolition of the positive charge of Lys-53, an invariant residue that contributes to AcChoR recognition. Therefore, the data indicate that Lys-53 more specifically recognizes the 128-142 region of AcChoR. Other monoderivatizations have no effect on toxin binding. The approach described in this paper may be of great help to identify toxin residues that establish direct contact with receptor fragments.

Minimal conformation of the alpha-conotoxin ImI for the alpha7 neuronal nicotinic acetylcholine receptor recognition: correlated CD, NMR and binding studies.

The alpha-ImI conotoxin, a selective potent inhibitor of the mammalian neuronal alpha7 nicotinic acetylcholine receptor (n-AchR), was shown by point mutation or by L-alanine scanning to display two regions essential for bioactivity: the active site Asp5-Pro6-Arg7 in the first loop and Trp10 in the second loop. The deletion of the Cys3,Cys12 disulfide bond in the alpha-ImI scaffold, e.g. peptide II, had no effect on its binding affinity. CD spectra, NMR studies and structure calculations were carried out on the wild type alpha-ImI, the weakest analog (R7A) and peptide II (equipotent to alpha-ImI) in order to point out the conformational differences between these compounds. Then, an attempt to correlate the conformational data and the affinity results was proposed. CD and NMR data were identical for the R7A analog and alpha-ImI, revealing the crucial functional role of the Arg7 side chain. On the other hand, the scaffold of the first loop in peptide II was shown by NMR to represent the minimal conformation for the optimal interaction of the toxin with the neuronal alpha7 n-AchR. Last, the beta-turn forming property of the 6th residue (Pro) in the active site of the alpha-ImI can be correlated with its affinity.

Functional determinants by which snake and cone snail toxins block the alpha 7 neuronal nicotinic acetylcholine receptors.

Snakes and cone snails produce toxins which block muscular and/or neuronal nicotinic acetylcholine receptors (AChRs). This paper mostly focuses on the determinants by which a snake long chain curaremimetic toxin and the cone snail toxin ImI bind specifically to the alpha 7 neuronal receptor. In both cases, the site involves a small turn-like structure constrained by two half-cystines.

Molecular characterization of the specificity of interactions of various neurotoxins on two distinct nicotinic acetylcholine receptors.

Snake curaremimetic toxins are currently classified as short-chain and long-chain toxins according to their size and their number of disulfide bonds. All these toxins bind with high affinity to muscular-type nicotinic acetylcholine receptor, whereas only long toxins recognize the alpha7 receptor with high affinity. On the basis of binding experiments with Torpedo or neuronal alpha7 receptors using wild-type and mutated neurotoxins, we characterized the molecular determinants involved in these different recognition processes. The functional sites by which long and short toxins interact with the muscular-type receptor include a common core of highly conserved residues and residues that are specific to each of toxin families. Furthermore, the functional sites through which alpha-cobratoxin, a long-chain toxin, interacts with muscular and alpha7 receptors share similarities but also marked differences. Our results reveal that the three-finger fold toxins have evolved toward various specificities by displaying distinct functional sites.

Particular ability of cytochrome P-450 CYP3A to reduce glyceryl trinitrate in rat liver microsomes: subsequent formation of nitric oxide.

Glyceryl trinitrate was denitrated in rat hepatic subcellular fractions, with formation of glyceryl dinitrates and glyceryl mononitrates. Among differently treated-rat liver microsomes, the highest microsomal activity was obtained under anaerobic conditions with microsomal preparations from dexamethasone-treated rats and NADPH. The reaction was inhibited by O2, CO, miconazole, dihydroergotamine and troleandomycin showing that it was catalyzed by cytochrome P-450 CYP3A isoforms. The formation of a transient cytochrome P-450 Fe(II)-NO complex during this reaction was shown by visible spectroscopy. The cytosolic activity was shown to be dependent on glutathione and glutathione transferase and was not inhibited by dioxygen. In the hepatic 9000 x g supernatant containing both NADPH and cytochrome P-450 and glutathione and glutathione transferase, the cytochrome P-450-dependent reaction accounts for 30-40% of the total denitration activity observed under anaerobic conditions, using 100 microM GTN.

