ABCG2 is a constitutively expressed ATP-binding cassette (ABC) transporter that protects many tissues against xenobiotic molecules. Its activity affects the pharmacokinetics of commonly used drugs ...and limits the delivery of therapeutics into tumour cells, thus contributing to multidrug resistance. Here we present the structure of human ABCG2 determined by cryo-electron microscopy, providing the first high-resolution insight into a human multidrug transporter. We visualize ABCG2 in complex with two antigen-binding fragments of the human-specific, inhibitory antibody 5D3 that recognizes extracellular loops of the transporter. We observe two cholesterol molecules bound in the multidrug-binding pocket that is located in a central, hydrophobic, inward-facing translocation pathway between the transmembrane domains. Combined with functional in vitro analyses, our results suggest a multidrug recognition and transport mechanism of ABCG2, rationalize disease-causing single nucleotide polymorphisms and the allosteric inhibition by the 5D3 antibody, and provide the structural basis of cholesterol recognition by other G-subfamily ABC transporters.
ABCG2 is a transporter protein of the ATP-binding-cassette (ABC) family that is expressed in the plasma membrane in cells of various tissues and tissue barriers, including the blood-brain, ...blood-testis and maternal-fetal barriers
. Powered by ATP, it translocates endogenous substrates, affects the pharmacokinetics of many drugs and protects against a wide array of xenobiotics, including anti-cancer drugs
. Previous studies have revealed the architecture of ABCG2 and the structural basis of its inhibition by small molecules and antibodies
. However, the mechanisms of substrate recognition and ATP-driven transport are unknown. Here we present high-resolution cryo-electron microscopy (cryo-EM) structures of human ABCG2 in a substrate-bound pre-translocation state and an ATP-bound post-translocation state. For both structures, we used a mutant containing a glutamine replacing the catalytic glutamate (ABCG2
), which resulted in reduced ATPase and transport rates and facilitated conformational trapping for structural studies. In the substrate-bound state, a single molecule of estrone-3-sulfate (E
S) is bound in a central, hydrophobic and cytoplasm-facing cavity about halfway across the membrane. Only one molecule of E
S can bind in the observed binding mode. In the ATP-bound state, the substrate-binding cavity has collapsed while an external cavity has opened to the extracellular side of the membrane. The ATP-induced conformational changes include rigid-body shifts of the transmembrane domains, pivoting of the nucleotide-binding domains (NBDs), and a change in the relative orientation of the NBD subdomains. Mutagenesis and in vitro characterization of transport and ATPase activities demonstrate the roles of specific residues in substrate recognition, including a leucine residue that forms a 'plug' between the two cavities. Our results show how ABCG2 harnesses the energy of ATP binding to extrude E
S and other substrates, and suggest that the size and binding affinity of compounds are important for distinguishing substrates from inhibitors.
Several systems, including contractile tail bacteriophages, the type VI secretion system and R-type pyocins, use a multiprotein tubular apparatus to attach to and penetrate host cell membranes. This ...macromolecular machine resembles a stretched, coiled spring (or sheath) wound around a rigid tube with a spike-shaped protein at its tip. A baseplate structure, which is arguably the most complex part of this assembly, relays the contraction signal to the sheath. Here we present the atomic structure of the approximately 6-megadalton bacteriophage T4 baseplate in its pre- and post-host attachment states and explain the events that lead to sheath contraction in atomic detail. We establish the identity and function of a minimal set of components that is conserved in all contractile injection systems and show that the triggering mechanism is universally conserved.
Aims/hypothesis
MicroRNAs regulate a broad range of biological mechanisms. To investigate the relationship between microRNA expression and type 2 diabetes, we compared global microRNA expression in ...insulin target tissues from three inbred rat strains that differ in diabetes susceptibility.
Methods
Using microarrays, we measured the expression of 283 microRNAs in adipose, liver and muscle tissue from hyperglycaemic (Goto–Kakizaki), intermediate glycaemic (Wistar Kyoto) and normoglycaemic (Brown Norway) rats (
n
= 5 for each strain). Expression was compared across strains and validated using quantitative RT-PCR. Furthermore, microRNA expression variation in adipose tissue was investigated in 3T3-L1 adipocytes exposed to hyperglycaemic conditions.
