Helix deformation is coupled to vectorial proton transport in the photocycle of bacteriorhodopsin

A wide variety of mechanisms are used to generate a proton-motive potential across cell membranes, a function lying at the heart of bioenergetics. Bacteriorhodopsin, the simplest known proton pump, provides a paradigm for understanding this process. Here we report, at 2.1 A resolution, the structural changes in bacteriorhodopsin immediately preceding the primary proton transfer event in its photocycle. The early structural rearrangements propagate from the protein's core towards the extracellular surface, disrupting the network of hydrogen-bonded water molecules that stabilizes helix C in the ground state. Concomitantly, a bend of this helix enables the negatively charged primary proton acceptor, Asp 85, to approach closer to the positively charged primary proton donor, the Schiff base. The primary proton transfer event would then neutralize these two groups, cancelling their electrostatic attraction and facilitating a relaxation of helix C to a less strained geometry. Reprotonation of the Schiff base by Asp 85 would thereby be impeded, ensuring vectorial proton transport. Structural rearrangements also occur near the protein's surface, aiding proton release to the extracellular medium.     

Structure of the dimerized hormone-binding domain of a guanylyl-cyclase-coupled receptor

The atrial natriuretic peptide (ANP) hormone is secreted by the heart in response to an increase in blood pressure. ANP exhibits several potent anti-hypertensive actions in the kidney, adrenal gland and vascular system. These actions are induced by hormone binding extracellularly to the ANP receptor, thereby activating its intracellular guanylyl cyclase domain for the production of cyclic GMP. Here we present the crystal structure of the glycosylated dimerized hormone-binding domain of the ANP receptor at 2.0-A resolution. The monomer comprises two interconnected subdomains, each encompassing a central beta-sheet flanked by alpha-helices, and exhibits the type I periplasmic binding protein fold. Dimerization is mediated by the juxtaposition of four parallel helices, arranged two by two, which brings the two protruding carboxy termini into close relative proximity. From affinity labelling and mutagenesis studies, the ANP-binding site maps to the side of the dimer crevice and extends to near the dimer interface. A conserved chloride-binding site is located in the membrane distal domain, and we found that hormone binding is chloride dependent. These studies suggest mechanisms for hormone activation and the allostery of the ANP receptor.     

A cholesteryl ester transfer protein inhibitor attenuates atherosclerosis in rabbits

Cholesteryl ester transfer protein (CETP) is a plasma protein that mediates the exchange of cholesteryl ester in high-density lipoprotein (HDL) for triglyceride in very low density lipoprotein (VLDL). This process decreases the level of anti-atherogenic HDL cholesterol and increases pro-atherogenic VLDL and low density lipoprotein (LDL) cholesterol, so CETP is potentially atherogenic. On the other hand, CETP could also be anti-atherogenic, because it participates in reverse cholesterol transport (transfer of cholesterol from peripheral cells through the plasma to the liver). Because the role of CETP in atherosclerosis remains unclear, we have attempted to develop a potent and specific CETP inhibitor. Here we describe CETP inhibitors that form a disulphide bond with CETP, and present one such inhibitor (JTT-705) that increases HDL cholesterol, decreases non-HDL cholesterol and inhibits the progression of atherosclerosis in rabbits. Our findings indicate that CETP may be atherogenic in vivo and that JTT-705 may be a potential anti-atherogenic drug.     

Fringe is a glycosyltransferase that modifies Notch

Notch receptors function in highly conserved intercellular signalling pathways that direct cell-fate decisions, proliferation and apoptosis in metazoans. Fringe proteins can positively and negatively modulate the ability of Notch ligands to activate the Notch receptor. Here we establish the biochemical mechanism of Fringe action. Drosophila and mammalian Fringe proteins possess a fucose-specific beta1,3 N-acetylglucosaminyltransferase activity that initiates elongation of O-linked fucose residues attached to epidermal growth factor-like sequence repeats of Notch. We obtained biological evidence that Fringe-dependent elongation of O-linked fucose on Notch modulates Notch signalling by using co-culture assays in mammalian cells and by expression of an enzymatically inactive Fringe mutant in Drosophila. The post-translational modification of Notch by Fringe represents a striking example of modulation of a signalling event by differential receptor glycosylation and identifies a mechanism that is likely to be relevant to other signalling pathways.     

Evolutionary origins of vertebrate appendicular muscle

The evolution of terrestrial tetrapod species heralded a transition in locomotor strategies. While most fish species use the undulating contractions of the axial musculature to generate propulsive force, tetrapods also rely on the appendicular muscles of the limbs to generate movement. Despite the fossil record generating an understanding of the way in which the appendicular skeleton has evolved to provide the scaffold for tetrapod limb musculature, there is, by contrast, almost no information as to how this musculature arose. Here we examine fin muscle formation within two extant classes of fish. We find that in the teleost, zebrafish, fin muscles arise from migratory mesenchymal precursor cells that possess molecular and morphogenetic identity with the limb muscle precursors of tetrapod species. Chondrichthyan dogfish embryos, however, use the primitive mechanism of direct epithelial somitic extensions to derive the muscles of the fin. We conclude that the genetic mechanism controlling formation of tetrapod limb muscles evolved before the Sarcopterygian radiation.     

Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors

Interaction between endothelial cells and mural cells (pericytes and vascular smooth muscle) is essential for vascular development and maintenance. Endothelial cells arise from Flk1-expressing (Flk1+) mesoderm cells, whereas mural cells are believed to derive from mesoderm, neural crest or epicardial cells and migrate to form the vessel wall. Difficulty in preparing pure populations of these lineages has hampered dissection of the mechanisms underlying vascular formation. Here we show that Flk1+ cells derived from embryonic stem cells can differentiate into both endothelial and mural cells and can reproduce the vascular organization process. Vascular endothelial growth factor promotes endothelial cell differentiation, whereas mural cells are induced by platelet-derived growth factor-BB. Vascular cells derived from Flk1+ cells can organize into vessel-like structures consisting of endothelial tubes supported by mural cells in three-dimensional culture. Injection of Flk1+ cells into chick embryos showed that they can incorporate as endothelial and mural cells and contribute to the developing vasculature in vivo. Our findings indicate that Flk1+ cells can act as 'vascular progenitor cells' to form mature vessels and thus offer potential for tissue engineering of the vascular system.