Copper(II) complexes with β-cyclodextrin-homocarnosine conjugates and their antioxidant activity

Copper(II) complexes of the β-cyclodextrin (β-CD) functionalized with homocarnosine (HC) in the primary (CDHC6) and secondary rim (CDHC3) were characterized by means of different spectroscopic techniques such as UV-Vis absorption, circular dichroism, electron paramagnetic resonance and electron-spray mass spectrometry. Taken together, all the spectroscopic parameters indicate the formation of different copper(II) complex species at various pH values. In the CDHC3 copper(II) complex species, a direct involvement of the secondary hydroxyl group 2 of functionalized β-CD’s ring has been pointed out.The antioxidant activity of the copper(II) complexes of the two derivatives was determined through pulse radiolysis measurements. The results obtained provide direct evidence for a high catalytic activity of both complexes towards the dismutation of the superoxide anion radical. It is also demonstrated that the complex formation is not detrimental to the excellent scavenger activity exhibited by the ligands alone towards hydroxyl radicals. These copper complexes then represent very intriguing antioxidant agents against well known toxic reactive oxygen species.     

Observation of the complex formation between Cu(II) and protein by capillary electrophoretic system incorporating an UV/CL dual detector

We investigated the complex formation between Cu(II) and human serum albumin (HSA) through a biuret reaction by use of capillary electrophoretic system incorporating an ultra-violet absorption (UV) and chemiluminescence (CL) dual detector. Cu(II)-tartrate complex and Cu(II)-human serum albumin complex were detected by UV detection (282 nm) with on-capillary, followed by CL detection (luminol-hydrogen peroxide CL reaction) with end-capillary. We examined the effects of the reaction time and temperature on the UV and CL responses. On the basis of the obtained results we considered the Cu(II)-human serum albumin complex formation processes and its catalytic activity for the CL reaction. The system easily, rapidly, and simultaneously produced useful information concerning the complex formation of Cu(II) and human serum albumin due to the presence of the both detectors.     

Subunit‐selective proteasome activity profiling uncovers uncoupled proteasome subunit activities during bacterial infections

The proteasome is a nuclear‐cytoplasmic proteolytic complex involved in nearly all regulatory pathways in plant cells. The three different catalytic activities of the proteasome can have different functions, but tools to monitor and control these subunits selectively are not yet available in plant science. Here, we introduce subunit‐selective inhibitors and dual‐color fluorescent activity‐based probes for studying two of the three active catalytic subunits of the plant proteasome. We validate these tools in two model plants and use this to study the proteasome during plant–microbe interactions. Our data reveal that Nicotiana benthamiana incorporates two different paralogs of each catalytic subunit into active proteasomes. Interestingly, both β1 and β5 activities are significantly increased upon infection with pathogenic Pseudomonas syringae pv. tomato DC3000 lacking hopQ1‐1 [PtoDC3000(ΔhQ)] whilst the activity profile of the β1 subunit changes. Infection with wild‐type PtoDC3000 causes proteasome activities that range from strongly induced β1 and β5 activities to strongly suppressed β5 activities, revealing that β1 and β5 activities can be uncoupled during bacterial infection. These selective probes and inhibitors are now available to the plant science community, and can be widely and easily applied to study the activity and role of the different catalytic subunits of the proteasome in different plant species.     

