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Biochimie
Edite par la Societe de Chimie Biologique avec le concours du CNRS et de l'INSERM
Biochimie

Edite par la Societe de Chimie Biologique avec le concours du CNRS et de l'INSERM

月刊

0300-9084

Biochimie/Journal BiochimieSCIISTP
正式出版
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    Antioxidant effects of resveratrol and other stilbene derivatives on oxidative stress and ·NO bioavailability: Potential benefits to cardiovascular diseases

    Matthieu FrombaumSolenn Le ClancheDominique Bonnefont-RousselotDidier Borderie...
    p.269-276页
    查看更多>>摘要：Oxidative stress plays an important part in the appearance and development of cardiovascular diseases. In this context, overproduction of reactive oxygen species leads to deregulation of metabolic pathways, such as cell proliferation or inflammation, which interferes with the homeostasis of vascular endothe-lium. Oxidative stress can decrease the bioavailability of nitric oxide (·NO) in vessels. This decrease is highly associated with endothelial dysfunction. The "French paradox" is a phenomenon that associates a diet rich in saturated fatty acids and a moderate consumption of wine to a low prevalence of cardiovascular diseases. During the past 10 years, the beneficial effects of wine on cardiovascular diseases have been attributed to the actions of resveratrol and other polyphenols. One of the mechanisms involved in these beneficial effects is the capacity of resveratrol and some other stilbene derivatives to maintain sufficient ·NO bioavailability in vascular endothelium. This review presents the latest findings on the molecular effects of resveratrol and other stilbene derivatives on the various actors that modulate ·NO bioavailability during oxidative stress.
      原文链接:
    • NETL
    • NSTL
    • Elsevier

    The stability of intramolecular DNA G-quadruplexes compared with other macromolecules

    Andrew N. Lane
    p.277-286页
    查看更多>>摘要：DNA quadruplexes are often conceived as very stable structures. However, most of the free energy of stabilization derives from specific ion binding via inner sphere coordination of the GO6 of the guanine residues comprising the basic quartet. When compared with other nucleic acid structures such as DNA or RNA duplexes and hairpins, or proteins of the same number of atoms, metal-coordinated intramolecular quadruplexes are found to be of comparable or lower thermodynamic stability under similar solution conditions. Furthermore, intramolecular quadruplexes are actually less stable kinetically, than DNA duplexes or hairpins of the same size. Although the literature is incomplete, it is clear that polyelectrolyte ion effects, the influence of solvation and steric crowding on stability are qualitatively different between intramolecular quadruplexes and DNA duplexes. For example, decreasing water activity destabilizes DNA duplexes, whereas quadruplexes are stabilized. The variety of folded conformations accessible to a single sequence further implies strong sensitivity of the conformational ensemble to the solution conditions, compared with DNA duplexes or small single domain proteins. These considerations may have relevance to the conditions prevailing inside cell nuclei and therefore the structures that potentially might form in vivo.
      原文链接:
    • NETL
    • NSTL
    • Elsevier

    SAHA/TRAIL combination induces detachment and anoikis of MDA-MB231 and MCF-7 breast cancer cells

    M. LauricellaA. CiraoloD. CarlisiR. Vento...
    p.287-299页
    查看更多>>摘要：SAHA, an inhibitor of histone deacetylase activity, has been shown to sensitize tumor cells to apoptosis induced by TRAIL, a member of TNF-family. In this paper we investigated the effect of SAHA/TRAIL combination in two breast cancer cell lines, the ERa-positive MCF-7 and the ERα-negative MDA-MB231. Treatment of MDA-MB231 and MCF-7 cells with SAHA in combination with TRAIL caused detachment of cells followed by anoikis, a form of apoptosis which occurs after cell detachment, while treatment with SAHA or TRAIL alone did not produce these effects. The effects were more evident in MDA-MB231 cells, which were chosen for ascertaining the mechanism of SAHA/TRAIL action. Our results show that SAHA decreased the level of c-FLIP, thus favouring the interaction of TRAIL with the specific death receptors DR4 and DR5 and the consequent activation of caspase-8. These effects increased when the cells were treated with SAHA/TRAIL combination. Because z-IEDT-fmk, an inhibitor of caspase-8, prevented both the cleavage of the focal adhesion-kinase FAK and cell detachment, we suggest that activation of caspase-8 can be responsible for both the decrement of FAK and the consequent cell detachment. In addition, treatment with SAHA/TRAIL combination caused dissipation of △Ψm, activation of caspase-3 and decrement of both phospho-EGFR and phospho-ERKl/2, a kinase which is involved in the phosphory-lation of BimEL. Therefore, co-treatment also induced decrement of phospho-BimEL and a concomitant increase in the dephosphorylated form of BimEL, which plays an important role in the induction of anoikis. Our findings suggest the potential application of SAHA in combination with TRAIL in clinical trials for breast cancer.
      原文链接:
    • NETL
    • NSTL
    • Elsevier

