Detection of protein–protein interactions by a combination of a novel cytoplasmic membrane targeting system of recombinant proteins and fluorescence resonance energy transfer

A novel protein molecular targeting system was created using a cytoplasmic face of a plasma membrane-targeting system in Saccharomyces cerevisiae. The technique involves a molecular display for the creation of a novel reaction site and interaction sites in the field of biotechnology. In a model system, a fluorescent protein was targeted as a reporter to the cytoplasmic face of the plasma membrane. The C-terminal transmembrane domain (CTM) of Ras2p and Snc2p was examined as the portions with anchoring ability to the cytoplasmic face of the plasma membrane. We found that the CTM of Snc2p targeted the enhanced cyan fluorescent protein (ECFP)–protein A fusion protein on the cytoplasmic face of the plasma membrane more strongly than that of Ras2p. To develop it for use as a detection system for protein–protein interactions, the Fc fragment of IgG (Fc) was genetically fused with the enhanced yellow fluorescent protein (EYFP) and expressed in the cytoplasm of the ECFP–protein A-anchored cell. A microscopic analysis showed that fluorescence resonance energy transfer (FRET) between ECFP–protein A and EYFP–Fc occurred, and the change in fluorescence was observed on the cytoplasmic face of the plasma membrane. The detection of protein–protein interactions at the cytoplasmic face of a plasma membrane using FRET combined with a cytoplasmic face-targeting system for proteins provides a novel method for examining the molecular interactions of cytoplasmic proteins, in addition to conventional methods, such as the two-hybrid method in the nuclei. S. Shibasaki and K. Kuroda equally contributed to this work     

Comparative evaluation of genotoxicity induced by nitrofurazone in two ciliated protozoa by detecting DNA strand breaks and DNA–protein crosslinks

Although organism-specific factors related to individual indicator organisms have hampered the use of bioassays for the evaluation of environmental risk in practice, the importance of understanding organism-specific factors when selecting model organisms has also not yet been fully recognized. In this work, genotoxicity was evaluated in the ciliated protozoa, Euplotes vannus and Pseudokeronopsis rubra, when exposed to graded doses of nitrofurazone for several discrete durations. Genotoxicity was expressed based on the LD₅₀ and was determined by assessing DNA strand breaks (through alkaline comet assay) and DNA–protein crosslinks (DPCs), by means of a KCl–SDS precipitation assay. It was found that E. vannus generally had lower LD₅₀'s than P. rubra (P < 0.05), and that the LD₅₀ values decreased in both ciliates as the exposure durations increased. Compared to the control groups, the nitrofurazone treated E. vannus generally produced more DNA strand breaks (P < 0.05), but for DPCs (P > 0.05). The relationship between these parameters was reversed in the case of P. rubra. Biphasic dose–response relationships were generally detected between nitrofurazone and genotoxicity parameters, however, parameters for DNA strand breaks presented significantly positive correlations between each other (P < 0.05), but showed nearly no significant correlations with DPC induction. In brief, our findings confirmed nitrofurazone-induced genotoxicity and the important role of organism-specific factors in the selection of model organisms from ciliated protozoa for environmental monitoring and risk assessment in aquaculture.     

Detection of an interaction between prion protein and neuregulin I-β1 by fluorescence resonance energy transfer analysis

Cellular prion protein (PrP) copurifies with neuregulin type I-β1 (NRG I-β1), but no interaction has been detected by a general immunoprecipitation study. We speculate that PrP interacts with NRG I-β1. Here, the interaction of PrP with NRG I-β1 was detected by measuring fluorescence resonance energy transfer (FRET) between enhanced blue (EBFP) and enhanced green (EGFP) fluorescent protein-fusion proteins. Full-length PrP interacted with EGFP in addition to NRG I-β1. From this result, we deduced that PrP interacts with EGFP through its unstructured N-terminal domain. We therefore detected FRET between PrP deleting the N-terminal domain and NRG I-β1. In contrast, the C-terminal domain of PrP interacted with NRG I-β1 and the proteins dissociated completely in the presence of sodium chloride. This interaction occurs at the nanomolar level, which is important for the reaction to be functional in organisms. We concluded that PrP interacted with NRG I-β1 through its C-terminal domain.     

