Our previous studies have shown that 17beta estradiol (E2) enhances the transcript levels of mitochondrial DNA (mtDNA)-encoded genes and mitochondrial respiratory chain (MRC) activity via estrogen receptors (ER). Others have reported the presence of putative estrogen responsive elements (ERE) in human mtDNA (mtEREs) and detection of ERs in mitochondria of rat uterine and ovary cells. Recently, we demonstrated the E2-enhanced mitochondrial localization of ERalpha and ERbeta, and E2-induced mtDNA transcript levels in MCF-7 cells. In this study, we applied electrophoresis mobility shift assays (EMSAs) and surface plasmon resonance (SPR) to determine if mitochondrial extracts, recombinant human ERalpha (rhERalpha), and rhERbeta interact with mtEREs. Using EMSAs, we observed that ER-containing mitochondrial extracts bound to mtEREs and the binding was enhanced by E2, whereas the binding of mitochondrial proteins from ERbeta-deficient cells was almost undetectable. Both rhERalpha and rhERbeta bound to the mtEREs and their binding was altered by their respective antibodies. However, the ERalpha antibodies did not alter the binding of MCF-7 cell mitochondrial extracts to mtEREs whereas the binding MCF-7 and MDA-MB-231 cell mitochondrial extracts to mtEREs was reduced by ERbeta antibody. These results suggest that the mtERE-bound mitochondrial protein is ERbeta. Using SPR, we observed the binding of both ERs to mtEREs and that the binding was increased by E2. These results indicate that the mitochondrial ERs can interact with mtEREs and suggest that they may be directly involved in E2 induction of mtDNA transcription.

Synthetic estrogens have diverse chemical structures and may either positively or negatively affect the estrogenic signaling pathways through interactions with the estrogen receptors (ERs). Modeling studies suggest that 4-(1-adamantyl)phenol (AdP) and 4,4 -(1,3-adamantanediyl)diphenol (AdDP) can bind in the ligand binding site of ER . We used fluorescence polarization (FP) to compare the binding affinities of AdP, AdDP and 2-(1-adamantyl)-4-methylphenol (AdMP) for human ER and ER with the binding affinities of the known ER ligands, diethylstilbestrol (DES) and 4-hydroxytamoxifen (4OHT). Competition binding experiments show that AdDP has greater affinity for both ERs than does AdP, while AdMP does not bind the receptor proteins. The relative binding affinities of AdDP and AdP are weaker than the affinity of DES or 4OHT for both ERs with the exception of AdDP, which binds ER with higher affinity than does 4OHT. We also found that AdDP and AdP cause differential conformational changes in ER and ER , which result in altered affinities of the ERs for fluorescein-labeled estrogen response elements (EREs) using a direct binding FP assay. The results show that ER liganded with either AdDP or AdP has greater affinity for human pS2 ERE than the ER -4OHT complex. The data suggest that synthetic molecules like adamantanes may function as biologically active ligands for human ERs. This demonstrates the importance of considering the potential of novel classes of synthetic compounds as selective ER modulators.

Protein recognition of RNA has been studied using Peptide Phage Display Libraries, but in the absence of RNA modifications. Peptides from two libraries, selected for binding the modified anticodon stem and loop (ASL) of human tRNA(LyS3) having 2-thiouridine (s(2)U34) and pseudouridine (psi39), bound the modified human ASL(Lys3)(s(2)U34;psi39) preferentially and had significant homology with RNA binding proteins. Selected peptides were narrowed to a manageable number using a less sensitive, but inexpensive assay before conducting intensive characterization. The affinity and specificity of the best binding peptide (with an N-terminal fluorescein) were characterized by fluorescence spectrophotometry. The peptide exhibited the highest binding affinity for ASL(LYS3)(s(2)U34; psi39), followed by the hypermodified ASL(Lys3) (mcm(5)s(2) U34; ms(2)t(6)A37) and the unmodified ASL(Lys3), but bound poorly to singly modified ASL(Lys3) constructs (psi39, ms(2)t(6)A37, s(2)34), ASL(Lys1,2) (t(6)A37) and Escherichia coli ASL(Glu) (s(2)U34). Thus, RNA modifications are potentially important recognition elements for proteins and can be targets for selective recognition by peptides.

Biodegradable Nanoparticles (NPs) are under intense investigation due to their potential application in targeted drug delivery. Upon their entry to the biological system, they encounter the immune system, which limits their availability at the intended site. Most importantly, the innate immune system is the one that acts as the first line of defense against foreign materials. It can be activated by collectin proteins which recognize the structural pattern of polysaccharide on the surface of microorganisms. NPs may interact with these proteins in a similar way, and the interaction may lead to beneficial outcomes in vaccine delivery. On the other hand, in targeted drug delivery, it is desirable for the NPs not to be recognized as foreign material as this may lead to their fast elimination from the system through mechanism such as opsonization. We investigated the interaction of PEGylated and un-PEGylated PLGA NPs with Recombinant Human Mannose-Binding Protein (HMBP) in an effort to understand the effect of surface modification on their binding to the protein.Results show that both PLGA-COOH and PLGA-PEG-NH 2 bind to HMBP as studied using dynamic light scattering (DLS), fluoresce and UV-vis spectroscopy. However, their binding is shown to have different effect on the structure of the protein. Study done using fluorescence spectroscopy displayed a decrease in fluorescence emission of the protein upon binding to PLGA-COOH. On the other hand the fluorescence emission of the protein increased upon binding to the PLGA-PEG-NH 2 indicating conformational changes in the protein structure.