    Characterization of human nicotinate phosphoribosyltransferase: Kinetic studies, structure prediction and functional analysis by site-directed mutagenesis

    Lucia GalassiMichele Di StefanoLucia BrunettiGiuseppe Orsomando...
    p.300-309页
    查看更多>>摘要：Nicotinate phosphoribosyltransferase (NaPRT, EC 2.4.2.11) catalyzes the conversion of nicotinate (Na) to nicotinate mononucleotide, the first reaction of the Preiss-Handler pathway for the biosynthesis of NAD~+. Even though NaPRT activity has been described to be responsible for the ability of Na to increase NAD~+ levels in human cells more effectively than nicotinamide (Nam), so far a limited number of studies on the human NaPRT have appeared. Here, extensive characterization of a recombinant human NaPRT is reported. We determined its major kinetic parameters and assayed the influence of different compounds on its enzymatic activity. In particular, ATP showed an apparent dual stimulation/inhibition effect at low/high substrates saturation, respectively, consistent with a negative cooperativity model, whereas inorganic phosphate was found to act as an activator. Among other metabolites assayed, including nucleotides, nucleosides, and intermediates of carbohydrates metabolism, some showed inhibitory properties, i.e. CoA, several acyl-CoAs, glyceraldehyde 3-phosphate, phosphoenolpyruvate, and fructose 1,6-bisphosphate, whereas dihy-droxyacetone phosphate and pyruvate exerted a stimulatory effect. Furthermore, in light of the absence of crystallographic data, we performed homology modeling to predict the protein three-dimensional structure, and molecular docking simulations to identify residues involved in the recognition and stabilization of several ligands. Most of these residues resulted universally conserved among NaPRTs, and, in this study, their importance for enzyme activity was validated through site-directed mutagenesis.
      原文链接:
    • NETL
    • NSTL
    • Elsevier

    Deciphering the molecular structure of cryptolepain in organic solvents

    N.K. Prasanna KumariM.V. Jagannadham
    p.310-317页
    查看更多>>摘要：Solvent composition plays a major role in stabilizing/destabilizing the forces that are responsible for the native structure of a protein. Often, the solvent composition drives the protein into non-native conformations. Elucidation of such non-native structures provides valuable information about the molecular structure of the protein, which is unavailable otherwise. Inclusion of methanol (non-fluorinated alcohol) or TFE (fluorinated alcohol) in the solvent composition drove cryptolepain, a serine protease and an all-P-protein, into a non-native structure with an enhanced β-sheet or induction of a-helix. These solvents did not much affect cryptolepain under neutral conditions, even at higher concentrations, but the effects were predominant at lower pH, when the protein molecule is under stress. The organic solvent-induced state is partially unfolded with similar characteristics to the molten globule state seen with protein under a variety of conditions. Chemical- or temperature-induced unfolding of cryptolepain in the presence of organic solvent is distinctly different from that in the absence of organic solvent. Such different unfolding provided evidence of two structural variants in the molecular structure of the protein as well as the differential stabilization/destabilization of such structural variants and their sequential unfolding.
      原文链接:
    • NETL
    • NSTL
    • Elsevier

    Substrate specificity of Staphylococcus aureus cysteine proteases - Staphopains A, B and C

