Kazunari Taira

Distinct roles of the co-activators p300 and CBP in retinoic-acid-induced F9-cell differentiation

The related proteins p300 and CBP (cAMP-response-element-binding protein (CREB)-binding protein)) are transcriptional co-activators that act with other factors to regulate gene expression and play roles in many cell-differentiation and signal transduction pathways. Both proteins have intrinsic histone-acetyltransferase activity, and may act directly on chromatin, of which histone is a component, to facilitate transcription. They are also involved in growth control pathways, as shown by their interaction with the tumour suppressor p53 (refs 13–15) and the viral oncogenes E1A (refs 1, 2, 16) and SV40 T antigen. Here we report functional differences of p300 and CBP in vivo. We examined their roles during retinoic-acid-induced differentiation, cell-cycle exit and programmed cell death (apoptosis) of embryonal carcinoma F9 cells, using hammerhead ribozymes capable of cleaving either p300 or CBP messenger RNAs. F9 cells expressing a p300-specific ribozyme became resistant to retinoic-acid-induced differentiation, whereas cells expressing a CBP-specific ribozyme were unaffected. Similarly, retinoic-acid-induced transcriptional upregulation of the cell-cycle inhibitor p21Cip1 required normal levels of p300, but not CBP, whereas the reverse was true for p27Kip1. In contrast, both ribozymes blocked retinoic-acid-induced apoptosis, indicating that both co-activators are required for this process. Thus, despite their similarities, p300 and CBP have distinct functions during retinoic-acid-induced differentiation of F9 cells.

Extremely high and specific activity of DNA enzymes in cells with a Philadelphia chromosome

BACKGROUNDnChronic myelogenous leukemia (CML) results from chromosome 22 translocations (the Philadelphia chromosome) that creates BCR-ABL fusion genes, which encode two abnormal mRNAs (b3a2 and b2a2). Various attempts to design antisense oligonucleotides that specifically cleave abnormal L6 BCR-ABL fusion mRNA have not been successful. Because b2a2 mRNA cannot be effectively cleaved by hammerhead ribozymes near the BCR-ABL junction, it has proved very difficult to engineer specific cleavage of this chimeric mRNA. Nonspecific effects associated with using antisense molecules make the use of such antisense molecules questionable.nnnRESULTSnThe usefulness of DNA enzymes in specifically suppressing expression of L6 BCR-ABL mRNA in mammalian cells is demonstrated. Although the efficacy of DNA enzymes with natural linkages decreased 12 hours after transfection, partially modified DNA enzymes, with either phosphorothioate or 2-O-methyl groups at both their 5 and 3 ends, remained active for much longer times in mammalian cells. Moreover, the DNA enzyme with only 2-O-methyl modifications was also highly specific for abnormal mRNA.nnnCONCLUSIONSnDNA enzymes with 2-O-methyl modifications are potentially useful as gene-inactivating agents in the treatment of diseases such as CML. In contrast to conventional antisense DNAs, some of the DNA enzymes used in this study were highly specific and cleaved only abnormal BCR-ABL mRNA.

PCR cloning and expression of the F/10 family xylanase gene from Streptomyces olivaceoviridis E-86

Abstract Using a simple long-range inverse PCR method, we cloned the GC-rich gene (68%) for an F 10 xylanase from Streptomyces olivaceoviridis E-86. The open reading frame of the cloned gene, fxyn, contained 1431 bp and encoded 477 amino acid residues. FXYN resembled a xylanase of the F 10 family and had two functional domains (a catalytic domain and a substrate-binding domain). Unique triple repeat sequence regions (CLD-C) were located in the substrate-binding domain, which was similar to the xylan-binding domains of xylanase A and that of arabinofuranosidase B from S. lividans. FXYN with a tag that consisted of six histidine residues at the carboxy-terminus was expressed at high levels in Escherichia coli and had the same properties as the native xylanase produced by S. olivaceoviridis. Moreover, the xylan-binding domain of FXYN significantly enhanced hydrolysis of insoluble xylan whereas it had minimal effect on the hydrolysis of soluble xylan.

