Cell-Wall-Bound Proteinase of Lactobacillus delbrueckii subsp. lactis ACA-DC 178: Characterization and Specificity for β-Casein

Lactobacillus delbrueckii subsp. lactis ACA-DC 178, which was isolated from Greek Kasseri cheese, produces a cell-wall-bound proteinase. The proteinase was removed from the cell envelope by washing the cells with a Ca²⁺-free buffer. The crude proteinase extract shows its highest activity at pH 6.0 and 40°C. It is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the enzyme is similar to the lactococcal P_I-type proteinases, since it hydrolyzes β-casein mainly and α- and κ-caseins to a much lesser extent. The cell-wall-bound proteinase from L. delbrueckii subsp. lactis ACA-DC 178 liberates four main peptides from β-casein, which have been identified.     

Partial Isolation and Degradation of Caseins by Cell Wall Proteinase(s) of Streptococcus cremoris HP

The cell wall proteinase fraction of Streptococcus cremoris HP has been isolated. This preparation did not exhibit any activity due to either specific peptidases known to be located near the outside surface of and in the membrane or intracellular proteolytic enzymes. By using thin-layer chromatography for the detection of relatively small hydrolysis products which remain soluble at pH 4.6, it was shown that β-casein is preferentially attacked by the cell wall proteinase. This was also the case when whole casein or micelles were used as the substrate. κ-casein hydrolysis is a relatively slow process, and α_s-casein degradation appeared to proceed at an extremely low rate. These results could be confirmed by using ¹⁴CH₃-labeled caseins. A relatively fast and linear initial progress of ¹⁴CH₃-labeled β-casein degradation is not inhibited by α_s-casein and only slightly by κ-casein at concentrations of these components which reflect their stoichiometry in the micelles. Possible implications of β-casein degradation for growth of the organism in milk are discussed.     

The Contribution of Caseins to the Amino Acid Supply for Lactococcus lactis Depends on the Type of Cell Envelope Proteinase

The ability of caseins to fulfill the amino acid requirements of Lactococcus lactis for growth was studied as a function of the type of cell envelope proteinase (P_I versus P_III type). Two genetically engineered strains of L. lactis that differed only in the type of proteinase were grown in chemically defined media containing α_s1-, β-, and κ-caseins (alone or in combination) as the sources of amino acids. Casein utilization resulted in limitation of the growth rate, and the extent of this limitation depended on the type of casein and proteinase. Adding different mixtures of essential amino acids to the growth medium made it possible to identify the nature of the limitation. This procedure also made it possible to identify the amino acid deficiency which was growth rate limiting for L. lactis in milk (S. Helinck, J. Richard, and V. Juillard, Appl. Environ. Microbiol. 63:2124–2130, 1997) as a function of the type of proteinase. Our results were compared with results from previous in vitro experiments in which casein degradation by purified proteinases was examined. The results were in agreement only in the case of the P_I-type proteinase. Therefore, our results bring into question the validity of the in vitro approach to identification of casein-derived peptides released by a P_III-type proteinase.     

Construction and Characterization of Salmonella typhimurium Strains That Accumulate and Excrete α- and β- Isopropylmalate

Two Salmonella typhimurium strains, which could be used as sources for the leucine biosynthetic intermediates α- and β-isopropylmalate were constructed by a series of P22-mediated transductions. One strain, JK527 [flr-19 leuA2010 Δ(leuD-ara)798 fol-162], accumulated and excreted α-isopropylmalate, whereas the second strain, JK553 (flr-19 leuA2010 leuB698), accumulated and excreted α- and β-isopropylmalate. The yield of α-isopropylmalate isolated from the culture medium of JK527 was more than five times the amount obtained from a comparable volume of medium in which Neurospora crassa strain FLR₉₂-1-216 (normally used as the source for α- and β-isopropylmalate) was grown. Not only was the yield greater, but S. typhimurium strains are much easier to handle and grow to saturation much faster than N. crassa strains. The combination of the two regulatory mutations flr-19, which results in constitutive expression of the leucine operon, and leuA2010, which renders the first leucine-specific biosynthetic enzyme insensitive to feedback inhibition by leucine, generated limitations in the production of valine and pantothenic acid. The efficient, irreversible, and unregulated conversion of α-ketoisovaleric acid into α-isopropylmalate (α-isopropylmalate synthetase K_m for α-ketoisovaleric acid, 6 × 10⁻⁵ M) severely restricted the amount of α-ketoisovaleric acid available for conversion into valine and pantothenic acid (ketopantoate hydroxymethyltransferase K_m for α-ketoisovaleric acid, 1.1 × 10⁻³ M; transaminase B K_m for α-ketoisovaleric acid, 2 × 10⁻³ M).     