    Magdalena KaliriskaTomasz KantykaDoron C. GreenbaumKatrine S. Larsen...
    p.318-327页
    查看更多>>摘要：Human strains of Staphylococcus aureus secrete two papain-like proteases, staphopain A and B. Avian strains produce another homologous enzyme, staphopain C. Animal studies suggest that staphopains B and C contribute to bacterial virulence, in contrast to staphopain A, which seems to have a virulence unrelated function. Here we present a detailed study of substrate preferences of all three proteases. The specificity of staphopain A, B and C substrate-binding subsites was mapped using different synthetic substrate libraries, inhibitor libraries and a protein substrate combinatorial library. The analysis demonstrated that the most efficiently hydrolyzed sites, using Schechter and Berger nomenclature, comprise a P2-GlyiAla(Ser) sequence motif, where P2 distinguishes the specificity of staphopain A (Leu) from that of both staphopains B and C (Phe/Tyr). However, we show that at the same time the overall specificity of staphopains is relaxed, insofar as multiple substrates that diverge from the sequences described above are also efficiently hydrolyzed.
      原文链接:
    • NETL
    • NSTL
    • Elsevier

    Two novel families of antimicrobial peptides from skin secretions of the Chinese torrent frog, Amolops jingdongensis

    Zhongming ChenXinwang YangZichao LiuLin Zeng...
    p.328-334页
    查看更多>>摘要：The characterization of new natural antimicrobial peptides (AMPs) can help to solve the serious problem of bacterial resistance to currently used antibiotics. In the current study, we analyzed two families of AMPs from the Chinese torrent frog Amolops jingdongensis with a range of bioactivities. The first family of peptides, named jindongenin-la, is 24 amino acids in length; a BLAST search of jindongenin-1a revealed no sequence similarity with other AMPs. The second family consists of two peptides containing 29 amino acid residues each. These peptides have high sequence similarity with the AMPs of palustrin-2 and are therefore designated palustrin-2AJl and palustrin-2AJ2. The cDNA sequences encoding these AMPs have been cloned and the deduced protein sequence of each AMP has been determined by protein sequencing. Sequence and structural analysis showed that each precursor is composed of a putative signal peptide, an N-terminal spacer, a processing site and a disulfide-bridged heptapeptide segment at the C-terminus. We synthesized jindongenin-la and palustrin-AJl to test their antimicrobial, hemolytic, antioxidative and cytotoxic activities. These two peptides showed broad-spectrum antimicrobial activity to standard and clinically isolated strains of bacteria. In addition, they exhibited weak hemolytic activity to human and rabbit erythrocytes under our experimental conditions. Moreover, these peptides also displayed cytotoxic activity against the K562 and HT29 mammalian cell lines and low anti-oxidant activity. These findings provide helpful insight that will be useful in the design of anti-infective peptide agents.
      原文链接:
    • NETL
    • NSTL
    • Elsevier

    Cloning, characterization and mutagenesis of Russell's viper venom L-amino acid oxidase: Insights into its catalytic mechanism

    Hong-Sen ChenYing-Ming WangWan-Ting HuangKai-Fa Huang...
    p.335-344页
    查看更多>>摘要：To investigate the structure-function relationships and geographic variations of L-amino acid oxidase (LAAO) from Daboia venoms, a single LAAO (designated as DrLAO) was purified from eastern Indian Daboia russelii venom and characterized. The purified DrLAO showed subunit molecular mass of 60-64 kDa; its N-terminal sequence (1-20) was identical to those of several true viper LAAOs. Its preferred substrates were hydrophobic L-amino acids and the kinetic specificities were ordered as follows: Phe, Tyr, Met, Leu, and Trp. Enzyme assay and Western blotting showed that the venom LAAO contents of D. russelii were higher than those of Daboia siamensis. DrLAO dose-dependently inhibited ADP- and collagen-induced platelet aggregation with IC50 values of 0.27 and 0.82 uM, respectively. Apparently, DrLAO may syner-gize with other venom components to prolong and enhance bleeding symptoms after Daboia envenoming. The full sequence of DrLAO was deduced from its cDNA sequence and then confirmed by peptide mass fingerprinting. Molecular phylogenetic analysis revealed that SV-LAAO family members could be differentiated not only by snake taxonomy but also by the variations at position 223, and they divided into H223, S223, N223, and D223 subclasses. We have further prepared recombinant DrLAO and mutants by the Pichia expression system. Mutagenic analyses of DrLAO His223 revealed that this residue bound substrates instead of serving as an essential base in the catalytic steps. Our results suggest a direct hydride transfer from substrate to FAD as the mechanism for SV-LAAOs.
      原文链接:
    • NETL
    • NSTL
    • Elsevier