Results
We found 29 significantly differentiated microRNAs (
p
adjusted
< 0.05): nine in adipose tissue, 18 in liver and two in muscle. Of these, five microRNAs had expression patterns that correlated with the strain-specific glycaemic phenotype. MiR-222 (
p
adjusted
= 0.0005) and miR-27a (
p
adjusted
= 0.006) were upregulated in adipose tissue; miR-195 (
p
adjusted
= 0.006) and miR-103 (
p
adjusted
= 0.04) were upregulated in liver; and miR-10b (
p
adjusted
= 0.004) was downregulated in muscle. Exposure of 3T3-L1 adipocytes to increased glucose concentration upregulated the expression of miR-222 (
p
= 0.008), miR-27a (
p
= 0.02) and the previously reported miR-29a (
p
= 0.02). Predicted target genes of these differentially expressed microRNAs are involved in pathways relevant to type 2 diabetes.
Conclusion
The expression patterns of miR-222, miR-27a, miR-195, miR-103 and miR-10b varied with hyperglycaemia, suggesting a role for these microRNAs in the pathophysiology of type 2 diabetes, as modelled by the Gyoto–Kakizaki rat. We observed similar patterns of expression of miR-222, miR-27a and miR-29a in adipocytes as a response to increased glucose levels, which supports our hypothesis that altered expression of microRNAs accompanies primary events related to the pathogenesis of type 2 diabetes.
ABCG2 is an ATP-binding cassette (ABC) transporter that protects tissues against xenobiotics, affects the pharmacokinetics of drugs and contributes to multidrug resistance. Although many inhibitors ...and modulators of ABCG2 have been developed, understanding their structure-activity relationship requires high-resolution structural insight. Here, we present cryo-EM structures of human ABCG2 bound to synthetic derivatives of the fumitremorgin C-related inhibitor Ko143 or the multidrug resistance modulator tariquidar. Both compounds are bound to the central, inward-facing cavity of ABCG2, blocking access for substrates and preventing conformational changes required for ATP hydrolysis. The high resolutions allowed for de novo building of the entire transporter and also revealed tightly bound phospholipids and cholesterol interacting with the lipid-exposed surface of the transmembrane domains (TMDs). Extensive chemical modifications of the Ko143 scaffold combined with in vitro functional analyses revealed the details of ABCG2 interactions with this compound family and provide a basis for the design of novel inhibitors and modulators.
Abstract
We describe data release 3 (DR3) of the Galaxy And Mass Assembly (GAMA) survey. The GAMA survey is a spectroscopic redshift and multiwavelength photometric survey in three equatorial regions ...each of 60.0 deg2 (G09, G12, and G15), and two southern regions of 55.7 deg2 (G02) and 50.6 deg2 (G23). DR3 consists of: the first release of data covering the G02 region and of data on H-ATLAS (Herschel – Astrophysical Terahertz Large Area Survey) sources in the equatorial regions; and updates to data on sources released in DR2. DR3 includes 154 809 sources with secure redshifts across four regions. A subset of the G02 region is 95.5 per cent redshift complete to r < 19.8 mag over an area of 19.5 deg2, with 20 086 galaxy redshifts, that overlaps substantially with the XXL survey (X-ray) and VIPERS (redshift survey). In the equatorial regions, the main survey has even higher completeness (98.5 per cent), and spectra for about 75 per cent of H-ATLAS filler targets were also obtained. This filler sample extends spectroscopic redshifts, for probable optical counterparts to H-ATLAS submillimetre sources, to 0.8 mag deeper (r < 20.6 mag) than the GAMA main survey. There are 25 814 galaxy redshifts for H-ATLAS sources from the GAMA main or filler surveys. GAMA DR3 is available at the survey website (www.gama-survey.org/dr3/).
We present a meta-analysis of star formation rate (SFR) indicators in the Galaxy And Mass Assembly (GAMA) survey, producing 12 different SFR metrics and determining the SFR–M
* relation for each. We ...compare and contrast published methods to extract the SFR from each indicator, using a well-defined local sample of morphologically selected spiral galaxies, which excludes sources which potentially have large recent changes to their SFR. The different methods are found to yield SFR–M
* relations with inconsistent slopes and normalizations, suggesting differences between calibration methods. The recovered SFR–M
* relations also have a large range in scatter which, as SFRs of the targets may be considered constant over the different time-scales, suggests differences in the accuracy by which methods correct for attenuation in individual targets. We then recalibrate all SFR indicators to provide new, robust and consistent luminosity-to-SFR calibrations, finding that the most consistent slopes and normalizations of the SFR–M
* relations are obtained when recalibrated using the radiation transfer method of Popescu et al. These new calibrations can be used to directly compare SFRs across different observations, epochs and galaxy populations. We then apply our calibrations to the GAMA II equatorial data set and explore the evolution of star formation in the local Universe. We determine the evolution of the normalization to the SFR–M
* relation from 0 < z < 0.35 – finding consistent trends with previous estimates at 0.3 < z < 1.2. We then provide the definitive z < 0.35 cosmic star formation history, SFR–M
* relation and its evolution over the last 3 billion years.
