Specificity of novel allosterically trans- and cis-activated connected maxizymes that are designed to suppress BCR-ABL expression

AbstractChronic myelogenous leukemia (CML) is associated with the presence of the Philadelphia chromosome, which is generated by the reciprocal translocation of chromosomes 9 and 22. In the case of L6 (b2a2) mRNA, it is difficult to cleave the abnormal mRNA specifically because the mRNA includes no sequences that can be cleaved efficiently by conventional hammerhead ribozymes near the BCR-ABL junction. We recently succeeded in designing a novel maxizyme, which specifically cleaves BCR-ABL fusion mRNA, as a result of the formation of a dimeric structure. As an extension of our molecular engineering of maxizymes, as well as to improve their potential utility, we examined whether an analogous conformational change could be induced within a single molecule when two maxizymes were connected via a linker sequence. An active conformation was achieved by binding of the construct to the BCR-ABL junction in trans, with part of the linker sequence then acting as an antisense modulator in cis (within the complex) to adjust the overall structure. Results of studies in vitro in the presence of cetyltrimethylammonium bromide (CTAB) (but not in its absence) suggested that a certain kind of connected maxizyme (cMzB) might be able to undergo a desired conformational change and, indeed, studies in vivo confirmed this prediction. Therefore, we successfully created a fully functional, connected maxizyme and, moreover, we found that the activity and specificity of catalytic RNAs in vivo might be better estimated if their reactions are monitored in vitro in the presence of CTAB

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

AbstractDivalent 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

Structural Basis of the Highly Efficient Trapping of the HIV Tat Protein by an RNA Aptamer

AbstractAn RNA aptamer containing two binding sites exhibits extremely high affinity to the HIV Tat protein. We have determined the structure of the aptamer complexed with two argininamide molecules. Two adjacent U:A:U base triples were formed, which widens the major groove to make space for the two argininamide molecules. The argininamide molecules bind to the G bases through hydrogen bonds. The binding is stabilized through stacking interactions. The structure of the aptamer complexed with a Tat-derived arginine-rich peptide was also characterized. It was suggested that the aptamer structure is similar for both complexes and that the aptamer interacts with two different arginine residues of the peptide simultaneously at the two binding sites, which could explain the high affinity to Tat

Myosin motor Myo1c and its receptor NEMO/IKK-γ promote TNF-α–induced serine307 phosphorylation of IRS-1

Tumor necrosis factor-α (TNF-α) signaling through the IκB kinase (IKK) complex attenuates insulin action via the phosphorylation of insulin receptor substrate 1 (IRS-1) at Ser307. However, the precise molecular mechanism by which the IKK complex phosphorylates IRS-1 is unknown. In this study, we report nuclear factor κB essential modulator (NEMO)/IKK-γ subunit accumulation in membrane ruffles followed by an interaction with IRS-1. This intracellular trafficking of NEMO requires insulin, an intact actin cytoskeletal network, and the motor protein Myo1c. Increased Myo1c expression enhanced the NEMO–IRS-1 interaction, which is essential for TNF-α– induced phosphorylation of Ser307–IRS-1. In contrast, dominant inhibitory Myo1c cargo domain expression diminished this interaction and inhibited IRS-1 phosphorylation. NEMO expression also enhanced TNF-α–induced Ser307–IRS-1 phosphorylation and inhibited glucose uptake. In contrast, a deletion mutant of NEMO lacking the IKK-β–binding domain or silencing NEMO blocked the TNF-α signal. Thus, motor protein Myo1c and its receptor protein NEMO act cooperatively to form the IKK–IRS-1 complex and function in TNF-α–induced insulin resistance

Defective repair of radiation-induced DNA damage is complemented by a CHORI-230-65K18 BAC clone on rat chromosome 4

AbstractThe Long Evans cinnamon (LEC) rat is highly susceptible to X-irradiation due to defective DNA repair and is thus a model for hepatocellular carcinogenesis. We constructed a bacterial artificial chromosome (BAC) contig of rat chromosome 4 completely covering the region associated with radiation susceptibility. We used transient and stable transfections to demonstrate that defective DNA repair in LEC cells is fully complemented by a 200-kb BAC, CHORI-230-65K18. Further analysis showed that the region associated with radiation susceptibility is located in a 128,543-bp region of 65K18 that includes the known gene Rpn1. However, neither knockdown nor overexpression of Rpn1 indicated that this gene is associated with radiation susceptibility. We also mapped three ESTs (TC523872, TC533727, and CB607546) in the 128,543-bp region, suggesting that 65K18 contains an unknown gene associated with X-ray susceptibility in the LEC rat

THEORETICAL AND EXPERIMENTAL EVIDENCE FOR THE STEREOELECTRONIC EFFECT (STEREOELECTRONIC, NUCLEOPHILICITY).

THEORETICAL AND EXPERIMENTAL EVIDENCE FOR THE STEREOELECTRONIC EFFECT (STEREOELECTRONIC, NUCLEOPHILICITY)