Secreted proteins produced by fungi associated with Botryosphaeria dieback trigger distinct defense responses in Vitis vinifera and Vitis rupestris cells.

Grapevine trunk diseases (Eutypa dieback, esca and Botryosphaeria dieback) are caused by a complex of xylem-inhabiting fungi, which severely reduce yields in vineyards. Botryosphaeria dieback is associated with Botryosphaeriaceae. In order to develop effective strategies against Botryosphaeria dieback, we investigated the molecular basis of grapevine interactions with a virulent species, Neofusicoccum parvum, and a weak pathogen, Diplodia seriata. We investigated defenses induced by purified secreted fungal proteins within suspension cells of Vitis (Vitis rupestris and Vitis vinifera cv. Gewurztraminer) with putative different susceptibility to Botryosphaeria dieback. Our results show that Vitis cells are able to detect secreted proteins produced by Botryosphaeriaceae, resulting in a rapid alkalinization of the extracellular medium and the production of reactive oxygen species. Concerning early defense responses, N. parvum proteins induced a more intense response compared to D. seriata. Early and late defense responses, i.e., extracellular medium alkalinization, cell death, and expression of PR defense genes were stronger in V. rupestris compared to V. vinifera, except for stilbene production. Secreted Botryosphaeriaceae proteins triggered a high accumulation of δ-viniferin in V. vinifera suspension cells. Artificial inoculation assays on detached canes with N. parvum and D. seriata showed that the development of necrosis is reduced in V. rupestris compared to V. vinifera cv. Gewurztraminer. This may be related to a more efficient induction of defense responses in V. rupestris, although not sufficient to completely inhibit fungal colonization. Overall, our work shows a specific signature of defense responses depending on the grapevine genotype and the fungal species.

Toxicity of extracellular proteins from Diplodia seriata and Neofusicoccum parvum involved in grapevine Botryosphaeria dieback.

Botryosphaeria dieback, esca and Eutypa dieback are three economic major grapevine trunk diseases that cause severe yield reduction in vineyards worldwide. The frequency of disease symptoms has increased considerably over the past decade, and no efficient treatment is currently available to control these diseases. The different fungi associated with grapevine trunk diseases mainly induce necrotic wood and characteristic foliar symptoms. In this context, fungi virulence factors and host invasion are not well understood. We hypothesise that extracellular proteins produced by Diplodia seriata and Neofusicoccum parvum, two causal agents associated with Botryosphaeria dieback, are virulence factors responsible for the pathogenicity. In our previous work, we demonstrated that the total extracellular compounds produced by N. parvum induced more necrosis on Chardonnay calli and triggered a different defence gene expression pattern than those produced by D. seriata. Furthermore, this aggressiveness was not clearly correlated with the production of mellein, a characteristic phytotoxin of Botryosphaeriaceae, in our in vitro calli model. To characterise other potential virulence factors and to understand the mechanisms of host invasion by the fungus, we evaluated the profile, quantity and the impact of extracellular proteins produced by these fungi on Vitis vinifera calli necrosis and defence gene expression. Our results reveal that, under the same conditions, N. parvum produces more extracellular proteins and in higher concentrations than D. seriata. With Vitis vinifera cv. Chardonnay cells, we showed that equivalent concentrations of proteins secreted by N. parvum were more aggressive than those of D. seriata in producing necrosis and that they clearly induced more grapevine defence genes.

A possible role for cathepsins D, E, and B in the processing of beta-amyloid precursor protein in Alzheimer's disease.

Formation of the 4-kDa peptides, which are essential constituents of the extracellular plaques in Alzheimer's disease, involves the sequential cleavage of the amyloid precursor protein (APP) by beta- and gamma-secretases. The carboxy-terminal 99-amino-acid peptide which is liberated from APP by beta-secretase was used as a potential native substrate of the gamma-secretase(s). With the addition of an initiator Met and a FLAG sequence at the C-terminus (betaAPP100-FLAG), it was expressed in Escherichia coli under the control of the T7 promotor. The preferred site(s) of cleavage in the N-terminal 40-amino-acid beta-amyloid peptide and betaAPP100-FLAG by potential gamma-secretase(s) were rapidly identified using matrix-assisted laser-desorption/ionization time-of-flight mass spectroscopy in addition to peptide mapping followed by protein sequence analysis. Since gamma-secretases seem to be active at acidic pH, three cathepsins (D, E and B) were selected for testing. Studies using different detergents indicated that the cleavage preference of cathepsin D for the betaAPP100-FLAG is highly dependent on the surfactant used to solubilize this substrate. All three cathepsins were found to be capable of catabolizing both beta-amyloid peptides and the betaAPP100-FLAG. As cathepsin D was found to cleave the betaAPP100-FLAG in the vicinity of the C-terminus of the beta-amyloid peptides and cathepsin B has a high carboxypeptidase activity at low pH, the possibility cannot be excluded that cathepsins D and B are involved in the amyloidogenic processing of APP.

