Site-specific Reaction Research Articles

Site-Specific Modification Of Proteins Mediated By Transglutaminase

AbstractTransglutaminase (TGase) catalyzes an acyl transfer reaction between the [gamma]-carboxamide group of glutamine (Gln) and the [epsilon]-amino group of lysine (Lys) residues to form a stable amide bond. The TGase reaction can be used for bioconjugation of an amino-derivative of poly-ethylene glycol (PEG) to protein drugs, leading to PEGylated proteins that display increased bioactivity and stability. The procedure was shown to lead to site-specific bioconjugation of few proteins, thus offering a valid alternative to the chemical methods of PEGylation in current use (1). Moreover, TGase can be used for site-specifically labeling proteins with fluorescent groups at the level of Gln or Lys for diagnostic applications. The TGase attack is not entirely dependent on a specific consensus sequence around the Gln or Lys residues. However, a correlation between the TGase-mediated sites of PEGylation and the chain flexibility has been observed (2). Our study was focused to elucidate the molecular features favoring the TGase mediated site-specific reactions on two extensively studied model proteins, i.e. apomyoglobin (apoMb) and lysozyme. Besides amino-PEG, we used dansyl-cadaverine as an acyl acceptor and N-carbobenzoxy-Gln-Gly-OH as acyl donor for the TGase reactions. The sites of protein modification were determined by fingerprinting and ESI mass spectrometry. Myoglobin in its holo form is not susceptible to TGase reactions due to its rigid conformation,but the apo form was conjugated with PEG and fluorescent labels at the level of helix F (chain segment 82–99). The NMR study on apoMb had earlier demonstrated increased flexibility of the helix F (3) and, moreover, several proteases cleave the 153-residue chain of apoMb at the level of helix F only (4). Therefore, the chain region attacked by both TGase and proteases is a flexible or unfolded site(s). Lysozyme in its disulfide crosslinked native state is highly resistant to proteases and TGase attack. However, the more dynamic three-disulphide derivative of lysozyme, lacking the Cys6–Cys127 disulfide bridge, is susceptible to TGase-mediated reactions, as well as limited proteolysis. These results indicate that the sites of TGase reactions and the sites of limited proteolysis have a clear analogy for their presence in flexible/disordered regions of protein substrates. Overall, our studies clearly demonstrate that TGase-mediated reactions occur only at disordered chain regions, as evidenced by the correlation between sites of the TGase reaction and sites of enhanced chain flexibility, the latter deduced from the crystallographic Bfactor.

Paclitaxel-induced Neutrophilic Adverse Reaction and Acral Erythema

Paclitaxel, a microtubule-stabilizing anti-neoplastic agent that belongs to the family of taxanes, is used in the treatment of advanced and/or refractory lung, breast, head, neck and other epithelial cancers. Site-specific reactions, such as inflammation at injection sites or cellulitis after extravasation (1), have been reported. Adverse cutaneous reactions associated with paclitaxel treatment include bullous fixed drug eruption (2), erythema multiforme (3), pustular eruption (4), scleroderma-like cutaneous lesions (5), onycholysis (6), and acral erythema (one case) (7). Of note, acral erythema (erythrodysesthesia) has been reported with multi-drug chemotherapy protocols that included paclitaxel (8, 9). We report here the first case of paclitaxel-induced neutrophilic adverse reaction with concomitant acral erythema, and highlight the mechanisms that may be involved.

Antioxidant Effects of the Essential Oils of Different Parts of Platycladus orientalis L. (Franco) and Their Components

The essential oils of different parts of Platycladus orientalis (Cupressuceae) were examined for their potential antioxidant activity. The compositions of the essential oils of the plant were studied qualitatively and quantitatively by GC and GC-MS. The main components of the essential oils as well as positive controls were subjected to antioxidant testing. Different methods of, rapid TLC screening, diphenylpicrylhydrazyl (DPPH) assay, desoxyribose assay, assay for site-specific reaction and non-enzymatic lipid peroxidation were used to evaluate the antioxidant activity of the oils and various Positive controls. Positive controls were quercetin, vitamin E, vitamin C, dimethylsulfoxide (DMSO) and buthylhydroxytoluene (BHT). a-Pinene, ß-pinene, sabinene, limonene, À-3-carene and terpinolene were the major constituents of these essential oils. The tested compounds mainly showed antioxidant activity when two different rapid TLC screening methods used to evaluation the antioxidants activity. The abilities of the volatile oils to act as nonspecific donors for hydrogen atoms were checked in the diphenylpicrylhydrazyl, DPPH, assay. In this method the strongest effects were shown by the oil of leaves of P. orientalis. In deoxyribose degradation assay, most of the tested compounds showed some antioxidant activity. In non-enzymatic lipid peroxidation test, the oil obtained from leaves of P. orientalis showed strongest antioxidant activity. The results of the study indicate that the essential oils extracted from different parts of P. orientalis, main components of the oils and positive controls show different antioxidant activity when they tested by different antioxidant methods.

