The conformation and dynamics of P-glycoprotein in a lipid bilayer investigated by atomic force microscopy.

The membrane-bound P-glycoprotein (Pgp) transporter plays a major role in human disease and drug disposition because of its ability to efflux a chemically diverse range of drugs through ATP hydrolysis and ligand-induced conformational changes. Deciphering these structural changes is key to understanding the molecular basis of transport and to developing molecules that can modulate efflux. Here, atomic force microscopy (AFM) is used to directly image individual Pgp transporter molecules in a lipid bilayer under physiological pH and ambient temperature. Analysis of the Pgp AFM images revealed "small" and "large" protrusions from the lipid bilayer with significant differences in protrusion height and volume. The geometry of these "small" and "large" protrusions correlated to the predicted extracellular (EC) and cytosolic (C) domains of the Pgp X-ray crystal structure, respectively. To assign these protrusions, simulated AFM images were produced from the Pgp X-ray crystal structures with membrane planes defined by three computational approaches, and a simulated 80 ŠAFM cantilever tip. The theoretical AFM images of the EC and C domains had similar heights and volumes to the "small" and "large" protrusions in the experimental AFM images, respectively. The assignment of the protrusions in the AFM images to the EC and C domains was confirmed by changes in protrusion volume by Pgp-specific antibodies. The Pgp domains showed a considerable degree of conformational dynamics in time resolved AFM images. With this information, a model of Pgp conformational dynamics in a lipid bilayer is proposed within the context of the known Pgp X-ray crystal structures.

H3K9 methyltransferases and demethylases control lung tumor-propagating cells and lung cancer progression.

Epigenetic regulators are attractive anticancer targets, but the promise of therapeutic strategies inhibiting some of these factors has not been proven in vivo or taken into account tumor cell heterogeneity. Here we show that the histone methyltransferase G9a, reported to be a therapeutic target in many cancers, is a suppressor of aggressive lung tumor-propagating cells (TPCs). Inhibition of G9a drives lung adenocarcinoma cells towards the TPC phenotype by de-repressing genes which regulate the extracellular matrix. Depletion of G9a during tumorigenesis enriches tumors in TPCs and accelerates disease progression metastasis. Depleting histone demethylases represses G9a-regulated genes and TPC phenotypes. Demethylase inhibition impairs lung adenocarcinoma progression in vivo. Therefore, inhibition of G9a is dangerous in certain cancer contexts, and targeting the histone demethylases is a more suitable approach for lung cancer treatment. Understanding cellular context and specific tumor populations is critical when targeting epigenetic regulators in cancer for future therapeutic development.