Interaction of anthracyclines with iron responsive element mRNAs

Double-stranded sections of mRNA are often inviting sites of interaction for a wide variety of proteins and small molecules. Interactions at these sites can serve to regulate, or disrupt, the homeostasis of the encoded protein products. Such ligand target sites exist as hairpin–loop structures in the mRNAs of several of the proteins involved in iron homeostasis, including ferritin heavy and light chains, and are known as iron responsive elements (IREs). These IREs serve as the main control mechanism for iron metabolism in the cell via their interaction with the iron regulatory proteins (IRPs). Disruption of the IRE/IRP interaction could greatly affect iron metabolism. Here, we report that anthracyclines, a class of clinically useful chemotherapeutic drugs that includes doxorubicin and daunorubicin, specifically interact with the IREs of ferritin heavy and light chains. We characterized this interaction through UV melting, fluorescence quenching and drug–RNA footprinting. Results from footprinting experiments with wild-type and mutant IREs indicate that anthracyclines preferentially bind within the UG wobble pairs flanking an asymmetrically bulged C-residue, a conserved base that is essential for IRE–IRP interaction. Additionally, drug–RNA affinities (apparent Kds) in the high nanomolar range were calculated from fluorescence quenching experiments, while UV melting studies revealed shifts in melting temperature (ΔTm) as large as 10°C. This anthracycline–IRE interaction may contribute to the aberration of intracellular iron homeostasis that results from anthracycline exposure

Phase shift cavity ring down absorption spectroscopy

Contains fulltext : 99005.pdf (publisher's version ) (Open Access

Environmental factors that enhance the action of the cell penetrating peptide pep-1: A spectroscopic study using lipidic vesicles

Pep-1 is a cell penetrating peptide (CPP) derived from the nuclear localization sequence of Simian Virus 40 large antigen T and from reverse transcriptase of Human Immunodeficiency Virus. Although it has been successfully used to transport proteins into cells, its action at the molecular level is not yet clear, mainly the local environmental factors that condition partition and translocation. Characterization in aqueous medium and quantification of partition into bilayers were carried out. Dynamic light scattering studies show that pep-1 self-associates in aqueous medium. The role of the bilayer phase, anionic lipids, ionic strength of the medium, reducing agents and pep-1 concentration on the extent and kinetics of partition were studied. Unlike others cationic CPP (e.g. penetratin) pep-1 has a high affinity to neutral vesicles (K-p=2.8 x 10(3)), which is enhanced by anionic lipids. In a reduction environment partition is strongly inhibited (K-p=2. 2 x 10(2)), which might be a key-feature in the biological action of pep-1, Peptide incorporation takes place in the millisecond time-range to the lipidic interfaces. These environmental factors are systematized to enlighten how they help cellular uptake. (C) 2005 Elsevier B.V. All rights reserved