Atrial fibrillation (AF) is a common cardiac arrhythmia whose molecular etiology is poorly understood. We studied a family with hereditary persistent AF and identified the causative mutation (S140G) in the KCNQ1 (KvLQT1) gene on chromosome 11p15.5. The KCNQ1 gene encodes the pore-forming alpha subunit of the cardiac I(Ks) channel (KCNQ1/KCNE1), the KCNQ1/KCNE2 and the KCNQ1/KCNE3 potassium channels. Functional analysis of the S140G mutant revealed a gain-of-function effect on the KCNQ1/KCNE1 and the KCNQ1/KCNE2 currents, which contrasts with the dominant negative or loss-of-function effects of the KCNQ1 mutations previously identified in patients with long QT syndrome. Thus, the S140G mutation is likely to initiate and maintain AF by reducing action potential duration and effective refractory period in atrial myocytes.

Atrial fibrillation (AF) is the most common cardiac arrhythmia encountered in clinical practice. We first reported an S140G mutation of KCNQ1, an alpha subunit of potassium channels, in one Chinese kindred with AF. However, the molecular defects and cellular mechanisms in most patients with AF remain to be identified. We evaluated 28 unrelated Chinese kindreds with AF and sequenced eight genes of potassium channels (KCNQ1, HERG, KCNE1, KCNE2, KCNE3, KCNE4, KCNE5, and KCNJ2). An arginine-to-cysteine mutation at position 27 (R27C) of KCNE2, the beta subunit of the KCNQ1-KCNE2 channel responsible for a background potassium current, was found in 2 of the 28 probands. The mutation was present in all affected members in the two kindreds and was absent in 462 healthy unrelated Chinese subjects. Similar to KCNQ1 S140G, the mutation had a gain-of-function effect on the KCNQ1-KCNE2 channel; unlike long QT syndrome-associated KCNE2 mutations, it did not alter HERG-KCNE2 current. The mutation did not alter the functions of the HCN channel family either. Thus, KCNE2 R27C is a gain-of-function mutation associated with the initiation and/or maintenance of AF.

Increased fracture risk is commonly reported in cancer patients receiving radiotherapy, particularly at sites within the field of treatment. The direct and systemic effects of ionizing radiation on bone at a therapeutic dose are not well characterized in clinically relevant animal models. Using twenty-week male C57Bl/6 mice, effects of irradiation (right hindlimb; 2 Gy) on bone volume and microarchitecture were evaluated prospectively by microcomputed tomography and histomorphometry and compared to contralateral-shielded bone (left hindlimb) and non-irradiated control bone. One-week post-irradiation, trabecular bone volume declined in irradiated tibiae (−22%; p<0.0001) and femora (−14%; p=0.0586) and microarchitectural parameters were compromised. Trabecular bone volume declined in contralateral tibiae (−17%; p=0.003), and no loss was detected at the femur. Osteoclast number, apoptotic osteocyte number and marrow adiposity were increased in irradiated bone relative to contralateral and non-irradiated bone, while osteoblast number was unchanged. Despite no change in osteoblast number one-week post-irradiation, dynamic bone formation indices revealed a reduction in mineralized bone surface and a concomitant increase in unmineralized osteoid surface area in irradiated bone relative to contralateral and non-irradiated control bone. Further, dose- and time-dependent calvarial culture and in vitro assays confirmed that calvarial osteoblasts and osteoblast-like MC3T3 cells were relatively radioresistant, while calvarial osteocyte and osteocyte-like MLO-Y4 cell apoptosis was induced as early as 48h post-irradiation (4 Gy). In osteoclastogenesis assays, radiation exposure (8 Gy) stimulated murine macrophage RAW264.7 cell differentiation and co-culture of irradiated RAW264.7 cells with MLO-Y4 or murine bone marrow cells enhanced this effect. These studies highlight the multi-faceted nature of radiation-induced bone loss by demonstrating direct and systemic effects on bone and its many cell types using clinically relevant doses and have important implications for bone health in patients treated with radiation therapy.