Processing of beta-amyloid precursor protein by cathepsin D.

The events leading to the formation of beta-amyloid (betaA4) from its precursor (betaAPP) involve proteolytic cleavages that produce the amino and carboxyl termini of betaA4. The enzyme activities responsible for these cleavages have been termed beta- and gamma-secretase, respectively, although these protease(s) have not been identified. Since betaA4 is known to possess heterogeneity at both the amino and carboxyl termini, beta- and gamma-secretases may actually be a collection of proteolytic activities or perhaps a single proteolytic enzyme with broad amino acid specificity. We investigated the role of cathepsin D in the processing of betaAPP since this enzyme has been widely proposed as a gamma-secretase candidate. Treatment of a synthetic peptide that spans the gamma-secretase site of betaAPP with human cathepsin D resulted in the cleavage of this substrate at Ala42-Thr43. A sensitive liquid chromatography/mass spectrometry technique was also developed to further investigate the ability of cathepsin D to process longer recombinant betaAPP substrates (156 and 100 amino acids of betaAPP carboxyl terminus) in vitro. The precise cathepsin D cleavage sites within these recombinant betaAPP substrates were identified using this technique. Both recombinant substrates were cleaved at the following sites: Leu49-Val50, Asp68-Ala69, Phe93-Phe94. No cleavages were observed at putative gamma-secretase sites: Val40-Ile41 or Ala42-Thr43, suggesting that cathepsin D is not gamma-secretase as defined by these betaA4 termini. Under conditions where the betaAPP156 substrate was first denatured prior to cathepsin D digestion, two additional cleavage sites near the amino terminus of betaA4, Glu-3-Val-2 and Glu3-Phe4, were observed, indicating that cathepsin D cleavage of betaAPP is influenced by the structural integrity of the substrate. Taken together, these results indicate that in vitro, cathepsin D is unlikely to function as gamma-secretase; however, the ability of this enzyme to efficiently cleave betaAPP substrates at nonamyloidogenic sites within the molecule may reflect a role in betaAPP catabolism.

3-Amino-2-hydroxy-propionaldehyde and 3-amino-1-hydroxy-propan-2-one derivatives: new classes of aminopeptidase inhibitors.

3-Amino-2-hydroxy-propionaldehydes [H2NCH(R)CHOHCHO with R = H, i-Bu, CH2Ph] were designed as metallo-aminopeptidase inhibitors based on the metal active site chelation concept. These compounds were found to be micromolar inhibitors of aminopeptidase-M (AP-M, EC 3.4.11.2) with potencies similar to bestatin (Ki = 3.5 microM). Notably, compound 5a (R = H) is a selective inhibitor of AP-M (Ki = 7 microM) with respect to cytosolic leucine aminopeptidase (LAPc, EC 3.4.11.1) (Ki = 385 microM). However, due to their easy oligomerization, these compounds are low practical value. In contrast, the corresponding isomeric 3-amino-1-hydroxy-propan-2-one derivatives [H2NCH(R)COCH2OH with R = H, i-Bu, CH2Ph, i-Pr, CH2Biph] are well defined structures. These hydroxymethylketones also exhibit micromolar affinities on AP-M. Compound 6c (R = CH2Ph) was the most potent (Ki = 1 microM). Selectivity studies of 6a (R = H) and 6b (R=i-Bu) show a preference for AP-M. Compound 6a is moderately active on AP-M (Ki = 25 microM) and inactive on LAPc. This new class of inhibitors is proposed to bind as bidentates, analogous to hydroxamates.

The structure of the Aeromonas proteolytica aminopeptidase complexed with a hydroxamate inhibitor. Involvement in catalysis of Glu151 and two zinc ions of the co-catalytic unit.

The structure of the complex of Aeromonas proteolytica aminopeptidase, a two-zinc exopeptidase, with the inhibitor p-iodo-D-phenylalanine hydroxamate has been determined by X-ray crystallography. Refinement of the structure, which includes 220 water molecules, using data at 0.80-0.23-nm resolution resulted in a crystallographic residual R value of 16%. The hydroxamate group adopts a planar conformation whereby the two oxygen atoms interact with the zinc ions. The N-hydroxyl group of the inhibitor is located between the two zinc ions, a position which is close to that occupied by a water molecule in the native structure. The carbonyl oxygen of the inhibitor binds to Zn1, which becomes pentacoordinated while Zn2 remains tetracoordinated, in contrast to the native protein where both zinc ions were shown to be tetracoordinated and structurally equivalent. Interactions of the carboxylate oxygens of Glu151 with the hydroxamate group play an important role in the stabilization of the complex.

