Quadruplex-Templated and Catalyzed Ligation of Nucleic Acids

Template-guided chemical reactions between nucleic acid strands are an important process in biomedical research. However, almost all of these reactions employ an oligonucleotide-templated approach that is based on the double-helix alignment. The moderate stability of the double helix makes this approach unsuitable for many chemical reactions, so alternative nucleic acid alignment mechanisms, demonstrating higher thermal and chemical stability, are desirable. Earlier, we described a noncovalent coupling mechanism between DNA strands through a quadruplex-and-Mg²⁺ connection (QMC). QMC is based on G-quadruplexes and allows unusually stable and specific interactions. Herein, a novel catalytic nucleic acid reaction, based on QMC, is described. This approach uses G-tetrads as a structural and recognition element without employing Watson-Crick complementarity rules at any stage of substrate/catalyst formation or interaction between them. Quadruplex-templated ligation can be achieved through the self-ligation of two nucleic acid strands, or through a quadruplex catalyst, which forms a G-triplex and specifically connects the strands. The process is extraordinarily robust and efficient. For instance, the ligation of carbodiimide-activated substrates can proceed in boiling solutions, and complete ligation is demonstrated within a minute. The quadruplex-templated and catalyzed reactions will create new opportunities for chemical reactions requiring harsh experimental conditions.     

Flame Retardant Functionalization of Microcrystalline Cellulose by Phosphorylation Reaction with Phytic Acid

The functionalization of microcrystalline cellulose (MCC) is an important strategy for broadening its application fields. In the present work, MCC was functionalized by phosphorylation reaction with phytic acid (PA) for enhanced flame retardancy. The conditions of phosphorylation reaction including PA concentration, MCC/PA weight ratio and temperature were discussed, and the thermal degradation, heat release and char-forming properties of the resulting PA modified MCC were studied by thermogravimetric analysis and pyrolysis combustion flow calorimetry. The PA modified MCC, which was prepared at 90 °C, 50%PA and 1:3 weight ratio of MCC to PA, exhibited early thermal dehydration with rapid char formation as well as low heat release capability. This work suggests a novel strategy for the phosphorylation of cellulose using PA and reveals that the PA phosphorylated MCC can act as a promising flame retardant material.     

Towards Efficient Nonenzymatic DNA Ligation: Comparing Key Parameters for Maximizing Ligation Rates and Yields with Carbodiimide Activation**

Chemical ligation is an important tool for the generation of synthetic DNA structures, which are used for a wide range of applications. Surprisingly, reported chemical ligation yields can range from 30 to 95 % for the same chemical activating agent and comparable DNA structures. We report a systematic study of DNA ligation by using a well-defined bimolecular test system and a water-soluble carbodiimide (EDC) as a phosphate-activating agent. Our results emphasize the interplay between template-substrate complex stability and the rates of the chemical steps of ligation, with 3′ phosphate substrates providing yields near 100 % after 24 hours for particularly favorable reaction conditions. Ligation rates are also shown to be sensitive to the identity of the base pairs flanking a nick site, with as much as threefold variation. Finally, the observation that DNA substrates are modified by EDC at rates that can be comparable with ligation rates emphasizes the importance of considering side reactions when designing protocols to maximize ligation yields.     

Different Enzymatic Processing of γ‐Phosphoramidate and γ‐Phosphoester‐Modified ATP Analogues

Monitoring the activity of ATP‐consuming enzymes provides the basis for elucidating their modes of action and regulation. Although a number of ATP analogues have been developed for this, their scope is restricted because of the limited acceptance by respective enzymes. In order to clarify which kind of phosphate‐modified ATP analogues are accepted by the α‐β‐phosphoanhydride‐cleaving ubiquitin‐activating enzyme 1 (UBA1) and the β‐γ‐phosphoanhydride‐cleaving focal adhesion kinase (FAK), we tested phosphoramidate‐ and phosphoester‐modified ATP analogues. UBA1 and FAK were able to convert phosphoramidate‐modified ATP analogues, even with a bulky modification like biotin. In contrast, a phosphoester‐modified analogue was poorly accepted. These results demonstrate that minor variations in the design of ATP analogues for monitoring ATP utilization have a significant impact on enzymatic acceptance.     