Ifitm3 was previously identified as an endosomal protein that blocks viral infection 1 – 3 . Studying clinical cohorts of B-cell leukemia and lymphoma patients, we identified IFITM3 as a strong predictor of poor outcome. In normal resting B-cells, Ifitm3 was minimally expressed and mainly localized in endosomes. However, B-cell receptor (BCR) engagement induced expression of Ifitm3 and phosphorylation at Y20, resulting in accumulation at the cell surface. In B-cell leukemia, oncogenic kinases phosphorylate IFITM3-Y20, causing constitutive plasma membrane localization. Ifitm3 ˉ / ˉ naïve B-cells developed at normal numbers; however, germinal center formation and production of antigen-specific antibodies were compromised. Oncogenes that induce development of leukemia and lymphoma failed to transform Ifitm3 ˉ / ˉ B-cells. Conversely, the phospho-mimetic IFITM3-Y20E induced oncogenic PI3K-signaling and initiated transformation of pre-malignant B-cells. Mechanistic experiments revealed that Ifitm3 functions as PIP3-scaffold and central amplifier of PI3K signaling. PI3K signal-amplification depends on Ifitm3 scaffolding PIP3-accumulation via two lysine residues (K83 and K104) in its conserved intracellular loop. In Ifitm3 ˉ / ˉ B-cells, lipid rafts were depleted of PIP3, resulting in defective expression of >60 lipid raft-associated surface receptors, impaired BCR-signaling and cellular adhesion. We conclude that phosphorylation of IFITM3 upon B-cell antigen-encounter induces a dynamic switch from antiviral effector functions in endosomes to a PI3K-amplification loop at the cell surface. IFITM3-dependent amplification of PI3K-signaling in part downstream of the BCR is critical to enable rapid expansion of B-cells with high affinity to antigen. In addition, multiple oncogenes depend on IFITM3 to assemble PIP3-dependent signaling complexes and amplify PI3K-signaling for malignant transformation.

Photophysical measurements are reported for Cy3-DNA constructs in which both Cy3 nitrogen atoms are attached to the DNA backbone by short linkers. While this linking was thought to rigidify the orientation of the dye and hinder cis-isomerization, the relatively low fluorescence quantum yield and the presence of a short component in the time-resolved fluorescence decay of the dye indicated that cis-isomerization remained possible. Fluorescence correlation spectroscopy and transient absorption experiments showed that photoisomerization occurred with high efficiency. Molecular dynamics simulations of the trans dye system indicated the presence of stacked and unstacked states, and free energy simulations showed that the barriers for stacking/unstacking were low. In addition, simulations showed that the ground cis state was feasible without DNA distortions. Based on these observations, a model is put forward in which the doubly linked dye can photoisomerize in the unstacked state.

G protein-coupled receptors (GPCRs) are divided phylogenetically into six classes, A-F 1 , 2 . Over 370 structures of vertebrate GPCRs (classes A, B, C and F) have been determined, leading to a substantial understanding of their function 3 . In contrast, there are no structures of Class D GPCRs, which are found exclusively in fungi where they regulate survival and reproduction. We have determined the first structure of a family D GPCR, the Saccharomyces cerevisiae pheromone receptor Ste2, in an active state coupled to the heterotrimeric G protein Gpa1-Ste4-Ste18. Ste2 was purified as a homodimer that coupled to two G proteins. The dimer interface of Ste2 is formed by the N-terminus, transmembrane helices H1, H2, H7 and the first extracellular loop ECL1. We established a Class D1 generic residue numbering system (CD1) to enable comparisons with orthologues and other GPCR classes. The structure of Ste2 bears similarities in overall topology to Class A GPCRs, but H4 is shifted by over 20 Å and the G protein binding site is a shallow groove rather than a cleft. The structure provides a template for the design of novel drugs targeting fungal GPCRs that could be utilised to treat a number of intractable fungal diseases 4 .

No abstract

The development of peptidomimetic helical foldamers with a wide repertoire of functions is of significant interest. Herein, we report the X-ray crystal structures of a series of homogeneous l-sulfono-γ-AA foldamers and elucidate their folding conformation at the atomic level. Single-crystal X-ray crystallography revealed that this class of oligomers fold into unprecedented dragon-boat-shaped and unexpected left-handed helices, which are stabilized by the 14-hydrogen-bonding pattern present in all sequences. These l-sulfono-γ-AApeptides have a helical pitch of 5.1 Å and exactly four side chains per turn, and the side chains lie perfectly on top of each other along the helical axis. 2D NMR spectroscopy, computational simulations, and CD studies support the folding conformation in solution. Our results provide a structural basis at the atomic level for the design of novel biomimetics with a precise arrangement of functional groups in three dimensions.