Quaternary and domain structure of glycoprotein processing glucosidase II

Glucose trimming from newly synthesized glycoproteins regulates their interaction with the calnexin/calreticulin chaperone system. We have recently proposed that glucosidase II consisted of two different subunits, alpha and beta. The alpha subunit is the catalytic component, and deletion of its homologue in yeast obliterates glucosidase II activity. Deletion of the homologue of the noncatalytic beta subunit in Schizosaccharomices pombe drastically reduces glucosidase II activity, but the role of the beta subunit in glucosidase II activity has not been established. Furthermore, a direct interaction between alpha and beta subunits has not been demonstrated. Using chemical cross-linking and hydrodynamic analysis by analytical ultracentrifugation, we found that the two subunits form a defined complex, composed of one catalytic subunit and one accessory subunit (alpha(1)beta(1)) with a molecular mass of 161 kDa. The complex had an s value of 6.3 S, indicative of a highly nonglobular shape. The asymmetric shape of the alpha(1)beta(1) complex was confirmed by its high susceptibility to proteases. The beta subunit could be proteolytically removed from the alpha(1)beta(1) complex without affecting catalysis, demonstrating that it is not required for glucosidase II activity in vitro. Furthermore, we isolated a monomeric C-terminal fragment of the alpha subunit, which retained full glucosidase activity. We conclude that the catalytic core of glucosidase II resides in a globular domain of the alpha subunit, which can function independently of the beta subunit, while the complete alpha and beta subunits assemble in a defined heterodimeric complex with a highly extended conformation, which may favor interaction with other proteins in the endoplasmic reticulum (ER). Through its C-terminal HDEL signal, the beta subunit may retain the complete alpha(1)beta(1) complex in the ER.     

P-loop catalytically assisting the enzymatic cleavage of single-stranded DNA

We demonstrated that a P-loop, a looped complex formed inside duplex DNA by adding peptide nucleic acids (PNA), acts catalytically as a template for enzymatic cleavage of single-stranded probe oligodeoxynucleotides (ODN). A PD-loop complex formed from P-loop and probe ODN was digested efficiently by a restriction enzyme, and the truncated probe ODN was released. The P-loop nicked by the enzyme can form PD-loop again with another probe ODN, and then assisted the enzymatic cleavage of an excess of probe ODN. In addition, by using dumbbell-formed ODN as a probe ODN, the efficiency of the P-loop-assisted ODN cleavage was enhanced considerably as compared with that of linear ODN. Thus, the method utilizing P-loop will make it possible to amplify the sequence information of duplex DNA via a catalytic cleavage of probe ODNs.     

Calpain-7 binds to CHMP1B at its second α-helical region and forms a ternary complex with IST1

Some intracellular proteins involved in the endosomal sorting complex required for transport (ESCRT) system have microtubule interacting and transport (MIT) domains and bind to ESCRT-III protein family members named charged multivesicular body proteins (CHMPs) at their C-terminal regions containing MIT-interacting motifs (MIMs). While two types of MIMs (MIM1 and MIM2) have been reported, CHMP1B has MIM1 and IST1 has both MIM1 and MIM2. Previously, we demonstrated that CHMP1B and IST1 directly interacted with a tandem repeat of MIT domains of calpain-7 (CL7MIT) and that autolytic activity of calpain-7 was enhanced by IST1 in vitro but not by overexpression of IST1 in HEK293T cells. In this study, we detected enhancement of autolysis of mGFP-fused calpain-7 by coexpression with CHMP1B and observed further activation by additional coexpression of IST1 in HEK293T cells. We found that CL7MIT interacted with the second α-helical region of CHMP1B but not with the canonical C-terminal region containing MIM1 in vitro. Co-immunoprecipitation assays demonstrated that the interaction between CL7MIT and CHMP1B and between CL7MIT and IST1 became stronger when IST1 or CHMP1B was additionally coexpressed, suggesting formation of ternary complex of calpain-7, IST1 and CHMP1B. Moreover, subcellular fractionation analyses revealed increase of calpain-7 in membrane/organelle fractions by concomitant overexpression of these ESCRT-III family member proteins.     

