Positional dependence of non-native polar mutations on folding of CFTR helical hairpins

Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) cause CF disease by altering the biosynthesis, maturation, folding and ion conductance of this protein. Our laboratory has focused on expression and structural analysis of the CFTR transmembrane (TM) domains using two-TM segments (i.e., helix-loop-helix constructs) which we term ‘helical hairpins’; these represent the minimal model of tertiary contacts between two helices in a membrane. Previous studies on a library of TM3/4 hairpins of the first CFTR TM domain suggested that introduction of non-native polar residues into TM4 can compromise CFTR function through side chain-side chain H-bonding interactions with native Q207 in TM3 [Choi, M. Y., Cardarelli, L., Therien, A. G., and Deber, C. M. Non-native interhelical hydrogen bonds in the cystic fibrosis transmembrane conductance regulator domain modulated by polar mutations, Biochemistry 43 (2004) 8077-8083]. In the present work, we combine gel shift assays with a series of NMR experiments for comparative structural characterization of the wild type TM3/4 hairpin and its mutants V232D, I231D, Q207N/V232E. Over 95% of the backbone resonances of a ¹⁵N,¹³C-labelled V232D-TM3/4 construct in the membrane-mimetic environment of perfluorooctanoate (PFO) micelles were successfully assigned, and the presence and boundaries of helical segments within TM3 and TM4 were defined under these conditions. Comparative analysis of ¹⁵N and ¹H chemical shift variations among HSQC spectra of WT-, V232D-, I231D- and Q207N/V232E-TM3/4 indicated that hairpin conformations vary with the position of a polar mutation (i.e., V232D and I231D vs. WT), but remain similar when hairpins with identically-positioned polar partners are compared (i.e., V232D vs. Q207N-V232E). The overall findings suggest that a polar mutation in a TM helix can potentially distort native interfacial packing determinants in membrane proteins such as CFTR, with consequences that may lead to disease.     

Arachidonic acid promotes FAK activation and migration in MDA-MB-231 breast cancer cells

Arachidonic acid (AA) is a common dietary n-6 polyunsaturated fatty acid that is present in an esterified form in cell membrane phospholipids, and it might be present in the extracellular microenvironment. In particular, AA promotes MAPK activation and mediates the adhesion of MDA-MB-435 breast cancer cells to type IV collagen. However, the signal transduction pathways mediated by AA have not been studied in detail. Our results demonstrate that stimulation of MDA-MB-231 breast cancer cells with AA promotes an increase in the phoshorylation of Src and FAK, as revealed by site-specific antibodies that recognized the phosphorylation state of Src at Tyr-418, and of FAK at tyrosine-397 and in vitro kinase assays. In addition, AA also induces an increase in the migration of MDA-MB-231 cells. In contrast, AA does not induce phosphorylation of FAK and an increase in cell migration of non-tumorigenic epithelial cells MCF10A. Inhibition of Gi/Go proteins, LOX and Src activity prevent FAK activation and cell migration. In conclusion, our results demonstrate, for the first time, that Gi/Go proteins, LOX and Src play an important role in FAK activation and cell migration induced by AA in MDA-MB-231 breast cancer cells.     

The polybasic N-terminal region of the prion protein controls the physical properties of both the cellular and fibrillar forms of PrP

Individual variations in structure and morphology of amyloid fibrils produced from a single polypeptide are likely to underlie the molecular origin of prion strains and control the efficiency of the species barrier in the transmission of prions. Previously, we observed that the shape of amyloid fibrils produced from full-length prion protein (PrP 23-231) varied substantially for different batches of purified recombinant PrP. Variations in fibril morphology were also observed for different fractions that corresponded to the highly pure PrP peak collected at the last step of purification. A series of biochemical experiments revealed that the variation in fibril morphology was attributable to the presence of miniscule amounts of N-terminally truncated PrPs, where a PrP encompassing residue 31-231 was the most abundant of the truncated polypeptides. Subsequent experiments showed that the presence of small amounts of recombinant PrP 31-231 (0.1-1%) in mixtures with full-length PrP 23-231 had a dramatic impact on fibril morphology and conformation. Furthermore, the deletion of the short polybasic N-terminal region 23-30 was found to reduce the folding efficiency to the native α-helical forms and the conformational stability of α-PrP. These findings are very surprising considering that residues 23-30 are very distant from the C-terminal globular folded domain in α-PrP and from the prion folding domain in the fibrillar form. However, our studies suggest that the N-terminal polybasic region 23-30 is essential for effective folding of PrP to its native cellular conformation. This work also suggests that this region could regulate diversity of prion strains or subtypes despite its remote location from the prion folding domain.     

Comparative Effects of Boric Acid and Calcium Fructoborate on Breast Cancer Cells

Recent studies suggested that boron has a chemo-preventive role in prostate cancer. In the present report, we investigated the effects of calcium fructoborate (CF) and boric acid (BA) on activation of the apoptotic pathway in MDA-MB-231 human breast cancer cells. Exposure to BA and CF inhibited the proliferation of breast cancer cells in a dose-dependent manner. Treatment with CF but not BA resulted in a decrease in p53 and bcl-2 protein levels. Furthermore, after the treatment with CF, augmentation of pro-caspase-3 protein expression, cytosolic cytochrome c level, and caspase-3 activity were observed, indicating apoptotic cell death induction. This was also demonstrated by terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate nick-end-labeling assay. In conclusion, our data provide arguments to the fact that both BA and CF inhibited the growth of breast cancer cells, while only CF induced apoptosis. Additional studies will be needed to identify the underlying mechanism responsible for the observed cellular responses to these compounds and to determine if BA and CF may be further evaluated as chemotherapeutic agents for human cancer.     