細胞内遺伝子発現制御を目指した遺伝子に対するピンポイント反応の開発

The site-specific reaction with high efficiency and specificity to DNA or RNA has become of great interests, as such modification may have possibility to induce point mutation of a genetic code. The cross-linking reagents within complementary duplexes and triplexes are one of the tools for site-specific modification by forming covalently bond. However, the existing alkylating agents still need further improvement for application in either in vitro or in vivo study. Previously, we have designed a new cross-linking motif, 2-amino-6-vinylpurine(1), toward cytosine by expecting a proximity effect within the complex resembling a natural G-C pair. In this paper, we wish to describe the development of the novel cross-linking reagents based on the structure of 1.

An intein-mediated site-specific click conjugation strategy for improved tumor targeting of nanoparticle systems.

The ability to modify and directly target nanoparticulate carriers has greatly increased their applicability in diagnostic and therapeutic studies. Generally essential to the targeting of nanoparticles is the bioconjugation of targeting ligands to the agent's surface. While bioconjugation techniques have steadily improved in recent years, the field is still plagued with inefficient conjugations reactions and/or the lack of site-specific coupling. To overcome these limitations, click chemistry and expressed protein ligation (EPL) are combined to produce a highly efficient, site-specific reaction. This new EPL-click conjugation strategy is applied to create superparamagnetic iron oxide nanoparticles (SPIO) labeled with HER2/neu affibodies. These HER2-SPIO nanoparticles prove to be highly potent and receptor-specific in both in vitro cell studies and murine tumor models. Moreover, when EPL-click-derived HER2-SPIO are compared with SPIO that had been labeled with HER2 affibodies using other popular bioconjugation methods, they produce a statistically significant improvement in contrast enhancement upon cell binding. The EPL-click system is also successfully extended to other nanoparticle platforms (i.e., liposomes and dendrimers) highlighting the versatility of the approach.

In situstudy of pentacene interaction with archetypal hybrid contacts: Fluorinated versus alkane thiols on gold

One approach developed to improve the performance of bottom contact source/drain electrodes is to treat the contacts with thiols before deposition of the semiconductor. There is evidence indicating that improvement is due to both morphological effects and improved work function matching. Especially suggestive evidence shows that thiols that increase the effective work function of the contacts (e.g., fluorinated thiols) yield better device performance than work function decreasing thiols (e.g., alkane thiols). Here, we compare two technologically relevant thiol treatments, an alkane thiol (1-hexadecanethiol), and a fluorinated thiol (pentafluorobenzenethiol), in pentacene organic field effect transistors. Using in situ semiconductor deposition, x-ray photoemission, and x-ray absorption spectroscopy, we are able to directly observe the interaction between the semiconductor and the thiol-treated gold layers. Our spectroscopic analysis suggests that there is not a site-specific chemical reaction between the pentacene and the thiol molecules. A homogeneous standing-up pentacene orientation was observed in both treated substrates, consistent with the morphological improvement expected from thiol treatment in both samples. Our study shows that both the highest occupied molecular orbital--Fermi level offset and $\text{C}\text{ }1s$ binding energy are shifted in the two thiol systems, which can be explained by varied dipole direction within the two thiols, causing a change in surface potential. The additional improvement of the electrical performance in the pentafluorobenzenethiol case is originated by a reduced hole injection barrier that is also associated with an increase in the density of states in the lowest unoccupied molecular orbital.