Proteasome activity imaging and profiling characterizes bacterial effector syringolin A

Syringolin A (SylA) is a nonribosomal cyclic peptide produced by the bacterial pathogen Pseudomonas syringae pv syringae that can inhibit the eukaryotic proteasome. The proteasome is a multisubunit proteolytic complex that resides in the nucleus and cytoplasm and contains three subunits with different catalytic activities: β1, β2, and β5. Here, we studied how SylA targets the plant proteasome in living cells using activity-based profiling and imaging. We further developed this technology by introducing new, more selective probes and establishing procedures of noninvasive imaging in living Arabidopsis (Arabidopsis thaliana) cells. These studies showed that SylA preferentially targets β2 and β5 of the plant proteasome in vitro and in vivo. Structure-activity analysis revealed that the dipeptide tail of SylA contributes to β2 specificity and identified a nonreactive SylA derivative that proved essential for imaging experiments. Interestingly, subcellular imaging with probes based on epoxomicin and SylA showed that SylA accumulates in the nucleus of the plant cell and suggests that SylA targets the nuclear proteasome. Furthermore, subcellular fractionation studies showed that SylA labels nuclear and cytoplasmic proteasomes. The selectivity of SylA for the catalytic subunits and subcellular compartments is discussed, and the subunit selectivity is explained by crystallographic data.     

Selective modification of the catalytic subunit of cAMP-dependent protein kinase with sulfhydryl-specific fluorescent probes

The catalytic subunit of cAMP-dependent protein kinase contains only two cysteine residues, and the side chains of both Cys 199 and Cys 343 are accessible. Modification of the catalytic subunit by a variety of sulfhydryl-specific reagents leads to the loss of enzymatic activity. The differential reactivity of the two sulfhydryl groups at pH 6.5 has been utilized to selectively modify each cysteine with the following fluorescent probes: 3,6,7-trimethyl-4-(bromomethyl)-1,5-diazabicyclo[3.3.0]octa-3,6-diene- 2,8-dione, N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine, and 4-[N-[(iodoacetoxy)ethyl]-N-methyl-amino]-7-nitrobenz-2-oxa-1,3-diazole. The most reactive cysteine is Cys 199, and exclusive modification of this residue was achieved with each reagent at pH 6.5. Modification of Cys 343 required reversible blocking of Cys 199 with 5,5'-dithiobis(2-nitrobenzoic acid) followed by reaction of Cys 343 with the fluorescent probe at pH 8.3. Treatment of this modified catalytic subunit with reducing reagent restored catalytic activity by unblocking Cys 199. In contrast, catalytic subunit that was selectively labeled at Cys 199 by the fluorescent probes was catalytically inactive. Even though Cys 199 is presumably close to the interaction site between the regulatory subunit and the catalytic subunit, all of the modified C-subunits retained the capacity to aggregate with the type II regulatory subunit in the absence of cAMP, and the resulting holoenzymes were dissociated in the presence of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth hormone-dependent changes in the rat lung proteome during alveorization

Growth hormone (GH) mRNA and protein have recently been demonstrated in the rat lung throughout the period of alveolarization (day 4-14 postnatally). The functional significance of this finding was therefore assessed, by determining the effects of GH mRNA knockout using aerosolized antisense oligodeoxynucleotides (ODN) directed against the GH gene. In a preliminary experiment, the effectiveness of the antisense GH ODN was demonstrated in a lung Type II epithelial cell line (L2 cells), in which constitutive GH mRNA expression was completely abolished by GH ODN transfection. Administration of the aerosolized GH ODN to 4-day-old rats for 10 days was accompanied by a widespread presence of its delivery liposomes within lung cells. Aerosolized GH ODN treatment decreased lung concentrations of IGF (insulin-like growth factor)-1 and increased concentrations of albumin, calcyclin binding protein, superoxide dismutase, RNA binding protein motif 3, and the alpha- and beta-subunits of ATP synthase and electron transfer flavoprotein. At least 32 other proteins (identified by 2D gel electrophoresis) were also significantly affected by the antisense GH ODN treatment. By changing the lung proteome, these results indicate hitherto unsuspected autocrine/paracrine actions of GH in developmental lung function.     

