Antimitochondrial activity displayed by the antimicrobial peptide microcin J25

In this report we studied the effect of the antimicrobial peptide, microcin J25, on the rat heart mitochondria. This peptide induced an important inhibition of the ATP synthesis with the concomitant enhancement of the ATP degradation. These effects were the result of two processes: on one hand, microcin J25 was able to insert into the membrane and hence alter its permeability with the consequent dissipation of the proton motive force. On the other, microcin J25 inhibited the enzymatic activity of the cytochrome c reductase (complex III) of the respiratory chain. The relevance of this study to the potential use of microcin J25 as an anti-tumoral agent is discussed.     

Antifungal activity displayed by cereulide, the emetic toxin produced by Bacillus cereus

In this study, the fungistatic activity of Bacillus cereus cereulide-producing strains was demonstrated against nine fungal species. The role of cereulide was confirmed using plasmid-cured derivatives and ces knockout mutants. The fungistatic spectra of cereulide and valinomycin, a chemically related cyclododecadepsipeptide, were also compared and found to be similar but distinct.     

Functional expression and characterization of the Epstein-Barr virus DNA polymerase catalytic subunit.

A recombinant baculovirus containing the complete sequence for the Epstein-Barr virus (EBV) DNA polymerase catalytic subunit, BALF5 gene product, under the control of the baculovirus polyhedrin promoter was constructed. Insect cells infected with the recombinant virus produced a protein of 110 kDa, recognized by anti-BALF5 protein-specific polyclonal antibody. The expressed EBV DNA polymerase catalytic polypeptide was purified from the cytosolic fraction of the recombinant virus-infected insect cells. The purified protein exhibited both DNA polymerase and 3'-to-5' exonuclease activities, which were neutralized by the anti-BALF5 protein-specific antibody. These results indicate that the 3'-to-5' exonuclease activity associated with the EBV DNA polymerase (T. Tsurumi, Virology 182:376-381, 1991) is an inherent feature of the polymerase catalytic polypeptide. The DNA polymerase and the exonuclease activities of the EBV DNA polymerase catalytic subunit were sensitive to ammonium sulfate in contrast to those of the polymerase complex purified from EBV-producing lymphoblastoid cells, which were stimulated by salt. Furthermore, the template-primer preference for the polymerase catalytic subunit was different from that for the polymerase complex. These observations strongly suggest that the presence of EBV DNA polymerase accessory protein, BMRF1 gene product, does influence the enzymatic properties of EBV DNA polymerase catalytic subunit.     

Comparison of two forms of catalytic antibody displayed on yeast-cell surface

Two forms of the Fab fragment of the catalytic antibody 6D9 were individually displayed on yeast-cell surface in fusion to the C-terminal half of -agglutinin: one was 6D9 Fab1, in which the light chain of the Fab (Lc) fragment is displayed on cell surface and the heavy chain of the Fab (Fd) fragment is secreted and linked to the Lc fragment with a disulfide bond; the other was 6D9 Fab2, in which the Fd fragment is displayed on cell surface and the Lc fragment is secreted and linked to the Fd fragment with a disulfide bond. Analysis by flow cytometry indicated that some 6D9 Fab2 fragments were unable to construct an appropriate conformation, and that most of the 6D9 Fab1 fragments displayed on yeast-cell surface exhibited higher binding affinity, stability, and catalytic activity. Conformation of the surface-displayed hetero-dimeric Fab fragment mainly depended on the intermolecular disulfide bond between the Lc and Fd fragments. The conformation of 6D9 Fab1 was more stable than that of Fab2. In the reducing environment of solution containing 25 nM DTT, the function of 6D9 Fab2 was almost completely lost. The successful display of 6D9 Fab1 on yeast-cell surface provides a novel approach to the engineering of catalytic antibodies.     