Site-Specific DNA Photocleavage and Photomodulation by Oligonucleotide Conjugates

Triplex-forming oligonucleotides have been explored in the last 3 decades as highly specific gene-modifying agents. Such agents have been showed to either upregulate or shut down gene expression in vitro, an outcome that depends on the specifically designed biological system. One interesting approach is to tether to the oligonucleotide a photoreactive moiety. After hybridization to the DNA target (typically single- or double-stranded DNA), a site-specific photoinduced reaction may take place at a timely manner. Further, as the light source may be focused at a certain area, one has control on the location at which the phototriggered DNA modification takes place. In this regard, the use of tissue-penetrating photons (typically 630-950 nm) would be most relevant for in vivo applications as photoactivation may lead to site-specific DNA modification at a specific tissue/organ. In this review we highlight the advances made in this field and discuss the hurdles that lay ahead for the realization of phototriggered triplex-forming oligonucleotides as a solid therapeutic approach.

Crystal Structures of the Substrate-Bound Forms of Red Chlorophyll Catabolite Reductase: Implications for Site-Specific and Stereospecific Reaction

Red chlorophyll catabolite reductase (RCCR) catalyzes the ferredoxin-dependent reduction of the C20/C1 double bond of red chlorophyll catabolite (RCC), the catabolic intermediate produced in chlorophyll degradation. The crystal structure of substrate-free Arabidopsis thaliana RCCR (AtRCCR) demonstrated that RCCR folds into a characteristic α/β/α sandwich, similar to that observed in the ferredoxin-dependent bilin reductase (FDBR) family. Here we have determined the crystal structures of RCC-bound AtRCCR, RCC-bound F218V AtRCCR, and substrate-free F218V AtRCCR, a mutant protein that produces the stereoisomer of primary fluorescent chlorophyll catabolites at the C1 position. RCC is bound to the pocket between the β-sheet and the C-terminal α-helices, as seen in substrate-bound FDBRs, but RCC binding to RCCR is much looser than substrate binding to FDBRs. The loose binding seems beneficial to the large conformational change in RCC upon reduction. Two conserved acidic residues, Glu154 and Asp291, sandwich the C20/C1 double bond of RCC, suggesting that these two residues are involved in site-specific reduction. The RCC in F218V AtRCCR rotates slightly compared with that in wild type to fill in the space generated by the substitution of Phe218 with valine. Concomitantly, the two carboxy groups of Glu154 and Asp291 move slightly away from the C20/C1 double bond. The geometrical arrangement of RCC and the carboxy groups of Glu154 and Asp291 in RCCR would appear to be essential for the stereospecificity of the RCCR reaction.

Small molecule functional analogs of peptides that inhibit λ site-specific recombination and bind Holliday junctions

Our lab has isolated hexameric peptides that are structure-selective ligands of Holliday junctions (HJ), central intermediates of several DNA recombination reactions. One of the most potent of these inhibitors, WRWYCR, has shown antibacterial activity in part due to its inhibition of DNA repair proteins. To increase the therapeutic potential of these inhibitors, we searched for small molecule inhibitors with similar activities. We screened 11 small molecule libraries comprising over nine million individual compounds and identified a potent N-methyl aminocyclic thiourea inhibitor that also traps HJs formed during site-specific recombination reactions in vitro. This inhibitor binds specifically to protein-free HJs and can inhibit HJ resolution by RecG helicase, but only showed modest growth inhibition of bacterial with a hyperpermeable outer membrane; nonetheless, this is an important step in developing a functional analog of the peptide inhibitors.

Elucidating the tertiary structure of protein ions in vacuo with site specific photoinitiated radical reactions.

A new method for identifying residue specific through space contacts as a function of protein secondary and tertiary structure in the gas phase is presented. Photodissociation of a non-native carbon-iodine bond incorporated into Tyr59 of ubiquitin yields a radical site specifically at that residue. The subsequent radical migration is shown to be highly dependent on the structure of the protein. Radical-directed dissociation (RDD) of low charge states, which adopt compact structures, generates backbone fragmentation that is prominently distributed throughout the protein sequence, including residues that are distant in sequence from Tyr59. Higher charge states of ubiquitin, which adopt elongated, unfolded structures, yield RDD that is primarily nearby in sequence to Tyr59. Regardless of which structure is probed, information at the residue-level is obtained by examining specific radical-donor and radical-acceptor pairs. The relative importance of a particular interaction pair for a specific conformation can be revealed by tracking the charge state dependence of the dissociation. Structurally important contact pairs exhibit strong and concerted changes in relative intensities as a function of charge state and can also be used to reveal structural features which persist among different protein structures. Moreover, incorporation of distance constraint information into molecular mechanics conformational searches can be used to drive the search toward relevant conformational space. Implementation of this approach has revealed highly stable, previously undiscovered structures for the +4 and +6 charge states of ubiquitin, which bear little resemblance to the crystal structure.