An investigation of the nature and function of module 10 in a family F/10 xylanase FXYN of Streptomyces olivaceoviridis E-86 by module shuffling with the Cex of Cellulomonas fimi and by site-directed mutagenesis

Although the amino acid homology in the catalytic domain of FXYN xylanase from Streptomyces olivaceoviridis E‐86 and Cex xylanase from Cellulomonas fimi is only 50%, an active chimeric enzyme was obtained by replacing module 10 in FXYN with module 10 from Cex. In the family F/10 xylanases, module 10 is an important region as it includes an acid/base catalyst and a substrate binding residue. In FXYN, module 10 consists of 15 amino acid residues, while in Cex it consists of 14 amino acid residues. The K m and k cat values of the chimeric xylanase FCF‐C10 for PNP‐xylobioside (PNP‐X2) were 10‐fold less than those for FXYN. CD spectral data indicated that the structure of the chimeric enzyme was similar to that of FXYN. Based on the comparison of the amino acid sequences of FXYN and Cex in module 10, we constructed four mutants of FXYN. When D133 or S135 of FXYN was deleted, the kinetic properties were not changed from those of FXYN. By deletion of both D133 and S135, the K m value for PNP‐X2 decreased from the 2.0 mM of FXYN to 0.6 mM and the k cat value decreased from the 20 s−1 of FXYN to 8.7 s−1. Insertion of Q140 into the doubly deleted mutant further reduced the K m value to 0.3 mM and the k cat value to 3.8 s−1. These values are close to those for the chimeric enzyme FCF‐C10. These results indicate that module 10 itself is able to accommodate changes in the sequence position of amino acids which are critical for enzyme function. Since changes of the spatial position of these amino acids would be expected to result in enzyme inactivation, module 10 must have some flexibility in its tertiary structure. The structure of module 10 itself also affects the substrate specificity of the enzyme.

Significant enhancement in the binding of p-nitrophenyl-β-d-xylobioside by the E128H mutant F/10 xylanase from Streptomyces olivaceoviridis E-86

Mutagenesis studies were carried out to examine the effects of replacement of either the nucleophile Glu‐236 or the acid/base Glu‐128 residue of the F/10 xylanase by a His residue. To our surprise, the affinity for the p‐nitrophenyl‐β‐D‐xylobioside substrate was increased by 103‐fold in the case of the mutant E128H enzyme compared with that of the wild‐type F/10 xylanase. The catalytic activity of the mutant enzymes was low, despite the fact that the distance between the nucleophilic atom (an oxygen in the native xylanase and a nitrogen in the mutant) and the α‐carbon was barely changed. Thus, the alteration of the acid/base functionality (Glu‐128 to His mutation) provided a significantly favorable interaction within the E128H enzyme/substrate complex in the ground state, accompanying a reduction in the stabilization effect in the transition state.

Explanation by a putative triester-like mechanism for the thio effects and Mn2+ rescues in reactions catalyzed by a hammerhead ribozyme

Divalent metal ion‐dependent hammerhead ribozymes can cleave any RNA with a NUX triplet, wherein the N can be any residue and X can be C, U or A. In recent literature on the mechanism of action of hammerhead ribozymes, one important role of divalent metal ions is generally suggested to be an electrophilic catalyst by directly coordinating with the pro‐Rp oxygen of the scissile phosphate to stabilize the transition state. This proposal was made on the basis of thio effects and the proposed electrophilic catalyst is very attractive as an explanation for the catalytic activity of metalloenzymes. Reexamination of thio effects with substrates having a GUA triplet at the cleavage site shows that, in agreement with the previous finding, the cleavage rate, in the presence of Mg2+ ions, is significantly reduced in the case of the phosphorothioate substrate (RpS), wherein the pro‐Rp oxygen at the scissile phosphate is replaced by sulfur, while the cleavage rate is reduced to a much lesser extent for the other isomer (SpS), wherein the pro‐Sp oxygen at the scissile phosphate is replaced by sulfur. However, more careful examination of the rescue ability of Mn2+ ions with these isomers demonstrates that more thiophilic Mn2+ ions rescue the reaction not only with the RpS isomer but also with the SpS isomer and, importantly, to a greater extent for the SpS isomer. These results argue against the previous conclusion that a metal ion is directly coordinating with the pro‐Rp oxygen of the scissile phosphate to stabilize the transition state. In this paper we try to elucidate the possible origin of the thio effects and propose a `triester‐like mechanism in reactions catalyzed by hammerhead ribozymes.