The Autoproteolysis of Lactococcus lactis Lactocepin III Affects Its Specificity towards β-Casein

The effect of autoproteolysis of Lactococcus lactis lactocepin III on its specificity towards β-casein was investigated. β-Casein degradation was performed by using either an autolysin-defective derivative of L. lactis MG1363 carrying the proteinase genes of L. lactis SK11, which was unable to transport oligopeptides, or autoproteolyzed enzyme purified from L. lactis SK11. Comparison of the peptide pools by high-performance liquid chromatography analysis revealed significant differences. To analyze these differences in more detail, the peptides released by the cell-anchored proteinase were identified by on-line coupling of liquid chromatography to mass spectrometry. More than 100 oligopeptides were released from β-casein by the cell-anchored proteinase. Analysis of the cleavage sites indicated that the specificity of peptide bond cleavage by the cell-anchored proteinase differed significantly from that of the autoproteolyzed enzyme.     

Specificity of a cell-envelope-located proteinase (PIII-type) from Lactococcus lactis subsp. cremoris AM1 in its action on bovine β-casein

Summary The action of the cell-envelope proteinase (P_III-type) from Lactococcus lactis ssp. cremoris AM₁ on bovine -casein was studied. The results were compared with those obtained earlier with (P_I-type) proteinases from the cell envelope of other L. lactis strains. From a 4-h digest (pH 6.2; 15°C) of -casein made with the P_III-type proteinase, 24 peptides were isolated and purified by selective precipitation followed by semi-preparative reversed-phase HPLC. Altogether, these peptides accounted for the preferential splitting of 16 peptide bonds in -casein by the P_III-type proteinase. In nine cases the primary cleavage site (P₁-P₁) was a Glx-X or X-Glx peptide bond. In ten cases at least one large hydrophobic residue (Met, Leu, Tyr, Phe) formed part of the cleavable bond. The P₂-P₃ and/or P₂-P₃ regions of the substrate consisted of hydrophobic and/or negatively charged side chains or of side chains potentially involved in hydrogen bonds. Nine of the peptide bonds split were reported previously to be also susceptible to cleavage by P_I-type proteinases, although the kinetics may be different. The P_III-type proteinase shows a broader specificity in its initial cleavage of -casein than does the P_I-type. Offprint requests to: S. Visser     

NF-κB Mediates αvβ3 Integrin-induced Endothelial Cell Survival

The α_vβ₃ integrin plays a fundamental role during the angiogenesis process by inhibiting endothelial cell apoptosis. However, the mechanism of inhibition is unknown. In this report, we show that integrin-mediated cell survival involves regulation of nuclear factor-kappa B (NF-κB) activity. Different extracellular matrix molecules were able to protect rat aorta- derived endothelial cells from apoptosis induced by serum withdrawal. Osteopontin and β₃ integrin ligation rapidly increased NF-κB activity as measured by gel shift and reporter activity. The p65 and p50 subunits were present in the shifted complex. In contrast, collagen type I (a β₁-integrin ligand) did not induce NF-κB activity. The α_vβ₃ integrin was most important for osteopontin-mediated NF-κB induction and survival, since adding a neutralizing anti-β₃ integrin antibody blocked NF-κB activity and induced endothelial cell death when cells were plated on osteopontin. NF-κB was required for osteopontin- and vitronectin-induced survival since inhibition of NF-κB activity with nonphosphorylatable IκB completely blocked the protective effect of osteopontin and vitronectin. In contrast, NF-κB was not required for fibronectin, laminin, and collagen type I–induced survival. Activation of NF-κB by osteopontin depended on the small GTP-binding protein Ras and the tyrosine kinase Src, since NF-κB reporter activity was inhibited by Ras and Src dominant-negative mutants. In contrast, inhibition of MEK and PI3-kinase did not affect osteopontin-induced NF-κB activation. These studies identify NF-κB as an important signaling molecule in α_vβ₃ integrin-mediated endothelial cell survival.     