Recent improvements in the speed, cost and accuracy of next generation sequencing are revolutionizing the discovery of single nucleotide polymorphisms (SNPs). SNPs are increasingly being used as an ...addition to the molecular ecology toolkit in nonmodel organisms, but their efficient use remains challenging. Here, we discuss common issues when employing SNP markers, including the high numbers of markers typically employed, the effects of ascertainment bias and the inclusion of nonneutral loci in a marker panel. We provide a critique of considerations specifically associated with the application and population genetic analysis of SNPs in nonmodel taxa, focusing specifically on some of the most commonly applied methods.
Whole genome duplication (leading to polyploidy) is widely accepted as an important evolutionary force in plants, but it is less recognized as a driver of animal diversification. Nevertheless, it ...occurs across a wide range of animals; this review investigates why it is particularly common in fish and amphibians, while rare among other vertebrates. We review the current geographic, ecological and phylogenetic distributions of sexually reproducing polyploid taxa before focusing more specifically on what factors drive polyploid formation and establishment. In summary, (1) polyploidy is phylogenetically restricted in both amphibians and fishes, although entire fish, but not amphibian, lineages are derived from polyploid ancestors. (2) Although mechanisms such as polyspermy are feasible, polyploid formation appears to occur principally through unreduced gamete formation, which can be experimentally induced by temperature or pressure shock in both groups. (3) External reproduction and fertilization in primarily temperate freshwater environments potentially exposes zygotes to temperature stress, which can promote increased production of unreduced gametes. (4) Large numbers of gametes and group breeding in relatively confined areas could increase the probability of compatible gamete combinations in both groups. (5) Both fish and amphibians have a propensity to form reproductively successful hybrids; although the relative frequency of autopolyploidy versus allopolyploidy is difficult to ascertain, multiple origins involving hybridization have been confirmed for a number of species in both groups. (6) Problems with establishment of polyploid lineages associated with minority cytotype exclusion could be overcome in amphibians via assortative mating by acoustic recognition of the same ploidy level, but less attention has been given to chemical or acoustic mechanisms that might operate in fish. (7) There is no strong evidence that polyploid fish or amphibians currently exist in more extreme environments than their diploid progenitors or have broader ecological ranges. (8) Although pathogens could play a role in the relative fitness of polyploid species, particularly given duplication of genes involved in immunity, this remains an understudied field in both fish and amphibians. (9) As in plants, many duplicate copies of genes are retained for long periods of time, indicative of selective maintenance of the duplicate copies, but we find no physiological or other reasons that could explain an advantage for allelic or genetic complexity. (10) Extant polyploid species do not appear to be more or less prone to extinction than related diploids in either group. We conclude that, while polyploid fish and amphibians share a number of attributes facilitating polyploidy, clear drivers of genome duplication do not emerge from the comparison. The lack of a clear association of sexually reproducing polyploids with range expansion, harsh environments, or risk of extinction could suggest that stronger correlations in plants may be driven by shifts in mating system more than ploidy. However, insufficient data currently exist to provide rigorous tests of these hypotheses and we make a plea for zoologists to also consider polyploidy as a possibility in continuing taxonomic surveys.
We present an analysis of the mid-infrared Wide-field Infrared Survey Explorer (WISE) sources seen within the equatorial GAMA G12 field, located in the North Galactic Cap. Our motivation is to study ...and characterize the behavior of WISE source populations in anticipation of the deep multiwavelength surveys that will define the next decade, with the principal science goal of mapping the 3D large-scale structures and determining the global physical attributes of the host galaxies. In combination with cosmological redshifts, we identify galaxies from their WISE W1 (3.4 m) resolved emission, and we also perform a star-galaxy separation using apparent magnitude, colors, and statistical modeling of star counts. The resulting galaxy catalog has 590,000 sources in 60 deg2, reaching a W1 5 depth of 31 Jy. At the faint end, where redshifts are not available, we employ a luminosity function analysis to show that approximately 27% of all WISE extragalactic sources to a limit of 17.5 mag (31 Jy) are at high redshift, . The spatial distribution is investigated using two-point correlation functions and a 3D source density characterization at 5 Mpc and 20 Mpc scales. For angular distributions, we find that brighter and more massive sources are strongly clustered relative to fainter sources with lower mass; likewise, based on WISE colors, spheroidal galaxies have the strongest clustering, while late-type disk galaxies have the lowest clustering amplitudes. In three dimensions, we find a number of distinct groupings, often bridged by filaments and superstructures. Using special visualization tools, we map these structures, exploring how clustering may play a role with stellar mass and galaxy type.
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