    Ruthenium methylimidazole complexes induced apoptosis in lung cancer A549 cells through intrinsic mitochondrial pathway

    Xiaoxin YangLanmei ChenYanan LiuYongguang Yang...
    p.345-353页
    查看更多>>摘要：Ruthenium(II) methylimidazole complexes, with the general formula [Ru(MeIm)4(N⌒N)]~(2+) (N⌒N = tip (RMC1), iip (RMC2), dppz (RMC3), dpq (RMC4); Melm = 1-methylimidazole, tip = 2-(thiophene-2-yl)-lH-imidazo [4,5-f] [1,10]phenanthroline, iip = 2-(lH-imidazol-4-yl)-1H-imidazo [4,5-f] [1,10]phenanthroline, dppz = dipyrido[3,2-a:2',3'-c]phenazine, dpq = pyrazino [2,3-f] [1,10]phenanthroline), were synthesized and characterized. As determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, these complexes displayed potent anti-proliferation activity against various cancer cells. RMC1 inhibited the growth of A549 (human lung adenocarcinoma) lung cells through induction of apoptotic cell death, as evidenced by the accumulation of cell population in sub-G1 phase. RMC1 also induced the depletion of mitochondrial membrane potential in A549 cells by regulating the expression of pro-survival and pro-apoptotic Bcl-2 family members. Another experiment showed that Bid protein was also activated by RMC1, which implied that RMC1 could existed two pathways crosstalk, namely, have exogenous death receptor signaling pathway. These results demonstrated that RMC1 induced cancer cell death by acting on both mitochondrial and death receptor apoptotic pathways, suggesting that RMC1 could be a candidate for further evaluation as a chemotherapeutic agent against human cancers.
      原文链接:
    • NETL
    • NSTL
    • Elsevier

    Motifs in the C-terminal region of the Penicillium chrysogenum ACV synthetase are essential for valine epimerization and processivity of tripeptide formation

    Xiaobin WuCarlos Garcia-EstradaInmaculada VacaJuan-Francisco Martin...
    p.354-364页
    查看更多>>摘要：The first step in the penicillin biosynthetic pathway is the non-ribosomal condensation of L-α-aminoadipic acid, L-cysteine and L-valine into the tripeptide δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (ACV). This reaction is catalysed by the multienzyme ACV synthetase (ACVS), which is encoded in the filamentous fungus Penicillium chrysogenum by the pcbAB gene. This enzyme contains at least ten catalytic domains. The precise role of the C-terminal domain of this multidomain NRPS still remains obscure. The C-terminal region of ACVS bears the epimerase and the thioesterase domains and may be involved in the epimerization of LLL-ACV to LLD-ACV and in the hydrolysis of the thioester bond. In this work, the conserved motifs ~(3371)EGHGRE~(3376) (located in the putative epimerase domain) and ~(3629)GWSFG~(3633) (located in the thioesterase domain) were changed by site-directed-mutagenesis to LGFGLL and GWAFG, respectively. In addition, the whole thioesterase domain (230 amino acids) and the different parts of this domain were deleted. The activity of these mutant enzymes was assessed in vivo by two different procedures: i) through the quantification of bisACV produced by the fungus and ii) by quantifying the benzylpenicillin production using tailored strains of P. chrysogenum, which lack the pcbAB gene, as host strains. All indicated mutant enzymes showed lower or null activity than the control strain confirming that E3371, H3373, R3375 and E3376 belong to the epimerase active centre. Different fragments included in the C-terminal region of ACVS control thioester hydrolysis. Overexpression of the sequence encoding the ACVS integrated thioesterase domain as a separate (stand-alone) transcriptional unit complemented mutants lacking the integrated thioesterase domain, although with low ACV releasing activity, suggesting that the stand-alone thio-esterease interacts with the other ACVS domains.
      原文链接:
    • NETL
    • NSTL
    • Elsevier