Purification,characterization and catalytic activity of anionic zucchini peroxidase

The isolation and purification, by preparative electrofocusing, of the major anionic (ZPOA) and cationic (ZPOC) isoenzymes, collected from young zucchini squash, are reported. The M _r and sugar content are similar to those found previously for the major isoenzymes from the ripe fruits and in the range commonly observed for plant peroxidases. The amount of the two cationic enzymes was very low compared with that of anionic ZPOA. The anionic enzyme has been characterized by electronic, circular dichroism, proton NMR and electron paramagnetic resonance spectroscopy. The spectra are qualitatively similar to those of the corresponding anionic horseradish peroxidase (HRPA) derivatives, with minor differences attributable to the particular protein environment around the heme. The kinetics of the enzymatic oxidation of a series of phenols by H₂O₂ have been studied. ZPOA shows a parallel behavior to HRPA, but it is systematically more active than HRPA, indicating that the zucchini enzymes have a marked tendency to carry out oxidation of this type of compounds.     

Functional characterization of starvation-induced lysosomal activity in Saccharomyces cerevisiae

Starvation induces significant alterations in lysosomal enzymes, and reduced concentrations of glucose increases the activity of several lysosomal enzymes. Therefore, to evaluate the lysosomal antimicrobial activity under starvation conditions, we added 0, 5, 10, 20, or 40 g/l of glucose (0%, 0.5%, 1%, 2%, or 4% glucose) supplemented YP medium to cultured Saccharomyces cerevisiae, and lysosomal fractions were isolated from S. cerevisiae grown under the various culture conditions. The lysosomes isolated from each condition exhibited increased antimicrobial activity against Escherichia coli as determined by a decrease in glucose concentration. In addition, a starvation-dependent increase in lysosomal activity coincided with increased lysosome intensity at the cytosol and distinct protein expression from lysosomes in S. cerevisiae. It also was determined found that the lysosomes have antimicrobial activity against seven different microorganisms, including E. coli, and starvation-induced lysosomes showed enhanced antimicrobial activity compared to those from normal lysosomes. These results suggest the possibility that lysosomal alterations during starvation may induce conditions that activate lysosomes for future development of efficient antimicrobial agents.     

Functional characterization of a ClC transporter by solid-supported membrane electrophysiology

EcClC, a prokaryotic member of the ClC family of chloride channels and transporters, works as coupled H⁺/Cl⁻ exchanger. With a known structure and the possibility of investigating its behavior with different biochemical and biophysical techniques, the protein has become an important model system for the family. Although many aspects of its function have been previously characterized, it was difficult to measure transport on the same sample under different environmental conditions. To overcome this experimental limitation, we have studied EcClC by solid-supported membrane electrophysiology. The large transport-related transient currents and a simple way of relating transport rates to the measured signal have allowed a thorough investigation of ion selectivity, inhibition, and the dependence of transport on changes in ion concentration and pH. Our results confirm that the protein transports larger anions with about similar rates, whereas the smaller fluoride is not a substrate. We also show that 4,4′-diisothiocyano-2,2’-stilbenedisulfonic acid (DIDS), a known inhibitor of other anion transport protein, irreversibly inhibits EcClC from the intracellular side. The chloride dependence shows an apparent saturation at millimolar concentrations that resembles a similar behavior in eukaryotic ClC channels. Our experiments have also allowed us to quantify the pH dependence of transport. EcClC shows a strong activation at low pH with an apparent pKa of 4.6. The pronounced pH dependence is lost by the mutation of a conserved glutamate facing the extracellular solution that was previously shown to be an acceptor for transported protons, whereas it is largely retained by the mutation of an equivalent residue at the intracellular side. Our results have provided a quantitative basis for the transport behavior of EcClC, and they will serve as a reference for future investigations of novel electrogenic transporters with still-uncharacterized properties.     

Molecular characterization of Hydra acetylcholinesterase and its catalytic activity

A full-length cDNA encoding an acetylcholinesterase (AChE) from Hydra magnipapillata was isolated. All of the important aromatic residues that line a catalytic gorge in cholinesterases of other species were conserved, but the sequences of peripheral anionic and choline binding sites were not. Hydra AChE, expressed in Xenopus oocytes, showed AChE activity. The gene was expressed in both ectodermal and endodermal epithelial cells except for the tentacles and basal disk. AChE gene expression was not detected in the regenerating tips in either the head or the foot, indicating that regeneration is controlled by the non-neuronal cholinergic system in Hydra.     