Microbial Conversion of α-Ionone, α-Methylionone, and α-Isomethylionone

α-Ionone, α-methylionone, and α-isomethylionone were converted by Aspergillus niger JTS 191. The individual bioconversion products from α-ionone were isolated and identified by spectrometry and organic synthesis. The major products were cis-3-hydroxy-α-ionone, trans-3-hydroxy-α-ionone, and 3-oxo-α-ionone. 2,3-Dehydro-α-ionone, 3,4-dehydro-β-ionone, and 1-(6,6-dimethyl-2-methylene-3-cyclohexenyl)-buten-3-one were also identified. Analogous bioconversion products from α-methylionone and α-isomethylionone were also identified. From results of gas-liquid chromatographic analysis during the fermentation, we propose a metabolic pathway for α-ionones and elucidation of stereochemical features of the bioconversion.     

Hydrolysis of Casein-Derived Peptides αS1-Casein(f1-9) and β-Casein(f193-209) by Lactobacillus helveticus Peptidase Deletion Mutants Indicates the Presence of a Previously Undetected Endopeptidase

Peptides derived from hydrolysis of α_S1-casein(f1-9) [α_S1-CN(f1-9)] and β-CN(f193-209) with cell extracts of Lactobacillus helveticus CNRZ32 and single-peptidase mutants (ΔpepC, ΔpepE, ΔpepN, ΔpepO, and ΔpepX) were isolated by using reverse-phase high-performance liquid chromatography and were characterized by mass spectrometry. The peptides identified suggest that there was activity of an endopeptidase, distinct from previously identified endopeptidases (PepE and PepO), with specificity for peptide bonds C terminal to Pro residues. Identification of hydrolysis products derived from a carboxyl-blocked form of β-CN(f193-209) confirmed that the peptides were derived from the activity of an endopeptidase.     

Force Measurements of the α5β1 Integrin–Fibronectin Interaction

The interaction of the α₅β₁ integrin and its ligand, fibronectin (FN), plays a crucial role in the adhesion of cells to the extracellular matrix. An important intrinsic property of the α₅β₁/FN interaction is the dynamic response of the complex to a pulling force. We have carried out atomic force microscopy measurements of the interaction between α₅β₁ and a fibronectin fragment derived from the seventh through tenth type III repeats of FN (i.e., FN7-10) containing both the arg-gly-asp (RGD) sequence and the synergy site. Direct force measurements obtained from an experimental system consisting of an α₅β₁ expressing K562 cell attached to the atomic force microscopy cantilever and FN7-10 adsorbed on a substrate were used to determine the dynamic response of the α₅β₁/FN7-10 complex to a pulling force. The experiments were carried out over a three-orders-of-magnitude change in loading rate and under conditions that allowed for detection of individual α₅β₁/FN7-10 interactions. The dynamic rupture force of the α₅β₁/FN7-10 complex revealed two regimes of loading: a fast loading regime (>10,000 pN/s) and a slow loading regime (<10,000 pN/s) that characterize the inner and outer activation barriers of the complex, respectively. Activation by TS2/16 antibody increased both the frequency of adhesion and elevated the rupture force of the α₅β₁/wild type FN7-10 complex to higher values in the slow loading regime. In experiments carried out with a FN7-10 RGD deleted mutant, the force measurements revealed that both inner and outer activation barriers were suppressed by the mutation. Mutations to the synergy site of FN, however, suppressed only the outer barrier activation of the complex. For both the RGD and synergy deletions, the frequency of adhesion was less than that of the wild type FN7-10, but was increased by integrin activation. The rupture force of these mutants was only slightly less than that of the wild type, and was not increased by activation. These results suggest that integrin activation involved a cooperative interaction with both the RGD and synergy sites.     