A Versatile Strategy for the Semisynthetic Production of Ser65 Phosphorylated Ubiquitin and Its Biochemical and Structural Characterisation

Ubiquitin phosphorylation is emerging as an important regulatory layer in the ubiquitin system. This is exemplified by the phosphorylation of ubiquitin on Ser65 by the Parkinson's disease‐associated kinase PINK1, which mediates the activation of the E3 ligase Parkin. Additional phosphorylation sites on ubiquitin might also have important cellular roles. Here we report a versatile strategy for preparing phosphorylated ubiquitin. We biochemically and structurally characterise semisynthetic phospho‐Ser65‐ubiquitin. Unexpectedly, we observed disulfide bond formation between ubiquitin molecules, and hence a novel crystal form. The method outlined provides a direct approach to study the combinatorial effects of phosphorylation on ubiquitin function. Our analysis also suggests that disulfide engineering of ubiquitin could be a useful strategy for obtaining alternative crystal forms of ubiquitin species thereby facilitating structural validation.     

Designed RNAs with Two Peptide‐Binding Units as Artificial Templates for Native Chemical Ligation of RNA‐Binding Peptides

The template effect plays important roles not only in modern synthetic and enzymatic catalysis but also in the ancient “RNA‐polypeptide (RNP) world,” which has been postulated to be a crucial stage in the origin of life. To mimic primitive template catalysis of peptide ligations by RNAs, we previously reported the design and synthesis of a ternary RNP complex in which the ligation of two peptides was significantly facilitated by a template RNA with two peptide‐binding units. However, RNA molecules also promoted the ligation reaction in a nonspecific manner through electrostatic interactions between RNA and basic peptides. In this study, we suppressed this effect by reducing the length of the original template derived from the Tetrahymena intron RNA. This modification, however, decreased the template ability for the specific reaction. As an alternative RNA that was as effective as the original template, we found that a self‐dimerizing RNA was a promising template for peptide ligation without a nonspecific effect.     

Evolutionary Divergence of Phosphorylation to Regulate Interactive Protein Networks in Lower and Higher Species

The phosphorylation of proteins affects their functions in extensively documented circumstances. However, the role of phosphorylation in many interactive networks of proteins remains very elusive due to the experimental limits of exploring the transient interaction in a large complex of assembled proteins induced by stimulation. Previous studies have suggested that phosphorylation is a recent evolutionary process that differently regulates ortholog proteins in numerous lineages of living organisms to create new functions. Despite the fact that numerous phospho-proteins have been compared between species, little is known about the organization of the full phospho-proteome, the role of phosphorylation to orchestrate large interactive networks of proteins, and the intertwined phospho-landscape in these networks. In this report, we aimed to investigate the acquired role of phosphate addition in the phenomenon of protein networking in different orders of living organisms. Our data highlighted the acquired status of phosphorylation in organizing large, connected assemblages in Homo sapiens. The protein networking guided by phosphorylation turned out to be prominent in humans, chaotic in yeast, and weak in flies. Furthermore, the molecular functions of GO annotation enrichment regulated by phosphorylation were found to be drastically different between flies, yeast, and humans, suggesting an evolutionary drift specific to each species.     

