Impact of cholesterol and Lumacaftor on the folding of CFTR helical hairpins.

Cystic fibrosis (CF) is caused by mutations in the gene that codes for the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). Recent advances in CF treatment have included use of small-molecule drugs known as modulators, such as Lumacaftor (VX-809), but their detailed mechanism of action and interplay with the surrounding lipid membranes, including cholesterol, remain largely unknown. To examine these phenomena and guide future modulator development, we prepared a set of wild type (WT) and mutant helical hairpin constructs consisting of CFTR transmembrane (TM) segments 3 and 4 and the intervening extracellular loop (termed TM3/4 hairpins) that represent minimal membrane protein tertiary folding units. These hairpin variants, including CF-phenotypic loop mutants E217G and Q220R, and membrane-buried mutant V232D, were reconstituted into large unilamellar phosphatidylcholine (POPC) vesicles, and into corresponding vesicles containing 70 mol% POPC +30 mol% cholesterol, and studied by single-molecule FRET and circular dichroism experiments. We found that the presence of 30 mol% cholesterol induced an increase in helicity of all TM3/4 hairpins, suggesting an increase in bilayer cross-section and hence an increase in the depth of membrane insertion compared to pure POPC vesicles. Importantly, when we added the corrector VX-809, regardless of the presence or absence of cholesterol, all mutants displayed folding and helicity largely indistinguishable from the WT hairpin. Fluorescence spectroscopy measurements suggest that the corrector alters lipid packing and water accessibility. We propose a model whereby VX-809 shields the protein from the lipid environment in a mutant-independent manner such that the WT scaffold prevails. Such 'normalization' to WT conformation is consistent with the action of VX-809 as a protein-folding chaperone.

Lipid membranes modulate the activity of RNA through sequence-dependent interactions.

RNA is a ubiquitous biomolecule that can serve as both catalyst and information carrier. Understanding how RNA bioactivity is controlled is crucial for elucidating its physiological roles and potential applications in synthetic biology. Here, we show that lipid membranes can act as RNA organization platforms, introducing a mechanism for riboregulation. The activity of R3C ribozyme can be modified by the presence of lipid membranes, with direct RNA-lipid interactions dependent on RNA nucleotide content, base pairing, and length. In particular, the presence of guanine in short RNAs is crucial for RNA-lipid interactions, and G-quadruplex formation further promotes lipid binding. Lastly, by artificially modifying the R3C substrate sequence to enhance membrane binding, we generated a lipid-sensitive ribozyme reaction with riboswitch-like behavior. These findings introduce RNA-lipid interactions as a tool for developing synthetic riboswitches and RNA-based lipid biosensors and bear significant implications for RNA world scenarios for the origin of life.

Tail-Oxidized Cholesterol Enhances Membrane Permeability for Small Solutes.

Cholesterol renders mammalian cell membranes more compact by reducing the amount of voids in the membrane structure. Because of this, cholesterol is known to regulate the ability of cell membranes to prevent the permeation of water and water-soluble molecules through the membranes. Meanwhile, it is also known that even seemingly tiny modifications in the chemical structure of cholesterol can lead to notable changes in membrane properties. The question is, how significantly do these small changes in cholesterol structure affect the permeability barrier function of cell membranes? In this work, we applied fluorescence methods as well as atomistic molecular dynamics simulations to characterize changes in lipid membrane permeability induced by cholesterol oxidation. The studied 7ß-hydroxycholesterol (7ß-OH-chol) and 27-hydroxycholesterol (27-OH-chol) represent two distinct groups of oxysterols, namely, ring- and tail-oxidized cholesterols, respectively. Our previous research showed that the oxidation of the cholesterol tail has only a marginal effect on the structure of a lipid bilayer; however, oxidation was found to disturb membrane dynamics by introducing a mechanism that allows sterol molecules to move rapidly back and forth across the membrane-bobbing. Herein, we show that bobbing of 27-OH-chol accelerates fluorescence quenching of NBD-lipid probes in the inner leaflet of liposomes by dithionite added to the liposomal suspension. Systematic experiments using fluorescence quenching spectroscopy and microscopy led to the conclusion that the presence of 27-OH-chol increases membrane permeability to the dithionite anion. Atomistic molecular dynamics simulations demonstrated that 27-OH-chol also facilitates water transport across the membrane. The results support the view that oxysterol bobbing gives rise to successive perturbations to the hydrophobic core of the membrane, and these perturbations promote the permeation of water and small water-soluble molecules through a lipid bilayer. The observed impairment of permeability can have important consequences for eukaryotic organisms. The effects described for 27-OH-chol were not observed for 7ß-OH-chol which represents ring-oxidized sterols.

Endometriosis in a post-laparoscopic scar--case report and literature review.

Endometriosis is an estrogen-dependent, chronic disease consisting in implantation and hyperplasia of the endometrium outside of the uterine cavity Endometriosis in post-laparoscopic scars applies to approx. 0.5-7% of the extraorganic locations of the disease. The purpose of the paper was to describe medical management and literature review for endometriosis in a post-laparoscopic scar. Two lesions located near the insertion site for the lower trocars were removed along with the border of healthy tissue. At the time of publication of this paper the patient did not report any complaints. In conclusion, each limited lesion in the subcutaneous tissue, with pain intensifying during menstruation, should suggest an initial diagnosis of scar endometriosis, regardless of patient age and type of surgery

Pleomorphic rhabdomyosarcoma of the uterine corpus--a case report.

Pleomorphic rhabdomyosarcoma of the uterus is a rare malignant tumor. It is connected with postmenopausal abnormal vaginal bleeding and abdominal pain. We report a case of a 66-year-old postmenopausal woman diagnosed with abnormal vaginal bleeding and abdominal pain. Vaginal ultrasonography showed enlarged uterus, 82 mm x 64 mm in size. The patient underwent total abdominal hysterectomy and bilateral salpingo-oophorectomy with postoperative chemotherapy due to pleomorphic rhabdomyosarcoma of the uterus. The patient died 2.5 years after the surgery as a result of a rapid spread of the neoplastic process. The case of rhabdomyosarcoma, together with the review of the literature, is presented in the following work. We find that the rarity of this histological entity makes it particularly worthy of publication.