TEMPERATURE-DEPENDENT CHANGE IN THE RATE-DETERMINING STEP IN A REACTION CATALYZED BY A HAMMERHEAD RIBOZYME

To characterize the reaction catalyzed by a hammerhead ribozyme, the dependence on temperature of the reaction was examined. An Arrhenius plot revealed a transition that indicated a temperature‐dependent change in the activation energy at around 25°C. Thermodynamic parameters of the reaction were estimated at 10 and 35°C. The analyses led to the following conclusions. At 25–50°C. the chemical cleavage step (k cleav was the rate‐determining step, and the cleaved fragments dissociated from the ribozyme at a higher rate than the rate of the chemical reaction. When the temperature was below 25°C, the cleaved fragments adhered to the ribozyme more tightly and the product dissociation step became the rate‐determining step. Above 50°C, the rate of the reaction decreased because, at such high temperatures, the formation of the Michaelis‐Menten complex (duplex formation) was hampered by thermal melting. A conformational change in the ribozyme‐substrate complex was not the rate‐determining step at any of the temperatures examined.

Catalytic activities of hammerhead ribozymes with a triterpenoid linker instead of stem/loop II

A minizyme is a hammerhead ribozyme with short oligonucleotide linkers instead of stem/loop II. In a previous study we demonstrated that a minizyme with high‐level activity forms a dimeric structure with a common stem II (Amontov and Taira, J. Am. Chem. Soc., 118 (1996) 1624–1628). As a continuation of this study, we recently examined whether a short oligonucleotide linker at stem/loop II could be replaced by a triterpenoid linker and whether the resulting minizymes with bulky hydrophobic groups would form dimeric structures. In contrast to the minizyme with high‐level activity that we characterized in the previous study, minizymes that contained a triterpenoid group had low cleavage activities. The nature of the dependence of the activity on the concentration of ribozyme revealed that these minizymes with a triterpenoid group do not form dimeric structures. Thus, the low activities of these structures can be attributed to their failure to form dimers.

CTAB-mediated enrichment for active forms of novel dimeric maxizymes

We demonstrated previously that shortened forms of (stem II‐deleted) hammerhead ribozymes with low intrinsic activity form very active dimers with a common stem II (very active short ribozymes capable of forming dimers were designated maxizymes). As a result of such a dimeric structure, heterodimeric maxizymes are potentially capable of cleaving a substrate at two different sites simultaneously. In this case, active heterodimers are in equilibrium with inactive homodimers. Longer forms of common stem II can lead to enrichment of the active heterodimers in vitro. In this study, we investigated whether the cationic detergent CTAB, which is known to enhance strand displacement of nucleic acids, might inhibit the dimerization of maxizymes. Significantly, under all conditions examined, CTAB instead enhanced the activity of a variety of maxizymes, with the extent of enhancement depending on the conditions. The activity of our least stable, least active maxizyme was enhanced 100‐fold by CTAB. The strand displacement activity of CTAB thus appears to enhance the conversion of alternative conformations of inactive maxizymes, with intra‐ and inter‐molecular hydrogen bonds, to active forms. Thus, our smallest maxizyme can also be considered a potential candidate for a gene‐inactivating agent in vivo, in view of the fact that various facilitators of strand displacement reactions are known to exist in vivo (indeed, a separate experiment in cell culture supported the conclusion that our smallest maxizyme is a good gene‐inactivating agent). Although activities of ribozymes in vitro do not necessarily reflect their activities in vivo, our findings suggest that the activity of ribozymes in vivo can be better estimated by running ribozyme kinetics in the presence of CTAB in vitro.

AB INITIO INVESTIGATION ON NUCLEOPHILIC RING OPENING OF 1,3,2-OXATHIAPHOSPHOLANE : NUCLEOPHILIC SUBSTITUTION AT PHOSPHORUS COUPLED WITH PSEUDOROTATION

Abstract Ab initio investigations on the reaction profiles for the base-catalyzed methanolysis of amino-2-thiono-1,3,2-oxathiophospholane suggested that the ring opening with retention of configuration at phosphorus would be energetically most favorable. This provides the rational interpretation for the chemo- and stereoselectivity ascertained experimentally for the reaction. The pseudorotation process is strongly coupled with the reaction coordinate for the substitution pathway, which suggests that substitution with retention of configuration can occur in general even though the trigonal bipyramidal species resulting from pseudorotation is significantly unstable and fails to exist as an intermediate.