Cyclization of the Antimicrobial Peptide Gomesin with Native Chemical Ligation: Influences on Stability and Bioactivity

Gomesin is an 18‐residue peptide originally isolated from the hemocytes of the Brazilian spider Acanthoscurria gomesiana. A broad spectrum of bioactivities have been attributed to gomesin, including in vivo and in vitro cytotoxicity against tumour cells, antimicrobial, antifungal, anti‐Leishmania and antimalarial effects. Given the potential therapeutic applications of gomesin, it was of interest to determine if an engineered version with a cyclic backbone has improved stability and bioactivity. Cyclization has been shown to confer enhanced stability and activity to a range of bioactive peptides and, in the case of a cone snail venom peptide, confer oral activity in a pain model. The current study demonstrates that cyclization improves the in vitro stability of gomesin over a 24 hour time period and enhances cytotoxicity against a cancer cell line without being toxic to a noncancerous cell line. In addition, antimalarial activity is enhanced upon cyclization. These findings provide additional insight into the influences of backbone cyclization on the therapeutic potential of peptides.     

A Quantitative Comparison of Wild‐Type and Gatekeeper Mutant Cdk2 for Chemical Genetic Studies with ATP Analogues

Mutant kinase kinetics : Protein kinases with enlarged ATP binding sites are increasingly being used as tools to probe the functioning signal transduction cascades. Using human cyclin‐dependent kinase 2 as a model system, we demonstrate that enlargement of the ATP binding site does not substantially alter either the catalysis kinetics nor substrate or phosphorylation site selection.

     

Application of the Thioacid-Azide Ligation (TAL) for the Preparation of Glycosylated and Fluorescently Labeled Amino Acids

The reaction of thiocarboxylic acids with azides (thioacid-azide ligation, TAL) is a chemoselective transformation, leading to the formation of amide bonds. Especially in the case of electron-deficient sulfonyl azides, the reaction occurs in excellent yields, within minutes at room temperature, in a broad range of solvents. In this article, the application of TAL for the synthesis of glycosylated and dansylated amino acids is reported. 2-Azidosulfonylethyl glycosides were prepared from peracetylated monosaccharides and reacted with thioacid-containing derivatives of aspartic acid and glutamic acid. Likewise, aromatic sulfonyl azides were employed. Ligation products were obtained in yields of up to 96 %. Different conditions for the generation of the thioacids were compared. Whereas reaction of succinimidyl esters with sodium hydrogen sulfide led to concomitant formation of diacyl disulfides, trityl thioesters turned out to be suitable precursors for the in situ preparation of thioacids by mild acidolysis.     

Phosphorylation of the WWOX Protein Regulates Its Interaction with p73

We describe a molecular characterization of the interaction between the cancer-related proteins WWOX and p73. This interaction is mediated by the first of two WW domains (WW1) of WWOX and a PPXY-motif-containing region in p73. While phosphorylation of Tyr33 of WWOX and association with p73 are known to affect apoptotic activity, the quantitative effect of phosphorylation on this specific interaction is determined here for the first time. Using ITC and fluorescence anisotropy, we measured the binding affinity between WWOX domains and a p73 derived peptide, and showed that this interaction is regulated by Tyr phosphorylation of WW1. Chemical synthesis of the phosphorylated domains of WWOX revealed that the binding affinity of WWOX to p73 is decreased when WWOX is phosphorylated. This result suggests a fine-tuning of binding affinity in a differential, ligand-specific manner: the decrease in binding affinity of WWOX to p73 can free both partners to form new interactions.     

Structural Insights into the Interaction of Heme with Protein Tyrosine Kinase JAK2**

Janus kinase 2 (JAK2) is the most important signal-transducing tyrosine kinase in erythropoietic precursor cells. Its malfunction drives several myeloproliferative disorders. Heme is a small metal-ion-carrying molecule that is incorporated into hemoglobin in erythroid precursor cells to transport oxygen. In addition, heme is a signaling molecule and regulator of various biochemical processes. Here, we show that heme exposure leads to hyperphosphorylation of JAK2 in a myeloid cancer cell line. Two peptides identified in JAK2 are heme-regulatory motifs and show low-micromolar affinities for heme. These peptides map to the kinase domain of JAK2, which is essential for downstream signaling. We suggest these motifs to be the interaction sites of heme with JAK2, which drive the heme-induced hyperphosphorylation. The results presented herein could facilitate the development of heme-related pharmacological tools to combat myeloproliferative disorders.     