Detection of β-thalassemia and hemoglobin E genes in Thai by a DNA amplification technique

Summary Enzymatic DNA amplification and polyacrylamide gel electrophoresis, which demonstrate different sizes of DNA fragments, were used to detect the common mutations causing -thalassemia and hemoglobin (Hb) E in Thai people. The 4-bp deletion at codons 41 and 42 can be detected directly by polyacrylamide gel electrophoresis and ethidium bromide staining. Whereas the nonsense mutations at codon 17 (AAG TAG) and Hb E (GAGAAG at codon 26) were detected after digestion of the amplified DNA with the enzymes MaeI and MnlI, respectively.     

Comparison of procainamide and 2-aminobenzamide labeling for profiling and identification of glycans by liquid chromatography with fluorescence detection coupled to electrospray ionization–mass spectrometry

One of the most widely used methods for glycan analysis is fluorescent labeling of released glycans followed by hydrophilic interaction chromatography–(ultra-)high-performance liquid chromatography [HILIC–(U)HPLC]. Here, we compare the data obtained by (U)HPLC–fluorescence (FLR) coupled to electrospray ionization–mass spectrometry (ESI–MS) for procainamide and 2-aminobenzamide (2-AB)-labeled N-glycans released from human immunoglobulin G (IgG). Fluorescence profiles from procainamide show comparable chromatographic separation to those obtained for 2-AB but gave higher fluorescence intensity as well as significantly improved ESI efficiency (up to 30 times that of 2-AB). Thus, labeling with procainamide increases the ability to identify minor glycan species that may have significant biological activity.     

Interactions of primary neuroepithelial progenitor and brain endothelial cells： distinct effect on neural progenitor maintenance and differentiation by soluble factors and direct contact

Neurovascular interactions are crucial for the normal development of the central nervous system. To study suchinteractions in primary cultures, we developed a procedure to simultaneously isolate neural progenitor and endothelialcell fractions from embryonic mouse brains. Depending on the culture conditions endothelial cells were found to favormaintenance of the neuroprogenitor phenotype through the production of soluble factors, or to promote neuronal differ-entiation of neural progenitors through direct contact. These apparently opposing effects could reflect differential cellularinteractions needed for the proper development of the brain.     

Comparative Study of Modification of DNA and RNA by Oligo(2′-O-Methylribonucleotide) Derivatives

Abstract

The results of modification of the model DNA and RNA targets by the alkylating derivatives of 2′-O-methylribo-, ribo-, and deoxyhexanucleotides in the presence and absence of effectors (N-(2-hydroxyethyl)phenazinium derivatives of the same type of octanucleotides) are presented. It has been shown that the alkylating 4(N-methyl-N-2-chloroethyl-)benzylmethylamidophosphate derivatives of oligo(2′-O-methylribonucleotides) are the high effective reagents for the site specific modification of nucleic acids especially RNA.

     

Structures and stabilities of ScBn (n = 1–12) clusters: an ab initio investigation

The geometries, stabilities, and electronic properties of ScB_n (n?=?1–12) clusters have been systematically investigated by using density functional theory B3LYP method and coupled–cluster theory CCSD(T) method. It is found that the ground state isomers of ScB_n have planar or quasi–planar structure when n?≤?6, which can be viewed as a B atom of the corresponding B_n+1 cluster is substituted by a Sc atom. From n?≥?7, the ground state isomers favor nest–like structure, in which the Sc atom sits on a nest–like B_n cluster. The calculated second–order differences of energies manifest that the magic numbers of stability are n?=?3, 7, 8, 9 and 11 for the ScB _n clusters. Further analysis indicates that the ScB₇ cluster with C _6v symmetry represents the outstanding stable ScB_n cluster, as confirmed by its electronic structure and molecular orbitals.     

