Fgf8 signalling from the AER is essential for normal limb development

Vertebrate limb development depends on signals from the apical ectodermal ridge (AER), which rims the distal tip of the limb bud. Removal of the AER in chick results in limbs lacking distal skeletal elements. Fibroblast growth factor (FGF) proteins can substitute for the AER (refs 4-7), suggesting that FGF signalling mediates AER activity. Of the four mouse Fgf genes (Fgf4 , Fgf8, Fgf9, Fgf17) known to display AER-specific expression domains within the limb bud (AER-Fgfs), only Fgf8 is expressed throughout the AER. Moreover, Fgf8 expression precedes that of other AER-Fgfs (refs 8-13), suggesting that Fgf8 may perform unique functions early in limb development. In mice, loss of function of Fgf4 (refs 13,14), Fgf9 (D. Ornitz, pers. comm.) or Fgf17 (ref. 15) has no effect on limb formation. We report here that inactivating Fgf8 in early limb ectoderm causes a substantial reduction in limb-bud size, a delay in Shh expression, misregulation of Fgf4 expression, and hypoplasia or aplasia of specific skeletal elements. Our data identify Fgf8 as the only known AER-Fgf individually necessary for normal limb development, and provide insight into the function of Fgf signalling from the AER in the normal outgrowth and patterning of the limb.     

Parameter Ecology for Feedback Vertex Set

This paper deals with the FEEDBACK VERTEX SET problem on undirected graphs, which asks for the existence of a vertex set of bounded size that intersects all cycles. Due it is theoretical and practical importance,the problem has been the subject of intensive study. Motivated by the parameter ecology program we attempt to classify the parameterized and kernelization complexity of FEEDBACK VERTEX SET for a wide range of parameters.We survey known results and present several new complexity classifications. For example, we prove that FEEDBACK VERTEX SET is fixed-parameter tractable parameterized by the vertex-deletion distance to a chordal graph. We also prove that the problem admits a polynomial kernel when parameterized by the vertex-deletion distance to a pseudo forest, a graph in which every connected component has at most one cycle. In contrast, we prove that a slightly smaller parameterization does not allow for a polynomial kernel unless NP coNP=poly and the polynomial-time hierarchy collapses.     

A hierarchy of timescales in protein dynamics is linked to enzyme catalysis

The synergy between structure and dynamics is essential to the function of biological macromolecules. Thermally driven dynamics on different timescales have been experimentally observed or simulated, and a direct link between micro- to milli-second domain motions and enzymatic function has been established. However, very little is understood about the connection of these functionally relevant, collective movements with local atomic fluctuations, which are much faster. Here we show that pico- to nano-second timescale atomic fluctuations in hinge regions of adenylate kinase facilitate the large-scale, slower lid motions that produce a catalytically competent state. The fast, local mobilities differ between a mesophilic and hyperthermophilic adenylate kinase, but are strikingly similar at temperatures at which enzymatic activity and free energy of folding are matched. The connection between different timescales and the corresponding amplitudes of motions in adenylate kinase and their linkage to catalytic function is likely to be a general characteristic of protein energy landscapes.     

Intrinsic motions along an enzymatic reaction trajectory

The mechanisms by which enzymes achieve extraordinary rate acceleration and specificity have long been of key interest in biochemistry. It is generally recognized that substrate binding coupled to conformational changes of the substrate-enzyme complex aligns the reactive groups in an optimal environment for efficient chemistry. Although chemical mechanisms have been elucidated for many enzymes, the question of how enzymes achieve the catalytically competent state has only recently become approachable by experiment and computation. Here we show crystallographic evidence for conformational substates along the trajectory towards the catalytically competent 'closed' state in the ligand-free form of the enzyme adenylate kinase. Molecular dynamics simulations indicate that these partially closed conformations are sampled in nanoseconds, whereas nuclear magnetic resonance and single-molecule fluorescence resonance energy transfer reveal rare sampling of a fully closed conformation occurring on the microsecond-to-millisecond timescale. Thus, the larger-scale motions in substrate-free adenylate kinase are not random, but preferentially follow the pathways that create the configuration capable of proficient chemistry. Such preferred directionality, encoded in the fold, may contribute to catalysis in many enzymes.     

Bone metastasis: can osteoclasts be excluded?

The RANK/RANKL signalling mechanism is the final common pathway of osteoclast formation and activity. Inhibitors of RANK ligand (RANKL) that bind to RANK (for 'receptor activator of NF-kappaB'), such as osteoprotegerin (OPG), neutralizing antibodies against RANKL and soluble RANK antagonists, are well described inhibitors of bone metastasis in preclinical and clinical models, presumably because of their effects on osteoclasts. Jones et al. show that OPG inhibits bone metastasis after intracardiac injection of B16F10 murine melanoma cells, but claim that bone metastases are entirely independent of osteoclast formation and bone resorption: rather, they are caused by an effect on cell migration through RANK. However, we question whether these surprising conclusions are rigorously supported by their data.     

Structural basis for AMP binding to mammalian AMP-activated protein kinase

AMP-activated protein kinase (AMPK) regulates cellular metabolism in response to the availability of energy and is therefore a target for type II diabetes treatment. It senses changes in the ratio of AMP/ATP by binding both species in a competitive manner. Thus, increases in the concentration of AMP activate AMPK resulting in the phosphorylation and differential regulation of a series of downstream targets that control anabolic and catabolic pathways. We report here the crystal structure of the regulatory fragment of mammalian AMPK in complexes with AMP and ATP. The phosphate groups of AMP/ATP lie in a groove on the surface of the gamma domain, which is lined with basic residues, many of which are associated with disease-causing mutations. Structural and solution studies reveal that two sites on the gamma domain bind either AMP or Mg.ATP, whereas a third site contains a tightly bound AMP that does not exchange. Our binding studies indicate that under physiological conditions AMPK mainly exists in its inactive form in complex with Mg.ATP, which is much more abundant than AMP. Our modelling studies suggest how changes in the concentration of AMP ([AMP]) enhance AMPK activity levels. The structure also suggests a mechanism for propagating AMP/ATP signalling whereby a phosphorylated residue from the alpha and/or beta subunits binds to the gamma subunit in the presence of AMP but not when ATP is bound.