How do snake curaremimetic toxins discriminate between nicotinic acetylcholine receptor subtypes.

Curaremimetic toxins from snake venoms form a large family of small proteins that adopt a similar fold and which bind to Torpedo nicotinic acetylcholine receptors with high affinity. Notwithstanding its apparent homogeneity, the toxin family is subdivided into short-chain (60-62 residues and four disulfide bonds) and long-chain toxins (66-74 residues and five disulfide bonds). In agreement with this structurally-based distinction we recently showed that only long-chain toxins bind with high affinity to the neuronal nicotinic acetylcholine alpha7 receptor. We suggested that a small loop cyclized by a disulfide bond and uniquely present in long-chain toxins may act as a major discriminative element. To assess the validity of this proposal we prepared various derivatives of a long-chain toxin, using stepwise solid-phase synthesis. We found that replacement of both half cystines of the small loop by a serine caused a 35-fold affinity decrease for the neuronal receptor and only a 6-fold affinity decrease for Torpedo receptor. In addition, insertion of this loop at a homologous position of a short-chain toxin caused a 20-fold affinity increase for the neuronal receptor whereas it did not modify its affinity for the Torpedo receptor. Our findings, therefore, reveal that a small structural deviation from a toxin fold can generate exquisite discriminative recognition for some receptor subtypes.

Identification, structural and pharmacological characterization of τ-CnVA, a conopeptide that selectively interacts with somatostatin sst3 receptor.

Conopeptides are a diverse array of small linear and reticulated peptides that interact with high potency and selectivity with a large diversity of receptors and ion channels. They are used by cone snails for prey capture or defense. Recent advances in venom gland transcriptomic and venom peptidomic/proteomic technologies combined with bioactivity screening approaches lead to the identification of new toxins with original pharmacological profiles. Here, from transcriptomic/proteomic analyses of the Conus consors cone snail, we identified a new conopeptide called τ-CnVA, which displays the typical cysteine framework V of the T1-conotoxin superfamily. This peptide was chemically synthesized and its three-dimensional structure was solved by NMR analysis and compared to that of TxVA belonging to the same family, revealing very few common structural features apart a common orientation of the intercysteine loop. Because of the lack of a clear biological function associated with the T-conotoxin family, τ-CnVA was screened against more than fifty different ion channels and receptors, highlighting its capacity to interact selectively with the somatostatine sst3 receptor. Pharmacological and functional studies show that τ-CnVA displays a micromolar (Ki of 1.5µM) antagonist property for the sst3 receptor, being currently the only known toxin to interact with this GPCR subfamily.

Isolation and pharmacological characterization of AdTx1, a natural peptide displaying specific insurmountable antagonism of the alpha1A-adrenoceptor.

BACKGROUND AND PURPOSE: Venoms are a rich source of ligands for ion channels, but very little is known about their capacity to modulate G-protein coupled receptor (GPCR) activity. We developed a strategy to identify novel toxins targeting GPCRs. EXPERIMENTAL APPROACH: We studied the interactions of mamba venom fractions with alpha(1)-adrenoceptors in binding experiments with (3)H-prazosin. The active peptide (AdTx1) was sequenced by Edman degradation and mass spectrometry fragmentation. Its synthetic homologue was pharmacologically characterized by binding experiments using cloned receptors and by functional experiments on rabbit isolated prostatic smooth muscle. KEY RESULTS: AdTx1, a 65 amino-acid peptide stabilized by four disulphide bridges, belongs to the three-finger-fold peptide family. It has subnanomolar affinity (K(i)= 0.35 nM) and high specificity for the human alpha(1A)-adrenoceptor subtype. We showed high selectivity and affinity (K(d)= 0.6 nM) of radio-labelled AdTx1 in direct binding experiments and revealed a slow association constant (k(on)= 6 x 10(6).M(-1).min(-1)) with an unusually stable alpha(1A)-adrenoceptor/AdTx1 complex (t(1/2diss)= 3.6 h). AdTx1 displayed potent insurmountable antagonism of phenylephrine's actions in vitro (rabbit isolated prostatic muscle) at concentrations of 10 to 100 nM. CONCLUSIONS AND IMPLICATIONS: AdTx1 is the most specific and selective peptide inhibitor for the alpha(1A)-adrenoceptor identified to date. It displays insurmountable antagonism, acting as a potent relaxant of smooth muscle. Its peptidic nature can be exploited to develop new tools, as a radio-labelled-AdTx1 or a fluoro-labelled-AdTx1. Identification of AdTx1 thus offers new perspectives for developing new drugs for treating benign prostatic hyperplasia.