Design of a serine protease-like catalytic triad on an antibody light chain displayed on the yeast cell surface

Lc-WT, the wild-type light chain of antibody, and Lc-Triad, its double mutant with E1D and T27aS designing for the construction of catalytic triad within Asp1, Ser27a, and original His93 residues, were displayed on the cell surface of the protease-deficient yeast strain BJ2168. When each cell suspension was reacted with BODIPY FL casein and seven kinds of peptide-MCA substrates, respectively, a remarkable difference in hydrolytic activities toward Suc-GPLGP-MCA (succinyl-Gly-Pro-Leu-Gly-Pro-MCA), a substrate toward collagenase-like peptidase, was observed between the constructs: Lc-Triad-displaying cells showed higher catalytic activity than Lc-WT-displaying cells. The difference disappeared in the presence of the serine protease inhibitor diisopropylfluorophosphate, suggesting that the three amino acid residues, Ser27a, His93, and Asp1, functioned as a catalytic triad responsible for the proteolytic activity in a similar way to the anti-vasoactive intestinal peptide (VIP) antibody light chain. A serine protease-like catalytic triad (Ser, His, and Asp) is considered to be directly involved in the catalytic mechanism of the anti-VIP antibody light chain, which moderately catalyzes the hydrolysis of VIP. These results suggest the possibility of new approach for the creation of tailor-made proteases beyond limitations of the traditional immunization approach.     

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

TRIM E3 ubiquitin ligases regulate a wide variety of cellular processes and are particularly important during innate immune signalling events. They are characterized by a conserved tripartite motif in their N‐terminal portion which comprises a canonical RING domain, one or two B‐box domains and a coiled‐coil region that mediates ligase dimerization. Self‐association via the coiled‐coil has been suggested to be crucial for catalytic activity of TRIMs; however, the precise molecular mechanism underlying this observation remains elusive. Here, we provide a detailed characterization of the TRIM ligases TRIM25 and TRIM32 and show how their oligomeric state is linked to catalytic activity. The crystal structure of a complex between the TRIM25 RING domain and an ubiquitin‐loaded E2 identifies the structural and mechanistic features that promote a closed E2~Ub conformation to activate the thioester for ubiquitin transfer allowing us to propose a model for the regulation of activity in the full‐length protein. Our data reveal an unexpected diversity in the self‐association mechanism of TRIMs that might be crucial for their biological function.     

Isolation of novel catalytic antibody clones from combinatorial library displayed on yeast-cell surface

A combinatorial library of the Fab fragment of a catalytic antibody able to hydrolyze a non-bioactive chloramphenicol monoester derivative to produce chloramphenicol was constructed on yeast-cell surface. Interesting clones were selected using fluorescence-activated cell sorting (FACS). When binding affinity to a transition-state analog was detected, evolution of the catalytic antibody was carried out in vitro on yeast-cell surface. A number of variants with enhanced catalytic activity and binding affinity were obtained. The results showed that the improvement of catalytic antibody, which can be performed easily on yeast-cell surface using the cell-surface engineering system, is a good example of the application of protein library construction.     

From structure to function: insights into the catalytic substrate specificity and thermostability displayed by Bacillus subtilis mannanase BCman

BCman, a β-mannanase from the plant root beneficial bacterium Bacillus subtilis Z-2, has a potential to be used in the production of mannooligosaccharide, which shows defense induction activity on both melon and tobacco, and plays an important role in the biological control of plant disease. Here we report the biochemical properties and crystal structure of BCman-GH26 enzyme. Kinetic analysis reveals that BCman is an endo-β-mannanase, specific for mannan, and has no activity on mannooligosaccharides. The catalytic acid/base Glu167 and nucleophile Glu266 are positioned on the β4 and β7 strands, respectively. The 1.45-Å crystal structure reveals that BCman is a typical (β/α)₈ folding type. One large difference from the saddle-shaped active center of other endo-β-mannanases is the presence of a shallow-dish-shaped active center and substrate-binding site that are both unique to BCman. These differences are mainly due to important changes in the length and position of loop 1 (Phe37-Met47), loop 2 (Ser103-Ala134), loop3 (Phe162-Asn185), loop 4 (Tyr215-Ile236), loop 5 (Pro269-Tyr278), and loop 6 (Trp298-Gly309), all of which surround the active site. Data from isothermal titration calorimetry and crystallography indicated only two substrate-binding subsites (+ 1 and − 1) within the active site of BCman. These two sites are involved in the enzyme's mannan degradation activity and in restricting the binding capacity for mannooligosaccharides. Binding and catalysis of BCman to mannan is mediated mainly by a surface containing a strip of solvent-exposed aromatic rings of Trp302, Trp298, Trp172, and Trp72. Additionally, BCman contains a disulfide bond (Cys66Cys86) and a special His1-His23-Glu336 metal-binding site. This secondary structure is a key factor in the enzyme's stability.     