Protein binding sites involved in the assembly of the KplE1 prophage intasome

The organization of the recombination regions of the KplE1 prophage in Escherichia coli K12 differs from that observed in the λ prophage. Indeed, the binding sites characterized for the IntS integrase, the TorI recombination directionality factor (RDF) and the integration host factor (IHF) vary in number, spacing and orientation on the attL and attR regions. In this paper, we performed site-directed mutagenesis of the recombination sites to decipher if all sites are essential for the site-specific recombination reaction and how the KplE1 intasome is assembled. We also show that TorI and IntS form oligomers that are stabilized in the presence of their target DNA. Moreover, we found that IHF is the only nucleoid associated protein (NAP) involved in KplE1 recombination, although it is dispensable. This is consistent with the presence of only one functional IHF site on attR and none on attL.

Site-selective dual modification of periplasmic binding proteins for sensing applications

We have developed three sensitive and specific amino acid sensors based on bacterial periplasmic solute binding proteins. A site-specific amino-terminal transamination reaction provides a useful complement to cysteine chemistry for the covalent modification of biomolecules in this application. We demonstrate this combination to attach two different chromophores to a single biomolecule in two locations. The periplasmic glutamine binding protein from E. coli was modified with a pair of dyes suitable for fluorescence resonance energy transfer, and this conjugate exhibited an l-glutamine dependent optical response. Two periplasmic binding proteins from the thermophilic organism Thermotoga maritima, for arginine and aliphatic amino acids, were modified and evaluated similarly. All three conjugates manifested signal changes mediated by resonant energy transfer upon binding their respective ligands, with nanomolar dissociation constants and stereochemical specificity. This represents a readily generalizable method for construction of reagentless biosensors. The double-labeling strategy was also exploited for the surface attachment of a dye-labeled glutamine binding protein via a biotin–streptavidin interaction.

Chemical Composition and Antioxidant Activities of the Essential Oils of Different Parts of Cupressus arizonica Greene

The essential oils of different parts of Cupressus arizonica were examined for their potential radical scavenging activity. Fresh leaves and fruits of C. arizonica were steam distilled and the oils were analyzed qualitatively and quantitatively by GC and GC/MS. Different methods of rapid TLC screening, DPPH assay, desoxyribose assay, assay for site-specific reaction and non-enzymatic lipid peroxidation were employed to evaluate possible antioxidant activity of the oils and their main components, as well as the positive controls. The main components of these essential oils were α-pinene, cymene, myrcene, limonene, sabinene hydrate, cedrane and germacrene D. In rapid TLC screening, the tested compounds mainly showed antioxidant activity. In DPPH assay the stronger antioxidant effects were shown by the fruit oil of C. arizonia. In deoxyribose degradation assay test, most of the tested compounds showed some antioxidant effects. In non-enzymatic lipid peroxidation test, most antioxidant activity was shown by the fruit oil.

Site specific ligand substitution in cubane-type Mo3FeS44+ clusters: Kinetics and mechanism of reaction and isolation of mixed ligand Cl/SPh complexes