Long-term bat study on an island in the southeastern Brazilian coast with comparison of richness and composition of species among sites

Although Rio de Janeiro is one of the best sampled states of Brazil for bats, there are still some poorly studied islands on its coast. Hence, we inventoried the bat species of an island on the southwestern coast of Rio de Janeiro, compared five sites sampled within this island, and compared its bat community with seven other sites in the same region. We carried out sampling in July 1995 and from September 2006 to August 2010, comprising 58 samplings. We captured a total of 1502 individuals of 25 bat species with mist nets set far from roosts, or close to or inside roosts. The most abundant species were Artibeus lituratus, Molossus molossus, and Carollia perspicillata. Total sampling effort was 89,400 m² h. The present study was one out of five inventories carried out in the southwestern coast of Rio de Janeiro that registered more than 1000 bat individuals from more than 24 bat species. Long-term inventories on islands are still needed, because they can result in a large number of species and captures as observed in the present study.     

Homocysteine pre-treatment increases redox capacity in both endothelial and tumor cells

Objective: We studied the modulatory effects of homocysteine pre-treatment on the disulfide reduction capacity of tumor and endothelial cells.

Methods: Human MDA-MB-231 breast carcinoma and bovine aorta endothelial cells were pre-treated for 1–24 hours with 0.5–5 mM homocysteine or homocysteine thiolactone. After washing to eliminate any rest of homocysteine or homocysteine thiolactone, cell redox capacity was determined by using a method for measuring disulfide reduction.

Results: Homocysteine pre-treatments for 1–4 hours at a concentration of 0.5–5 mM increase the disulfide reduction capacity of both tumor and endothelial cells. This effect cannot be fully mimicked by either cysteine or homocysteine thiolactone pre-treatments of tumor cells.

Discussion: Taken together, our data suggest that homocysteine can behave as an anti-oxidant agent by increasing the anti-oxidant capacity of tumor and endothelial cells.     

Invasiveness of breast carcinoma cells and transcript profile: Eph receptors and ephrin ligands as molecular markers of potential diagnostic and prognostic application

The Eph family of receptors, with 14 members in humans, makes up the largest group of receptor tyrosine kinases. These Eph receptors, along with their ligands, the 8 members of the ephrin family of ligands are involved in diverse developmental functions, including hindbrain development in vertebrates, tissue patterning, and angiogenesis. These Eph receptors and ephrin ligands have also been identified as important regulators in the development and progression of cancer. We have presented here a systematic and comprehensive investigation of the Eph/ephrin expression profiles of MCF-10A, MCF-7, and MDA-MB-231 cells representing normal breast, non-invasive breast tumor, and invasive tumor, respectively, based on their characteristic phenotypes in Matrigel matrix. The data have allowed us to correlate the gene expression profile with the cell phenotype that has potential application in tumor diagnostics. We demonstrate here that upregulation of EphA2, A7, A10, and ephrinA2 and B3 is likely involved in tumorigenesis and/or invasiveness, while downregulation of EphA1, A3, A4, A8, B3, B4, B6, and ephrinA1 and B1 may be particularly important in invasiveness. Based on these results we discuss the role of EphA2 and ephrinA1 combination in malignancy. The data have provided clues as to the importance of these molecules in the progression of breast cancer and specifically identified EphB6, a kinase-deficient receptor, which is downregulated in the most aggressive cell line, as reported for several other cancer types including neuroblastoma and melanoma suggesting its potential as a prognostic indicator in breast cancer as well.     

Plasma membrane redox system activity in tumour cell lines of mammary origins with different proliferation rates

Summary Plasma membrane redox systems seem to play a role in the control of cell growth. In fact, we have found that in mammary tumour cell lines the increase in the proliferation rate is accompanied by a decrease in the plasma membrane redox activity. The oxygen consumption rates, the glycolytic fluxes and other bioenergetic parameters have been studied in two cell strains of Ehrlich ascites tumour with different proliferation rates. In the more proliferative Ehrlich cell strain, the decrease in plasma membrane redox system activity is accompanied by decreased oxygen consumption and glycolytic flux and to a generally less energised status.     

Synthesis and antiproliferative activity of two diastereomeric lignan amides serving as dimeric caffeic acid-l-DOPA hybrids

Two new diastereomeric lignan amides (4 and 5) serving as dimeric caffeic acid-l-DOPA hybrids were synthesized. The synthesis involved the FeCl₃-mediated phenol oxidative coupling of methyl caffeate to afford trans-diester 1a as a mixture of enantiomers, protection of the catechol units, regioselective saponification, coupling with a suitably protected l-DOPA derivative, separation of the two diastereomers thus obtained by flash column chromatography and finally global chemoselective deprotection of the catechol units. The effect of hybrids 4 and 5 and related compounds on the proliferation of two breast cancer cell lines with different metastatic potential and estrogen receptor status (MDA-MB-231 and MCF-7) and of one epithelial lung cancer cell line, namely A-549, was evaluated for concentrations ranging from 1 to 256 μM and periods of treatment of 24, 48 and 72 h. Both hybrids showed interesting and almost equipotent antiproliferative activities (IC₅₀ 64–70 μM) for the MDA-MB-231 cell line after 24–48 h of treatment, but they were more selective and much more potent (IC₅₀ 4–16 μM) for the MCF-7 cells after 48 h of treatment. The highest activity for both hybrids and both breast cancer lines was observed after 72 h of treatment (IC₅₀ 1–2 μM), probably as the result of slow hydrolysis of their methyl ester functions.