Molecular interaction between bleomycin and amsacrine in the presence of cupric ions

The antineoplastic activity of m-AMSA [4'-(9-acridinylamino)-methanesulfon-m-anisidide] has been related to its ability to produce oxygenated free radical during its oxidation to a quinonimine form, in the presence of cupric ions. It has been demonstrated here that the rate of the oxidation is greatly increased by the addition of bleomycin (Blm), another antitumor agent, which is able to complex metallic ions. The catalytic role of Blm has been established on the basis of kinetics measurements and the occurrence of an intermediary ternary complex Blm-m-AMSA-Cu(II) has been demonstrated by circular dichroism and polarography experiments.     

Structure-dependent modulation of alpha interferon production by porcine circovirus 2 oligodeoxyribonucleotide and CpG DNAs in porcine peripheral blood mononuclear cells

DNA sequences containing CpG motifs are recognized as immunomodulators in several species. Phosphodiester oligodeoxyribonucleotides (ODNs) representing sequences from the genome of porcine circovirus type 2 (PCV2) have been identified as potent inducers (ODN PCV2/5) or inhibitors (ODN PCV2/1) of alpha interferon (IFN-alpha) production by porcine peripheral blood mononuclear cells (poPBMCs) in vitro. In this study, the IFN-alpha-inducing or -inhibitory activities of specific phosphodiester ODNs were demonstrated to be dependent on their ability to form secondary structures. When a poly(G) sequence was added to a stimulatory self-complementary ODN, high levels of IFN-alpha were elicited, and the induction was not dependent on pretreatment with the transfecting agent Lipofectin. In addition, the IFN-alpha-inducing ODN required the presence of an intact CpG dinucleotide, whereas the inhibitory activity of ODN PCV2/1 was not affected by methylation or removal of the central CpG dinucleotide. Of particular significance, the IFN-alpha inhibition elicited by ODN PCV2/1 was only effective against induction stimulated by DNA control inducers and not RNA control inducers, indicating activity directed to TLR9 signaling. The PCV2 genome as a whole was demonstrated to induce IFN-alpha in cultures of poPBMCs, and the presence of immune modulatory sequences within the genome of PCV2 may, therefore, have implications with regard to the immune evasion mechanisms utilized by PCV2.     

Nitric oxide inhibits peroxidase activity of cytochrome c.cardiolipin complex and blocks cardiolipin oxidation

The increased production of NO during the early stages of apoptosis indicates its potential involvement in the regulation of programmed cell death through yet to be identified mechanisms. Recently, an important role for catalytically competent peroxidase form of pentacoordinate cytochrome c (cyt c) in a complex with a mitochondria-specific phospholipid, cardiolipin (CL), has been demonstrated during execution of the apoptotic program. Because the cyt c.CL complex acts as CL oxygenase and selectively oxidizes CL in apoptotic cells in a reaction dependent on the generation of protein-derived (tyrosyl) radicals, we hypothesized that binding and nitrosylation of cyt c regulates CL oxidation. Here we demonstrate by low temperature electron paramagnetic resonance spectroscopy that CL facilitated interactions of ferro- and ferri-states of cyt c with NO and NO(-), respectively, to yield a mixture of penta- and hexa-coordinate nitrosylated cyt c. In the nitrosylated cyt c.CL complex, NO chemically reacted with H(2)O(2)-activated peroxidase intermediates resulting in their reduction. A dose-dependent quenching of H(2)O(2)-induced protein-derived radicals by NO donors was shown using direct electron paramagnetic resonance measurements as well as immuno-spin trapping with antibodies against protein 5,5-dimethyl-1-pyrroline N-oxide-nitrone adducts. In the presence of NO donors, H(2)O(2)-induced oligomeric forms of cyt c positively stained for 3-nitrotyrosine confirming the reactivity of NO toward tyrosyl radicals of cyt c. Interaction of NO with the cyt c.CL complex inhibited its peroxidase activity with three different substrates: CL, etoposide, and 3,3'-diaminobenzidine. Given the importance of CL oxidation in apoptosis, mass spectrometry analysis was utilized to assess the effects of NO on oxidation of 1,1'2,2'-tertalinoleoyl cardiolipin. NO effectively inhibited 1,1'2,2'-tertalinoleoyl cardiolipin oxidation catalyzed by the peroxidase activity of cyt c. Thus, NO can act as a regulator of peroxidase activity of cyt c.CL complexes.     