Role of alpha7-nicotinic acetylcholine receptor in human non-small cell lung cancer proliferation.

OBJECTIVES: Lung cancer is the most common cause of cancer death in the world. Cigarette smoking represents the major risk factor. Nicotine, an active component of cigarettes, can induce cell proliferation, angiogenesis and apoptosis resistance. All these events are mediated through the nicotinic acetylcholine receptor (nAChR) expressed on lung cancer cells. We speculate that new insights into the pathophysiological roles of nAChR may lead to new therapeutic avenues to reduce non-small cell lung cancer (NSCLC) tumour growth. MATERIALS AND METHODS: Human samples of NSCLC, cell lines and mouse models were utilized in Western blotting, reverse transcriptase polymerase chain reaction and apoptosis studies. RESULTS: Human NSCLC tissues expressed alpha7-nAChR. This expression was higher in smoking patients with squamous carcinomas than those with adenocarcinomas and in male smoking patients than in females. All the data support the hypothesis that major expression of alpha7-nAChR is related to major activation of the Rb-Raf-1/phospho-ERK/phospho-p90RSK pathway. alpha7-nAChR antagonists, via mitochondria associated apoptosis, inhibited proliferation of human NSCLC primary and established cells. Nicotine stimulates tumour growth in a murine model, A549 cells orthotopically grafted. The effects of nicotine were associated with increases in phospho-ERK in tumours. Proliferation effects of nicotine could be blocked by inhibition of alpha7-nAChR by the high affinity ligand alpha-cobratoxin. CONCLUSION: These results showed that alpha7-nAChR plays an important role in NSCLC cell growth and tumour progression as well as in cell death.

Variability among the sites by which curaremimetic toxins bind to torpedo acetylcholine receptor, as revealed by identification of the functional residues of alpha-cobratoxin.

alpha-Cobratoxin, a long chain curaremimetic toxin from Naja kaouthia venom, was produced recombinantly (ralpha-Cbtx) from Escherichia coli. It was indistinguishable from the snake toxin. Mutations at 8 of the 29 explored toxin positions resulted in affinity decreases for Torpedo receptor with DeltaDeltaG higher than 1.1 kcal/mol. These are R33E > K49E > D27R > K23E > F29A >/= W25A > R36A >/= F65A. These positions cover a homogeneous surface of approximately 880 A(2) and mostly belong to the second toxin loop, except Lys-49 and Phe-65 which are, respectively, on the third loop and C-terminal tail. The mutations K23E and K49E, and perhaps R33E, induced discriminative interactions at the two toxin-binding sites. When compared with the short toxin erabutoxin a (Ea), a number of structurally equivalent residues are commonly implicated in binding to muscular-type nicotinic acetylcholine receptor. These are Lys-23/Lys-27, Asp-27/Asp-31, Arg-33/Arg-33, Lys-49/Lys-47, and to a lesser and variable extent Trp-25/Trp-29 and Phe-29/Phe-32. In addition, however, the short and long toxins display three major differences. First, Asp-38 is important in Ea in contrast to the homologous Glu-38 in alpha-Cbtx. Second, all of the first loop is insensitive to mutation in alpha-Cbtx, whereas its tip is functionally critical in Ea. Third, the C-terminal tail may be specifically critical in alpha-Cbtx. Therefore, the functional sites of long and short curaremimetic toxins are not identical, but they share common features and marked differences that might reflect an evolutionary pressure associated with a great diversity of prey receptors.