Coumarins effectively inhibit bacterial α-carbonic anhydrases

Coumarins are known to act as prodrug inhibitors of mammalian α-carbonic anhydrases (CAs, EC 4.2.1.1) but they were not yet investigated for the inhibition of bacterial α-CAs. Here we demonstrate that such enzymes from the bacterial pathogens Neisseria gonorrhoeae (NgCAα) and Vibrio cholerae (VchCAα) are inhibited by a panel of simple coumarins incorporating hydroxyl, amino, ketone or carboxylic acid ester moieties in various positions of the ring system. The nature and the position of the substituents in the coumarin ring were the factors which strongly influenced inhibitory efficacy. NgCAα was inhibited with K_Is in the range of 28.6–469.5 µM, whereas VchCAα with K_Is in the range of 39.8–438.7 µM. The two human (h)CA isoforms included for comparison reason in the study, hCA I and II, were less prone to inhibition by these compounds, with K_Is of 137–948.9 µM for hCA I and of 296.5–961.2 µM for hCA II, respectively. These findings are relevant for discovering coumarin bacterial CA inhibitors with selectivity for the bacterial over human isoform, with potential applications as novel antibacterial agents.     

Bile Salt 3α- and 12α-Hydroxysteroid Dehydrogenases from Eubacterium lentum and Related Organisms

Thirty-two strains of Eubacterium lentum and phenotypically similar anaerobic gram-positive bacilli were screened for intracellular bile salt 3α- and 12α-hydroxysteroid dehydrogenase (HSDHase) activities. These organisms were categorized into four groups: (A) those containing 12α-HSDHase only (10 strains), (B) those containing 3α- and 12α-HSDHase (13 strains), (C) those containing 3α-HSDHase only (2 strains), and (D) those devoid of any measurable HSDHase activity (7 strains). Of the respective four groups, 9/10, 13/13, 0/2, and 0/7 were like the neotype strain of E. lentum (ATCC 25559) in that they produced H₂S in a triple sugar iron agar butt, reduced nitrate to nitrite, and weakly decomposed hydrogen peroxide. The other strains were variable for nitrate reduction and activity on hydrogen peroxide, but all the organisms in the first three categories (with one exception) were H₂S producers (triple sugar iron agar butt) and all (with one exception) were designated E. lentum, whereas the organisms of category B were non-H₂S producers (triple sugar iron agar butt). Five of these seven were not stimulated by arginine and are designated “phenotypically similar organisms.” Thin-layer chromatography of extracted spent bacterial medium of four representative strains from each group grown in the presence of cholate revealed the presence of (A) 12-oxo product, (B) 12-oxo and 3-oxo products, (C) 3-oxo product, and (D) the absence of any of these products. The 12α-HSDHase of category B organisms was unstable unless 10⁻³ M dithioerythritol was added to the buffer. With the exception of 3 out of 32 strains, there was a positive correlation between the production of measurable amounts of 12α-HSDHase and H₂S production. Growth curves and the effect of arginine on growth and the production of 3α- and 12α-HSDHase were examined in representative strains of categories A, B, and C. Both enzymes were shown to bind onto a nicotinamide adenine dinucleotide-Sepharose column and could be eluted by high-ionic-strength buffer, resulting in approximately 25-fold and 18-fold purification, respectively. Molecular weight estimations by Sephadex G-200 gave values of 205,000 and 125,000 for the 3α- and 12α-HSDHase, respectively. Purified 12α-HSDHase was investigated with respect to pH requirement, substrate specificity, and enzyme kinetics.     

Identification of Pyruvate Carboxylase Genes in Pseudomonas aeruginosa PAO1 and Development of a P. aeruginosa-Based Overexpression System for α4- and α4β4-Type Pyruvate Carboxylases