Functional implications of the presenilin dimerization: reconstitution of gamma-secretase activity by assembly of a catalytic site at the dimer interface of two catalytically inactive presenilins

Presenilins are the catalytic components of gamma-secretase, an intramembrane-cleaving protease whose substrates include beta-amyloid precursor protein (betaAPP) and the Notch receptors. These type I transmembrane proteins undergo two distinct presenilin-dependent cleavages within the transmembrane region, which result in the production of Abeta and APP intracellular domain (from betaAPP) and the Notch intracellular domain signaling peptide. Most cases of familial Alzheimer's disease are caused by presenilin mutations, which are scattered throughout the coding sequence. Although the underlying molecular mechanism is not yet known, the familial Alzheimer's disease mutations produce a shift in the ratio of the long and short forms of the Abeta peptide generated by the gamma-secretase. We and others have previously shown that presenilin homodimerizes and suggested that a presenilin dimer is at the catalytic core of gamma-secretase. Here, we demonstrate that presenilin transmembrane domains contribute to the formation of the dimer. In-frame substitution of the hydrophilic loop 1, located between transmembranes I and II, which modulates the interactions within the N-terminal fragment/N-terminal fragment dimer, abolishes both presenilinase and gamma-secretase activities. In addition, by reconstituting gamma-secretase activity from two catalytically inactive presenilin aspartic mutants, we provide evidence of an active diaspartyl group assembled at the interface between two presenilin monomers. Under our conditions, this catalytic group mediates the generation of APP intracellular domain and Abeta but not Notch intracellular domain, therefore suggesting that specific diaspartyl groups within the presenilin catalytic core of gamma-secretase mediate the cleavage of different substrates.     

Modulation of the catalytic activity of pyridoxal kinase by metallothionein

Pyridoxal kinase displays high catalytic activity in the presence of metallothionein. The apoprotein of metallothionein as well as the peptide LYS-CYS-THR-CYS-CYS-ALA exert a strong inhibitory effect upon pyridoxal kinase by sequestering free Zn ions. Several steps intervene in the process of pyridoxal kinase activation, i.e. binding of Zn ions by ATP and interaction of Zn-ATP with the enzyme; but direct interaction between metallothionein and pyridoxal kinase (protein association) could not be detected by emission anisotropy measurements. Since the concentration of free Zn++ in mammalian tissues is lower than 10(-9)M, it is postulated that the concentration of metallothionein regulates the catalytic activity of pyridoxal kinase. The mechanism of reconstitution of the metalloenzyme yeast aldolase in the presence of metallothionein was also investigated.     

Spinach ferredoxin-nitrite reductase: characterization of catalytic activity and interaction of the enzyme with substrates

The steady-state kinetic parameters of the enzymatic reduction of nitrite by spinach ferredoxin-nitrite reductase [EC 1.7.7.1] were measured under anaerobic conditions. The maximum velocity of ferredoxin-linked activity was essentially the same as for the methyl viologen-linked activity of the enzyme. The initial velocity patterns of the oxidation of reduced ferredoxin suggested a sequential reaction scheme by which nitrite and reduced ferredoxin bind to the free enzyme. The binding of nitrite and ferredoxin to the enzyme was also investigated by different spectra produced by the complex formed by the enzyme with the substrates. Nitrite and ferredoxin each gave a 1: 1 complex with the enzyme. The dissociation constant (Kd) of the enzyme-nitrite complex agreed well with the Km value for the ferredoxin-linked activity, whereas the Kd of the enzyme-ferredoxin complex differed from the Km value for the enzyme activity. It was concluded that our preparation of spinach ferredoxin-nitrite reductase differs from both the complex (Mr = 85,000) and the modified (Mr = 61,000) forms of the enzyme reported by Hirasawa et al. [J. Biol. Chem. 262, 12428-12433 (1987)].     

