Strong evidence supports the idea that specific metabolites of estrogens, mainly catechol estrogen-3,4-quinones, can react with DNA to become endogenous initiators of breast, prostate, and other human cancers. Oxidation of the catechol estrogen metabolites 4-hydroxyestradiol (4-OHE 2 ) and 2-OHE 2 leads to the quinones, estradiol-3,4-quinone (E 2 -3,4-Q) and estradiol-2,3-quinone (E 2 -2,3-Q), respectively. The reaction of E 2 -3,4-Q with DNA affords predominantly the depurinating adducts 4-OHE 2 -1-N3Ade and 4-OHE 2 -1-N7Gua, whereas the reaction of E 2 -2,3-Q with DNA yields the newly synthesized depurinating adduct 2-OHE 2 -6-N3Ade. The N3Ade adducts are lost from DNA by rapid depurination, while the N7Gua adduct is lost from DNA with a half-life of ∼3 h at 37°C. To compare the relative reactivity of E 2 -3,4-Q and E 2 -2,3-Q, the compounds were reacted individually with DNA for 0.5-20 h at 37°C, as well as in mixtures (3:1, 1:1, 1:3, and 5:95) for 10 h at 37°C. Depurinating and stable adducts were analyzed. In similar experiments, the relative reactivity of 4-OHE 2 and 2-OHE 2 with DNA was determined after activation by lactoperoxidase, tyrosinase, prostaglandin H synthase (PHS), or 3-methylcholanthreneinduced rat liver microsomes. Starting with the quinones, the levels of depurinating adducts formed from E 2 -3,4-Q were much higher than that of the depurinating adduct from E 2 -2,3-Q. Similar results were obtained with lactoperoxidase or tyrosinase-catalyzed oxidation of 4-OHE 2 and 2-OHE 2 , whereas with activation by PHS or microsomes, a relatively higher amount of the depurinating adduct from E 2 -2,3-Q was detected. These results demonstrate that the E 2 -3,4-Q is much more reactive with DNA than E 2 -2,3-Q. The relative reactivities of E 2 -3,4-Q and E 2 -2,3-Q to form depurinating adducts correlate with the carcinogenicity, mutagenicity, and cell-transforming activity of their precursors, the catechol estrogens 4-OHE 2 and 2-OHE 2 . This is essential information for understanding the cancer risk posed by oxidation of the two catechol estrogens.

Background: Ubiquitin (E3) ligases interact with specific ubiquitin conjugating (E2) enzymes to ubiquitinate particular substrate proteins. As the combination of E2 and E3 dictates the type and biological consequence of ubiquitination, it is important to understand the basis of specificity in E2:E3 interactions. The E3 ligase CHIP interacts with Hsp70 and Hsp90 and ubiquitinates client proteins that are chaperoned by these heat shock proteins. CHIP interacts with two types of E2 enzymes, UbcH5 and Ubc13-Uev1a. It is unclear, however, why CHIP binds these E2 enzymes rather than others, and whether CHIP interacts preferentially with UbcH5 or Ubc13-Uev1a, which form different types of polyubiquitin chains.

Cochaperones are essential for Hsp70/Hsc70-mediated folding of proteins and include nucleotide exchange factors (NEF) that assist protein folding by accelerating ADP/ATP exchange on Hsp70. The cochaperone Bag2 binds misfolded Hsp70 clients and also acts as a NEF, but the molecular basis of its functions is unclear. We show that, rather than being a member of the Bag domain family, Bag2 contains a new type of Hsp70 NEF domain, which we call the “Brand New Bag” (BNB) domain. Free and Hsc70-bound crystal structures of Bag2-BNB show its dimeric structure in which a flanking linker helix and loop bind to Hsc70 to promote nucleotide exchange. NMR analysis demonstrates that the client-binding sites and Hsc70 interaction sites of Bag2-BNB overlap, and that Hsc70 can displace clients from Bag2-BNB, indicating a distinct mechanism for the regulation of Hsp-70-mediated protein folding by Bag2.

Activation of cytotoxic nucleoside analogs in vivo depends primarily on their cell-specific phosphorylation. Anticancer chemotherapy using nucleoside analogs may be significantly enhanced by intracellular administration of active phosphorylated drugs. However, the cellular transport of anionic compounds is very ineffective and restricted by many drug efflux transporters. Recently developed cationic nanogel carriers can encapsulate large amounts of nucleoside 5′-triphosphates that form polyionic complexes with protonated amino groups on the polyethylenimine backbone of the nanogels. In this paper, 5′-triphosphate of an antiviral nucleoside analog, 3′-azido-2′,3′-dideoxythymidine (AZT), was efficiently synthesized and its complexes with nanogels were obtained and evaluated as potential cytotoxic drug formulations for treatment of human breast carcinoma cells. A selective phosphorylating reagent, tris-imidazolylphosphate, was used to convert AZT into the nucleoside analog 5′-triphosphate using a one-pot procedure. The corresponding 3′-azidothymidine 5′-triphosphate (AZTTP) was isolated with high yield (75%). Nanogels encapsulated up to 30% of AZTTP by weight by mixing solutions of the carrier and the drug. The AZTTP/nanogel formulation showed enhanced cytotoxicity in two breast cancer cell lines, MCF-7 and MDA-MB-231, demonstrating the IC 50 values 130-200 times lower than those values for AZT alone. Exact mechanism of drug release from nanogels remains unclear. One mechanism could involve interaction with negatively charged counterions. A high affinity of nanogels to isolated cellular membranes has been observed, especially for nanogels made of amphiphilic block copolymer, Pluronic® P85. Cellular trafficking of nanogel particles, contrasted by PEI-coordinated copper(II) ions, was studied by transmission electron microscopy (TEM), which revealed membranotropic properties of nanogels. A substantial release of encapsulated drug was observed following interactions of drug-loaded nanogels with cellular membranes. A drug release mechanism triggered by interaction of the drugloaded nanogels with phospholipid bilayer is proposed. The results illustrate therapeutic potential of the phosphorylated nucleoside analogs formulated in nanosized crosslinked polymeric carriers for cancer chemotherapy.