Pyruvate carboxylase (PYC) is an ecologically, medically, and industrially important enzyme. It is widespread in all three domains of life, the archaea, bacteria, and eukarya. PYC catalyzes ATP-dependent carboxylation of pyruvate to oxaloacetate. Detailed structure-function studies of this enzyme have been hampered due to the unavailability of a facile recombinant overexpression system. Except for the α₄ enzyme from a thermophilic Bacillus species, Escherichia coli has been unsuitable for overexpression of PYCs. We show that a Pseudomonas aeruginosa strain carrying the T7 polymerase gene can serve as a host for the overexpression of Mycobacterium smegmatis α₄ PYC and Pseudomonas aeruginosa α₄β₄ PYC under the control of the T7 promoter from a broad-host-range conjugative plasmid. Overexpression occurred both in aerobic (LB medium) and nitrate-respiring anaerobic (LB medium plus glucose and nitrate) cultures. The latter system presented a simpler option because it involved room temperature cultures in stationary screw-cap bottles. We also developed a P. aeruginosa Δpyc strain that allowed the expression of recombinant PYCs in the absence of the native enzyme. Since P. aeruginosa can be transformed genetically and lysed for cell extract preparation rather easily, our system will facilitate site-directed mutagenesis, kinetics, X-ray crystallographic, and nuclear magnetic resonance-based structure-function analysis of PYCs. During this work we also determined that, contrary to a previous report (C. K. Stover et al., Nature 406:959-964, 2000), the open reading frame (ORF) PA1400 does not encode a PYC in P. aeruginosa. The α₄β₄ PYC of this organism was encoded by the ORFs PA5436 and PA5435.     

Action of a cell-envelope proteinase (CEPIII-type) from Lactococcus lactis subsp. cremoris AM1 on bovine κ-casein

The specificity of the cell-envelope proteinase (CEP_III-type) from Lactococcus lactis subsp. cremoris AM₁ in its action on bovine -casein was studied. A 4-h digest (pH 6.2, 15°C) of -casein was made with the purified proteinase. The pH-4.6 soluble fraction, representing more than 95% of the whole hydrolysate, was ultrafiltered to obtain a high-molecular-mass (HMM) and a low-molecular-mass (LMM) fraction, which were separately further purified by electrophoretic and chromatographic techniques. Isolated HMM and LMM products were identified by amino acid analysis, end-group determination and mass spectrometry. On-line HPLC/mass spectrometry was also used for the separation of an LMM peptide mixture and the identification of its components. The HMM products formed were the fragments 1–160, 1–151, 1–95 and 1–79 of -casein, whereas the main LMM products found were the 161–169 and 152–160 fragments. The enzyme specificity was concluded to be primarily directed towards the C-terminal region of the substrate molecule by cleavage of the 160–161 and 151–152 peptide bonds. Two minor LMM products were identified as the fragments 96–104 and 103–106, indicating additional cleavage at positions 102–103, 104–105 and 106–107 of the sequence. Also several peptide bonds within the 161–169 sequence were found to be subject to secondary cleavage by the proteinase. From electrophoretic and identification data it is concluded that the lactococaal CEP_I, CEP_III and several mixed-type proteinases all act on the peptide bonds at positions 79–80 and 95–96. However, the C-terminal region of the -casein sequence is the exclusive target of the CEP_III-aand, to variable extents, of the mixed-type enzymes.     

Comparison of bovine β-casein hydrolysis by PI and PIII-type proteinases from Lactobacillus lactis subsp. cremoris

Summary The action of the cell-wall-associated proteinases from Lactococcus lactis subsp. cremoris strains H2 and SK112 on bovine -casein was compared. The proteinase from the H2 strain was characterised as a P_I-type proteinase since it did not hydrolyse _s1-casein and the initial trifluoroacetic acid-soluble products of -casein hydrolysis were identical to those previously identified as hydrolysis products of P_I-type lactococcal proteinase action. The time-course of product formation by the proteinase from the H2 strain indicated that the bonds Tyr₁₉₃-Gln₁₉₄ and Gln₁₈₂-Arg₁₈₃ were the first to be hydrolysed. Cleavage of the bonds Gln₁₇₅-Lys₁₇₆, Ser₁₆₈-Lys₁₆₉, Ser₁₆₆-Gln₁₆₇ and Leu₁₆₃-Ser₁₆₄ was also very rapid. Four of the five bonds in -casein most susceptible to hydrolysis by the P_III-type proteinase from strain SK112 were different from those cleaved by the P_I-type proteinase, initial hydrolysis being at the sites Tyr₁₉₃-Gln₁₉₄, Leu₁₉₂-Tyr₁₉₃, Asp₄₃-Glu₄₄, Gln₄₆-Asp₄₇ and Phe₅₂-Ala₅₃. Early hydrolysis at the three sites in the N-terminal region of -casein, leading to cleavage of the N-terminal phosphopeptide and rapid precipitation of the residual fragment, represents a marked contrast to the action of P_I-type proteinases where cleavage at sites in the N-terminal region occurs only very slowly. Offprint requests to: G. G. Pritchard     