Recognition of β-calcineurin by the domains of calmodulin: thermodynamic and structural evidence for distinct roles

Calcineurin (CaN, PP2B, PPP3), a heterodimeric Ca²⁺‐calmodulin‐dependent Ser/Thr phosphatase, regulates swimming in Paramecia, stress responses in yeast, and T‐cell activation and cardiac hypertrophy in humans. Calcium binding to CaN_B (the regulatory subunit) triggers conformational change in CaN_A (the catalytic subunit). Two isoforms of CaN_A (α, β) are both abundant in brain and heart and activated by calcium‐saturated calmodulin (CaM). The individual contribution of each domain of CaM to regulation of calcineurin is not known. Hydrodynamic analyses of (Ca²⁺)₄‐CaM_1–148 bound to βCaNp, a peptide representing its CaM‐binding domain, indicated a 1:1 stoichiometry. βCaNp binding to CaM increased the affinity of calcium for the N‐ and C‐domains equally, thus preserving intrinsic domain differences, and the preference of calcium for sites III and IV. The equilibrium constants for individual calcium‐saturated CaM domains dissociating from βCaNp were ～1 μM. A limiting K_d ≤ 1 nM was measured directly for full‐length CaM, while thermodynamic linkage analysis indicated that it was approximately 1 pM. βCaNp binding to ¹⁵N‐(Ca²⁺)₄‐CaM_1–148 monitored by ¹⁵N/¹HN HSQC NMR showed that association perturbed the N‐domain of CaM more than its C‐domain. NMR resonance assignments of CaM and βCaNp, and interpretation of intermolecular NOEs observed in the ¹³C‐edited and ¹²C‐¹⁴N‐filtered 3D NOESY spectrum indicated anti‐parallel binding. The sole aromatic residue (Phe) located near the βCaNp C‐terminus was in close contact with several residues of the N‐domain of CaM outside the hydrophobic cleft. These structural and thermodynamic properties would permit the domains of CaM to have distinct physiological roles in regulating activation of βCaN. Proteins 2011. © 2010 Wiley‐Liss, Inc.     

Construction and characterization of novel chimeric β-glucosidases with Cellvibrio gilvus (CG) and Thermotoga maritima (TM) by overlapping PCR

In order to create novel β-glucosidase constructs, 8 kinds of chimeric β-glucosidases were constructed using overlapping polymerase chain reaction (PCR) based on Cellvibrio gilvus (CG) and Thermotoga maritima (TM) genes. Two kinds of novel chimeric β-glucosidases (No. 6 and No. 8 type) were selected and their properties characterized. SDS-PAGE analysis showed that both constructs had a molecular mass of 80 kDa. The optimum pH of No. 6 chimeric β-glucosidase was found to be 3.0 and 5.0, showing varying maximum activity according to the buffer used. No. 8 chimeric enzyme was found to be optimally active at a pH of 4.5 and the optimum temperature of No.6 and No.8 chimeric β-glucosidases was reported to be 60°C, respectively. The Km values of both novel chimeric enzymes were calculated to be 0.012 mM and 0.0082 mM, respectively and the characteristics of the novel chimeric enzymes were to lie between those of the parental enzymes.     

Comparison Between poly(dG-dC)·poly(dG-dC) and DNA Modified by cis-diamminedichloroplatinum (II): Immunological and Spectroscopic Studies

Abstract

The importance of the base composition and of the conformation of nucleic acids in the reaction with the drug cis-diamminedichloroplatinum(II) has been studied by competition experiments between the drug and several double-stranded polydeoxyribonucleotides. Binding to poly(dG)·poly(dC) is larger than to poly (dG-dC)·poly(dG-dC). There is no preferential binding in the competition between poly(dG-dC) ·poly(dG-dC), poly(dA-dC) ·poly(dG-dT) and poly(dA-dG)·poly(dC-dT). In the competition between poly(dG-dC) ·poly (dG-dC) (B conformation) and poly(dG-br⁵dC) ·poly(dG-br⁵dC) (Z conformation), the drug binds equally well to both polynucleotides. In natural DNA, modification of guanine residues in (GC)_n·(GC)_nsequences by the drug has been revealed by the inhibition of cleavage of these sequences by the restriction enzyme BssHII. By means of antibodies to platinated poly(dG-dC), it is shown that some of the adducts formed in platinated poly(dG-dC) are also formed in platinated pBR322 DNA. The type of adducts recognized by the antibodies is not known. Thin layer chromatography of the products after chemical and enzymatic hydrolysis of platinated poly(dG-dC) suggests that interstrand cross-links are formed. Finally, the conformations of poly(dG-m⁵dC) modified either by cis-diamminedichloroplatinum(II) or by trans-diammine- dichloroplatinum(II) have been compared by circular dichroism. Both the cis-isomer and the trans-isomer stabilize the Z conformation when they bind to poly(dG-m⁵dC) in the Z conformation. When they bind to poly(dG-m⁵dC) in the B conformation, the conformations of poly(dG-m⁵dC) modified by the cis or the trans-isomer are different. Moreover, the cis-isomer facilitates the B form-Z form transition of the unplatinated regions while the trans-isomer makes it more difficult.     