The synthesis, crystal structure and solution characterization of the cubane-type [Mo(3)(FeCl)S(4)(dmpe)(3)Cl(3)] (1) (dmpe = 1,2-bis(dimethylphophane-ethane)) cluster are reported and the ligand substitution processes of chloride by thiophenolate investigated. The kinetics and the intimate mechanism of these substitutions reveal that compound 1 undergoes a number of Fe and Mo site specific ligand substitution reactions in acetonitrile solutions. In particular, PhS(-) coordination at the tetrahedral Fe site proceeds in a single resolved kinetic step whereas such substitutions at the Mo sites proceed more slowly. The effect of the presence of acids in the reaction media is also investigated and reveals that an acid excess hinders substitution reactions both at the Fe and Mo sites; however, an acid-promoted solvolysis of the Fe-Cl bonds is observed. Electrospray ionization (ESI) and tandem (ESI-MS/MS) mass spectrometry allow the identification of all the reaction intermediates proposed on the basis of stopped-flow measurements. The distinctive site specific reactivity made it possible to isolate two new clusters of the Mo(3)FeS(4)(4+) family featuring mixed chlorine/thiophenolate ligands, namely Mo(3)S(4)(FeSPh)(dmpe)(3)Cl(3) (2) and [Mo(3)S(4)(FeSPh)(dmpe)(3)(SPh)(3)] (3). A detailed computational study has also been carried out to understand the details of the mechanism of substitution at the M-Cl (M = Mo and Fe) bonds as well as the solvolysis at the Fe-Cl sites, with particular emphasis on the role of acids on the substitution process. The results of the calculations are in agreement with the experimental observations, thus justifying the non-existence of an accelerating effect of acids on the thiophenolate substitution reaction, which differs from previous proposals for the Fe(4)S(4) and MoFe(3)S(4) clusters and some related compounds.

DNA relaxation‐dependent phase biasing of the fim genetic switch in Escherichia coli depends on the interplay of H‐NS, IHF and LRP

Reversible inversion of the DNA element fimS is responsible for the phase variable expression of type 1 fimbriae in Escherichia coli. The FimB tyrosine integrase site-specific recombinase inverts fimS in the on-to-off and off-to-on directions with approximately equal efficiencies. However, when DNA supercoiling is relaxed, fimS adopts predominantly the on orientation. This orientational bias is known to require binding of the nucleoid-associated protein LRP within fimS. Here we show that binding of the IHF protein to a site immediately adjacent to fimS is also required for phase-on orientational bias. In the absence of both LRP and IHF binding, fimS adopts the off orientation and the H-NS protein is required to maintain this alternative orientational bias. Thus, fimS has three Recombination Directionality Factors, H-NS, IHF and LRP. The relevant H-NS binding site straddles the left inverted repeat in phase-off fimS and this site is disrupted when fimS inverts to the on orientation. The inversion of fimS with the associated creation and removal of an H-NS binding site required for DNA inversion biasing represents a novel mechanism for modulating the interaction of H-NS with a DNA target and for influencing a site-specific recombination reaction.

Structural Features of Single-Stranded Integron Cassette attC Sites and Their Role in Strand Selection

We recently showed that cassette integration and deletion in integron platforms were occurring through unconventional site-specific recombination reactions involving only the bottom strand of attC sites. The lack of sequence conservation among attC sites led us to hypothesize that sequence-independent structural recognition determinants must exist within attC sites. The structural data obtained from a synaptic complex of the Vibrio cholerae integrase with the bottom strand of an attC site has shown the importance of extra helical bases (EHB) inside the stem-loop structure formed from the bottom strand. Here, we systematically determined the contribution of three structural elements common to all known single-stranded attC site recombination substrates (the EHBs, the unpaired central spacer (UCS), and the variable terminal structure (VTS)) to strand choice and recombination. Their roles have been evaluated in vivo in the attI×attC reaction context using the suicide conjugation assay we previously developed, but also in an attC×attC reaction using a deletion assay. Conjugation was used to deliver the attC sites in single-stranded form. Our results show that strand choice is primarily directed by the first EHB, but the presence of the two other EHBs also serves to increase this strand selection. We found that the structure of the central spacer is essential to achieve high level recombination of the bottom strand, suggesting a dual role for this structure in active site exclusion and for hindering the reverse reaction after the first strand exchange. Moreover, we have shown that the VTS has apparently no role in strand selectivity.

Site-Specific Recombination in the Cyanobacterium Anabaena sp. Strain PCC 7120 Catalyzed by the Integrase of Coliphage HK022

The integrase (Int) of the lambda-like coliphage HK022 catalyzes the site-specific integration and excision of the phage DNA into and from the chromosome of its host, Escherichia coli. Int recognizes two different pairs of recombining sites attP x attB and attL x attR for integration and excision, respectively. This system was adapted to the cyanobacterium Anabaena sp. strain PCC 7120 as a potential tool for site-specific gene manipulations in the cyanobacterium. Two plasmids were consecutively cointroduced by conjugation into Anabaena cells, one plasmid that expresses HK022 Int recombinase and the other plasmid that carries the excision substrate P(glnA)-attL-T1/T2-attR-lacZ, where T1/T2 are the strong transcription terminators of rrnB, to prevent expression of the lacZ reporter under the constitutive promoter P(glnA). The Int-catalyzed site-specific recombination reaction was monitored by the expression of lacZ emanating as a result of T1/T2 excision. Int catalyzed the site-specific excision reaction in Anabaena cells when its substrate was located either on the plasmid or on the chromosome with no need to supply an accessory protein, such as integration host factor and excisionase (Xis), which are indispensable for this reaction in its host, E. coli.

