Cleavage of pre-tRNAs by the splicing endonuclease requires a composite active site

Splicing is required for the removal of introns from a subset of transfer RNAs in all eukaryotic organisms. The first step of splicing, intron recognition and cleavage, is performed by the tRNA-splicing endonuclease, a tetrameric enzyme composed of the protein subunits Sen54, Sen2, Sen34 and Sen15. It has previously been demonstrated that the active sites for cleavage at the 5' and 3' splice sites of precursor tRNA are contained within Sen2 and Sen34, respectively. A recent structure of an archaeal endonuclease complexed with a bulge-helix-bulge RNA has led to the unexpected hypothesis that catalysis requires a critical 'cation-pi sandwich' composed of two arginine residues that serve to position the RNA substrate within the active site. This motif is derived from a cross-subunit interaction between the two catalytic subunits. Here we test the role of this interaction within the eukaryotic endonuclease and show that catalysis at the 5' splice site requires the conserved cation-pi sandwich derived from the Sen34 subunit in addition to the catalytic triad of Sen2. The catalysis of pre-tRNA by the eukaryotic tRNA-splicing endonuclease therefore requires a previously unrecognized composite active site.     

Blockade of Dll4 inhibits tumour growth by promoting non-productive angiogenesis

Tumour growth requires accompanying expansion of the host vasculature, with tumour progression often correlated with vascular density. Vascular endothelial growth factor (VEGF) is the best-characterized inducer of tumour angiogenesis. We report that VEGF dynamically regulates tumour endothelial expression of Delta-like ligand 4 (Dll4), which was previously shown to be absolutely required for normal embryonic vascular development. To define Dll4 function in tumour angiogenesis, we manipulated this pathway in murine tumour models using several approaches. Here we show that blockade resulted in markedly increased tumour vascularity, associated with enhanced angiogenic sprouting and branching. Paradoxically, this increased vascularity was non-productive-as shown by poor perfusion and increased hypoxia, and most importantly, by decreased tumour growth-even for tumours resistant to anti-VEGF therapy. Thus, VEGF-induced Dll4 acts as a negative regulator of tumour angiogenesis; its blockade results in a striking uncoupling of tumour growth from vessel density, presenting a novel therapeutic approach even for tumours resistant to anti-VEGF therapies.     

Structural diversity in binary nanoparticle superlattices

Assembly of small building blocks such as atoms, molecules and nanoparticles into macroscopic structures--that is, 'bottom up' assembly--is a theme that runs through chemistry, biology and material science. Bacteria, macromolecules and nanoparticles can self-assemble, generating ordered structures with a precision that challenges current lithographic techniques. The assembly of nanoparticles of two different materials into a binary nanoparticle superlattice (BNSL) can provide a general and inexpensive path to a large variety of materials (metamaterials) with precisely controlled chemical composition and tight placement of the components. Maximization of the nanoparticle packing density has been proposed as the driving force for BNSL formation, and only a few BNSL structures have been predicted to be thermodynamically stable. Recently, colloidal crystals with micrometre-scale lattice spacings have been grown from oppositely charged polymethyl methacrylate spheres. Here we demonstrate formation of more than 15 different BNSL structures, using combinations of semiconducting, metallic and magnetic nanoparticle building blocks. At least ten of these colloidal crystalline structures have not been reported previously. We demonstrate that electrical charges on sterically stabilized nanoparticles determine BNSL stoichiometry; additional contributions from entropic, van der Waals, steric and dipolar forces stabilize the variety of BNSL structures.     

Exploiting a natural conformational switch to engineer an interleukin-2 'superkine'

The immunostimulatory cytokine interleukin-2 (IL-2) is a growth factor for a wide range of leukocytes, including T cells and natural killer (NK) cells. Considerable effort has been invested in using IL-2 as a therapeutic agent for a variety of immune disorders ranging from AIDS to cancer. However, adverse effects have limited its use in the clinic. On activated T cells, IL-2 signals through a quaternary 'high affinity' receptor complex consisting of IL-2, IL-2Rα (termed CD25), IL-2Rβ and IL-2Rγ. Naive T cells express only a low density of IL-2Rβ and IL-2Rγ, and are therefore relatively insensitive to IL-2, but acquire sensitivity after CD25 expression, which captures the cytokine and presents it to IL-2Rβ and IL-2Rγ. Here, using in vitro evolution, we eliminated the functional requirement of IL-2 for CD25 expression by engineering an IL-2 'superkine' (also called super-2) with increased binding affinity for IL-2Rβ. Crystal structures of the IL-2 superkine in free and receptor-bound forms showed that the evolved mutations are principally in the core of the cytokine, and molecular dynamics simulations indicated that the evolved mutations stabilized IL-2, reducing the flexibility of a helix in the IL-2Rβ binding site, into an optimized receptor-binding conformation resembling that when bound to CD25. The evolved mutations in the IL-2 superkine recapitulated the functional role of CD25 by eliciting potent phosphorylation of STAT5 and vigorous proliferation of T cells irrespective of CD25 expression. Compared to IL-2, the IL-2 superkine induced superior expansion of cytotoxic T cells, leading to improved antitumour responses in vivo, and elicited proportionally less expansion of T regulatory cells and reduced pulmonary oedema. Collectively, we show that in vitro evolution has mimicked the functional role of CD25 in enhancing IL-2 potency and regulating target cell specificity, which has implications for immunotherapy.     