Synthesis and comparison of tripeptidylfluoroalkane thrombin inhibitors.

Fluorinated ketone thrombin inhibitors based on the peptide sequence methyl-(D)-Phe-Pro-Arg-CF2R were synthesized: MDL 73,446 (1, R = F); MDL 73,775 (2, R = CF3); and MDL 75,579 (3, R = CH2CH2CH3). These were shown to be highly effective, slow binding inhibitors of thrombin. Anticoagulant activity was dose-dependent with 3 > 2 > 1 at doubling thrombin time and APTT, respectively. Anticoagulant activity corresponded with efficacy in a platelet-dependent (FeCl3-induced) rat carotid artery thrombosis model. Arterial occlusion was dose-dependently prolonged with 3 > 2 > 1 at doubling the occlusion time.

Design, synthesis, and conformational analysis of a novel macrocyclic HIV-protease inhibitor.

Design modifications to the lead HIV-PR inhibitor 1 (MDL 73,669, Ki = 5 nM) have been postulated based on a computational model of the 1/HIV-PR complex. A novel macrocyclic inhibitor 8 (MDL 104,168) wherein the P1 and P3 side chains of the original acyclic inhibitor have been joined retains good biological activity (Ki = 20 nM). NMR analysis of the precursor alcohol (S)-7 shows the conformation of the cyclic region to be very similar to that observed in the enzyme-bound 8 as determined by the computational model. Consistency of the computational model with NMR data and in vacuo molecular dynamics simulations provide the basis for postulating further modifications of the cyclic inhibitor expected to optimize its interactions with HIV-PR.

3-Amino-2-tetralone derivatives: novel potent and selective inhibitors of aminopeptidase-M (EC 3.4.11.2).

Derivatives of 3-amino-2-tetralone were evaluated for their ability to selectively inhibit the membrane-bound zinc-dependent aminopeptidase (EC 3.4.11.2), isolated from porcine kidney. These novel nonpeptidic compounds are potent competitive inhibitors of the enzyme. Some of them have Ki values in the nanomolar range (g, Ki = 80 nM). Moreover, these inhibitors are selective for aminopeptidase-M (AP-M) since they do not inhibit aspartate aminopeptidase and arginine aminopeptidase and only poorly inhibit cytosolic leucine aminopeptidase at high concentrations (g, Ki = 70 microM). The availability of inhibitors which are selective for AP-M with respect to other mammalian aminopeptidases may aid in identifying new endogenous substrates and thus clarify the physiological or pathophysiological role(s) of AP-M.

Crystal structure of Aeromonas proteolytica aminopeptidase: a prototypical member of the co-catalytic zinc enzyme family.

BACKGROUND: Aminopeptidases specifically cleave the amino-terminal residue from polypeptide chains and are involved in the metabolism of biologically active peptides. The family includes zinc-dependent enzymes possessing either one or two zinc ions per active site. Structural studies providing a detailed view of the metal environment may reveal whether the one-zinc and two-zinc enzymes constitute structurally and mechanistically distinct subclasses, and what role the metal ions play in the catalytic process. RESULTS: We have solved the crystal structure of the monomeric aminopeptidase from Aeromonas proteolytica at 1.8 A resolution. The protein is folded into a single alpha/beta globular domain. The active site contains two zinc ions (3.5 A apart) with shared ligands and symmetrical coordination spheres. We have compared it with the related bovine lens leucine aminopeptidase and the cobalt-containing Escherichia coli methionine aminopeptidase. CONCLUSIONS: The environment and coordination of the two zinc ions in A. proteolytica aminopeptidase strongly support the view that the two metal ions constitute a co-catalytic unit and play equivalent roles during catalysis. This conflicts with the conclusions drawn from the related bovine leucine aminopeptidase and early biochemical studies. In addition, the known specificity of the aminopeptidase for hydrophobic amino-terminal residues is reflected in the hydrophobicity of the active site cleft.

Calf spleen NAD+ glycohydrolase: solubilization, purification, and properties of the intact form of the enzyme.

NAD+ glycohydrolase was solubilized from calf spleen microsomes with emulphogene, a nonionic detergent, and purified to apparent homogeneity by ion-exchange chromatographies and by affinity chromatography on Affi-Gel blue gel. In contrast to the hydrosoluble form of the enzyme, which can be obtained by a proteolytic treatment of the microsomes with steapsin, the intact form of NAD+ glycohydrolase is characterized by its high hydrophobicity; i.e., the enzyme interacts very strongly with hydrophobic gels such as octyl-Sepharose and partitions into Triton X-114-rich phases. The apparent M(r) of the intact form of calf spleen NAD+ glycohydrolase is about 30 kDa, as compared to 24 kDa for the hydrosoluble form. This difference in molecular mass could account for the polypeptide moiety which allows the anchoring of the enzyme to the membranes. Both forms of the enzyme are strongly adsorbed by immobilized concanavalin A gels and biospecifically eluted with alpha-methylmannoside; this glycoprotein (mannosylated) nature of NAD+ glycohydrolase is in agreement with the previous demonstration that it is an ectoenzyme.

