Solution structure of a protein denatured state and folding intermediate

The most controversial area in protein folding concerns its earliest stages. Questions such as whether there are genuine folding intermediates, and whether the events at the earliest stages are just rearrangements of the denatured state or progress from populated transition states, remain unresolved. The problem is that there is a lack of experimental high-resolution structural information about early folding intermediates and denatured states under conditions that favour folding because competent states spontaneously fold rapidly. Here we have solved directly the solution structure of a true denatured state by nuclear magnetic resonance under conditions that would normally favour folding, and directly studied its equilibrium and kinetic behaviour. We engineered a mutant of Drosophila melanogaster Engrailed homeodomain that folds and unfolds reversibly just by changing ionic strength. At high ionic strength, the mutant L16A is an ultra-fast folding native protein, just like the wild-type protein; however, at physiological ionic strength it is denatured. The denatured state is a well-ordered folding intermediate, poised to fold by docking helices and breaking some non-native interactions. It unfolds relatively progressively with increasingly denaturing conditions, and so superficially resembles a denatured state with properties that vary with conditions. Such ill-defined unfolding is a common feature of early folding intermediate states and accounts for why there are so many controversies about intermediates versus compact denatured states in protein folding.     

Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells

A long-standing hypothesis on tumorigenesis is that cell division failure, generating genetically unstable tetraploid cells, facilitates the development of aneuploid malignancies. Here we test this idea by transiently blocking cytokinesis in p53-null (p53-/-) mouse mammary epithelial cells (MMECs), enabling the isolation of diploid and tetraploid cultures. The tetraploid cells had an increase in the frequency of whole-chromosome mis-segregation and chromosomal rearrangements. Only the tetraploid cells were transformed in vitro after exposure to a carcinogen. Furthermore, in the absence of carcinogen, only the tetraploid cells gave rise to malignant mammary epithelial cancers when transplanted subcutaneously into nude mice. These tumours all contained numerous non-reciprocal translocations and an 8-30-fold amplification of a chromosomal region containing a cluster of matrix metalloproteinase (MMP) genes. MMP overexpression is linked to mammary tumours in humans and animal models. Thus, tetraploidy enhances the frequency of chromosomal alterations and promotes tumour development in p53-/- MMECs.     

Global pattern of trends in streamflow and water availability in a changing climate

Water availability on the continents is important for human health, economic activity, ecosystem function and geophysical processes. Because the saturation vapour pressure of water in air is highly sensitive to temperature, perturbations in the global water cycle are expected to accompany climate warming. Regional patterns of warming-induced changes in surface hydroclimate are complex and less certain than those in temperature, however, with both regional increases and decreases expected in precipitation and runoff. Here we show that an ensemble of 12 climate models exhibits qualitative and statistically significant skill in simulating observed regional patterns of twentieth-century multidecadal changes in streamflow. These models project 10-40% increases in runoff in eastern equatorial Africa, the La Plata basin and high-latitude North America and Eurasia, and 10-30% decreases in runoff in southern Africa, southern Europe, the Middle East and mid-latitude western North America by the year 2050. Such changes in sustainable water availability would have considerable regional-scale consequences for economies as well as ecosystems.     

ATP is a mediator of chemosensory transduction in the central nervous system

Extracellular signalling by the purine nucleotide ATP has long been associated with sensory function. In the periphery, ATP mediates nociception, mechanosensitivity, thermal sensitivity and O2 chemosensitivity. These processes share a common mechanism that involves the release of ATP to excite afferent fibres via activation of ionotropic P2X and/or metabotropic P2Y receptors. Chemosensors located in the brainstem are crucial for the maintenance of physiological levels of blood gases through the regulation of breathing. Here we show that an increase in pCO2 in the arterial blood triggers the immediate release of ATP from three chemosensitive regions located on the ventral surface of the medulla oblongata. Blockade of ATP receptors at these sites diminishes the chemosensory control of breathing, suggesting that ATP release constitutes a key step in central chemosensory transduction. These new data suggest that ATP, a phylogenetically ancient, unique and simple molecule, has been widely used in the evolution of afferent systems to mediate distinct forms of sensory transduction not only in the periphery but also within the central nervous system.     