Proteomic characterization of postmortem amyloid plaques isolated by laser capture microdissection

The presence of amyloid plaques in the brain is one of the pathological hallmarks of Alzheimer's disease (AD). We report here a comprehensive proteomic analysis of senile plaques from postmortem AD brain tissues. Senile plaques labeled with thioflavin-S were procured by laser capture microdissection, and their protein components were analyzed by liquid chromatography coupled with tandem mass spectrometry. We identified a total of 488 proteins co-isolated with the plaques, and we found multiple phosphorylation sites on the neurofilament intermediate chain, implicating the complexity and diversity of cellular processes involved in the plaque formation. More significantly, we identified 26 proteins enriched in the plaques of two AD cases by quantitative comparison with surrounding non-plaque tissues. The localization of several proteins in the plaques was further confirmed by the approach of immunohistochemistry. In addition to previously identified plaque constituents, we discovered novel association of dynein heavy chain with the plaques in human postmortem brain and in a double transgenic AD mouse model, suggesting that neuronal transport may play a role in neuritic degeneration. Overall, our results revealed for the first time the sub-proteome of amyloid plaques that is important for further studies on disease biomarker identification and molecular mechanisms of AD pathogenesis.     

Functional characterization of a prokaryotic Kir channel

The Kir gene family encodes inward rectifying K+ (Kir) channels that are widespread and critical regulators of excitability in eukaryotic cells. A related gene family (KirBac) has recently been identified in prokaryotes. While a crystal structure of one member, Kir-Bac1.1, has been solved, there has been no functional characterization of any KirBac gene products. Here we present functional characterization of KirBac1.1 reconstituted in liposomes. Utilizing a 86Rb+ uptake assay, we demonstrate that KirBac1.1 generates a K+ -selective permeation path that is inhibited by extraliposomal Ba2+ and Ca2+ ions. In contrast to KcsA (an acid-activated bacterial potassium channel), KirBac1.1 is inhibited by extraliposomal acid (pKa approximately 6). This characterization of KirBac1.1 activity now paves the way for further correlation of structure and function in this model Kir channel.     

Improvement of catalytic antibody activity by protease processing

An immunoglobulin L chain (HIR) was treated with lysyl-endopeptidase. Gel filtration chromatography of the digestion mix identified a peak displaying a significantly higher specific catalytic activity than that of the original sample. The protein in the peak was 11 kDa in size and constituted the VL fragment of HIR. The Km and Kcat values of Chromozym TRY hydrolysis for HIR were 1.5 x 10(-4) M and 6.2 min(-1), and for the VL fragment 7.3 x 10(-4) M and 4.8 x 10(2) min(-1), respectively. Three out of the five BJPs studied in this paper displayed elevated catalytic activity after processing with lysyl-endopeptidase. Similar results were also obtained for the complete antibody.     

Functional characterization of a maize purine transporter by expression in Aspergillus nidulans

We have characterized the function of Leaf Permease1 (LPE1), a protein that is necessary for proper chloroplast development in maize, by functional expression in the filamentous fungus Aspergillus nidulans. The choice of this ascomycete was dictated by the similarity of its endogenous purine transporters to LPE1 and by particular genetic and physiological features of purine transport and metabolism in A. nidulans. When Lpe1 was expressed in a purine transport-deficient A. nidulans strain, the capacity for uric acid and xanthine transport was acquired. This capacity was directly dependent on Lpe1 copy number and expression level. Interestingly, overexpression of LPE1 from >10 gene copies resulted in transformants with pleiotropically reduced growth rates on various nitrogen sources and the absolute inability to transport purines. Kinetic analysis established that LPE1 is a high-affinity (K(m) = 30 +/- 2.5 microM), high-capacity transporter specific for the oxidized purines xanthine and uric acid. Competition studies showed that high concentrations of ascorbic acid (>30 mM) competitively inhibit LPE1-mediated purine transport. This work defines the biochemical function of LPE1, a plant representative of a large and ubiquitous transporter family. In addition, A. nidulans is introduced as a novel model system for the cloning and/or functional characterization of transporter genes.