Gilbert Briand

Bovine hemoglobin : An attractive source of antibacterial peptides

A peptic hemoglobin hydrolysate was fractioned by a semi-preparative reversed-phase HPLC and some fractions have an antibacterial activity against four bacteria strains: Micrococcus luteus A270, Listeria innocua, Escherichia coli and Salmonella enteritidis. These fractions were analyzed by ESI/MS and ESI/MS/MS, in order to characterize the peptides in these fractions. Each fraction contains at least three peptides and some fractions contain five peptides. All these fractions were purified several times by HPLC to obtain pure peptides. Thirty antibacterial peptides were identified. From the isolated antibacterial peptides, 24 peptides were derived from the alpha chains of hemoglobin and 6 peptides were derived from the beta chains of hemoglobin. The lowest concentration of these peptides (minimum inhibitory concentration (MIC)) necessary to completely inhibit the growth of four bacteria strain was determined. The cell population of all of the tested bacteria species decreased by at least 97% after a 24-h incubation with any of the peptides at the minimum inhibitory concentration.

New antibacterial peptide derived from bovine hemoglobin

Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields an intermediate peptide fraction exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli and Salmonella enteritidis after separation by reversed-phase HPLC. From this fraction a pure peptide was isolated and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). This peptide correspond to the 107-136 fragment of the alpha chain of bovine hemoglobin. The minimum inhibitory concentrations (MIC) towards the four strains and its hemolytic activity towards bovine erythrocytes were determined. A MIC of 38 microM was reported against L. innocua and 76 microM for other various bacterial species. This peptide had no hemolytic activity up to 380 microM concentration.

Inhibition of mitochondrial respiration mediates apoptosis induced by the anti-tumoral alkaloid lamellarin D

Lamellarin D (Lam D), a marine alkaloid, exhibits a potent cytotoxicity against many different tumors. The pro-apoptotic function of Lam D has been attributed to its direct induction of mitochondrial permeability transition (MPT). This study was undertaken to explore the mechanisms through which Lam D promotes changes in mitochondrial function and as a result apoptosis. The use of eight Lam derivatives provides useful structure-apoptosis relationships. We demonstrate that Lam D and structural analogues induce apoptosis of cancer cells by acting directly on mitochondria inducing reduction of mitochondrial membrane potential, swelling and cytochrome c release. Cyclosporin A, a well-known inhibitor of MPT, completely prevents mitochondrial signs of apoptosis. The drug decreases calcium uptake by mitochondria but not by microsomes indicating that Lam D-dependent permeability is specific to mitochondrial membranes. In addition, upon Lam D exposure, a rapid decline of mitochondrial respiration and ATP synthesis occurs in isolated mitochondria as well as in intact cells. Evaluation of the site of action of Lam D on the electron-transport chain revealed that the activity of respiratory chain complex III is reduced by a half. To determine whether Lam D could induce MPT-dependent apoptosis by inhibiting mitochondrial respiration, we generated respiration-deficient cells (ρ0) derived from human melanoma cells. In comparison to parental cells, ρ0 cells are totally resistant to the induction of MPT-dependent apoptosis by Lam D. Our results indicate that functional mitochondria are required for Lam D-induced apoptosis. Inhibition of mitochondrial respiration is responsible for MPT-dependent apoptosis of cancer cells induced by Lam-D.

Isolation and characterization of four antibacterial peptides from bovine hemoglobin.

Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields several intermediate peptide fractions after separation by reversed phase HPLC exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli, and Salmonella enteritidis. From these fractions, four new antibacterial peptides were isolated and analyzed by ESI-MS/MS. Three of these peptides correspond to fragments of the alpha-chain of bovine hemoglobin: alpha107-141, alpha137-141, and alpha133-141, and one peptide to the beta-chain: beta126-145. The minimum inhibitory concentrations (MIC) of these peptides towards the four strains and their hemolytic activity towards bovine erythrocytes were determined.