A Three-dimensional Collagen Lattice Induces Protein Kinase C-ζ Activity: Role in α2 Integrin and Collagenase mRNA Expression

A three-dimensional collagen lattice can provide skin fibroblasts with a cell culture environment that simulates normal dermis. Such a collagen matrix environment regulates interstitial collagenase (type I metalloproteinase [MMP-1], collagenase-1) and collagen receptor α₂ subunit mRNA expression in both unstimulated or platelet-derived growth factor–stimulated dermal fibroblasts (Xu, J., and R.A.F. Clark. 1996. J. Cell Biol. 132:239–249). Here we report that the collagen gel can signal protein kinase C (PKC)-ζ activation in human dermal fibroblasts. An in vitro kinase assay demonstrated that autophosphorylation of PKC-ζ immunoprecipitates was markedly increased by a collagen matrix. In contrast, no alteration in PKC-ζ protein levels or intracellular location was observed. DNA binding activity of nuclear factor κB (NF-κB), a downstream regulatory target of PKC-ζ, was also increased by fibroblasts grown in collagen gel. The composition of the NF-κB/Rel complexes that contained p50, was not changed. The potential role of PKC-ζ in collagen gel–induced mRNA expression of collagen receptor α₂ subunit and human fibroblast MMP-1 was assessed by the following evidence. Increased levels of α₂ and MMP-1 mRNA in collagen gel–stimulated fibroblasts were abrogated by bisindolylmaleimide GF 109203X and calphostin C, chemical inhibitors for PKC, but retained when cells were depleted of 12-myristate 13-acetate (PMA)–inducible PKC isoforms by 24 h of pretreatment with phorbol PMA. Antisense oligonucleotides complementary to the 5′ end of PKC-ζ mRNA sequences significantly reduced the collagen lattice–stimulated α₂ and MMP-1 mRNA levels. Taken together, these data indicate that PKC-ζ, a PKC isoform not inducible by PMA or diacylglycerol, is a component of collagen matrix stimulatory pathway for α₂ and MMP-1 mRNA expression. Thus, a three-dimensional collagen lattice maintains the dermal fibroblast phenotype, in part, through the activation of PKC-ζ.     

Characterization of Amylolytic Enzymes, Having Both α-1,4 and α-1,6 Hydrolytic Activity, from the Thermophilic Archaea Pyrococcus furiosus and Thermococcus litoralis

Extracellular pullulanases were purified from cell-free culture supernatants of the marine thermophilic archaea Thermococcus litoralis (optimal growth temperature, 90°C) and Pyrococcus furiosus (optimal growth temperature, 98°C). The molecular mass of the T. litoralis enzyme was estimated at 119,000 Da by electrophoresis, while the P. furiosus enzyme exhibited a molecular mass of 110,000 Da under the same conditions. Both enzymes tested positive for bound sugar by the periodic acid-Schiff technique and are therefore glycoproteins. The thermoactivity and thermostability of both enzymes were enhanced in the presence of 5 mM Ca²⁺, and under these conditions, enzyme activity could be measured at temperatures of up to 130 to 140°C. The addition of Ca²⁺ also affected substrate binding, as evidenced by a decrease in K_m for both enzymes when assayed in the presence of this metal. Each of these enzymes was able to hydrolyze, in addition to the α-1,6 linkages in pullulan, α-1,4 linkages in amylose and soluble starch. Neither enzyme possessed activity against maltohexaose or other smaller α-1,4-linked oligosaccharides. The enzymes from T. litoralis and P. furiosus appear to represent highly thermostable amylopullulanases, versions of which have been isolated from less-thermophilic organisms. The identification of these enzymes further defines the saccharide-metabolizing systems possessed by these two organisms.     