Combination of Thiol‐Additive‐Free Native Chemical Ligation/Desulfurization and Intentional Replacement of Alanine with Cysteine

We report a novel strategy for native chemical ligation (NCL). Alanines not located at a ligation site are temporarily replaced with cysteines, and this enables efficient thiol‐additive‐free NCL, with subsequent desulfurization to regenerate the target peptide. We synthesized stresscopin‐related peptide and neuroendocrine regulatory peptide‐2 (NERP‐2) by this method. We confirmed that both conventional alkyl thioester and thioester‐equivalent N‐acyl‐N′‐methyl‐benzimidazolinone (MeNbz) can be adopted as thioester components for thiol‐additive‐free NCL of multi‐Cys‐containing peptides.     

On the Enzymatic Formation of Metal Base Pairs with Thiolated and pKa-Perturbed Nucleotides

The formation of artificial metal base pairs is an alluring and versatile method for the functionalization of nucleic acids. Access to DNA functionalized with metal base pairs is granted mainly by solid-phase synthesis. An alternative, yet underexplored method, envisions the installation of metal base pairs through the polymerization of modified nucleoside triphosphates. Herein, we have explored the possibility of using thiolated and pK_a-perturbed nucleotides for the enzymatic construction of artificial metal base pairs. The thiolated nucleotides S2C, S6G, and S4T as well as the fluorinated analogue 5FU are readily incorporated opposite a templating S4T nucleotide through the guidance of metal cations. Multiple incorporation of the modified nucleotides along with polymerase bypass of the unnatural base pairs are also possible under certain conditions. The thiolated nucleotides S4T, S4T, S2C, and S6G were also shown to be compatible with the synthesis of modified, high molecular weight single-stranded (ss)DNA products through TdT-mediated tailing reactions. Thus, sulfur-substitution and pK_a perturbation represent alternative strategies for the design of modified nucleotides compatible with the enzymatic construction of metal base pairs.     

反胶团酶反应-膜分离技术研究进展

将反胶团酶反应与膜分离相结合被认为是使反胶团酶反应技术工业化的最有前途的方法。文中介绍反胶团酶反应－膜分离技术的研究现状,探讨了今后进一步研究开发的方向。     

Bile Duct Ligation Impairs Function and Expression of Mrp1 at Rat Blood–Retinal Barrier via Bilirubin-Induced P38 MAPK Pathway Activations

Liver injury is often associated with hepatic retinopathy, resulting from accumulation of retinal toxins due to blood–retinal barrier (BRB) dysfunction. Retinal pigment epithelium highly expresses MRP1/Mrp1. We aimed to investigate whether liver injury affects the function and expression of retinal Mrp1 using bile duct ligation (BDL) rats. Retinal distributions of fluorescein and 2,4-dinitrophenyl-S-glutathione were used for assessing Mrp1 function. BDL significantly increased distributions of the two substrates and bilirubin, downregulated Mrp1 protein, and upregulated phosphorylation of p38 and MK2 in the retina. BDL neither affected the retinal distribution of FITC-dextran nor expressions of ZO-1 and claudin-5, demonstrating intact BRB integrity. In ARPE-19 cells, BDL rat serum or bilirubin decreased MRP1 expression and enhanced p38 and MK2 phosphorylation. Both inhibiting and silencing p38 significantly reversed the bilirubin- and anisomycin-induced decreases in MRP1 protein. Apparent permeability coefficients of fluorescein in the A-to-B direction (P_{app, A-to-B}) across the ARPE-19 monolayer were greater than P_{app, B-to-A}. MK571 or bilirubin significantly decreased P_{app, A-to-B} of fluorescein. Bilirubin treatment significantly downregulated Mrp1 function and expression without affecting integrity of BRB and increased bilirubin levels and phosphorylation of p38 and MK2 in rat retina. In conclusion, BDL downregulates the expression and function of retina Mrp1 by activating the p38 MAPK pathway due to increased bilirubin levels.     