Alkylation of guanine in DNA by S23906-1, a novel potent antitumor compound derived from the plant alkaloid acronycine

The discovery of a new DNA-targeted antitumor agent is a challenging enterprise, and the elucidation of its mechanism of action is an essential first step in investigating the structural and biological consequences of DNA modification and to guide the rational design of analogues. Here, we have dissected the mode of action of the newly discovered antitumor agent S23906-1. Gel retardation experiments reveal that the diacetate compound S23906-1 and its monoacetate analogue S28687 form highly stable covalent adducts with DNA. The covalent adducts formed between S23906-1 and a 7-bp hairpin oligonucleotide duplex were identified by spectrometry. In contrast, the inactive compound S23907, lacking the two acetate groups of S23906-1, fails to yield covalent DNA adducts, indicating that the C1-C2 functionality is the DNA reactive moiety. DNase I footprinting and DNA alkylation experiments indicate that S23906-1 reacts primarily with guanine residues. A 30-mer oligonucleotide containing only G.C bp forms highly stable complexes with S23906-1 and S28687, whereas the equivalent A.T oligonucleotide is not a good substrate for these two drugs. The use of an oligonucleotide duplex containing inosines instead of guanosines identifies the guanine 2-amino group exposed in the minor groove of DNA as the potential reactive site. The reactivity of S23906-1 toward the guanine-N2 group was independently confirmed by fluorescence spectroscopy. Covalent DNA adducts were also identified in the genomic DNA of B16 melanoma cells exposed to S23906-1, and the specific accumulation of the drug in the nucleus of the cells was visualized by confocal microscopy. The elucidation of the mechanism of action of this highly potent anticancer agent opens a new field for future drug design.

Exploiting Mitochondrial Dysfunction for Effective Elimination of Imatinib-Resistant Leukemic Cells

Challenges today concern chronic myeloid leukemia (CML) patients resistant to imatinib. There is growing evidence that imatinib-resistant leukemic cells present abnormal glucose metabolism but the impact on mitochondria has been neglected. Our work aimed to better understand and exploit the metabolic alterations of imatinib-resistant leukemic cells. Imatinib-resistant cells presented high glycolysis as compared to sensitive cells. Consistently, expression of key glycolytic enzymes, at least partly mediated by HIF-1α, was modified in imatinib-resistant cells suggesting that imatinib-resistant cells uncouple glycolytic flux from pyruvate oxidation. Interestingly, mitochondria of imatinib-resistant cells exhibited accumulation of TCA cycle intermediates, increased NADH and low oxygen consumption. These mitochondrial alterations due to the partial failure of ETC were further confirmed in leukemic cells isolated from some imatinib-resistant CML patients. As a consequence, mitochondria generated more ROS than those of imatinib-sensitive cells. This, in turn, resulted in increased death of imatinib-resistant leukemic cells following in vitro or in vivo treatment with the pro-oxidants, PEITC and Trisenox, in a syngeneic mouse tumor model. Conversely, inhibition of glycolysis caused derepression of respiration leading to lower cellular ROS. In conclusion, these findings indicate that imatinib-resistant leukemic cells have an unexpected mitochondrial dysfunction that could be exploited for selective therapeutic intervention.

Determination of glycan structures and molecular masses of the glycovariants of serum transferrin from a patient with carbohydrate deficient syndrome type II

Serum transferrin from a child with carbohydrate deficient syndrome type II was isolated by immunoaffinity chromatography and separated into minor and major fractions by fast protein liquid chromatography. The structure of the glycans released from the major fraction by hydrazinolysis was established by application of methanolysis and 1H-NMR spectroscopy. The results led to the identification of an N-acetyllactosamininic type monosialylated, monoantennary Man(α1-3) linked glycan. By electrospray-mass spectrometry analysis, the whole serum transferrin was separated into at least seven species (I to VII) with molecular masses ranging from 77 958 to 79 130 Da. On the basis of a polypeptide chain molecular mass of 75 143 Da, it was calculated that the major transferrin species III (78 247 Da) contains two monosialylated monoantennary glycans. The molecular mass of transferrin species V and VI (78 678 and 78 971 Da) suggests that one of their two glycans contains an additional N-acetyllactosamine and a sialylated N-acetyllactosamine units, respectively. Transferrin species I and V were found to correspond to the desialylated forms of species III and VI. The abnormal glycan structures can be explained by a defect in the N-acetylglucosaminyltransferase II activity [Charuk et al. (1995) Eur J Biochem 230: 797-805].