On-line preparation of peroxymonocarbonate and its application for the study of energy transfer chemiluminescence to lanthanide inorganic coordinate complexes.

It has been shown that peroxymonocarbonate ion (HCO(4) (-)) is a potent oxidant. In this study, a flow-injection system was developed in order to prepare on-line HCO(4) (-) ion and the optimum conditions for the on-line preparation of HCO(4) (-) were studied in detail. We used 99% (13)C-enriched NaHCO(3) to examine peroxymonocarbonate by (13)C-NMR at 25 degrees C. An ultra-weak chemiluminescence (CL) was observed after mixing H(2)O(2) and sodium bicarbonate in an organic co-solvent that can accelerate the formation of HCO(4) (-) ion. When lanthanide inorganic coordinate complex, Eu(II)-EDTA, was added into this HCO(4) (-) system, the CL intensity was significantly enhanced. The CL mechanism was investigated by various methods. The experimental results indicate that peroxymonocarbonate oxidizes Eu(II) to Eu(III) and produces singlet oxygen; meanwhile, the energy originating from dimers of singlet oxygen is accepted by the Eu(III)-EDTA(-) complex. The excited Eu(III) ions undergo radiative deactivation and emit CL.     

Nanogels for oligonucleotide delivery to the brain

Systemic delivery of oligonucleotides (ODN) to the central nervous system is needed for development of therapeutic and diagnostic modalities for treatment of neurodegenerative disorders. Macromolecules injected in blood are poorly transported across the blood-brain barrier (BBB) and rapidly cleared from circulation. In this work we propose a novel system for ODN delivery to the brain based on nanoscale network of cross-linked poly(ethylene glycol) and polyethylenimine ("nanogel"). The methods of synthesis of nanogel and its modification with specific targeting molecules are described. Nanogels can bind and encapsulate spontaneously negatively charged ODN, resulting in formation of stable aqueous dispersion of polyelectrolyte complex with particle sizes less than 100 nm. Using polarized monolayers of bovine brain microvessel endothelial cells as an in vitro model this study demonstrates that ODN incorporated in nanogel formulations can be effectively transported across the BBB. The transport efficacy is further increased when the surface of the nanogel is modified with transferrin or insulin. Importantly the ODN is transported across the brain microvessel cells through the transcellular pathway; after transport, ODN remains mostly incorporated in the nanogel and ODN displays little degradation compared to the free ODN. Using mouse model for biodistribution studies in vivo, this work demonstrated that as a result of incorporation into nanogel 1 h after intravenous injection the accumulation of a phosphorothioate ODN in the brain increases by over 15 fold while in liver and spleen decreases by 2-fold compared to the free ODN. Overall, this study suggests that nanogel is a promising system for delivery of ODN to the brain.     

Catabolism of the octadecaneuropeptide ODN by prolyl endopeptidase: identification of an unusual cleavage site