Therapies including nucleoside analogs are associated with severe toxic side effects and acquirement of drug resistance. We have previously reported the drug delivery in the form of 5'-triphosphates (NTP) encapsulated in cross-linked cationic networks of polyethylenimine (PEI) and PEG/Pluronic polymers (Nanogels). In this study, Nanogels, containing biodegradable PEI that could easily dissociate in reducing cytosolic environment and form products with minimal toxicity, were synthesized and displayed low cytotoxicity. Toxicity of Nanogels was clearly dependent on the total positive charge of carriers and was 5-6 fold lower for carriers loaded with NTP. Though intracellular ATP level was immediately reduced by ca. 50% following the treatment with Nanogels, it was largely restored 24 h later. Effect of Nanogels on various respiratory components of cells was reversible too, and, therefore, resulted in low immediate cell death. Nanogel alone and formulations with AZT-TP demonstrated a much lower mitochondrial toxicity than AZT. As an example of potential antiviral applications of low-toxic Nanogel carriers, a 5'-triphosphorylated Ribavirin-Nanogel formulation was prepared that demonstrated a 30-fold decrease in effective drug concentration (EC(90)) and, totally, a 10-fold increase in selectivity index compared to the drug alone in MDCK cells infected with influenza A virus.

Nanogels composed of Pluronic F68 and P123 were shown to display certain advanced properties compared to NG(PEG) as a drug delivery system for NTP analogs. Formulations of nucleoside analogs in active NTP form with these nanogels will improve the delivery of these cytotoxic drugs to cancer cells and the therapeutic potential of this anticancer chemotherapy.

Mutations in the chloride channel cystic fibrosis transmembrane regulator (CFTR) cause cystic fibrosis, a genetic disorder characterized by defects in CFTR biosynthesis, localization to the cell surface, or activation by regulatory factors. It was discovered recently that surface localization of CFTR is stabilized by an interaction between the CFTR N terminus and the multidomain cytoskeletal protein filamin. The details of the CFTRfilamin interaction, however, are unclear. Using x-ray crystallography, we show how the CFTR N terminus binds to immunoglobulin-like repeat 21 of filamin A (FlnA-Ig21). CFTR binds to ␤-strands C and D of FlnA-Ig21 using backbone-backbone hydrogen bonds, a linchpin serine residue, and hydrophobic side-chain packing. We use NMR to determine that the CFTR N terminus also binds to several other immunoglobulinlike repeats from filamin A in vitro. Our structural data explain why the cystic fibrosis-causing S13F mutation disrupts CFTRfilamin interaction. We show that FlnA-Ig repeats transfected into cultured Calu-3 cells disrupt CFTR-filamin interaction and reduce surface levels of CFTR. Our findings suggest that filamin A stabilizes surface CFTR by anchoring it to the actin cytoskeleton through interactions with multiple filamin Ig repeats. Such an interaction mode may allow filamins to cluster multiple CFTR molecules and to promote colocalization of CFTR and other filamin-binding proteins in the apical plasma membrane of epithelial cells.Cystic fibrosis (CF) 4 is a genetic disorder caused by mutations in an apical chloride channel, cystic fibrosis transmembrane regulator (CFTR). This disorder is characterized by high sweat chloride concentration, pulmonary disease with high production of dehydrated viscous secretions, and pancreatic insufficiency (1). CF affects all exocrine epithelia, with morbidity and mortality primarily caused by bacterial infection and inflammation in the lung. CF affects ϳ30,000 individuals in North America, of whom about 70% carry one copy of the mutation ⌬F508, the most common of Ͼ1,000 CF-associated mutations. ⌬F508 is a folding mutation that leads to rapid degradation at the endoplasmic reticulum. The small fraction of ⌬F508-CFTR that is not degraded is characterized by inefficient trafficking to the apical plasma membrane and reduced residency in the plasma membrane (2, 3).Although the levels of ⌬F508-CFTR in the apical plasma membrane are low, ⌬F508-CFTR retains partial function as a cAMP-activated chloride channel (4, 5). This justifies therapeutic approaches to promote delivery of ⌬F508-CFTR and other functionally impaired CFTR mutants to the plasma membrane. A detailed understanding of factors that stabilize and regulate CFTR at the plasma membrane is important for the development of new therapies to correct CF-causing defects in vivo.CFTR is regulated by intracellular cAMP levels and phosphorylated at multiple sites by cAMP-activated protein kinase, which modulates CFTR trafficking (6) and activity (7,8). We and others have identified and characterized addit...

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