Rapid purification of the Aeromonas proteolytica aminopeptidase: crystallization and preliminary X-ray data.

The heat-stable aminopeptidase from Aeromonas proteolytica has been purified using two new procedures, with the aim of preparing large single crystals for X-ray analysis. In a first procedure, we tried to avoid any drastic conditions capable of inducing microheterogeneities in the protein sample. The enzyme was purified through two chromatographic steps based on hydrophobic interactions and ion exchange. In a second procedure a heat treatment of the protein to a temperature of 70 degrees C over 5 to 8 h was performed. Both procedures led to an electrophoretically homogeneous and crystallizable aminopeptidase; however, unexpectedly, the crystals obtained through the first procedure contained, in addition to the native aminopeptidase, a cleaved form of the enzyme which has been characterized. Only the native protein was present when the second procedure was used. Large crystals obtained with the native protein form, having an approximate size of 0.4 x 0.4 x 0.6 mm, produced an X-ray diffraction pattern that exhibited the symmetry associated with the hexagonal space group P6(1)22 (or its enantiomorph P6(5)22). The unit cell parameters were a = 109.1 A and c = 97.8 A. Assuming one molecule/asymmetric unit, a value of VM = 2.6 A3/Da and an approximate solvent content of 45% could be estimated. Measurable diffraction intensities were observed at a resolution of 2.5 A.

New fluoroketones as human renin inhibitors.

Renin inhibition has been evaluated for a new class of fluorinated ketones, true analogues of peptides that have been retroinverted at the C-terminal position. The readily formed hydrate of the ketone is proposed to mimic the tetrahedral intermediate that occurs during the enzyme-catalyzed hydrolysis of amide linkage. From this series of compounds it appears that the number of reverted amide bonds is crucial in terms of activity. Furthermore, a shortening of the C-terminal part of our peptide analogues and the replacement of the leucine residue in P1 by a cyclohexylalanine leads to the tripeptide analogue 12 a potent renin inhibitor (IC50 = 3.5 x 10(-9) M).

Targeting of liposomes by covalent coupling with ecto-NAD+-glycohydrolase ligands.

We have tested the feasibility of targeting liposomes via interaction with specific ecto-enzymes, i.e., enzymes which have their active site oriented to the external surface of the cell. 3,4-Dimethylpyridine adenine dinucleotide, a competitive inhibitor of ecto-NAD+-glycohydrolase, was substituted at N6 with a hydrophilic spacer arm, functionalized with a sulfhydryl group, and covalently linked to performed liposomes containing 4-(p-maleimidophenyl)butyryl phosphatidylethanolamine. We show that compared to control vesicles, the binding of the conjugated liposomes was greatly increased (up to 5-fold) to cells presenting ecto-NAD+-glycohydrolase activity (Swiss 3T3 fibroblasts, mouse peritoneal macrophages); in contrast, no specific binding was detected with hepatoma tissue culture cells, which lack this enzyme. Specific binding was found to depend on the ligand/lipid molar ratio of the vesicles and on the length of the arm. High concentrations of free 3,4-dimethylpyridine adenine dinucleotide virtually abolished the specific binding to cells of the targeted liposomes. Analysis of binding revealed that the ligand conjugated to the liposomes presented a functional affinity for 3T3 fibroblasts 15-fold superior to that of the free ligand.

Preparation of analogues of NAD+ as substrates for a sensitive fluorimetric assay of nucleotide pyrophosphatase.

1,N6-Etheno derivatives of pyridine analogues of NAD+ were synthesized, characterized and tested as substrates for a fluorimetric assay of nucleotide pyrophosphatase (EC 3.6.1.9). Upon cleavage of their pyrophosphate bond, the fluorescence of pyridine-1,N6-ethenoadenine dinucleotide (epsilon PdAD+) and of 4-hydrazinocarbonyl-pyridine-1,N6-ethenoadenine dinucleotide (epsilon hy4PdAD+) increased respectively 15-and 73-fold, at pH 7.4. This property allows a convenient steady-state assay of nucleotide pyrophosphatase by continuous monitoring of reaction progress. Both compounds were good substrates of this class of enzyme. The relative insensitivity of the fluorescence of epsilon PdAD+ and epsilon hy4PdAD+ to pH changes allowed assays under conditions preserving cellular integrity. epsilon PdAD+ is useful as a substrate for measuring nucleotide pyrophosphatase activity on the outside of mammalian cells because it is not a substrate for the external NAD+ glycohydrolase. epsilon Hy4PdAD+ proved useful when high sensitivity was needed.