Identification of an Imino Group Indispensable for Cleavage by a Small Ribozyme

The hairpin ribozyme is a small, noncoding RNA (ncRNA) that catalyzes a site-specific phosphodiester bond cleavage reaction. Prior biochemical and structural analyses pinpointed the amidine moiety of base Ade38 as a key functional group in catalysis, but base changes designed to probe function resulted in localized misfolding of the active site. To define the requirements for chemical activity using a conservative modification, we synthesized and incorporated N1-deazaadenosine into the full-length ribozyme construct. This single-atom variant severely impairs activity, although the active-site fold remains intact in the accompanying crystal structures. The results demonstrate the essentiality of the imino moiety as well as the importance of its interaction with the substrate in the precatalytic and transition-state conformations. This work demonstrates the efficacy of single-atom approaches in the analysis of ncRNA structure-function relationships.

Preparation of Recombinant Baculoviruses with the BVboost System

INTRODUCTIONWe have developed an improved transposition-based system (BVboost) for the generation of recombinant baculoviruses. This system bypasses the disadvantages of the original transposition-based generation of baculoviral genomes in Escherichia coli while remaining a simple, rapid, and convenient viral production technique. The method is based on the modified donor vector (pBVboost) and an improved selection scheme of the baculoviral genomes (bacmids) in E. coli with a mutated sacB gene. Recombinant bacmids can be generated at a frequency of ~107/μg of donor vector with a negligible background. The BVboost system also allows efficient setups for high-throughput screening and gene expression purposes. After cloning the desired gene/cDNA/library into a BVboost system-compatible donor plasmid, the recombinant baculoviral genome is prepared simply by transforming electrocompetent DH10BacΔTn7 E. coli cells with the donor. Transfer from the donor vector into the baculoviral genome (bacmid) occurs via a Tn7-mediated site-specific transposition reaction in E. coli cells. The selection scheme guarantees that virtually all colonies are correct. The recombinant baculoviral genome is subsequently extracted from E. coli culture using a modified isolation procedure for large plasmids. To generate the recombinant viruses, insect cells are transfected with the isolated recombinant bacmid. This protocol provides instructions on how to prepare recombinant baculoviruses by the BVboost system in order to express the desired gene(s) in insect and/or vertebrate cells.

A Non-Sequence-Specific DNA Binding Mode of RAG1 Is Inhibited by RAG2

RAG1 and RAG2 proteins catalyze site-specific DNA cleavage reactions in V(D)J recombination, a process that assembles antigen receptor genes from component gene segments during lymphocyte development. The first step towards the DNA cleavage reaction is the sequence-specific association of the RAG proteins with the conserved recombination signal sequence (RSS), which flanks each gene segment in the antigen receptor loci. Questions remain as to the contribution of each RAG protein to recognition of the RSS. For example, while RAG1 alone is capable of recognizing the conserved elements of the RSS, it is not clear if or how RAG2 may enhance sequence-specific associations with the RSS. To shed light on this issue, we examined the association of RAG1, with and without RAG2, with consensus RSS versus non-RSS substrates using fluorescence anisotropy and gel mobility shift assays. The results indicate that while RAG1 can recognize the RSS, the sequence-specific interaction under physiological conditions is masked by a high-affinity non-sequence-specific DNA binding mode. Significantly, addition of RAG2 effectively suppressed the association of RAG1 with non-sequence-specific DNA, resulting in a large differential in binding affinity for the RSS versus the non-RSS sites. We conclude that this represents a major means by which RAG2 contributes to the initial recognition of the RSS and that, therefore, association of RAG1 with RAG2 is required for effective interactions with the RSS in developing lymphocytes.