Mass spectrometry–based relative quantification of proteins in precatalytic and catalytically active spliceosomes by metabolic labeling (SILAC), chemical labeling (iTRAQ), and label-free spectral count

The spliceosome undergoes major changes in protein and RNA composition during pre-mRNA splicing. Knowing the proteins—and their respective quantities—at each spliceosomal assembly stage is critical for understanding the molecular mechanisms and regulation of splicing. Here, we applied three independent mass spectrometry (MS)–based approaches for quantification of these proteins: (1) metabolic labeling by SILAC, (2) chemical labeling by iTRAQ, and (3) label-free spectral count for quantification of the protein composition of the human spliceosomal precatalytic B and catalytic C complexes. In total we were able to quantify 157 proteins by at least two of the three approaches. Our quantification shows that only a very small subset of spliceosomal proteins (the U5 and U2 Sm proteins, a subset of U5 snRNP-specific proteins, and the U2 snRNP-specific proteins U2A′ and U2B′′) remains unaltered upon transition from the B to the C complex. The MS-based quantification approaches classify the majority of proteins as dynamically associated specifically with the B or the C complex. In terms of experimental procedure and the methodical aspect of this work, we show that metabolically labeled spliceosomes are functionally active in terms of their assembly and splicing kinetics and can be utilized for quantitative studies. Moreover, we obtain consistent quantification results from all three methods, including the relatively straightforward and inexpensive label-free spectral count technique.     

N-(4-Substituted-benzoyl)-N'-(β-d-glucopyranosyl)ureas as inhibitors of glycogen phosphorylase: Synthesis and evaluation by kinetic, crystallographic, and molecular modelling methods

N-(4-Substituted-benzoyl)-N'-(β-d-glucopyranosyl) ureas (substituents: Me, Ph, Cl, OH, OMe, NO(2), NH(2), COOH, and COOMe) were synthesised by ZnCl(2) catalysed acylation of O-peracetylated β-d-glucopyranosyl urea as well as in reactions of O-peracetylated or O-unprotected glucopyranosylamines and acyl-isocyanates. O-deprotections were carried out by base or acid catalysed transesterifications where necessary. Kinetic studies revealed that most of these compounds were low micromolar inhibitors of rabbit muscle glycogen phosphorylase b (RMGPb). The best inhibitor was the 4-methylbenzoyl compound (K(i)=2.3μM). Crystallographic analyses of complexes of several of the compounds with RMGPb showed that the analogues exploited, together with water molecules, the available space at the β-pocket subsite and induced a more extended shift of the 280s loop compared to RMGPb in complex with the unsubstituted benzoyl urea. The results suggest the key role of the water molecules in ligand binding and structure-based ligand design. Molecular docking study of selected inhibitors was done to show the ability of the binding affinity prediction. The binding affinity of the highest scored docked poses was calculated and correlated with experimentally measured K(i) values. Results show that correlation is high with the R-squared (R(2)) coefficient over 0.9.     