Reaction of cytidine nucleotides with cyanoacetylene: support for the intermediacy of nucleoside-2',3'-cyclic phosphates in the prebiotic synthesis of RNA

A robust and prebiotically plausible synthesis of RNA is a key requirement of the "RNA World" hypothesis, but, to date, no such synthesis has been demonstrated. Monomer synthesis strategies involving attachment of preformed nucleobases to sugars have failed, and, even if activated 5'-nucleotides could be made, the hydrolysis of these intermediates in water makes their efficient oligomerisation appear unlikely. We recently reported a synthesis of cytidine-2',3'-cyclic phosphate 1 (C>p) in which the nucleobase was assembled in stages on a sugar-phosphate template. However, 2',3'-cyclic nucleotides (N>p's) also undergo hydrolysis, in this case giving a mixture of the 2'- and 3'-monophosphates. This hydrolysis has previously been seen as making the, otherwise promising, oligomerisation of N>p's seem as unlikely as that of the 5'-activated nucleotides. We now find that cyanoacetylene, the reagent used for the second stage of nucleobase assembly in the synthesis of C>p, also reverses the effect of the hydrolysis by driving efficient cyclisation of C2'p and C3'p back to C>p. Excess cyanoacetylene also derivatises the nucleobase, but this modification is reversible at neutral pH. These findings significantly strengthen the case for N>p's in a prebiotic synthesis of RNA.     

G-Protein Phosphorylation: Aspects of Binding Specificity and Function in the Plant Kingdom

Plant survival depends on adaptive mechanisms that constantly rely on signal recognition and transduction. The predominant class of signal discriminators is receptor kinases, with a vast member composition in plants. The transduction of signals occurs in part by a simple repertoire of heterotrimeric G proteins, with a core composed of α-, β-, and γ-subunits, together with a 7-transmembrane Regulator G Signaling (RGS) protein. With a small repertoire of G proteins in plants, phosphorylation by receptor kinases is critical in regulating the active state of the G-protein complex. This review describes the in vivo detected phosphosites in plant G proteins and conservation scores, and their in vitro corresponding kinases. Furthermore, recently described outcomes, including novel arrestin-like internalization of RGS and a non-canonical phosphorylation switching mechanism that drives G-protein plasticity, are discussed.     

Identification of Nrl1 Domains Responsible for Interactions with RNA-Processing Factors and Regulation of Nrl1 Function by Phosphorylation

Pre-mRNA splicing is a key process in the regulation of gene expression. In the fission yeast Schizosaccharomyces pombe, Nrl1 regulates splicing and expression of several genes and non-coding RNAs, and also suppresses the accumulation of R-loops. Here, we report analysis of interactions between Nrl1 and selected RNA-processing proteins and regulation of Nrl1 function by phosphorylation. Bacterial two-hybrid system (BACTH) assays revealed that the N-terminal region of Nrl1 is important for the interaction with ATP-dependent RNA helicase Mtl1 while the C-terminal region of Nrl1 is important for interactions with spliceosome components Ctr1, Ntr2, and Syf3. Consistent with this result, tandem affinity purification showed that Mtl1, but not Ctr1, Ntr2, or Syf3, co-purifies with the N-terminal region of Nrl1. Interestingly, mass-spectrometry analysis revealed that in addition to previously identified phosphorylation sites, Nrl1 is also phosphorylated on serines 86 and 112, and that Nrl1-TAP co-purifies with Cka1, the catalytic subunit of casein kinase 2. In vitro assay showed that Cka1 can phosphorylate bacterially expressed Nrl1 fragments. An analysis of non-phosphorylatable nrl1 mutants revealed defects in gene expression and splicing consistent with the notion that phosphorylation is an important regulator of Nrl1 function. Taken together, our results provide insights into two mechanisms that are involved in the regulation of the spliceosome-associated factor Nrl1, namely domain-specific interactions between Nrl1 and RNA-processing proteins and post-translational modification of Nrl1 by phosphorylation.