Chemical engineering of a three-fingered toxin with anti-alpha7 neuronal acetylcholine receptor activity.

Though it possesses four disulfide bonds the three-fingered fold is amenable to chemical synthesis, using a Fmoc-based method. Thus, we synthesized a three-fingered curaremimetic toxin from snake with high yield and showed that the synthetic and native toxins have the same structural and biological properties. Both were characterized by the same 2D NMR spectra, identical high binding affinity (K(d) = 22 +/- 5 pM) for the muscular acetylcholine receptor (AChR) and identical low affinity (K(d) = 2.0 +/- 0.4 microM) for alpha7 neuronal AchR. Then, we engineered an additional loop cyclized by a fifth disulfide bond at the tip of the central finger. This loop is normally present in longer snake toxins that bind with high affinity (K(d) = 1-5 nM) to alpha7 neuronal AchR. Not only did the chimera toxin still bind with the same high affinity to the muscular AchR but also it displayed a 20-fold higher affinity (K(d) = 100 nM) for the neuronal alpha7 AchR, as compared with the parental short-chain toxin. This result demonstrates that the engineered loop contributes, at least in part, to the high affinity of long-chain toxins for alpha7 neuronal receptors. That three-fingered proteins with four or five disulfide bonds are amenable to chemical synthesis opens new perspectives for engineering new activities on this fold.

Nitric oxide, a biological effector. Electron paramagnetic resonance detection of nitrosyl-iron-protein complexes in whole cells.

Nitric oxide has been used for more than 20 years as an electron paramagnetic resonance probe of oxygen binding sites in oxygen-carriers and oxygen-metabolizing metalloenzymes. The high reactivity of NO with oxygen and the superoxide anion and its high affinity for metalloproteins led biochemists to consider NO as a highly toxic compound for a living cell. This assertion has recently been reconsidered following a number of discoveries of great significance: the finding of the activation of guanylate cyclase by NO, the recognition that NO is the precursor of nitrite and nitrate ions released in the activation of macrophages by endotoxin and cytokines, evidence that NO is an Endothelium-Derived Relaxing Factor, and the discovery of NO-biosynthesis from L-arginine, a pathway common in various biological cell-to-cell signalling processes. It is now admitted that NO plays a key bioregulatory role within mammalian cells, between cells of different types and in the host defence response. In the present review we have attempted to give a general picture of what is known of the chemical, physical, biochemical and biophysical properties of NO among the various nitrogen oxides. We have focussed on the structural information that can be obtained by electron paramagnetic resonance spectroscopy of nitrosyl-metalloprotein complexes. Finally we have shown how molecular targets of nitric oxide can be characterized, within whole cells, by electron paramagnetic resonance spectroscopy.

Multiple enzymatic pathways involved in the metabolism of glyceryl trinitrate in Phanerochaete chrysosporium.

A study of glyceryl trinitrate metabolism by a filamentous fungus, Phanerochaete chrysosporium, carried out with the 14C-labeled substrate, provides evidence for a multienzymatic system leading to di- and mononitrate derivatives. At least two independent enzymatic activities were detected in the cytosolic fraction: an aerobic glutathione S-transferase activity and an anaerobic NADPH-dependent soluble cytochrome P450-like activity. Other hemoproteins with enzymatic activities dependent upon the presence of NADPH or ferrous ions were also detected in the microsomal fraction. Electron paramagnetic resonance spectra characteristic of an interaction between a hemoprotein and nitric oxide appeared in these two subcellular fractions during the anaerobic metabolism of glyceryl trinitrate.