Effects on the conformation of FVIIa by sTF and Ca²⁺ binding: studies of fluorescence resonance energy transfer and quenching

The apparent length of FVIIa in solution was estimated by a FRET analysis. Two fluorescent probes, fluorescein (Fl-FPR) and a rhodamine derivative (TMR), were covalently attached to FVIIa. The binding site of Fl-FPR was in the protease domain whereas TMR was positioned in the Gla domain, thus allowing a length measure over virtually the whole extension of the protein. From the FRET measurements, the distances between the two probes were determined to be 61.4 for free FVIIa and 65.5? for FVIIa bound to soluble tissue factor (sTF). These seemingly short distances, compared to those anticipated based on the complex crystal structure, require that the probes stretch towards each other. Thus, the apparent distance from the FRET analysis was shown to increase with 4? upon formation of a complex with sTF in solution. However, considering how protein dynamics, based on recent molecular dynamics simulations of FVIIa and sTF:FVIIa (Y.Z. Ohkubo, J.H. Morrissey, E. Tajkhorshid, J. Thromb. Haemost. 8 (2010) 1044-1053), can influence the apparent fluorescence signal our calculations indicated that the global average conformation of active-site inhibited FVIIa is nearly unaltered upon ligation to sTF. It is known from amidolytic activity measurements that Ca(2+) binding leads to activation of FVIIa, but we have for the first time directly demonstrated conformational changes in the environment of the active site upon Ca(2+) binding. Interestingly, this Ca(2+)-induced conformational change can be noted even in the presence of an inhibitor. Forming a complex with sTF further stabilized this conformational change, leading to a more inaccessible active-site located probe.     

Discovery of novel lipophilic inhibitors of OXA-10 enzyme (class D β-lactamase) by screening amino analogs and homologs of citrate and isocitrate

Aminocitrate (and homolog) derivatives have been prepared by bis-alkylation of glycinate Schiff bases with bromoacetates (and ethyl acrylate), followed by N-acylation and esters (partial or complete) deprotection. Aminoisocitrate was similarly obtained by mono-alkylation with diethyl fumarate. Evaluation against representative β-lactamases revealed that the free acid derivatives are modest inhibitors of class A enzymes, whilst their benzyl esters showed a good inhibition of OXA-10 (class D enzyme). A docking experiment featured hydrophobic interactions in the active site.     

Selective Gαi subunits as novel direct activators of transient receptor potential canonical (TRPC)4 and TRPC5 channels

The ubiquitous transient receptor potential canonical (TRPC) channels function as non-selective, Ca(2+)-permeable channels and mediate numerous cellular functions. It is commonly assumed that TRPC channels are activated by stimulation of Gα(q)-PLC-coupled receptors. However, whether the Gα(q)-PLC pathway is the main regulator of TRPC4/5 channels and how other Gα proteins may regulate these channels are poorly understood. We previously reported that TRPC4/TRPC5 can be activated by Gα(i). In the current work, we found that Gα(i) subunits, rather than Gα(q), are the primary and direct activators of TRPC4 and TRPC5. We report a novel molecular mechanism in which TRPC4 is activated by several Gα(i) subunits, most prominently by Gα(i2), and TRPC5 is activated primarily by Gα(i3). Activation of Gα(i) by the muscarinic M2 receptors or expression of the constitutively active Gα(i) mutants equally and fully activates the channels. Moreover, both TRPC4 and TRPC5 are activated by direct interaction of their conserved C-terminal SESTD (SEC14-like and spectrin-type domains) with the Gα(i) subunits. Two amino acids (lysine 715 and arginine 716) of the TRPC4 C terminus were identified by structural modeling as mediating the interaction with Gα(i2). These findings indicate an essential role of Gα(i) proteins as novel activators for TRPC4/5 and reveal the molecular mechanism by which G-proteins activate the channels.     

On the mechanism of direct conversion of cellulose to ethanol by Fusarium oxysporum: effect of cellulase and β-glucosidase

Summary The effects of the three main enzymes involved in cellulose saccharification, namely cellobiohydrolase, carboxymethylcellulase and -glucosidase, on the direct conversion of cellulose to ethanol by Fusarium oxysporum F3 were investigated. Ethanol production was not affected when the activity of the former two enzymes was varied within a wide range. By contrast, -glucosidase markedly affected ethanol production showing an optimum level of 0.7–0.8 unit/ml growth medium. A significant decrease of cellulose bioconversion time to ethanol was obtained when -glucosidase activity was adjusted to this optimal level at the beginning of the fermentation process. Offprint requests to: B. J. Macris