The octadecaneuropeptide ODN (QATVGDVNTDRPGLLDLK), a biologically active fragment of diazepam-binding inhibitor, exerts a number of behavioral and neurophysiological activities. The presence of a proline residue in the sequence of ODN led us to investigate the role of proline endopeptidase (PEP) in the catabolism of this neuropeptide. The effect of PEP on the breakdown of ODN and related analogs was studied by combining RP-HPLC analysis and MALDI-TOF MS characterization. Incubation of ODN with PEP generated two products, i.e. ODN3-18 and ODN5-18 which resulted from cleavage of the Ala-Thr and Val-Gly peptide bonds. S 17092, a specific PEP inhibitor, significantly reduced the PEP-induced cleavages of ODN. Similarly, [Ala2]OP showed S 17092-sensitive post-alanine cleavage, while [pGlu1]ODN and OP (ODN11-18) were not catabolized by the enzyme. For all these peptides, cleavage of the Pro-Gly peptide bond by PEP was never observed, even after prolonged incubation times. In contrast, PEP hydrolyzed human urotensin II at the canonical post-proline site. Collectively, these data suggest that the Ala2 residue is the preferential cleavage site of ODN and that the Pro-Gly bond of ODN is not hydrolyzed by PEP. In addition, this study reveals for the first time that the endoproteolytic activity of PEP can specifically take place after a valine moiety.     

Analysis of the spatial and temporal expression pattern directed by the Populus tomentosa 4-coumarate:CoA ligase Pto4CL2 promoter in transgenic tobacco

4-Coumarate:CoA ligase (4CL) is a key enzyme in the phenylpropanoid synthesis pathway. The Pto4CL2 promoter was cloned from Populus tomentosa Carr. and fused to the reporter gene encoding β-glucuronidase (GUS); the complex expression patterns directed by the Pto4CL2 promoter were then characterized in Nicotiana tabacum Xanthi by histochemical assays. The promoter 5′-deletion and histochemical assay conducted on transformants indicated that the ?317 to ?292 nt region supports Pto4CL2 expression in the epidermis and petals and the deletion of the ?266 to ?252 nt region resulted in the loss of tissue specificity and a dramatic reduction in GUS activity. Furthermore, electrophoretic mobility shift assays testified that an adenine and cytosine-rich element (?264 to ?255 nt) and an abscisic acid-responsive element (?242 to ?235 nt) in the Pto4CL2 promoter would have functions for the complex expression profiling and efficient basal expression, respectively. These results further clarify the mode of the regulatory expression of class II 4CL promoters in higher plants.     

Dissection study on the severe acute respiratory syndrome 3C-like protease reveals the critical role of the extra domain in dimerization of the enzyme: defining the extra domain as a new target for design of highly specific protease inhibitors

The severe acute respiratory syndrome (SARS) 3C-like protease consists of two distinct folds, namely the N-terminal chymotrypsin fold containing the domains I and II hosting the complete catalytic machinery and the C-terminal extra helical domain III unique for the coronavirus 3CL proteases. Previously the functional role of this extra domain has been completely unknown, and it was believed that the coronavirus 3CL proteases share the same enzymatic mechanism with picornavirus 3C proteases, which contain the chymotrypsin fold but have no extra domain. To understand the functional role of the extra domain and to characterize the enzyme-substrate interactions by use of the dynamic light scattering, circular dichroism, and NMR spectroscopy, we 1) dissected the full-length SARS 3CL protease into two distinct folds and subsequently investigated their structural and dimerization properties and 2) studied the structural and binding interactions of three substrate peptides with the entire enzyme and its two dissected folds. The results lead to several findings; 1) although two dissected parts folded into the native-like structures, the chymotrypsin fold only had weak activity as compared with the entire enzyme, and 2) although the chymotrypsin fold remained a monomer within a wide range of protein concentrations, the extra domain existed as a stable dimer even at a very low concentration. This observation strongly indicates that the extra domain contributes to the dimerization of the SARS 3CL protease, thus, switching the enzyme from the inactive form (monomer) to the active form (dimer). This discovery not only separates the coronavirus 3CL protease from the picornavirus 3C protease in terms of the enzymatic mechanism but also defines the dimerization interface on the extra helical domain as a new target for design of the specific protease inhibitors. Furthermore, the determination of the preferred solution conformation of the substrate peptide S1 together with the NMR differential line-broadening and transferred nuclear Overhauser enhancement study allows us to pinpoint the bound structure of the S1 peptide.