Endosomal proteolysis of insulin-like growth factor-I at its C-terminal D-domain by cathepsin B

IGF‐I is degraded within the endosomal apparatus as a consequence of receptor‐mediated endocytosis. However, the nature of the responsible protease and the position of the cleavage sites in the IGF‐I molecule remain undefined. In vitro proteolysis of IGF‐I using an endosomal lysate required an acidic pH and was sensitive to CA074, an inhibitor of the cathepsin B enzyme. By nondenaturing immunoprecipitation, the acidic IGF‐I‐degrading activity was attributed to the luminal species of endosomal cathepsin B with apparent molecular masses of 32‐ and 28‐kDa. The cathepsin B precursor, procathepsin B, was processed in vitro within isolated endosomes at pH 5 or at 7 in the presence of ATP, the substrate of the vacuolar H+‐ATPase. The rate of IGF‐I hydrolysis using an endosomal lysate or pure cathepsin B was found to be optimal at pH 5–6 and moderate at pH 4 and 7. Competition studies revealed that EGF and IGF‐I share a common binding site on the cathepsin B enzyme, with native IGF‐I displaying the lowest affinity for the protease (IC50 ≈ 1.5 μM). Hydrolysates of IGF‐I generated at low pH by endosomal IGF‐I‐degrading activity and analyzed by reverse‐phase HPLC and mass spectrometry revealed cleavage sites at Lys68‐Ser69, Ala67‐Lys68, Pro66‐Ala67 and Lys65‐Pro66 within the C‐terminal D‐domain of IGF‐I. Treatment of human HepG2 hepatoma cells with the cathepsin B proinhibitor CA074‐Me reduced, in vivo, the intracellular degradation of internalized [125I]IGF‐I and, in vitro, the degradation of exogenous [125I]IGF‐I incubated with the cell‐lysates at pH 5. Inhibitors of cathepsin B and pro‐cathepsin B processing, which abolish endosomal proteolysis of IGF‐I and alter tumor cell growth and IGF‐I receptor signalling, merit investigation as antimetastatic drugs.

Mitochondrial dysfunction and lipid homeostasis.

This review is aimed at illustrating that mitochondrial dysfunction and altered lipid homeostasis may concur in a variety of pathogenesis states, being either contributive or consecutive to primary disease events. Underlying mechanisms for this concurrence are far from being the exhaustive elements taking place in disease development. They may however complicate, contribute or cause the disease. In the first part of the review, physiological roles of mitochondria in coordinating lipid metabolism and in controlling reactive oxygen species (ROS), ATP and calcium levels are briefly presented. In a second part, clues for how mitochondria-driven alterations in lipid metabolism may induce toxicity are discussed. In the third part, it is illustrated how mitochondrial dysfunction and lipid homeostasis disruption may be associated (i) to complicate type 1 diabetes (pancreatic β-cell mitochondrial dysfunction in ATP yield induces reduced insulin secretion and hence disruption of glucose and lipid metabolism), (ii) to contribute to type 2 diabetes and other insulin resistant states (mitochondrial impairment may induce adipocyte dysfunction with subsequent increase in circulating free fatty acids and their abnormal deposit in non adipose tissues (pancreatic β-cells, skeletal muscle and liver) which results in lipotoxicity and mitochondrial dysfunction), (iii) to offer new clues in our understanding of how the brain controls feeding supply and energy expenditure, (iv) to promote cancer development notably via fatty acid oxidation/synthesis imbalance (in favor of synthesis) further strengthened in some cancers by a lipogenetic benefit induced by a HER2/fatty acid synthase cross-talk, and (v) to favor cardiovascular disorders by impacting heart function and arterial wall integrity.

Influence of the amino acid sequence on the MUC5AC motif peptide O-Glycosylation by Human gastric UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase(s)

The present work was carried out to study the role of the peptide moiety in the addition of O-linked N-acetylgalactosamine to human apomucin using human crude microsomal homogenates from gastric mucosa (as enzyme source) and a series of peptide acceptors representative of tandem repeat domains deduced from the MUC5AC mucin gene (expressed in the gastric mucosa). Being rich in threonine and serine placed in clusters, these peptides provided several potential sites for O-glycosylation. The glycosylated products were analysed by a combination of electrospray mass spectrometry and capillary electrophoresis in order to isolate the glycopeptides and to determine their sequence by Edman degradation. The O-glycosylation of our MUC5AC motif peptides gave information on the specificity and activity of the gastric microsomal UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase(s). The proline residues and the induced-conformations are of great importance for the recognition of MUC5AC peptides but they are not the only factors for the choice of the O-glycosylation sites. Moreover, for the di-glycosylated peptides, the flanking regions of the proline residues strongly influence the site of the second O-glycosylation.