Stereospecific binding of a disordered peptide segment mediates BK channel inactivation

A number of functionally important actions of proteins are mediated by short, intrinsically disordered peptide segments, but the molecular interactions that allow disordered domains to mediate their effects remain a topic of active investigation. Many K+ channel proteins, after initial channel opening, show a time-dependent reduction in current flux, termed 'inactivation', which involves movement of mobile cytosolic peptide segments (approximately 20-30 residues) into a position that physically occludes ion permeation. Peptide segments that produce inactivation show little amino-acid identity and tolerate appreciable mutational substitutions without disrupting the inactivation process. Solution nuclear magnetic resonance of several isolated inactivation domains reveals substantial conformational heterogeneity with only minimal tendency to ordered structures. Channel inactivation mechanisms may therefore help us to decipher how intrinsically disordered regions mediate functional effects. Whereas many aspects of inactivation of voltage-dependent K+ channels (Kv) can be described by a simple one-step occlusion mechanism, inactivation of the voltage-dependent large-conductance Ca2+-gated K+ (BK) channel mediated by peptide segments of auxiliary β-subunits involves two distinguishable kinetic steps. Here we show that two-step inactivation mediated by an intrinsically disordered BK β-subunit peptide involves a stereospecific binding interaction that precedes blockade. In contrast, blocking mediated by a Shaker Kv inactivation peptide is consistent with direct, simple occlusion by a hydrophobic segment without substantial steric requirement. The results indicate that two distinct types of molecular interaction between disordered peptide segments and their binding sites produce qualitatively similar functions.     

Contrasting patterns of early twenty-first-century glacier mass change in the Himalayas

Glaciers are among the best indicators of terrestrial climate variability, contribute importantly to water resources in many mountainous regions and are a major contributor to global sea level rise. In the Hindu Kush-Karakoram-Himalaya region (HKKH), a paucity of appropriate glacier data has prevented a comprehensive assessment of current regional mass balance. There is, however, indirect evidence of a complex pattern of glacial responses in reaction to heterogeneous climate change signals. Here we use satellite laser altimetry and a global elevation model to show widespread glacier wastage in the eastern, central and south-western parts of the HKKH during 2003-08. Maximal regional thinning rates were 0.66?±?0.09 metres per year in the Jammu-Kashmir region. Conversely, in the Karakoram, glaciers thinned only slightly by a few centimetres per year. Contrary to expectations, regionally averaged thinning rates under debris-mantled ice were similar to those of clean ice despite insulation by debris covers. The 2003-08 specific mass balance for our entire HKKH study region was -0.21?±?0.05?m?yr(-1) water equivalent, significantly less negative than the estimated global average for glaciers and ice caps. This difference is mainly an effect of the balanced glacier mass budget in the Karakoram. The HKKH sea level contribution amounts to one per cent of the present-day sea level rise. Our 2003-08 mass budget of -12.8?±?3.5 gigatonnes (Gt) per year is more negative than recent satellite-gravimetry-based estimates of -5?±?3?Gt?yr(-1) over 2003-10 (ref. 12). For the mountain catchments of the Indus and Ganges basins, the glacier imbalance contributed about 3.5% and about 2.0%, respectively, to the annual average river discharge, and up to 10% for the Upper Indus basin.     

A comparative analysis of the evolution of imperfect mimicry

Although exceptional examples of adaptation are frequently celebrated, some outcomes of natural selection seem far from perfect. For example, many hoverflies (Diptera: Syrphidae) are harmless (Batesian) mimics of stinging Hymenoptera. However, although some hoverfly species are considered excellent mimics, other species bear only a superficial resemblance to their models and it is unclear why this is so. To evaluate hypotheses that have been put forward to explain interspecific variation in the mimetic fidelity of Palearctic Syrphidae we use a comparative approach. We show that the most plausible explanation is that predators impose less selection for mimetic fidelity on smaller hoverfly species because they are less profitable prey items. In particular, our findings, in combination with previous results, allow us to reject several key hypotheses for imperfect mimicry: first, human ratings of mimetic fidelity are positively correlated with both morphometric measures and avian rankings, indicating that variation in mimetic fidelity is not simply an illusion based on human perception; second, no species of syrphid maps out in multidimensional space as being intermediate in appearance between several different hymenopteran model species, as the multimodel hypothesis requires; and third, we find no evidence for a negative relationship between mimetic fidelity and abundance, which calls into question the kin-selection hypothesis. By contrast, a strong positive relationship between mimetic fidelity and body size supports the relaxed-selection hypothesis, suggesting that reduced predation pressure on less profitable prey species limits the selection for mimetic perfection.     

Exercise-induced BCL2-regulated autophagy is required for muscle glucose homeostasis

Exercise has beneficial effects on human health, including protection against metabolic disorders such as diabetes. However, the cellular mechanisms underlying these effects are incompletely understood. The lysosomal degradation pathway, autophagy, is an intracellular recycling system that functions during basal conditions in organelle and protein quality control. During stress, increased levels of autophagy permit cells to adapt to changing nutritional and energy demands through protein catabolism. Moreover, in animal models, autophagy protects against diseases such as cancer, neurodegenerative disorders, infections, inflammatory diseases, ageing and insulin resistance. Here we show that acute exercise induces autophagy in skeletal and cardiac muscle of fed mice. To investigate the role of exercise-mediated autophagy in vivo, we generated mutant mice that show normal levels of basal autophagy but are deficient in stimulus (exercise- or starvation)-induced autophagy. These mice (termed BCL2 AAA mice) contain knock-in mutations in BCL2 phosphorylation sites (Thr69Ala, Ser70Ala and Ser84Ala) that prevent stimulus-induced disruption of the BCL2-beclin-1 complex and autophagy activation. BCL2 AAA mice show decreased endurance and altered glucose metabolism during acute exercise, as well as impaired chronic exercise-mediated protection against high-fat-diet-induced glucose intolerance. Thus, exercise induces autophagy, BCL2 is a crucial regulator of exercise- (and starvation)-induced autophagy in vivo, and autophagy induction may contribute to the beneficial metabolic effects of exercise.