Studies on human alpha(s)- and kappa-casein fractions and human caseinoglycomacropeptide

1. Fractions have been obtained from human whole casein closely resembling the α_s- and κ-fractions of cow casein. 2. The α_s-fraction (human α_s-casein) is calcium-sensitive, heterogeneous in zone analysis and inert towards rennin. 3. The κ-fraction (human κ-casein) is calcium-insensitive, heterogeneous in zone analysis, and forms a soluble glycopeptide when acted upon by rennin. 4. Human κ-casein stabilizes human α_s-casein in the presence of Ca²⁺ ions. 5. The glycopeptides released by rennin from human casein and from cow casein have been compared. There are important differences in both the peptide and non-peptide structures of the two compounds. 6. In both human and bovine glycopeptides some of the carbohydrate residues are joined to the peptide by O-glycosidic links with threonine, and possibly with serine.     

Synthesis and bioactivities evaluation of oleanolic acid oxime ester derivatives as α-glucosidase and α-amylase inhibitors

Different oleanolic acid (OA) oxime ester derivatives (3a-3t) were designed and synthesised to develop inhibitors against α-glucosidase and α-amylase. All the synthesised OA derivatives were evaluated against α-glucosidase and α-amylase in vitro. Among them, compound 3a showed the highest α-glucosidase inhibition with an IC₅₀ of 0.35 µM, which was ∼1900 times stronger than that of acarbose, meanwhile compound 3f exhibited the highest α-amylase inhibitory with an IC₅₀ of 3.80 µM that was ∼26 times higher than that of acarbose. The inhibition kinetic studies showed that the inhibitory mechanism of compounds 3a and 3f were reversible and mixed types towards α-glucosidase and α-amylase, respectively. Molecular docking studies analysed the interaction between compound and two enzymes, respectively. Furthermore, cytotoxicity evaluation assay demonstrated a high level of safety profile of compounds 3a and 3f against 3T3-L1 and HepG2 cells.

Highlights

1. Oleanolic acid oxime ester derivatives (3a–3t) were synthesised and screened against α-glucosidase and α-amylase.
2. Compound 3a showed the highest α-glucosidase inhibitory with IC50 of 0.35 µM.
3. Compound 3f presented the highest α-amylase inhibitory with IC50 of 3.80 µM.
4. Kinetic studies and in silico studies analysed the binding between compounds and α-glucosidase or α-amylase.

     

Affinity Modulation of Platelet Integrin αIIbβ3 by β3-Endonexin, a Selective Binding Partner of the β3 Integrin Cytoplasmic Tail

Platelet agonists increase the affinity state of integrin α_IIbβ₃, a prerequisite for fibrinogen binding and platelet aggregation. This process may be triggered by a regulatory molecule(s) that binds to the integrin cytoplasmic tails, causing a structural change in the receptor. β₃-Endonexin is a novel 111–amino acid protein that binds selectively to the β₃ tail. Since β₃-endonexin is present in platelets, we asked whether it can affect α_IIbβ₃ function. When β₃-endonexin was fused to green fluorescent protein (GFP) and transfected into CHO cells, it was found in both the cytoplasm and the nucleus and could be detected on Western blots of cell lysates. PAC1, a fibrinogen-mimetic mAb, was used to monitor α_IIbβ₃ affinity state in transfected cells by flow cytometry. Cells transfected with GFP and α_IIbβ₃ bound little or no PAC1. However, those transfected with GFP/β₃-endonexin and α_IIbβ₃ bound PAC1 specifically in an energy-dependent fashion, and they underwent fibrinogen-dependent aggregation. GFP/β₃-endonexin did not affect levels of surface expression of α_IIbβ₃ nor did it modulate the affinity of an α_IIbβ₃ mutant that is defective in binding to β₃-endonexin. Affinity modulation of α_IIbβ₃ by GFP/β₃-endonexin was inhibited by coexpression of either a monomeric β₃ cytoplasmic tail chimera or an activated form of H-Ras. These results demonstrate that β₃-endonexin can modulate the affinity state of α_IIbβ₃ in a manner that is structurally specific and subject to metabolic regulation. By analogy, the adhesive function of platelets may be regulated by such protein–protein interactions at the level of the cytoplasmic tails of α_IIbβ₃.