Reinforcement of pre-zygotic isolation and karyotype evolution in Agrodiaetus butterflies

The reinforcement model of evolution argues that natural selection enhances pre-zygotic isolation between divergent populations or species by selecting against unfit hybrids or costly interspecific matings. Reinforcement is distinguished from other models that consider the formation of reproductive isolation to be a by-product of divergent evolution. Although theory has shown that reinforcement is a possible mechanism that can lead to speciation, empirical evidence has been sufficiently scarce to raise doubts about the importance of reinforcement in nature. Agrodiaetus butterflies (Lepidoptera: Lycaenidae) exhibit unusual variability in chromosome number. Whereas their genitalia and other morphological characteristics are largely uniform, different species vary considerably in male wing colour, and provide a model system to study the role of reinforcement in speciation. Using comparative phylogenetic methods, we show that the sympatric distribution of 15 relatively young sister taxa of Agrodiaetus strongly correlates with differences in male wing colour, and that this pattern is most likely the result of reinforcement. We find little evidence supporting sympatric speciation: rather, in Agrodiaetus, karyotypic changes accumulate gradually in allopatry, prompting reinforcement when karyotypically divergent races come into contact.     

Disruption of Bardet-Biedl syndrome ciliary proteins perturbs planar cell polarity in vertebrates

The evolutionarily conserved planar cell polarity (PCP) pathway (or noncanonical Wnt pathway) drives several important cellular processes, including epithelial cell polarization, cell migration and mitotic spindle orientation. In vertebrates, PCP genes have a vital role in polarized convergent extension movements during gastrulation and neurulation. Here we show that mice with mutations in genes involved in Bardet-Biedl syndrome (BBS), a disorder associated with ciliary dysfunction, share phenotypes with PCP mutants including open eyelids, neural tube defects and disrupted cochlear stereociliary bundles. Furthermore, we identify genetic interactions between BBS genes and a PCP gene in both mouse (Ltap, also called Vangl2) and zebrafish (vangl2). In zebrafish, the augmented phenotype results from enhanced defective convergent extension movements. We also show that Vangl2 localizes to the basal body and axoneme of ciliated cells, a pattern reminiscent of that of the BBS proteins. These data suggest that cilia are intrinsically involved in PCP processes.     

Ipr1 gene mediates innate immunity to tuberculosis

An estimated eight million people are infected each year with the pathogen Mycobacterium tuberculosis, and more than two million die annually. Yet only about 10% of those infected develop tuberculosis. Genetic variation within host populations is known to be significant in humans and animals, but the nature of genetic control of host resistance to tuberculosis remains poorly understood. Previously we mapped a new genetic locus on mouse chromosome 1, designated sst1 (for supersusceptibility to tuberculosis 1). Here we show that this locus mediates innate immunity in sst1 congenic mouse strains and identify a candidate gene, Intracellular pathogen resistance 1 (Ipr1), within the sst1 locus. The Ipr1 gene is upregulated in the sst1 resistant macrophages after activation and infection, but it is not expressed in the sst1 susceptible macrophages. Expression of the Ipr1 transgene in the sst1 susceptible macrophages limits the multiplication not only of M. tuberculosis but also of Listeria monocytogenes and switches a cell death pathway of the infected macrophages from necrosis to apoptosis. Our data indicate that the Ipr1 gene product might have a previously undocumented function in integrating signals generated by intracellular pathogens with mechanisms controlling innate immunity, cell death and pathogenesis.     

Sodium channel mutation leading to saxitoxin resistance in clams increases risk of PSP

Bivalve molluscs, the primary vectors of paralytic shellfish poisoning (PSP) in humans, show marked inter-species variation in their capacity to accumulate PSP toxins (PSTs) which has a neural basis. PSTs cause human fatalities by blocking sodium conductance in nerve fibres. Here we identify a molecular basis for inter-population variation in PSP resistance within a species, consistent with genetic adaptation to PSTs. Softshell clams (Mya arenaria) from areas exposed to 'red tides' are more resistant to PSTs, as demonstrated by whole-nerve assays, and accumulate toxins at greater rates than sensitive clams from unexposed areas. PSTs lead to selective mortality of sensitive clams. Resistance is caused by natural mutation of a single amino acid residue, which causes a 1,000-fold decrease in affinity at the saxitoxin-binding site in the sodium channel pore of resistant, but not sensitive, clams. Thus PSTs might act as potent natural selection agents, leading to greater toxin resistance in clam populations and increased risk of PSP in humans. Furthermore, global expansion of PSP to previously unaffected coastal areas might result in long-term changes to communities and ecosystems.